U.S. patent application number 11/994996 was filed with the patent office on 2009-10-22 for polynucleotide marker genes and their expression, for diagnosis of endotoxemia.
This patent application is currently assigned to ATHLOMICS PTY LTD.. Invention is credited to Richard Bruce Brandon, Mervyn Rees Thomas.
Application Number | 20090264305 11/994996 |
Document ID | / |
Family ID | 37636667 |
Filed Date | 2009-10-22 |
United States Patent
Application |
20090264305 |
Kind Code |
A1 |
Brandon; Richard Bruce ; et
al. |
October 22, 2009 |
Polynucleotide Marker Genes and their Expression, for Diagnosis of
Endotoxemia
Abstract
The present invention discloses disease-associated molecules and
assays, which are useful for diagnosing and assessing those animals
with endotoxemia, and determining those animals at risk of
developing endotoxemia or its sequelae. The invention has practical
use in the early diagnosis of disease, in monitoring an animal's
immune response to the disease, and in enabling better treatment
and management decisions to be made in clinically and
sub-clinically affected animals.
Inventors: |
Brandon; Richard Bruce;
(Queensland, AU) ; Thomas; Mervyn Rees;
(Queensland, AU) |
Correspondence
Address: |
PROSKAUER ROSE LLP
1001 PENNSYLVANIA AVE, N.W.,, SUITE 400 SOUTH
WASHINGTON
DC
20004
US
|
Assignee: |
ATHLOMICS PTY LTD.
Toowong
AU
|
Family ID: |
37636667 |
Appl. No.: |
11/994996 |
Filed: |
July 7, 2006 |
PCT Filed: |
July 7, 2006 |
PCT NO: |
PCT/AU2006/000970 |
371 Date: |
June 9, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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60696776 |
Jul 7, 2005 |
|
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Current U.S.
Class: |
506/9 ;
435/320.1; 435/325; 435/6.17; 435/7.1; 536/23.5; 536/24.31 |
Current CPC
Class: |
G01N 2800/26 20130101;
C12Q 1/6883 20130101; C12Q 1/6809 20130101; C12Q 2600/158 20130101;
G01N 33/56911 20130101 |
Class at
Publication: |
506/9 ; 435/6;
435/7.1; 536/23.5; 435/320.1; 435/325; 536/24.31 |
International
Class: |
C40B 30/04 20060101
C40B030/04; C12Q 1/68 20060101 C12Q001/68; G01N 33/53 20060101
G01N033/53; C12N 15/11 20060101 C12N015/11; C12N 15/00 20060101
C12N015/00; C12N 5/06 20060101 C12N005/06; C07H 21/04 20060101
C07H021/04 |
Claims
1. A method for diagnosing the presence of an endotoxemia-related
condition in a test subject, comprising detecting in the test
subject aberrant expression of at least one endotoxemia marker gene
that is expressed in cells of the immune system and that is
selected from the group consisting of: (a) a gene having a
polynucleotide expression product comprising a nucleotide sequence
that shares at least 50% sequence identity with the sequence set
forth in any one of SEQ ID NO: 1, 3, 4, 5, 6, 7, 9, 10, 11, 13, 15,
16, 17, 18, 19, 21, 23, 25, 26, 27, 29, 31, 33, 35, 37, 38, 39, 41,
42, 43, 44, 45, 47, 49, 50, 52, 54, 56, 58, 60, 61, 63, 64, 66, 67,
68, 69, 70, 71, 73, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 86, 88,
90, 92, 93, 94, 96, 98, 100, 101, 102, 103, 104, 106, 107, 109,
110, 111, 113, 114, 115, 117, 119, 121, 122, 123, 124, 125, 126,
128, 130, 132, 134, 136, 137, 139, 141, 143, 145, 147, 149, 151,
153, 155, 157, 158, 160, 162, 164, 166, 168, 169, 170, 172, 173,
174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 193, 194, 195,
197, 199, 201, 203, 205, 206, 207, 209, 210, 211, 212, 214, 215,
216, 218, 220, 222, 223, 224, 225, 227, 229, 231, 233, 235, 236,
237, 239, 240, 242, 244, 245, 246, 248, 250, 252, 254, 255, 257,
259 260, 262, 264, 266, 268, 269, 270, 271, 272, 274, 276, 278,
279, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302,
304, 305, 3 06, 307, 309, 311, 312, 314, 315, 316, 318, 320 321,
323 or 325, or a complement thereof; (b) a gene having a
polynucleotide expression product comprising a nucleotide sequence
that encodes a polypeptide comprising the amino acid sequence set
forth in any one of SEQ ID NO: 2, 8, 12, 14, 20, 22, 24, 28, 30,
32, 34, 36, 40, 46, 48, 51, 53, 55, 57, 59, 62, 65, 72, 74, 78, 85,
87, 89, 91, 95, 97, 99, 105, 108, 112, 116, 118, 120, 127, 129,
131, 133, 135, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156,
159, 161, 163, 165, 167, 171, 175, 177, 179, 181, 183, 185, 187,
189, 191, 196, 198, 200, 202, 204, 208, 213, 217, 219, 221, 226,
228, 230, 232, 234, 236, 238, 241, 243, 247, 249, 251, 253, 256,
258, 261, 263, 265, 267, 273, 275, 277, 281, 283, 285, 287, 289,
291, 293, 295, 297, 299, 301, 303, 308, 310, 313, 317, 319, 322,
324 or 326; (c) a gene having a polynucleotide expression product
comprising a nucleotide sequence that encodes a polypeptide that
shares at least 50% sequence similarity with at least a portion of
the sequence set forth in SEQ ID NO: 2, 8, 12, 14, 20, 22, 24, 28,
30, 32, 34, 36, 40, 46, 48, 51, 53, 55, 57, 59, 62, 65, 72, 74, 78,
85, 87, 89, 91, 95, 97, 99, 105, 108, 112, 116, 118, 120, 127, 129,
131, 133, 135, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156,
159, 161, 163, 165, 167, 171, 175, 177, 179, 181, 183, 185, 187,
189, 191, 196, 198, 200, 202, 204, 208, 213, 217, 219, 221, 226,
228, 230, 232, 234, 236, 238, 241, 243, 247, 249, 251, 253, 256,
258, 261, 263, 265, 267, 273, 275, 277, 281, 283, 285, 287, 289,
291, 293, 295, 297, 299, 301, 303, 308, 310, 313, 317, 319, 322,
324 or 326, wherein the portion comprises at least 15 contiguous
amino acid residues of that sequence; and (d) a gene having a
polynucleotide expression product comprising a nucleotide sequence
that hybridizes to the sequence of (a), (b), (c) or a complement
thereof, under at least low stringency conditions.
2. A method according to claim 1, comprising detecting aberrant
expression of an endotoxemia marker polynucleotide selected from
the group consisting of (a) a polynucleotide comprising a
nucleotide sequence that shares at least 50% sequence identity with
the sequence set forth in any one of SEQ ID NO: 1, 3, 4, 5, 6, 7,
9, 10, 11, 13, 15, 16, 17, 18, 19, 21, 23, 25, 26,27, 29, 31, 33,
35, 37, 38, 39, 41, 42, 43, 44, 45, 47, 49, 50, 52, 54, 56, 58, 60,
61, 63, 64, 66, 67, 68, 69, 70, 71, 73, 75, 76, 77, 78, 79, 80, 81,
82, 83, 84, 86, 88, 90, 92, 93, 94, 96, 98, 100, 101, 102, 103,
104, 106, 107, 109, 110, 111, 113, 114, 115, 117, 119, 121, 122,
123, 124, 125, 126, 128, 130, 132, 134, 136, 137, 139, 141, 143,
145, 147, 149, 151, 153, 155, 157, 158, 160, 162, 164, 166, 168,
169, 170, 172, 173, 174, 176, 178, 180, 182, 184, 186, 188, 190,
192, 193, 194, 195, 197, 199, 201, 203, 205, 206, 207, 209, 210,
211, 212, 214, 215, 216, 218, 220, 222, 223, 224, 225, 227, 229,
231, 233, 235, 236, 237, 239, 240, 242, 244, 245, 246, 248, 250,
252, 254, 255, 257, 259 260, 262, 264, 266, 268, 269, 270, 271,
272, 274, 276, 278, 279, 280, 282, 284, 321, 323 or 325, or a
complement thereof, (b) a polynucleotide comprising a nucleotide
sequence that encodes a polypeptide comprising the amino acid
sequence set forth in any one of SEQ ID NO: 2, 8, 12, 14, 20, 22,
24, 28, 30, 32, 34, 36, 40, 46, 48, 51, 53, 55, 57, 59, 62, 65, 72,
74, 78, 85, 87, 89, 91, 95, 97, 99, 105, 108, 112, 116, 118, 120,
127, 129, 131, 133, 135, 138, 140, 142, 144, 146, 148, 150, 152,
154, 156, 159, 161, 163, 165, 167, 171, 175, 177, 179, 181, 183,
185, 187, 189, 191, 196, 198, 200, 202, 204, 208, 213, 217, 219,
221, 226, 228, 230, 232, 234, 236, 238, 241, 243, 247, 249, 251,
253, 256, 258, 261, 263, 265, 267, 273, 275, 277, 281, 283, 285,
287, 289, 291, 293, 295, 297, 299, 301, 303, 308, 310, 313, 317,
319, 322, 324 or 326; (c) a polynucleotide comprising a nucleotide
sequence that encodes a polypeptide that shares at least 50%
sequence similarity with at least a portion of the sequence set
forth in SEQ ID NO: 2, 8, 12, 14, 20, 22, 24, 28, 30, 32, 34, 36,
40, 46, 48, 51, 53, 55, 57, 59, 62, 65, 72, 74, 78, 85, 87, 89, 91,
95, 97, 99, 105, 108, 112, 116, 118, 120, 127, 129, 131, 133, 135,
138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 159, 161, 163,
165, 167, 171, 175, 177, 179, 181, 183, 185, 187, 189, 191, 196,
198, 200, 202, 204, 208, 213, 217, 219, 221, 226, 228, 230, 232,
234, 236, 238, 241, 243, 247, 249, 251, 253, 256, 258, 261, 263,
265, 267, 273, 275, 277, 281, 283, 285, 287, 289, 291, 293, 295,
297, 299, 301, 303, 308, 310, 313, 317, 319, 322, 324 or 326;
wherein the portion comprises at least 15 contiguous amino acid
residues of that sequence; and (d) a polynucleotide comprising a
nucleotide sequence that hybridizes to the sequence of (a), (b),
(c) or a complement thereof, under at least low stringency
conditions.
3. A method according to claim 1, comprising detecting aberrant
expression of an endotoxemia marker polypeptide selected from the
group consisting of: (i) a polypeptide comprising an amino acid
sequence that shares at least 50% (sequence similarity with the
sequence set forth in any one of SEQ ID NO: 2, 8, 12, 14, 20, 22,
24, 28,30, 32, 34, 36, 40, 46, 48, 51, 53, 55, 57, 59, 62, 65, 72,
74, 78,85, 87, 89, 91, 95, 97, 99, 105, 108, 112, 116, 118, 120,
127, 129, 131, 133, 135, 138, 140, 142, 144, 146, 148, 150, 152,
154, 156, 159, 161, 163, 165, 167, 171, 175, 177, 179, 181, 183,
185, 187, 189, 191, 196, 198, 200, 202, 204, 208, 213, 217, 219,
221, 226, 228, 230, 232, 234, 236, 238, 241, 243, 247, 249, 251,
253, 256, 258, 261, 263, 265, 267, 273, 275, 277, 281, 283, 285,
287, 289, 291, 293, 295, 297, 299, 301, 303, 308, 310, 313, 317,
319, 322, 324 or 326; (ii) a polypeptide comprising a portion of
the sequence set forth in any one of SEQ ID NO: 2, 8, 12, 14, 20,
22, 24, 28, 30, 32, 34, 36, 40, 46, 48, 51, 53, 55, 57, 59, 62, 65,
72, 74, 78, 85, 87, 89, 91, 95, 97, 99, 105, 108, 112, 116, 118,
120, 127, 129, 131, 133, 135, 138, 140, 142, 144, 146, 148, 150,
152, 154, 156, 159, 161, 163, 165, 167, 171, 175, 177, 179, 181,
183, 185, 187, 189, 191, 196, 198, 200, 202, 204, 208, 213, 217,
219, 221, 226, 228, 230, 232, 234, 236, 238, 241, 243, 247, 249,
251, 253, 256, 258, 261, 263, 265, 267, 273, 275, 277, 281, 283,
285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 308, 310, 313,
317, 319, 322, 324 or 326, wherein the portion comprises at least 5
contiguous amino acid residues of that sequence; (iii) a
polypeptide comprising an amino acid sequence that shares at least
30% similarity with at least 15 contiguous amino acid residues of
the sequence set forth in any one of SEQ ID NO: 2, 8, 12, 14, 20,
22, 24, 28, 30, 32, 34, 36, 40, 46, 48, 51, 53, 55, 57, 59,62, 65,
72, 74, 78, 85, 87, 89, 91, 95, 97, 99, 105, 108, 112, 116, 118,
120, 127, 129, 131, 133, 135, 138, 140, 142, 144, 146, 148, 150,
152, 154, 156, 159, 161, 163, 165, 167, 171, 175, 177, 179, 181,
183, 185, 187, 189, 191, 196, 198, 200, 202, 204, 208, 213, 217,
219, 221, 226, 228, 230, 232, 234, 236, 238, 241, 243, 247, 249,
251, 253, 256, 258, 261, 263, 265, 267, 273, 275, 277, 281, 283,
285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 308, 310, 313,
317, 319, 322, 324 or 326; and (iv) a polypeptide comprising a
portion of the sequence set forth in any one of SEQ ID: 2, 8, 12,
14, 20, 22, 24, 28, 30, 32, 34, 36, 40, 46, 48, 51, 53, 55, 57, 59,
62, 65, 72, 74, 78, 85, 87, 89, 91, 95, 97, 99, 105, 108, 112, 116,
118, 120, 127, 129, 131, 133, 135, 138, 140, 142, 144, 146, 148,
150, 152, 154, 156, 159, 161, 163, 165, 167, 171, 175, 177, 179,
181, 183, 185, 187, 189, 191, 196, 198, 200, 202, 204, 208, 213,
217, 219, 221, 226, 228, 230, 232, 234, 236, 238, 241, 243, 247,
249, 251, 253, 256, 258, 261, 263, 265, 267, 273, 275, 277, 281,
283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 308, 310,
313, 317, 319, 322, 324 or 326, wherein the portion comprises at
least 5 contiguous amino acid residues of that sequence and is
immuno-interactive with an antigen-binding molecule that is
immuno-interactive with a sequence of (i), (ii) or (iii).
4. A method according to claim 1, wherein the aberrant expression
is detected by: (1) measuring in a biological sample obtained from
the test subject the level or functional activity of an expression
product of at least one endotoxemia marker gene and (2) comparing
the measured level or functional activity of each expression
product to the level or functional activity of a corresponding
expression product in a reference sample obtained from one or more
normal subjects or from one or more subjects lacking an
endotoxemia-related condition, wherein a difference in the level or
functional activity of the expression product in the biological
sample as compared to the level or functional activity of the
corresponding expression product in the reference sample is
indicative of the presence of an endotoxemia-related condition in
the test subject.
5. A method according to claim 4, further comprising diagnosing the
presence, stage or degree of an endotoxemia-related condition in
the test subject when the measured level or functional activity of
the or each expression product is 10% higher than the measured
level or functional activity of the or each corresponding
expression product.
6. A method according to claim 5, wherein the presence of
endotoxemia-related condition is determined by detecting an
increase in the level or functional activity of at least one
endotoxemia marker polynucleotide selected from (a) a
polynucleotide comprising a nucleotide sequence that shares at
least 50% sequence identity with the sequence set forth in any one
of SEQ ID NO: 1, 3, 4, 5, 6, 7, 9, 10, 11, 13, 15, 16, 17, 18, 19,
21, 23, 25,26, 27, 29, 31, 33, 35, 37, 38, 39, 41, 42, 43, 44, 45,
47, 49, 50, 52, 54, 56, 58, 60, 61, 63, 64, 66, 67, 68, 69, 70, 71,
73, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 86, 88, 90, 92, 93, 94,
96, 98, 100, 101, 102, 103, 104, 106, 107, 109, 110, 111, 113, 114,
115, 117, 119, 121, 122, 123, 124, 125, 126, 128, 130, 132, 134,
136, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 158,
160, 162, 164, 166, 168, 169, 170, 172, 173, 174, 176, 178, 180,
182, 184, 186, 188, 190, 192, 193, 194, 195, 197, 199, 201, 203,
205, 206, 207, 209, 210, 211, 212, 214, 215, 216, 218, 220, 222,
223, 224, 225, 227, 229, 231, 233, 235, 236, 237, 239, 240, 242,
244, 245, 246, 248, 250, 252, 254, 255, 257, 259 260, 262, 264,
266, 268, 269, 270, 271, 272, 274, 276, 278, 279, 280, 282, 284,
286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 305, 306, 307,
309, 311, 312, 314, 315, 316, 318, 320 321, 323 or 325, or a
complement thereof, (b) a polynucleotide comprising a nucleotide
sequence that encodes a polypeptide comprising the amino acid
sequence set forth in any one of SEQ ID NO: 2, 8, 12, 14, 20, 22,
24, 28, 30, 32, 34, 36, 40, 46, 48, 51, 53, 55, 57, 59, 62, 65, 72,
74, 78, 85, 87, 89, 91, 95, 97, 99, 105, 108, 112, 116, 118, 120,
127, 129, 131, 133, 135, 138, 140, 142, 144, 146, 148, 150, 152,
154, 156, 159, 161, 163, 165, 167, 171, 175, 177, 179, 181, 183,
185, 187, 189, 191, 196, 198, 200, 202, 204, 208, 213, 217, 219,
221, 226, 228, 230, 232, 234, 236, 238, 241, 243, 247, 249, 251,
253, 256, 258, 261, 263, 265, 267, 273, 275, 277, 281, 283, 285,
287, 289, 291, 293, 295, 297, 299, 301, 303, 308, 310, 313, 317,
319, 322, 324 or 326; (c) a polynucleotide comprising a nucleotide
sequence that encodes a polypeptide that shares at least 50%
sequence similarity with at least a portion of the sequence set
forth in SEQ ID NO: 2, 8, 12, 14, 20, 22, 24, 28, 30, 32, 34, 36,
40, 46, 48, 51, 53, 55, 57, 59, 62, 65, 72, 74, 78, 85, 87, 89, 91,
95, 97, 99, 105, 108, 112, 116, 118, 120, 127, 129, 131, 133, 135,
138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 159, 161, 163,
165, 167, 171, 175, 177, 179, 181, 183, 185, 187, 189, 191, 196,
198, 200, 202, 204, 208, 213, 217, 219, 221, 226, 228, 230, 232,
234, 236, 238, 241, 243, 247, 249, 251, 253, 256, 258, 261, 263,
265, 267, 273, 275, 277, 281, 283, 285, 287, 289, 291, 293, 295,
297, 299, 301, 303, 308, 310, 313, 317, 319, 322, 324 or 326,
wherein the portion comprises at least 15 contiguous amino acid
residues of that sequence; and (d) a polynucleotide comprising a
nucleotide sequence that hybridizes to the sequence of (a), (b),
(c) or a complement thereof, under at least low stringency
conditions.
7. A method according to claim 4, further comprising diagnosing the
presence, stage or degree of an endotoxemia-related condition in
the test subject when the measured level or functional activity of
the or each expression product is 10% lower than the measured level
or functional activity of the or each corresponding expression
product.
8. A method according to claim 7, wherein the presence of an
endotoxemia-related condition is determined by detecting a decrease
in the level or functional activity of at least one endotoxemia
marker polynucleotide selected from (a) a polynucleotide comprising
a nucleotide sequence that shares at least 50% sequence identity
with the sequence set forth in any one of SEQ ID NO: 5, 6, 7, 11,
13, 15, 16, 17, 18, 19, 21, 25, 26,35, 37, 38, 41, 42, 43, 44, 50,
52, 54, 58, 60, 63, 64, 69, 70, 71, 73, 77, 79, 80, 81, 82, 83, 86,
92, 93, 96, 98, 100, 101, 102, 103, 104, 106, 115, 117, 128, 134,
137, 141, 143, 153, 155, 158, 162, 164, 166, 174, 182, 186, 188,
190, 195, 197, 199, 201, 205, 206, 207, 209, 210, 211, 214, 215,
216, 222, 223, 240, 244, 246, 248, 259, 260, 269, 272, 280, 282,
284, 286, 290, 298, 300, 302, 305, 306, 307, 309, 312, 314, 315,
316, 318, 320, 321, 323, or a complement thereof, (b) a
polynucleotide comprising a nucleotide sequence that encodes a
polypeptide comprising the amino acid sequence set forth in any one
of SEQ ID NO: 8, 12, 14, 20, 22, 36, 51, 53, 55, 59, 65, 72, 74,
78, 87, 97, 99, 105, 116, 118, 129, 135, 138, 142, 144, 154, 156,
159, 163, 165, 167, 175, 183, 187, 189, 191, 196, 198, 200, 202,
208, 217, 241, 247, 249, 261, 273, 281, 283, 285, 287, 291, 299,
301, 303, 308, 310, 313, 317, 319, 322, 324; (c) a polynucleotide
comprising a nucleotide sequence that encodes a polypeptide that
shares at least 50% sequence similarity with at least a portion of
the sequence set forth in SEQ ID NO: 8, 12, 14, 20, 22, 36, 51, 53,
55, 59, 65, 72, 74, 78, 87, 97, 99, 105, 116, 118, 129, 135, 138,
142, 144, 154, 156, 159, 163, 165, 167, 175, 183, 187, 189, 191,
196, 198, 200, 202, 208, 217, 241, 247, 249, 261, 273, 281, 283,
285, 287, 291, 299, 301, 303, 308, 310, 313, 317, 319, 322, 324,
wherein the portion comprises at least 15 contiguous amino acid
residues of that sequence; and (d) a polynucleotide comprising a
nucleotide sequence that hybridizes to the sequence of (a), (b),
(c) or a complement thereof, under at least low stringency
conditions.
9. A method according to claim 4, further comprising diagnosing the
absence of an endotoxemia-related condition when the measured level
or functional activity of the or each expression product is the
same as or similar to the measured level or functional activity of
the or each corresponding expression product.
10. A method according to claim 4, wherein the measured level or
functional activity of an individual expression product varies from
the measured level or functional activity of an individual
corresponding expression product by no more than about 5%.
11. A method according to claim 4, comprising measuring the level
or functional activity of individual expression products of at
least about two endotoxemia marker genes.
12. A method according to claim 4, comprising measuring the level
or functional activity of individual expression products of at
least one level one correlation endotoxemia marker gene selected
from: (a) a polynucleotide comprising a nucleotide sequence that
shares at least 50% sequence identity with the sequence set forth
in any one of SEQ ID NO: 11, 23, 29, 35, 43, 44, 68, 81, 82, 84,
104, 105, 107, 119, 130, 136, 147, 155, 174, 192, 193, 245, 254,
255, 262, 264, 270, 271, 279, 296, or 325, or a complement thereof;
(b) a polynucleotide comprising a nucleotide sequence that encodes
a polypeptide comprising the amino acid sequence set forth in any
one of SEQ ID NO: 12, 24, 30, 36, 85, 108, 120, 131, 148, 156, 175,
256, 263, 265, 297, or 326; (c) a polynucleotide comprising a
nucleotide sequence that encodes a polypeptide that shares at least
50% sequence similarity with at least a portion of the sequence set
forth in SEQ ID NO: 12, 24, 30, 36, 85, 108, 120, 131, 148, 156,
175, 256, 263, 265, 297, or 326, wherein the portion comprises at
least 15 contiguous amino acid residues of that sequence; and (d) a
polynucleotide comprising a nucleotide sequence that hybridizes to
the sequence of (a), (b), (c) or a complement thereof, under at
least low stringency conditions.
13. A method according to claim 4, comprising measuring the level
or functional activity of individual expression products of at
least one level two correlation endotoxemia marker gene selected
from: (a) a polynucleotide comprising a nucleotide sequence that
shares at least 50% (sequence identity with the sequence set forth
in any one of SEQ ID NO: 1, 7, 9, 10, 17, 18, 21, 25, 26, 33, 54,
61, 64, 79, 80, 90, 94, 115, 117, 121, 122, 125, 126, 143, 160,
162, 164, 172, 173, 178, 184, 186, 194, 199, 205, 206, 225, 229,
242, 244, 252, 257, 259, 274, 276, 282, 284, 288, 294, 306, 316, or
318, or a complement thereof; (b) a polynucleotide comprising a
nucleotide sequence that encodes a polypeptide comprising the amino
acid sequence set forth in any one of SEQ ID NO: 2, 8, 22, 34, 55,
62, 65, 91, 95, 116, 118, 127, 144, 161, 163, 165, 179, 185, 187,
200, 226, 230, 243, 253, 258, 275, 277, 283, 285, 289, 295, 317, or
319, (c) a polynucleotide comprising a nucleotide sequence that
encodes a polypeptide that shares at least 50% sequence similarity
with at least a portion of the sequence set forth in SEQ ID NO: 2,
8, 22, 34, 55, 62, 65, 91, 95, 116, 118, 127, 144, 161, 163, 165,
179, 185, 187, 200, 226, 230, 243, 253, 258, 275, 277, 283, 285,
289, 295, 317, or 319, wherein the portion comprises at least 15
contiguous amino acid residues of that sequence; and (d) a
polynucleotide comprising a nucleotide sequence that hybridizes to
the sequence of (a), (b), (c) or a complement thereof, under at
least low stringency conditions.
14. A method according to claim 4, comprising measuring the level
or functional activity of individual expression products of at
least one level three correlation endotoxemia marker gene selected
from: (a) a polynucleotide comprising a nucleotide sequence that
shares at least 50% sequence identity with the sequence set forth
in any one of SEQ ID NO: 3, 4, 5, 6, 13, 15, 16, 27, 31, 37, 38,
39, 41, 42, 45, 47, 49, 52, 56, 58, 63, 66, 67, 69, 70, 71, 77, 83,
86, 88, 96, 98, 100, 101, 106, 109, 110, 111, 113, 114, 128, 132,
134, 137, 139, 141, 145, 149, 151, 153, 157, 158, 166, 168, 169,
176, 180, 188, 190, 197, 203, 207, 209, 210, 211, 214, 215, 218,
220, 222, 223, 224, 231, 233, 236, 237, 239, 240, 241, 246, 250,
260, 266, 268, 269, 272, 278, 280, 286, 290, 292, 300, 304, 309,
312, 314, 315, 321, or 323, or a complement thereof; (b) a
polynucleotide comprising a nucleotide sequence that encodes a
polypeptide comprising the amino acid sequence set forth in any one
of SEQ ID NO: 14, 28, 32, 40, 46, 48, 53, 57, 59, 72, 78, 87, 89,
97, 99, 112, 129, 133, 135, 138, 140, 142, 146, 150, 152, 154, 159,
167, 177, 181, 189, 191, 198, 204, 208, 219, 221, 232, 234, 238,
247, 251, 261, 267, 273, 281, 287, 291, 293, 301, 310, 313, 322, or
324; (c) a polynucleotide comprising a nucleotide sequence that
encodes a polypeptide that shares at least 50% sequence similarity
with at least a portion of the sequence set forth in SEQ ID NO: 14,
28, 32, 40, 46, 48, 53, 57, 59, 72, 78, 87, 89, 97, 99, 112, 129,
133, 135, 138, 140, 142, 146, 150, 152, 154, 159, 167, 177, 181,
189, 191, 198, 204, 208, 219, 221, 232, 234, 238, 247, 251, 261,
267, 273, 281, 287, 291, 293, 301, 310, 313, 322, or 324, wherein
the portion comprises at least 15 contiguous amino acid residues of
that sequence; and (d) a polynucleotide comprising a nucleotide
sequence that hybridizes to the sequence of (a), (b), (c) or a
complement thereof, under at least low stringency conditions.
15. A method according to claim 4, comprising measuring the level
or functional activity of individual expression products of at
least one level four correlation endotoxemia marker gene selected
from: (a) a polynucleotide comprising a nucleotide sequence that
shares at least 50% sequence identity with the sequence set forth
in any one of SEQ ID NO: 19, 50, 60, 73, 75, 92, 93, 102, 103, 123,
124, 170, 182, 195, 201, 212, 216, 227, 235, 248, 298, 302, 305,
307, 311, or 320, or a complement thereof; (b) a polynucleotide
comprising a nucleotide sequence that encodes a polypeptide
comprising the amino acid sequence set forth in any one of SEQ ID
NO: 20, 51, 74, 76, 171, 183, 196, 202, 213, 217, 228, 249, 299,
303, or 308; (c) a polynucleotide comprising a nucleotide sequence
that encodes a polypeptide that shares at least 50% sequence
similarity with at least a portion of the sequence set forth in SEQ
ID NO: 20, 51, 74, 76, 171, 183, 196, 202, 213, 217, 228, 249, 299,
303, or 308, wherein the portion comprises at least 15 contiguous
amino acid residues of that sequence; and (d) a polynucleotide
comprising a nucleotide sequence that hybridizes to the sequence of
(a), (b), (c) or a complement thereof, under at least low
stringency conditions.
16. A method according to claim 4, wherein the biological sample
comprises blood.
17. A method according to claim 4, wherein the biological sample
comprises peripheral blood.
18. A method according to claim 4, wherein the biological sample
comprises leukocytes.
19. A method according to claim 4, wherein the expression product
is a RNA molecule.
20. A method according to claim 4, wherein the expression product
is a polypeptide.
21. A method according to claim 4, wherein the expression product
is the same as the corresponding expression product.
22. A method according to claim 4, wherein the expression product
is a variant of the corresponding expression product.
23. A method according to claim 4, wherein the expression product
or corresponding expression product is a target RNA or a DNA copy
of the target RNA whose level is measured using at least one
nucleic acid probe that hybridizes under at least low stringency
conditions to the target RNA or to the DNA copy, wherein the
nucleic acid probe comprises at least 15 contiguous nucleotides of
an endotoxemia marker polynucleotide.
24. A method according to claim 23, wherein the measured level or
abundance of the target RNA or its DNA copy is normalized to the
level or abundance of a reference RNA or a DNA copy of the
reference RNA that is present in the same sample.
25. A method according to claim 23, wherein the nucleic acid probe
is immobilized on a solid or semi-solid support.
26. A method according to claim 23, wherein the nucleic acid probe
forms part of a spatial array of nucleic acid probes.
27. A method according to claim 23, wherein the level of nucleic
acid probe that is bound to the target RNA or to the DNA copy is
measured by hybridization.
28. A method according to claim 23, wherein the level of nucleic
acid probe that is bound to the target RNA or to the DNA copy is
measured by nucleic acid amplification.
29. A method according to claim 23, wherein the level of nucleic
acid probe that is bound to the target RNA or to the DNA copy is
measured by nuclease protection assay.
30. A method according to claim 23, wherein the probe for detecting
the endotoxemia marker polynucleotide comprises a sequence as set
forth in any one of SEQ ID NO: 327-2317.
31. A method according to claim 23, wherein the expression product
or corresponding expression product is a target polypeptide whose
level is measured using at least one antigen-binding molecule that
is immuno-interactive with the target polypeptide.
32. A method according to claim 23, wherein the measured level of
the target polypeptide is normalized to the level of a reference
polypeptide that is present in the same sample.
33. A method according to claim 23, wherein the antigen-binding
molecule is immobilized on a solid or semi-solid support.
34. A method according to claim 23, wherein the antigen-binding
molecule forms part of a spatial array of antigen-binding
molecule.
35. A method according to claim 23, wherein the level of
antigen-binding molecule that is bound to the target polypeptide is
measured by immunoassay.
36. A method according to claim 4, wherein the expression product
or corresponding expression product is a target polypeptide whose
level is measured using at least one substrate for the target
polypeptide with which it reacts to produce a reaction product.
37. A method according to claim 36, wherein the measured functional
activity of the target polypeptide is normalized to the functional
activity of a reference polypeptide that is present in the same
sample.
38. A method according to claim 4, wherein a system is used to
perform the method, which comprises at least one end station
coupled to a base station., wherein the base station is caused (a)
to receive subject data from the end station via a communications
network, wherein the subject data represents parameter values
corresponding to the measured or normalized level or functional
activity of at least one expression product in the biological
sample, and (b) to compare the subject data with predetermined data
representing the measured or normalized level or functional
activity of at least one corresponding expression product in the
reference sample to thereby determine any difference in the level
or functional activity of the expression product in the biological
sample as compared to the level or functional activity of the
corresponding expression product in the reference sample.
39. A method according to claim 38, wherein the base station is
further caused to provide a diagnosis for the presence, absence or
degree of an endotoxemia-related condition.
40. A method according to claim 38, wherein the base station is
further caused to transfer an indication of the diagnosis to the
end station via the communications network.
41. A method according to claim 1, wherein detection of the
aberrant expression is indicative of the presence or risk of an
endotoxemia-related condition.
42. A method according to claim 1, wherein the test subject is a
horse.
43. A method for treating, preventing or inhibiting the development
of an endotoxemia-related condition in a subject, the method
comprising detecting aberrant expression of at least one
endotoxemia marker gene in the subject, and administering to the
subject an effective amount of an agent that treats or ameliorates
the symptoms or reverses or inhibits the development of the
endotoxemia-related condition in the subject, wherein the
endotoxemia marker gene is expressed in cells of the immune system
and is selected from the group consisting of: (a) a gene having a
polynucleotide expression product comprising a nucleotide sequence
that shares at least 50% sequence identity with the sequence set
forth in any one of SEQ ID NO: 1, 3, 4, 5, 6, 7, 9, 10, 11, 13, 15,
16, 17, 18, 19, 21, 23, 25, 26, 27, 29, 31, 33, 35, 37, 38, 39, 41,
42, 43, 44, 45, 47, 49, 50, 52, 54, 56, 58, 60, 61, 63, 64, 66, 67,
68, 69, 70, 71, 73, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 86, 88,
90, 92, 93, 94, 96, 98, 100, 101, 102, 103, 104, 106, 107, 109,
110, 111, 113, 114, 115, 117, 119, 121, 122, 123, 124, 125, 126,
128, 130, 132, 134, 136, 137, 139, 141, 143, 145, 147, 149, 151,
153, 155, 157, 158, 160, 162, 164, 166, 168, 169, 170, 172, 173,
174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 193, 194, 195,
197, 199, 201, 203, 205, 206, 207, 209, 210, 211, 212, 214, 215,
216, 218, 220, 222, 223, 224, 225, 227, 229, 231, 233, 235, 236,
237, 239, 240, 242, 244, 245, 246, 248, 250, 252, 254, 255, 257,
259 260, 262, 264, 266, 268, 269, 270, 271, 272, 274, 276, 278,
279, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302,
304, 305, 306, 307, 309, 311, 312, 314, 315, 316, 318, 320 321, 323
or 325; or a complement thereof; (b) a gene having a polynucleotide
expression product comprising a nucleotide sequence that encodes a
polypeptide comprising the amino acid sequence set forth in any one
of SEQ ID NO: 2, 8, 12, 14, 20, 22, 24, 28, 30, 32, 34, 36, 40, 46,
48, 51, 53, 55, 57, 59, 62, 65, 72, 74, 78, 85, 87, 89, 91, 95, 97,
99, 105, 108, 112, 116, 118, 120, 127, 129, 131, 133, 135, 138,
140, 142, 144, 146, 148, 150, 152, 154, 156, 159, 161, 163, 165,
167, 171, 175, 177, 179, 181, 183, 185, 187, 189, 191, 196, 198,
200, 202, 204, 208, 213, 217, 219, 221, 226, 228, 230, 232, 234,
236, 238, 241, 243, 247, 249, 251, 253, 256, 258, 261, 263, 265,
267, 273, 275, 277, 281, 283, 285, 287, 289, 291, 293, 295, 297,
299, 301, 303, 308, 310, 313, 317, 319, 322, 324 or 326; (c) a gene
having a polynucleotide expression product comprising a nucleotide
sequence that encodes a polypeptide that shares at least 50%
sequence similarity with at least a portion of the sequence set
forth in SEQ ID NO: 2, 8, 12, 14, 20, 22, 24, 28, 30, 32, 34, 36,
40, 46, 48, 51, 53, 55, 57, 59, 62, 65, 72, 74, 78, 85, 87, 89, 91,
95, 97, 99, 105, 108, 112, 116, 118, 120, 127, 129, 131, 133, 135,
138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 159, 161, 163,
165, 167, 171, 175, 177, 179, 181, 183, 185, 187, 189, 191, 196,
198, 200, 202, 204, 208, 213, 217, 219, 221, 226, 228, 230, 232,
234, 236, 238, 241, 243, 247, 249, 251, 253, 256, 258, 261, 263,
265, 267, 273, 275, 277, 281, 283, 285, 287, 289, 291, 293, 295,
297, 299, 301, 303, 308, 310, 313, 317, 319, 322, 324 or 326,
wherein the portion comprises at least 15 contiguous amino acid
residues of that sequence; and (d) a gene having a polynucleotide
expression product comprising a nucleotide sequence that hybridizes
to the sequence of (a), (b), (c) or a complement thereof, under at
least low stringency conditions.
44. An isolated endotoxemia marker polynucleotide selected from:
(a) a polynucleotide comprising a nucleotide sequence that shares
at least 50% sequence identity with the sequence set forth in any
one of SEQ ID NO: 1, 3, 4, 5, 6, 7, 9, 10, 11, 13, 15, 16, 17, 18,
19, 21, 23, 25, 26, 27, 29, 31, 33, 35, 37, 38, 39, 41, 42, 43, 44,
45, 47, 49, 50, 52, 54, 56, 58, 60, 61, 63, 64, 66, 67, 68, 69, 70,
71, 73, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 86, 88, 90, 92, 93,
94, 96, 98, 100, 101, 102, 103, 104, 106, 107, 109, 110, 111, 113,
114, 115, 117, 119, 121, 122, 123, 124, 125, 126, 128, 130, 132,
134, 136, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157,
158, 160, 162, 164, 166, 168, 169, 170, 172, 173, 174, 176, 178,
180, 182, 184, 186, 188, 190, 192, 193, 194, 195, 197, 199, 201,
203, 205, 206, 207, 209, 210, 211, 212, 214, 215, 216, 218, 220,
222, 223, 224, 225, 227, 229, 231, 233, 235, 236, 237,239, 240,
242, 244, 245, 246, 248, 250, 252, 254, 255, 257, 259 260, 262,
264, 266, 268, 269, 270, 271, 272, 274, 276, 278, 279, 280, 282,
284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 305, 306,
307, 309, 311, 312, 314, 315, 316, 318, 320 321, 323 or 325, or a
complement thereof, (b) a polynucleotide comprising a portion of
the sequence set forth in any one of SEQ ID NO: 1, 3, 4, 5, 6, 7,
9, 10, 11, 13, 15, 16, 17, 18, 19, 21, 23, 25, 26, 27, 29, 31, 33,
35, 37, 38, 39, 41, 42, 43, 44, 45, 47, 49, 50, 52, 54, 56, 58, 60,
61, 63, 64, 66, 67, 68, 69, 70, 71, 73, 75, 76, 77, 78, 79, 80, 81,
82, 83, 84, 86, 88, 90, 92, 93, 94, 96, 98, 100, 101, 102, 103,
104, 106, 107, 109, 110, 111, 113, 114, 115, 117, 119, 121, 122,
123, 124, 125, 126, 128, 130, 132, 134, 136, 137, 139, 141, 143,
145, 147, 149, 151, 153, 155, 157, 158, 160, 162, 164, 166, 168,
169, 170, 172, 173, 174, 176, 178, 180, 182, 184, 186, 188, 190,
192, 193, 194, 195, 197, 199, 201, 203, 205, 206, 207, 209, 210,
211, 212, 214, 215, 216, 218, 220, 222, 223, 224, 225, 227, 229,
231, 233, 235, 236, 237, 239, 240, 242, 244, 245, 246, 248, 250,
252, 254, 255, 257, 259 260, 262, 264, 266, 268, 269, 270, 271,
272, 274, 276, 278, 279, 280, 282, 284, 286, 288, 290, 292, 294,
296, 298, 300, 302, 304, 305, 306, 307, 309, 311, 312, 314, 315,
316, 318, 320 321, 323 or 325, or a complement thereof, wherein the
portion comprises at least 15 contiguous nucleotides of that
sequence or complement; (c) a polynucleotide that hybridizes to the
sequence of (a) or (b) or a complement thereof, under at least low
stringency conditions; and (d) a polynucleotide comprising a
portion of any one of SEQ ID NO: 1, 3, 4, 5, 6, 7, 9, 10, 11, 13,
15, 16, 17, 18, 19, 21, 23, 25,26, 27, 29, 31, 33, 35, 37, 38, 39,
41, 42, 43, 44, 45, 47, 49, 50, 52, 54, 56, 58, 60, 61, 63, 64, 66,
67, 68, 69, 70, 71, 73, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 86,
88, 90, 92, 93, 94, 96, 98, 100, 101, 102, 103, 104, 106, 107, 109,
110, 111, 113, 114, 115, 117, 119, 121, 122, 123, 124, 125, 126,
128, 130, 132, 134, 136, 137, 139, 141, 143, 145, 147, 149, 151,
153, 155, 157, 158, 160, 162, 164, 166, 168, 169, 170, 172, 173,
174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 193, 194, 195,
197, 199, 201, 203, 205, 206, 207, 209, 210, 211, 212, 214, 215,
216, 218, 220, 222, 223, 224, 225, 227, 229, 231, 233, 235, 236,
237, 239, 240, 242, 244, 245, 246, 248, 250, 252, 254, 255, 257,
259 260, 262, 264, 266, 268, 269, 270, 271, 272, 274, 276, 278,
279, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302,
304, 305, 306, 307, 309, 311, 312, 314, 315, 316, 318, 320321, 323
or 325, or a complement thereof, wherein the portion comprises at
least 15 contiguous nucleotides of that sequence or complement and
hybridizes to a sequence of (a), (b) or (c), or a complement
thereof, under at least low stringency conditions.
45. A nucleic acid construct comprising an endotoxemia marker
polynucleotide according to claim 44, in operable connection with a
regulatory element that is operable in a host cell.
46. An isolated host cell containing a nucleic acid construct
according to claim 45.
47. A probe comprising a nucleotide sequence that hybridizes under
at least low stringency conditions to a polynucleotide according to
claim 44.
48. A probe according to claim 47, consisting essentially of a
nucleic acid sequence that corresponds or is complementary to at
least a portion of a nucleotide sequence encoding the amino acid
sequence set forth in any one of SEQ ID NO: 2, 8, 12, 14, 20, 22,
24, 28, 30, 32, 34, 36, 40, 46, 48, 51, 53, 55, 57, 59, 62, 65, 72,
74, 78, 85, 87, 89, 91, 95, 97, 99, 105, 108, 112, 116, 118, 120,
127, 129, 131, 133, 135, 138, 140, 142, 144, 146, 148, 150, 152,
154, 156, 159, 161, 163, 165, 167, 171, 175, 177, 179, 181, 183,
185, 187, 189, 191, 196, 198, 200, 202, 204, 208, 213, 217, 219,
221, 226, 228, 230, 232, 234, 236, 238, 241, 243, 247, 249, 251,
253, 256, 258, 261, 263, 265, 267, 273, 275, 277, 281, 283, 285,
287, 289, 291, 293, 295, 297, 299, 301, 303, 308, 310, 313, 317,
319, 322, 324 or 326, wherein the portion is at least 15
nucleotides in length.
49. A probe according to claim 47, wherein the probe comprises a
nucleotide sequence which is capable of hybridizing to at least a
portion of a nucleotide sequence encoding the amino acid sequence
set forth in any one of SEQ ID NO: 2, 8, 12, 14, 20, 22, 24, 28,
30, 32, 34, 36, 40, 46, 48, 51, 53, 55, 57, 59, 62, 65, 72, 74, 78,
85, 87, 89, 91, 95, 97, 99, 105, 108, 112, 116, 118, 120, 127, 129,
131, 133, 135, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156,
159, 161, 163, 165, 167, 171, 175, 177, 179, 181, 183, 185, 187,
189, 191, 196, 198, 200, 202, 204, 208, 213, 217, 219, 221, 226,
228, 230, 232, 234, 236, 238, 241, 243, 247, 249, 251, 253, 256,
258, 261, 263, 265, 267, 273, 275, 277, 281, 283, 285, 287, 289,
291, 293, 295, 297, 299, 301, 303, 308, 310, 313, 317, 319, 322,
324 or 326; under at least low stringency conditions, wherein the
portion is at least 15 nucleotides in length.
50. A probe according to claim 47, wherein the probe comprise a
nucleotide sequence that is capable of hybridizing to at least a
portion of any one of SEQ ID NO: 1, 3, 4, 5, 6, 7, 9, 10, 11, 13,
15, 16, 17, 18, 19, 21, 23, 25, 26, 27, 29, 31, 33, 35, 37, 38, 39,
41, 42, 43, 44, 45, 47, 49, 50, 52, 54, 56, 58, 60, 61, 63, 64, 66,
67, 68, 69, 70, 71, 73, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 86,
88, 90, 92, 93, 94, 96, 98, 100, 101, 102, 103, 104, 106, 107, 109,
110, 111, 113, 114, 115, 117, 119, 121, 122, 123, 124, 125, 126,
128, 130, 132, 134, 136, 137, 139, 141, 143, 145, 147, 149, 151,
153, 155, 157, 158, 160, 162, 164, 166, 168, 169, 170, 172, 173,
174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 193, 194, 195,
197, 199, 201, 203, 205, 206, 207, 209, 210, 211, 212, 214, 215,
216, 218, 220, 222, 223, 224, 225, 227, 229, 231, 233, 235, 236,
237, 239, 240, 242, 244, 245, 246, 248, 250, 252, 254, 255, 257,
259 260, 262, 264, 266, 268, 269, 270, 271, 272, 274, 276, 278,
279, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302,
304, 305, 306, 307, 309, 311, 312, 314, 315, 316, 318, 320 321, 323
or 325, under at least low stringency conditions, wherein the
portion is at least 15 nucleotides in length.
51. A probe according to claim 47, comprising a sequence as set
forth in any one of SEQ ID NO: 145-2150.
52. A solid or semi-solid support comprising at least one probe
according to claim 47 immobilized thereon.
53. A kit comprising one or more endotoxemia marker polynucleotides
according to claim 44.
54. A method for diagnosing the presence of an endotoxemia-related
condition in a test subject, comprising detecting in the test
subject aberrant expression of at least one endotoxemia marker
polynucleotide that is expressed in cells of the immune system and
that is selected from the group consisting of: (a) a polynucleotide
comprising a nucleotide sequence that shares at least 50% sequence
identity with the sequence set forth in any one of SEQ ID NO: 1, 3,
4, 5, 6, 7, 9, 10, 11, 13, 15, 16, 17, 18, 19, 21, 23, 25, 26, 27,
29, 31, 33, 35, 37, 38, 39, 41, 42, 43, 44, 45, 47, 49, 50, 52, 54,
56, 58, 60, 61, 63, 64, 66, 67, 68, 69, 70, 71, 73, 75, 76, 77, 78,
79, 80, 81, 82, 83, 84, 86, 88, 90, 92, 93, 94, 96, 98, 100, 101,
102, 103, 104, 106, 107, 109, 110, 111, 113, 114, 115, 117, 119,
121, 122, 123, 124, 125, 126, 128, 130, 132, 134, 136, 137, 139,
141, 143, 145, 147, 149, 151, 153, 155, 157, 158, 160, 162, 164,
166, 168, 169, 170, 172, 173, 174, 176, 178, 180, 182, 184, 186,
188, 190, 192, 193, 194, 195, 197, 199, 201, 203, 205, 206, 207,
209, 210, 211, 212, 214, 215, 216, 218, 220, 222, 223, 224, 225,
227, 229, 231, 233, 235, 236, 237, 239, 240, 242, 244, 245, 246,
248, 250, 252, 254, 255, 257, 259 260, 262, 264, 266, 268, 269,
270, 271, 272, 274, 276, 278, 279, 280, 282, 284, 286, 288, 290,
292, 294, 296, 298, 300, 302, 304, 305, 306, 307, 309, 311, 312,
314, 315, 316, 318, 320 321, 323 or 325, or a complement thereof;
(b) a polynucleotide comprising a nucleotide sequence that encodes
a polypeptide comprising the amino acid sequence set forth in any
one of SEQ ID NO: 2, 8, 12, 14, 20, 22, 24, 28, 30, 32, 34, 36, 40,
46, 48, 51, 53, 55, 57, 59, 62, 65, 72, 74, 78, 85, 87, 89, 91, 95,
97, 99, 105, 108, 112, 116, 118, 120, 127, 129, 131, 133, 135, 138,
140, 142, 144, 146, 148, 150, 152, 154, 156, 159, 161, 163, 165,
167, 171, 175, 177, 179, 181, 183, 185, 187, 189, 191, 196, 198,
200, 202, 204, 208, 213, 217, 219, 221, 226, 228, 230, 232, 234,
236, 238, 241, 243, 247, 249, 251, 253, 256, 258, 261, 263, 265,
267, 273, 275, 277, 281, 283, 285, 287, 289, 291, 293, 295, 297,
299, 301, 303, 308, 310, 313, 317, 319, 322, 324 or 326; (c) a
polynucleotide comprising a nucleotide sequence that encodes a
polypeptide that shares at least 50% sequence similarity with at
least a portion of the sequence set forth in SEQ ID NO: 2, 8, 12,
14, 20, 22, 24, 28, 30, 32, 34, 36, 40, 46, 48, 51, 53, 55, 57, 59,
62, 65, 72, 74, 78, 85, 87, 89, 91, 95, 97, 99, 105, 108, 112, 116,
118, 120, 127, 129, 131, 133, 135, 138, 140, 142, 144, 146, 148,
150, 152, 154, 156, 159, 161, 163, 165, 167, 171, 175, 177, 179,
181, 183, 185, 187, 189, 191, 196, 198, 200, 202, 204, 208, 213,
217, 219, 221, 226, 228, 230, 232, 234, 236, 238, 241, 243, 247,
249, 251, 253, 256, 258, 261, 263, 265, 267, 273, 275, 277, 281,
283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 308, 310,
313, 317, 319, 322, 324 or 326, wherein the portion comprises at
least 15 contiguous amino acid residues of that sequence; and (d) a
polynucleotide comprising a nucleotide sequence that hybridizes to
the sequence of (a), (b), (c) or a complement thereof, under at
least low stringency conditions.
55. A kit comprising one or more probes according to claim 47.
56. A kit comprising one or more endotoxemia marker polypeptides
selected from the group consisting of: (i) a polypeptide comprising
an amino acid sequence that shares at least 50% sequence similarity
with the sequence set forth in any one of SEQ ID NO: 2, 8, 12, 14,
20, 22, 24, 28, 30, 32, 34, 36, 40, 46, 48, 51, 53, 55, 57, 59, 62,
65, 72, 74, 78, 85, 87, 89, 91, 95, 97, 99, 105, 108, 112, 116,
118, 120, 127, 129, 131, 133, 135, 138, 140, 142, 144, 146, 148,
150, 152, 154, 156, 159, 161, 163, 165, 167, 171, 175, 177, 179,
181, 183, 185, 187, 189, 191, 196, 198, 200, 202, 204, 208, 213,
217, 219, 221, 226, 228, 230, 232, 234, 236, 238, 241, 243, 247,
249, 251, 253, 256, 258, 261, 263, 265, 267, 273, 275, 277, 281,
283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 308, 310,
313, 317, 319, 322, 324 or 326; (ii) a polypeptide comprising an
amino acid sequence that shares at least 50% sequence similarity
with a polypeptide expression product of an endotoxemia marker gene
that comprises a sequence set forth in anyone of SEQ ID NO: 2, 8,
12, 14,20, 22, 24, 28, 30, 32, 34, 36, 40, 46, 48, 51, 53, 55, 57,
59, 62, 65, 72, 74, 78, 85, 87, 89, 91, 95, 97, 99, 105, 108, 112,
116, 118, 120, 127, 129, 131, 133, 135, 138, 140, 142, 144, 146,
148, 150, 152, 154, 156, 159, 161, 163, 165, 167, 171, 175, 177,
179, 181, 183, 185, 187, 189, 191, 196, 198, 200, 202, 204, 208,
213, 217, 219, 221, 226, 228, 230, 232, 234, 236, 238, 241, 243,
247, 249, 251, 253, 256, 258, 261, 263, 265, 267, 273, 275, 277,
281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 308,
310, 313, 317, 319, 322, 324 or 326; (iii) a portion of the
polypeptide according to (i) or (ii) wherein the portion comprises
at least 5 contiguous amino acid residues of that polypeptide; (iv)
a polypeptide comprising an amino acid sequence that shares at
least 30% similarity with at least 15 contiguous amino acid
residues of the polypeptide according to (i) or (ii); and (v) a
polypeptide comprising a portion of the polypeptide according to
(i) or (ii), wherein the portion comprises at least 5 contiguous
amino acid residues of the polypeptide according to (i) or (ii) and
is immuno-interactive with an antigen-binding molecule that is
immuno-interactive with a sequence of (i), (ii) or (iii), or the
use of one or more antigen-binding molecules that are
immuno-interactive with a said endotoxemia marker polypeptide, in
the manufacture of a kit for diagnosing the presence of an
endotoxemia-related condition in a subject.
Description
FIELD OF THE INVENTION
[0001] This invention relates generally to a method and apparatus
for the diagnosis, detection of host response, monitoring,
treatment and management of endotoxemia and endotoxemia-induced
conditions in mammals. The invention also relates to the use of
this method in monitoring, treatment and management of conditions
that can lead to endotoxemia. The invention has practical use in
early diagnosis, diagnosis of mild or sub-clinical endotoxemia, in
the detection of specific cell immune responses as part of active
or progressive disease, in monitoring clinically affected animals,
and in enabling better treatment and management decisions to be
made in clinically and sub-clinically affected animals.
Additionally, the invention has practical use in monitoring
patients in critical care or intensive care units for endotoxemia
and in predicting clinical outcome.
BACKGROUND OF THE INVENTION
[0002] Endotoxemia (also called septic shock and septic syndrome)
is generally considered to result from an inability of the host
defense mechanisms to cope with foreign organisms, including gram
positive and negative bacteria, viruses, fungi and parasites. The
majority of endotoxemia cases are caused by gram negative bacteria
(Glauser et al., 1991, Lancet 338 (Sept) 732-736), in particular a
product of gram negative bacteria called endotoxin or
lipopolysaccharide (LPS), which is a component of the bacterial
outer cell wall.
[0003] Because the main initiator of endotoxemia is endotoxin, many
tests have been developed to measure these molecules in fluids,
including the Limulus Amoebocyte Lysate (LAL) test (Cohen J., 2000,
Intensive Care Med. 26: S51-S56), rabbit pyrogen test, and
lethality in mice and chicken embryos (Hurley J C. Clin Microbiol
Reviews 8(2): 268-292). However, measurement of endotoxin in
fluids, especially blood, is a poor predictor of clinical outcome
in endotoxemia for a number of reasons:
[0004] The levels of endotoxin required to trigger the biological
cascade of events leading to endotoxemia varies widely from patient
to patient.
[0005] The bioavailability of endotoxin varies from patient to
patient depending upon the body's ability to detoxify or neutralize
it.
[0006] Some patients develop sensitivity to endotoxin, or are
tolerant to endotoxin.
[0007] Various biological fluids (and even fluid containers)
contain agents that bind endotoxin that are capable of enhancing or
limiting the biological effect of endotoxin, or can interfere with
the measurement of endotoxin.
[0008] Some endotoxins are more potent than others.
[0009] The specificity and sensitivity of the LAL assay is at its
limit when used for assaying endotoxin in blood or serum.
[0010] For these reasons, efforts have been made to develop assays
for the determination of the biological effects of endotoxin--as a
means of determining clinical outcome--including the measurement of
molecules such as Tumor Necrosis Factor (TNF), C3a, C5a, Factor XII
(Hageman Factor), interleukin-1 (IL-1), .gamma.-interferon and
various other cytokines, and the measurement of the level of
activation of leukocytes. Such measurements have contributed to a
"sepsis score" concept developed by Casey et al. (1993, Ann Intern
Med. 119: 771-778).
[0011] However, none of these tests have been sufficiently
sensitive, specific or practical enough to be used in routine
clinical practice. In addition, the efficacy of available
treatments also limits the practical use of such prognostic and
monitoring tests.
[0012] Despite this, there are a number of features of endotoxemia
that make early detection, monitoring, determination of clinical
outcome, prognosis determination, early intervention and informed
management of affected animals clinically and economically
important, viz:
[0013] Many and varied conditions can lead to endotoxemia.
[0014] Endotoxemia can lead to many other conditions.
[0015] Endotoxemia is often a peracute condition causing death if
not correctly managed.
[0016] It is estimated that 20-30% of human patients in intensive
care units in the USA are affected and that more than 100,000
humans die each year in the USA alone (Young L & Glauser M
(Eds) Gram negative septicemia and septic shock. WB Saunders
Philadelphia (1991); Parrillo J E. 1990, Ann Intern Med. 113:
227-242).
[0017] The extent of the condition in less developed countries is
likely to be far higher due to poor hygiene and medical
infrastructure.
[0018] In up to 50% of cases an etiological agent is not
determined.
[0019] The condition is most common in hospitals in patients with
other underlying diseases.
[0020] The extent of subclinical disease and its effects on human
health, animal husbandry, athletic performance, and ethical
management are not known.
[0021] Apart from the direct detection of endotoxin, there have
been many efforts to use secondary indicators of sepsis to diagnose
and monitor this condition, including measuring heart rate,
temperature, respiratory rate, cardiac output, systemic vascular
resistance, plasma IL-6 levels, macrophage inflammatory protein-2,
chemokine KC, protein C and C-reactive protein (Panacek et al.,
2004, Crit. Care Med. 32: 2173-2182; Ulloa and Tracey, 2005, Trends
Mol. Med. 1:56-63).
[0022] Currently no panel of biomarkers is used to define sepsis in
humans (Buras et al., 2006, Nature Reviews Drug Discovery, 4:
854-865). However, a cytokine profile has been suggested (Ulloa et
al., 2005, Trends Mol. Biol. 11: 56-63) Reasons for this are the
complexity of the disease, difficulty in defining the stage of
disease and the apparent existence of two distinct but not mutually
exclusive phases of inflammatory and anti-inflammatory responses
(Bone R C., 1996, Crit. Care Med, 24: 1125-1128).
[0023] Given the current limitations of diagnostic, monitoring and
prognostic procedures for endotoxemia, especially in sub-clinical
or early-stages, there is a need for more effective modalities for
early detection, diagnosis, monitoring, prognosis and management of
the various phases of sepsis including, acute, peracute, early
stage, advanced, and sub-clinical endotoxemia.
[0024] An example of a complication arising from endotoxemia is
laminitis that causes profound lameness in hoofed animals. It
occurs in perissodactyl and artiodactyl animals, including horses,
cattle, goats, sheep and other hoofed animals (ungulates). It is
believed the condition results from the action of endotoxin on
tissues and the lamellae of the inner hoof wall. Failure of the
lamellae results in separation of the inner hoof capsule from the
pedal bone and the subsequent (weight-bearing) driving of the pedal
bone through the hoof capsule, and crushing of the corium, sole and
coronet (Sloet van Oldruitenborgh-Oosterbaan M M., 1999, Vet
Quarterly 21(4) 121-127).
[0025] There are a number of features of laminitis that make early
detection, monitoring, early intervention and informed management
of affected animals clinically and economically important, viz:
[0026] The exact cause of laminitis is not known.
[0027] The extent of subclinical disease (often called the
developmental stage; Hood D M., 1999, Vet Clin Nth Amer Eq Pract
15(2): 287-294) and its effects on animal husbandry, athletic
performance, and ethical management are not known.
[0028] The first 72 hours of the disease (developmental and acute
stages) is the most critical period for monitoring. Animals that
have not suffered major mechanical or structural failures at this
stage are likely to recover.
[0029] The pathogenesis of the disease is poorly understood.
[0030] Laminitis is the largest killer of horses worldwide, usually
as a result of euthanasia due to progressive disease that causes
serious disability and pain.
[0031] Present diagnostics are only partially effective once the
disease is established, by which time preventative management or
ameliorating therapies have little effect.
[0032] There are few practical interventions available.
[0033] Thus, there is a need for more effective modalities for
early diagnosis, diagnosis of mild or sub-clinical laminitis, in
the detection of specific immune responses as part of active or
progressive disease, and in monitoring animals clinically affected
by laminitis. Such modalities would enable better treatment and
management decisions to be made in clinically and sub-clinically
affected animals prior to irreversible tissue damage.
[0034] Existing technology for diagnosing endotoxemia or for
monitoring conditions that lead to endotoxemia or for evaluating
sequelae of endotoxemia, is limited in that the detection of
bacterial endotoxin in body fluids does not correlate well with
clinical signs, and the sensitivity and specificity of these
technologies is insufficient to be clinically useful. In addition,
because the conditions are often peracute, advanced and
irreversible tissue damage may have occurred (and possibly death)
by the time endotoxin is able to be detected.
[0035] In addition, existing technologies for diagnosis or
evaluation of laminitis are limited and are almost entirely reliant
upon clinical evaluation and the detection of lameness. In many
instances the lameness can be very subtle or sub-clinical. In
addition, many of these clinical changes can only be observed in
advanced stages of disease, at which time irreversible tissue
damage has occurred, and where humane euthanasia is the only
recourse.
SUMMARY OF THE INVENTION
[0036] The present invention represents a significant advance over
current technologies for the management of affected animals. In
certain advantageous embodiments, it relies upon measuring the
level of certain markers in cells, especially circulating
leukocytes, of the host rather than detecting endotoxin. As such,
these methods are suitable for widespread screening of symptomatic
and asymptomatic animals. In certain embodiments where circulating
leukocytes are the subject of analysis, it is proposed that
detection of a host response to endotoxemia and its sequelae (also
referred to herein as "endotoxemia-related conditions") will be
feasible at very early stages of its progression before extensive
tissue damage has occurred.
[0037] Thus, the present invention addresses the problem of
diagnosing endotoxemia-related conditions by detecting a host
response that may be measured in host cells. Advantageous
embodiments involve monitoring the expression of certain genes in
peripheral leukocytes of the immune system, which may be reflected
in changing patterns of RNA levels or protein production that
correlate with the presence of active disease or response to
disease.
[0038] Accordingly, in one aspect, the present invention provides
methods for diagnosing the presence of an endotoxemia-related
condition in a test subject, especially in an equine test subject.
These methods generally comprise detecting in the test subject
aberrant expression of at least one gene (also referred to herein
as an "endotoxemia marker gene") selected from the group consisting
of: (a) a gene having a polynucleotide expression product
comprising a nucleotide sequence that shares at least 50% (and at
least 51% to at least 99% and all integer percentages in between)
sequence identity with the sequence set forth in any one of SEQ ID
NO: 1, 3, 4, 5, 6, 7, 9, 10, 11, 13, 15, 16, 17, 18, 19, 21, 23,
25, 26, 27, 29, 31, 33, 35, 37, 38, 39, 41, 42, 43, 44, 45, 47, 49,
50, 52, 54, 56, 58, 60, 61, 63, 64, 66, 67, 68, 69, 70, 71, 73, 75,
76, 77, 78, 79, 80, 81, 82, 83, 84, 86, 88, 90, 92, 93, 94, 96, 98,
100, 101, 102, 103, 104, 106, 107, 109, 110, 111, 113, 114, 115,
117, 119, 121, 122, 123, 124, 125, 126, 128, 130, 132, 134, 136,
137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 158, 160,
162, 164, 166, 168, 169, 170, 172, 173, 174, 176, 178, 180, 182,
184, 186, 188, 190, 192, 193, 194, 195, 197, 199, 201, 203, 205,
206, 207, 209, 210, 211, 212, 214, 215, 216, 218, 220, 222, 223,
224, 225, 227, 229, 231, 233, 235, 236, 237, 239, 240, 242, 244,
245, 246, 248, 250, 252, 254, 255, 257, 259 260, 262, 264, 266,
268, 269, 270, 271, 272, 274, 276, 278, 279, 280, 282, 284, 286,
288, 290, 292, 294, 296, 298, 300, 302, 304, 305, 306, 307, 309,
311, 312, 314, 315, 316, 318, 320 321, 323 or 325, or a complement
thereof, (b) a gene having a polynucleotide expression product
comprising a nucleotide sequence that encodes a polypeptide
comprising the amino acid sequence set forth in any one of SEQ ID
NO: 2, 8, 12, 14, 20, 22, 24, 28, 30, 32, 34, 36, 40, 46, 48, 51,
53, 55, 57, 59, 62, 65, 72, 74, 78, 85, 87, 89, 91, 95, 97, 99,
105, 108, 112, 116, 118, 120, 127, 129, 131, 133, 135, 138, 140,
142, 144, 146, 148, 150, 152, 154, 156, 159, 161, 163, 165, 167,
171, 175, 177, 179, 181, 183, 185, 187, 189, 191, 196, 198, 200,
202, 204, 208, 213, 217, 219, 221, 226, 228, 230, 232, 234, 236,
238, 241, 243, 247, 249, 251, 253, 256, 258, 261, 263, 265, 267,
273, 275, 277, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299,
301, 303, 308, 310, 313, 317, 319, 322, 324 or 326; (c) a gene
having a polynucleotide expression product comprising a nucleotide
sequence that encodes a polypeptide that shares at least 50% (and
at least 51% to at least 99% and all integer percentages in
between) sequence similarity with at least a portion of the
sequence set forth in SEQ ID NO: 2, 8, 12, 14, 20, 22, 24, 28, 30,
32, 34, 36, 40, 46, 48, 51, 53, 55, 57, 59, 62, 65, 72, 74, 78, 85,
87, 89, 91, 95, 97, 99, 105, 108, 112, 116, 118, 120, 127, 129,
131, 133, 135, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156,
159, 161, 163, 165, 167, 171, 175, 177, 179, 181, 183, 185, 187,
189, 191, 196, 198, 200, 202, 204, 208, 213, 217, 219, 221, 226,
228, 230, 232, 234, 236, 238, 241, 243, 247, 249, 251, 253, 256,
258, 261, 263, 265, 267, 273, 275, 277, 281, 283, 285, 287, 289,
291, 293, 295, 297, 299, 301, 303, 308, 310, 313, 317, 319, 322,
324 or 326, wherein the portion comprises at least 15 contiguous
amino acid residues of that sequence; and (d) a gene having a
polynucleotide expression product comprising a nucleotide sequence
that hybridizes to the sequence of (a), (b), (c) or a complement
thereof, under at least low, medium, or high stringency conditions.
In accordance with the present invention, these endotoxemia marker
genes are aberrantly expressed in animals with an
endotoxemia-related condition such as but not limited to an
endotoxemia-induced condition, illustrative examples of which
include shock, depression, abdominal discomfort, reduced pain
threshold, laminitis and idiopathic conditions.
[0039] As used herein, polynucleotide expression products of
endotoxemia marker genes are referred to herein as "endotoxemia
marker polynucleotides." Polypeptide expression products of the
endotoxemia marker genes are referred to herein as "endotoxemia
marker polypeptides."
[0040] Thus, in some embodiments, the methods comprise detecting
aberrant expression of an endotoxemia marker polynucleotide
selected from the group consisting of (a) a polynucleotide
comprising a nucleotide sequence that shares at least 50% (and at
least 51% to at least 99% and all integer percentages in between)
sequence identity with the sequence set forth in any one of SEQ ID
NO: 1, 3, 4, 5, 6, 7, 9, 10, 11, 13, 15, 16, 17, 18, 19, 21, 23,
25, 26, 27, 29, 31, 33, 35, 37, 38, 39, 41, 42, 43, 44, 45, 47, 49,
50, 52, 54, 56, 58, 60, 61, 63, 64, 66, 67, 68, 69, 70, 71, 73, 75,
76, 77, 78, 79, 80, 81, 82, 83, 84, 86, 88, 90, 92, 93, 94, 96, 98,
100, 101, 102, 103, 104, 106, 107, 109, 110, 111, 113, 114, 115,
117, 119, 121, 122, 123, 124, 125, 126, 128, 130, 132, 134, 136,
137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 158, 160,
162, 164, 166, 168, 169, 170, 172, 173, 174, 176, 178, 180, 182,
184, 186, 188, 190, 192, 193, 194, 195, 197, 199, 201, 203, 205,
206, 207, 209, 210, 211, 212, 214, 215, 216, 218, 220, 222, 223,
224, 225, 227, 229, 231, 233, 235, 236, 237, 239, 240, 242, 244,
245, 246, 248, 250, 252, 254, 255, 257, 259 260, 262, 264, 266,
268, 269, 270, 271, 272, 274, 276, 278, 279, 280, 282, 284, 286,
288, 290, 292, 294, 296, 298, 300, 302, 304, 305, 306, 307, 309,
311, 312, 314, 315, 316, 318, 320 321, 323 or 325, or a complement
thereof; (b) a polynucleotide comprising a nucleotide sequence that
encodes a polypeptide comprising the amino acid sequence set forth
in any one of SEQ ID NO: 2, 8, 12, 14, 20, 22, 24, 28, 30, 32, 34,
36, 40, 46, 48, 51, 53, 55, 57, 59, 62, 65, 72, 74, 78, 85, 87, 89,
91, 95, 97, 99, 105, 108, 112, 116, 118, 120, 127, 129, 131, 133,
135, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 159, 161,
163, 165, 167, 171, 175, 177, 179, 181, 183, 185, 187, 189, 191,
196, 198, 200, 202, 204, 208, 213, 217, 219, 221, 226, 228, 230,
232, 234, 236, 238, 241, 243, 247, 249, 251, 253, 256, 258, 261,
263, 265, 267, 273, 275, 277, 281, 283, 285, 287, 289, 291, 293,
295, 297, 299, 301, 303, 308, 310, 313, 317, 319, 322, 324 or 326;
(c) a polynucleotide comprising a nucleotide sequence that encodes
a polypeptide that shares at least 50% (and at least 51% to at
least 990% and all integer percentages in between) sequence
similarity with at least a portion of the sequence set forth in SEQ
ID NO: 2, 8, 12, 14, 20, 22, 24, 28, 30, 32, 34, 36, 40, 46, 48,
51, 53, 55, 57, 59, 62, 65, 72, 74, 78, 85, 87, 89, 91, 95, 97, 99,
105, 108, 112, 116, 118, 120, 127, 129, 131, 133, 135, 138, 140,
142, 144, 146, 148, 150, 152, 154, 156, 159, 161, 163, 165, 167,
171, 175, 177, 179, 181, 183, 185, 187, 189, 191, 196, 198, 200,
202, 204, 208, 213, 217, 219, 221, 226, 228, 230, 232, 234, 236,
238, 241, 243, 247, 249, 251, 253, 256, 258, 261, 263, 265, 267,
273, 275, 277, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299,
301, 303, 308, 310, 313, 317, 319, 322, 324 or 326, wherein the
portion comprises at least 15 contiguous amino acid residues of
that sequence; and (d) a polynucleotide comprising a nucleotide
sequence that hybridizes to the sequence of (a), (b), (c) or a
complement thereof, under at least low, medium, or high stringency
conditions.
[0041] In other embodiments, the methods comprise detecting
aberrant expression of an endotoxemia marker polypeptide selected
from the group consisting of: (i) a polypeptide comprising an amino
acid sequence that shares at least 50% (and at least 51% to at
least 99% and all integer percentages in between) sequence
similarity with the sequence set forth in any one of SEQ ID NO: 2,
8, 12, 14, 20, 22, 24, 28, 30, 32, 34, 36, 40, 46, 48, 51, 53, 55,
57, 59, 62, 65, 72, 74, 78, 85, 87, 89, 91, 95, 97, 99, 105, 108,
112, 116, 118, 120, 127, 129, 131, 133, 135, 138, 140, 142, 144,
146, 148, 150, 152, 154, 156, 159, 161, 163, 165, 167, 171, 175,
177, 179, 181, 183, 185, 187, 189, 191, 196, 198, 200, 202, 204,
208, 213, 217, 219, 221, 226, 228, 230, 232, 234, 236, 238, 241,
243, 247, 249, 251, 253, 256, 258, 261, 263, 265, 267, 273, 275,
277, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303,
308, 310, 313, 317, 319, 322, 324 or 326; (ii) a polypeptide
comprising a portion of the sequence set forth in any one of SEQ ID
NO: 2, 8, 12, 14, 20, 22, 24, 28, 30, 32, 34, 36, 40, 46, 48, 51,
53, 55, 57, 59, 62, 65, 72, 74, 78, 85, 87, 89, 91, 95, 97, 99,
105, 108, 112, 116, 118, 120, 127, 129, 131, 133, 135, 138, 140,
142, 144, 146, 148, 150, 152, 154, 156, 159, 161, 163, 165, 167,
171, 175, 177, 179, 181, 183, 185, 187, 189, 191, 196, 198, 200,
202, 204, 208, 213, 217, 219, 221, 226, 228, 230, 232, 234, 236,
238, 241, 243, 247, 249, 251, 253, 256, 258, 261, 263, 265, 267,
273, 275, 277, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299,
301, 303, 308, 310, 313, 317, 319, 322, 324 or 326, wherein the
portion comprises at least 5 contiguous amino acid residues of that
sequence; (iii) a polypeptide comprising an amino acid sequence
that shares at least 30% similarity with at least 15 contiguous
amino acid residues of the sequence set forth in any one of SEQ ID
NO: 2, 8, 12, 14, 20, 22, 24, 28, 30, 32, 34, 36, 40, 46, 48, 51,
53, 55, 57, 59, 62, 65, 72, 74, 78, 85, 87, 89, 91, 95, 97, 99,
105, 108, 112, 116, 118, 120, 127, 129, 131, 133, 135, 138, 140,
142, 144, 146, 148, 150, 152, 154, 156, 159, 161, 163, 165, 167,
171, 175, 177, 179, 181, 183, 185, 187, 189, 191, 196, 198, 200,
202, 204, 208, 213, 217, 219, 221, 226, 228, 230, 232, 234, 236,
238, 241, 243, 247, 249, 251, 253, 256, 258, 261, 263, 265, 267,
273, 275, 277, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299,
301, 303, 308, 310, 313, 317, 319, 322, 324 or 326; and (iv) a
polypeptide comprising a portion of the sequence set forth in any
one of SEQ ID NO: 2, 8, 12, 14, 20, 22, 24, 28, 30, 32, 34, 36, 40,
46, 48, 51, 53, 55, 57, 59, 62, 65, 72, 74, 78, 85, 87, 89, 91, 95,
97, 99, 105, 108, 112, 116, 118, 120, 127, 129, 131, 133, 135, 138,
140, 142, 144, 146, 148, 150, 152, 154, 156, 159, 161, 163, 165,
167, 171, 175, 177, 179, 181, 183, 185, 187, 189, 191, 196, 198,
200, 202, 204, 208, 213, 217, 219, 221, 226, 228, 230, 232, 234,
236, 238, 241, 243, 247, 249, 251, 253, 256, 258, 261, 263, 265,
267, 273, 275, 277, 281, 283, 285, 287, 289, 291, 293, 295, 297,
299, 301, 303, 308, 310, 313, 317, 319, 322, 324 or 326, wherein
the portion comprises at least 5 contiguous amino acid residues of
that sequence and is immuno-interactive with an antigen-binding
molecule that is immuno-interactive with a sequence of (i), (ii) or
(iii).
[0042] Typically, such aberrant expression is detected by: (1)
measuring in a biological sample obtained from the test subject the
level or functional activity of an expression product of at least
one endotoxemia marker gene and (2) comparing the measured level or
functional activity of each expression product to the level or
functional activity of a corresponding expression product in a
reference sample obtained from one or more normal subjects or from
one or more subjects lacking disease, wherein a difference in the
level or functional activity of the expression product in the
biological sample as compared to the level or functional activity
of the corresponding expression product in the reference sample is
indicative of the presence of an endotoxemia-related condition in
the test subject. In some embodiments, the methods further comprise
diagnosing the presence, stage or degree of an endotoxemia-related
condition in the test subject when the measured level or functional
activity of the or each expression product is different than the
measured level or functional activity of the or each corresponding
expression product. In these embodiments, the difference typically
represents an at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%
or 90%, or even an at least about 100%, 200%, 300%, 400%, 500%,
600%, 700%, 800%, 900% or 1000% increase, or an at least about 10%,
20%, 30% 40%, 50%, 60%, 70%, 80%, 90%, 92%, 94%, 96%, 97%, 98% or
99%, or even an at least about 99.5%, 99.9%, 99.95%, 99.99%,
99.995% or 99.999% decrease in the level or functional activity of
an individual expression product as compared to the level or
functional activity of an individual corresponding expression
product., which is hereafter referred to as "aberrant expression."
In illustrative examples of this type, the presence of an
endotoxemia-related condition is determined by detecting a decrease
in the level or functional activity of at least one endotoxemia
marker polynucleotide selected from (a) a polynucleotide comprising
a nucleotide sequence that shares at least 50% (and at least 51% to
at least 99% and all integer percentages in between) sequence
identity with the sequence set forth in any one of SEQ ID NO: 5, 6,
7, 11, 13, 15, 16, 17, 18, 19, 21, 25, 26, 35, 37, 38, 41, 42, 43,
44, 50, 52, 54, 58, 60, 63, 64, 69, 70, 71, 73, 77, 79, 80, 81, 82,
83, 86, 92, 93, 96, 98, 100, 101, 102, 103, 104, 106, 115, 117,
128, 134, 137, 141, 143, 153, 155, 158, 162, 164, 166, 174, 182,
186, 188, 190, 195, 197, 199, 201, 205, 206, 207, 209, 210, 211,
214, 215, 216, 222, 223, 240, 244, 246, 248, 259, 260, 269, 272,
280, 282, 284, 286, 290, 298, 300, 302, 305, 306, 307, 309, 312,
314, 315, 316, 318, 320, 321, 323, or a complement thereof; (b) a
polynucleotide comprising a nucleotide sequence that encodes a
polypeptide comprising the amino acid sequence set forth in any one
of SEQ ID NO: 8, 12, 14, 20, 22, 36, 51, 53, 55, 59, 65, 72, 74,
78, 87, 97, 99, 105, 116, 118, 129, 135, 138, 142, 144, 154, 156,
159, 163, 165, 167, 175, 183, 187, 189, 191, 196, 198, 200, 202,
208, 217, 241, 247, 249, 261, 273, 281, 283, 285, 287, 291, 299,
301, 303, 308, 310, 313, 317, 319, 322, 324; (c) a polynucleotide
comprising a nucleotide sequence that encodes a polypeptide that
shares at least 50% (and at least 51% to at least 99% and all
integer percentages in between) sequence similarity with at least a
portion of the sequence set forth in SEQ ID NO: 8, 12, 14,20, 22,
36, 51, 53, 55, 59, 65, 72, 74, 78, 87, 97, 99, 105, 116, 118, 129,
135, 138, 142, 144, 154, 156, 159, 163, 165, 167, 175, 183, 187,
189, 191, 196, 198, 200, 202, 208, 217, 241, 247, 249, 261, 273,
281, 283, 285, 287, 291, 299, 301, 303, 308, 310, 313, 317, 319,
322, 324, wherein the portion comprises at least 15 contiguous
amino acid residues of that sequence; and (d) a polynucleotide
comprising a nucleotide sequence that hybridizes to the sequence of
(a), (b), (c) or a complement thereof, under at least low, medium,
or high stringency conditions.
[0043] In other illustrative examples, the presence of an
endotoxemia-related condition is determined by detecting an
increase in the level or functional activity of at least one
endotoxemia marker polynucleotide selected from (a) a
polynucleotide comprising a nucleotide sequence that shares at
least 500/0 (and at least 51% to at least 99% and all integer
percentages in between) sequence identity with the sequence set
forth in any one of SEQ ID NO: 1, 3, 4, 9, 10, 23, 27, 29, 31, 33,
39, 45, 47, 49, 56, 61, 66, 67, 68, 75, 76, 84, 88, 90, 94, 107,
109, 110, 111, 113, 114, 119, 121, 122, 123, 124, 125, 126, 130,
132, 136, 139, 145, 147, 149, 151, 157, 160, 168, 169, 170, 172,
173, 176, 178, 180, 184, 192, 193, 194, 203, 212, 218, 220, 224,
225, 227, 229, 231, 233, 235, 236, 237, 239, 242, 245, 250, 252,
254, 255, 257, 262, 264, 266, 268, 270, 271, 274, 276, 278, 279,
288, 292, 294, 296, 304, 311, 325, or a complement thereof; (b) a
polynucleotide comprising a nucleotide sequence that encodes a
polypeptide comprising the amino acid sequence set forth in any one
of SEQ ID NO: 2, 24, 28, 30, 32, 34, 40, 46, 48, 57, 62, 85, 89,
91, 95, 108, 112, 120, 127, 131, 133, 140, 146, 148, 150, 152, 161,
171, 177, 179, 181, 185, 204, 213, 219, 221, 226, 228, 230, 232,
234, 238, 243, 251, 253, 256, 258, 263, 265, 267, 275, 277, 289,
293, 295, 297, 326; (c) a polynucleotide comprising a nucleotide
sequence that encodes a polypeptide that shares at least 50% (and
at least 51% to at least 99% and all integer percentages in
between) sequence similarity with at least a portion of the
sequence set forth in SEQ ID NO: 8, 12, 14, 20, 22, 36, 51, 53, 55,
59, 65, 72, 74, 78, 87,97, 99, 105, 116, 118, 129, 135, 138, 142,
144, 154, 156, 159, 163, 165, 167, 175, 183, 187, 189, 191, 196,
198, 200, 202, 208, 217, 241, 247, 249, 261, 273, 281, 283, 285,
287, 291, 299, 301, 303, 308, 310, 313, 317, 319, 322, 324 wherein
the portion comprises at least 15 contiguous amino acid residues of
that sequence; and (d) a polynucleotide comprising a nucleotide
sequence that hybridizes to the sequence of (a), (b), (c) or a
complement thereof, under at least low, medium, or high stringency
conditions.
[0044] In some embodiments, the method further comprises diagnosing
the absence of an endotoxemia-related condition when the measured
level or functional activity of the or each expression product is
the same as or similar to the measured level or functional activity
of the or each corresponding expression product. In these
embodiments, the measured level or functional activity of an
individual expression product varies from the measured level or
functional activity of an individual corresponding expression
product by no more than about 20%, 18%, 16%, 14%, 12%, 10%, 9%,
18%, 7%, 16%, 5%, 4%, 13%, 22%, 1% or 0.1%, which is hereafter
referred to as "normal expression.".
[0045] In some embodiments, the methods comprise measuring the
level or functional activity of individual expression products of
at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,
34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or
50 endotoxemia marker polynucleotides. For example, the methods may
comprise measuring the level or functional activity of an
endotoxemia marker polynucleotide either alone or in combination
with as much as 49, 48, 47, 46, 45, 44, 43, 42, 41,40, 39, 38, 37,
36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20,
19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1
other endotoxemia marker polynucleotide(s). In another example, the
methods may comprise measuring the level or functional activity of
an endotoxemia marker polypeptide either alone or in combination
with as much as 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37,
36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20,
19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1
other endotoxemia marker polypeptides(s). In illustrative examples
of this type, the methods comprise measuring the level or
functional activity of individual expression products of at least
1, 2, 3, 4, 5 or 6 endotoxemia marker genes that have a very high
correlation (p<0.001) with the presence or risk of an
endotoxemia-related condition (hereafter referred to as "level one
correlation endotoxemia marker genes"), representative examples of
which include, but are not limited to, (a) a polynucleotide
comprising a nucleotide sequence that shares at least 50% (and at
least 51% to at least 99% and all integer percentages in between)
sequence identity with the sequence set forth in any one of SEQ ID
NO: 11, 23, 29, 35, 43, 44, 68, 81, 82, 84, 104, 105, 107, 119,
130, 136, 147, 155, 174, 192, 193, 245, 254, 255, 262, 264, 270,
271, 279, 296, 325, or a complement thereof, (b) a polynucleotide
comprising a nucleotide sequence that encodes a polypeptide
comprising the amino acid sequence set forth in any one of SEQ ID
NO: 12, 24, 30, 36, 85, 108, 120, 131, 148, 156, 175, 256, 263,
265, 297, 326; (c) a polynucleotide comprising a nucleotide
sequence that encodes a polypeptide that shares at least 50% (and
at least 51% to at least 99% and all integer percentages in
between) sequence similarity with at least a portion of the
sequence set forth in SEQ ID NO: 12, 24, 30, 36, 85, 108, 120, 131,
148, 156, 175, 256, 263, 265, 297, 326, wherein the portion
comprises at least 15 contiguous amino acid residues of that
sequence; and (d) a polynucleotide comprising a nucleotide sequence
that hybridizes to the sequence of (a), (b), (c) or a complement
thereof, under at least low, medium, or high stringency
conditions.
[0046] In other illustrative examples, the methods comprise
measuring the level or functional activity of individual expression
products of at least 1, 2, 3, 4, 5, 6, 7 or 8 endotoxemia marker
genes that have a high correlation (p<0.005) with the presence
or risk of an endotoxemia-related condition (hereafter referred to
as "level two correlation endotoxemia marker genes"),
representative examples of which include, but are not limited to,
(a) a polynucleotide comprising a nucleotide sequence that shares
at least 50% (and at least 51% to at least 99% and all integer
percentages in between) sequence identity with the sequence set
forth in any one of SEQ ID NO: 1, 7, 9, 10, 17, 18, 21, 25, 26, 33,
54, 61, 64, 79, 80, 90, 94, 115, 117, 121, 122, 125, 126, 143, 160,
162, 164, 172, 173, 178, 184, 186, 194, 199, 205, 206, 225, 229,
242, 244, 252, 257, 259, 274, 276, 282, 284, 288, 294, 306, 316,
318, or a complement thereof, (b) a polynucleotide comprising a
nucleotide sequence that encodes a polypeptide comprising the amino
acid sequence set forth in any one of SEQ ID NO: 2, 8, 22, 34, 55,
62, 65, 91, 95, 116, 118, 127, 144, 161, 163, 165, 179, 185, 187,
200, 226, 230, 243, 253, 258, 275, 277, 283, 285, 289, 295, 317,
319; (c) a polynucleotide comprising a nucleotide sequence that
encodes a polypeptide that shares at least 50% (and at least 51% to
at least 99% and all integer percentages in between) sequence
similarity with at least a portion of the sequence set forth in SEQ
ID NO: 2, 8, 22, 34, 55, 62, 65, 91, 95, 116, 118, 127, 144, 161,
163, 165, 179, 185, 187, 200, 226, 230, 243, 253, 258, 275, 277,
283, 285, 289, 295, 317, 319, wherein the portion comprises at
least 15 contiguous amino acid residues of that sequence; and (d) a
polynucleotide comprising a nucleotide sequence that hybridizes to
the sequence of (a), (b), (c) or a complement thereof, under at
least low, medium, or high stringency conditions.
[0047] In still other illustrative examples, the methods comprise
measuring the level or functional activity of individual expression
products of at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 endotoxemia
marker genes that have a medium correlation (p<0.05) with the
presence or risk of an endotoxemia-related condition (hereafter
referred to as "level three correlation endotoxemia marker genes"),
representative examples of which include, but are not limited to,
(a) a polynucleotide comprising a nucleotide sequence that shares
at least 50% (and at least 51% to at least 99% and all integer
percentages in between) sequence identity with the sequence set
forth in any one of SEQ ID NO: 3, 4, 5, 6, 13, 15, 16, 27, 31, 37,
38, 39, 41, 42, 45, 47, 49, 52, 56, 58, 63, 66, 67, 69, 70, 71, 77,
83, 86, 88, 96, 98, 100, 101, 106, 109, 110, 111, 113, 114, 128,
132, 134, 137, 139, 141, 145, 149, 151, 153, 157, 158, 166, 168,
169, 176, 180, 188, 190, 197, 203, 207, 209, 210, 211, 214, 215,
218, 220, 222, 223, 224, 231, 233, 236, 237, 239, 240, 241, 246,
250, 260, 266, 268, 269, 272, 278, 280, 286, 290, 292, 300, 304,
309, 312, 314, 315, 321, 323, or a complement thereof; (b) a
polynucleotide comprising a nucleotide sequence that encodes a
polypeptide comprising the amino acid sequence set forth in any one
of SEQ ID NO: 14, 28, 32, 40, 46, 48, 53, 57, 59, 72, 78, 87, 89,
97, 99, 112, 129, 133, 135, 138, 140, 142, 146, 150, 152, 154, 159,
167, 177, 181, 189, 191, 198, 204, 208, 219, 221, 232, 234, 238,
247, 251, 261, 267, 273, 281, 287, 291, 293, 301, 310, 313, 322,
324; (c) a polynucleotide comprising a nucleotide sequence that
encodes a polypeptide that shares at least 50% (and at least 51% to
at least 99% and all integer percentages in between) sequence
similarity with at least a portion of the sequence set forth in SEQ
ID NO: 14, 28, 32, 40, 46, 48, 53, 57, 59, 72, 78, 87, 89, 97, 99,
112, 129, 133, 135, 138, 140, 142, 146, 150, 152, 154, 159, 167,
177, 181, 189, 191, 198, 204, 208, 219, 221, 232, 234, 238, 247,
251, 261, 267, 273, 281, 287, 291, 293, 301, 310, 313, 322, 324,
wherein the portion comprises at least 15 contiguous amino acid
residues of that sequence; and (d) a polynucleotide comprising a
nucleotide sequence that hybridizes to the sequence of (a), (b),
(c) or a complement thereof, under at least low, medium, or high
stringency conditions.
[0048] In still other illustrative examples, the methods comprise
measuring the level or functional activity of individual expression
products of at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 endotoxemia
marker genes that have a moderate correlation (significant at 72
hours post-induction only and p<0.05) with the presence or risk
of an endotoxemia-related condition (hereafter referred to as
"level four correlation endotoxemia marker genes"), representative
examples of which include, but are not limited to, (a) a
polynucleotide comprising a nucleotide sequence that shares at
least 50% (and at least 51% to at least 99% and all integer
percentages in between) sequence identity with the sequence set
forth in any one of SEQ ID NO: 19, 50, 60, 73, 75, 92, 93, 102,
103, 123, 124, 170, 182, 195, 201, 212, 216, 227, 235, 248, 298,
302, 305, 307, 311, 320, or a complement thereof; (b) a
polynucleotide comprising a nucleotide sequence that encodes a
polypeptide comprising the amino acid sequence set forth in any one
of SEQ ID NO: 20, 51, 74, 76, 171, 183, 196, 202, 213, 217, 228,
249, 299, 303, 308; (c) a polynucleotide comprising a nucleotide
sequence that encodes a polypeptide that shares at least 50% (and
at least 51% to at least 99% and all integer percentages in
between) sequence similarity with at least a portion of the
sequence set forth in SEQ ID NO: 20, 51, 74, 76, 171, 183, 196,
202, 213, 217, 228, 249, 299, 303, 308, wherein the portion
comprises at least 15 contiguous amino acid residues of that
sequence; and (d) a polynucleotide comprising a nucleotide sequence
that hybridizes to the sequence of (a), (b), (c) or a complement
thereof, under at least low, medium, or high stringency
conditions.
[0049] In some embodiments, the methods comprise measuring the
level or functional activity of an expression product of at least 1
level one correlation endotoxemia marker gene. In other
embodiments, the methods comprise measuring the level or functional
activity of an expression product of at least 2 level one
correlation endotoxemia marker genes. In still other embodiments,
the methods comprise measuring the level or functional activity of
an expression product of at least 1 level one correlation
endotoxemia marker gene and the level or functional activity of an
expression product of at least 1 level two endotoxemia marker gene.
In still other embodiments, the methods comprise measuring the
level or functional activity of an expression product of at least 2
level one correlation endotoxemia marker genes and the level or
functional activity of an expression product of at least 1 level
two correlation endotoxemia marker gene. In still other
embodiments, the methods comprise measuring the level or functional
activity of an expression product of at least 1 level one
correlation endotoxemia marker gene and the level or functional
activity of an expression product of at least 2 level two
correlation endotoxemia marker genes.
[0050] In some embodiments, the methods comprise measuring the
level or functional activity of an expression product of at least 1
level one correlation endotoxemia marker gene and the level or
functional activity of an expression product of at least 1 level
three correlation endotoxemia marker gene. In other embodiments,
the methods comprise measuring the level or functional activity of
an expression product of at least 2 level one correlation
endotoxemia marker genes and the level or functional activity of an
expression product of at least 1 level three correlation
endotoxemia marker gene. In still other embodiments, the methods
comprise measuring the level or functional activity of an
expression product of at least 1 level one correlation endotoxemia
marker gene and the level or functional activity of an expression
product of at least 2 level three correlation endotoxemia marker
genes. In still other embodiments, the methods comprise measuring
the level or functional activity of an expression product of at
least 1 level one correlation endotoxemia marker gene and the level
or functional activity of an expression product of at least 3 level
three correlation endotoxemia marker genes.
[0051] In some embodiments, the methods comprise measuring the
level or functional activity of an expression product of at least 1
level one correlation endotoxemia marker gene and the level or
functional activity of an expression product of at least 1 level
four correlation endotoxemia marker gene. In other embodiments, the
methods comprise measuring the level or functional activity of an
expression product of at least 2 level one correlation endotoxemia
marker genes and the level or functional activity of an expression
product of at least 1 level four correlation endotoxemia marker
gene. In still other embodiments, the methods comprise measuring
the level or functional activity of an expression product of at
least 1 level one correlation endotoxemia marker gene and the level
or functional activity of an expression product of at least 2 level
four correlation endotoxemia marker gene. In still other
embodiments, the methods comprise measuring the level or functional
activity of an expression product of at least 1 level one
correlation endotoxemia marker gene and the level or functional
activity of an expression product of at least 3 level four
correlation endotoxemia marker genes. In still other embodiments,
the methods comprise measuring the level or functional activity of
an expression product of at least 1 level one correlation
endotoxemia marker gene and the level or functional activity of an
expression product of at least 4 level four correlation endotoxemia
marker genes.
[0052] In some embodiments, the methods comprise measuring the
level or functional activity of an expression product of at least 1
level two correlation endotoxemia marker gene. In other
embodiments, the methods comprise measuring the level or functional
activity of an expression product of at least 2 level two
correlation endotoxemia marker genes. In still other embodiments,
the methods comprise measuring the level or functional activity of
an expression product of at least 1 level two correlation
endotoxemia marker gene and the level or functional activity of an
expression product of at least 1 level three correlation
endotoxemia marker gene. In other embodiments, the methods comprise
measuring the level or functional activity of an expression product
of at least 2 level two correlation endotoxemia marker genes and
the level or functional activity of an expression product of at
least 1 level three correlation endotoxemia marker gene. In still
other embodiments, the methods comprise measuring the level or
functional activity of an expression product of at least 1 level
two correlation endotoxemia marker gene and the level or functional
activity of an expression product of at least 2 level three
correlation endotoxemia marker genes. In still other embodiments,
the methods comprise measuring the level or functional activity of
an expression product of at least 1 level two correlation
endotoxemia marker gene and the level or functional activity of an
expression product of at least 3 level three correlation
endotoxemia marker genes. In still other embodiments, the methods
comprise measuring the level or functional activity of an
expression product of at least 1 level two correlation endotoxemia
marker gene and the level or functional activity of an expression
product of at least 4 level three correlation endotoxemia marker
genes.
[0053] In some embodiments, the methods comprise measuring the
level or functional activity of an expression product of at least 1
level two correlation endotoxemia marker gene and the level or
functional activity of an expression product of at least 1 level
four correlation endotoxemia marker gene. In other embodiments, the
methods comprise measuring the level or functional activity of an
expression product of at least 2 level two correlation endotoxemia
marker genes and the level or functional activity of an expression
product of at least 1 level four correlation endotoxemia marker
gene. In still other embodiments, the methods comprise measuring
the level or functional activity of an expression product of at
least 1 level two correlation endotoxemia marker gene and the level
or functional activity of an expression product of at least 2 level
four correlation endotoxemia marker genes. In still other
embodiments, the methods comprise measuring the level or functional
activity of an expression product of at least 1 level two
correlation endotoxemia marker gene and the level or functional
activity of an expression product of at least 3 level four
correlation endotoxemia marker genes. In still other embodiments,
the methods comprise measuring the level or functional activity of
an expression product of at least 1 level two correlation
endotoxemia marker gene and the level or functional activity of an
expression product of at least 4 level four correlation endotoxemia
marker genes. In still other embodiments, the methods comprise
measuring the level or functional activity of an expression product
of at least 1 level two correlation endotoxemia marker gene and the
level or functional activity of an expression product of at least 5
level four correlation endotoxemia marker genes.
[0054] In some embodiments, the methods comprise measuring the
level or functional activity of an expression product of at least 1
level two correlation endotoxemia marker gene. In other
embodiments, the methods comprise measuring the level or functional
activity of an expression product of at least 2 level two
correlation endotoxemia marker gene. In still other embodiments,
the methods comprise measuring the level or functional activity of
an expression product of at least 1 level two correlation
endotoxemia marker gene and the level or functional activity of an
expression product of at least 1 level five correlation endotoxemia
marker gene. In other embodiments, the methods comprise measuring
the level or functional activity of an expression product of at
least 2 level two correlation endotoxemia marker genes and the
level or functional activity of an expression product of at least 1
level five correlation endotoxemia marker gene. In still other
embodiments, the methods comprise measuring the level or functional
activity of an expression product of at least 1 level two
correlation endotoxemia marker gene and the level or functional
activity of an expression product of at least 2 level five
correlation endotoxemia marker genes. In still other embodiments,
the methods comprise measuring the level or functional activity of
an expression product of at least 1 level two correlation
endotoxemia marker gene and the level or functional activity of an
expression product of at least 3 level five correlation endotoxemia
marker genes. In still other embodiments, the methods comprise
measuring the level or functional activity of an expression product
of at least 1 level two correlation endotoxemia marker gene and the
level or functional activity of an expression product of at least 4
level five correlation endotoxemia marker genes. In still other
embodiments, the methods comprise measuring the level or functional
activity of an expression product of at least 1 level two
correlation endotoxemia marker gene and the level or functional
activity of an expression product of at least 5 level five
correlation endotoxemia marker genes.
[0055] In some embodiments, the methods comprise measuring the
level or functional activity of an expression product of at least 1
level three correlation endotoxemia marker gene. In other
embodiments, the methods comprise measuring the level or functional
activity of an expression product of at least 2 level three
correlation endotoxemia marker genes. In still other embodiments,
the methods comprise measuring the level or functional activity of
an expression product of at least 1 level three correlation
endotoxemia marker gene and the level or functional activity of an
expression product of at least 1 level four correlation endotoxemia
marker gene. In other embodiments, the methods comprise measuring
the level or functional activity of an expression product of at
least 2 level three correlation endotoxemia marker genes and the
level or functional activity of an expression product of at least 1
level four correlation endotoxemia marker gene. In still other
embodiments, the methods comprise measuring the level or functional
activity of an expression product of at least 1 level three
correlation endotoxemia marker gene and the level or functional
activity of an expression product of at least 2 level four
correlation endotoxemia marker genes. In still other embodiments,
the methods comprise measuring the level or functional activity of
an expression product of at least 1 level three correlation
endotoxemia marker gene and the level or functional activity of an
expression product of at least 3 level four correlation endotoxemia
marker genes. In still other embodiments, the methods comprise
measuring the level or functional activity of an expression product
of at least 1 level three correlation endotoxemia marker gene and
the level or functional activity of an expression product of at
least 4 level four correlation endotoxemia marker genes. In still
other embodiments, the methods comprise measuring the level or
functional activity of an expression product of at least 1 level
three correlation endotoxemia marker gene and the level or
functional activity of an expression product of at least 5 level
four correlation endotoxemia marker genes.
[0056] In some embodiments, the methods comprise measuring the
level or functional activity of an expression product of at least 1
level four correlation endotoxemia marker gene. In other
embodiments, the methods comprise measuring the level or functional
activity of an expression product of at least 2 level four
correlation endotoxemia marker genes. In other embodiments, the
methods comprise measuring the level or functional activity of an
expression product of at least 3 level four correlation endotoxemia
marker genes. In still other embodiments, the methods comprise
measuring the level or functional activity of an expression product
of at least 3 level four correlation endotoxemia marker genes. In
still other embodiments, the methods comprise measuring the level
or functional activity of an expression product of at least 4 level
four correlation endotoxemia marker genes. In still other
embodiments, the methods comprise measuring the level or functional
activity of an expression product of at least 5 level four
correlation endotoxemia marker genes. In still other embodiments,
the methods comprise measuring the level or functional activity of
an expression product of at least 6 level four correlation
endotoxemia marker genes.
[0057] Advantageously, the biological sample comprises blood,
especially peripheral blood, which suitably includes leukocytes.
Suitably, the expression product is selected from a RNA molecule or
a polypeptide. In some embodiments, the expression product is the
same as the corresponding expression product. In other embodiments,
the expression product is a variant (e.g., an allelic variant) of
the corresponding expression product.
[0058] In certain embodiments, the expression product or
corresponding expression product is a target RNA (e.g., mRNA) or a
DNA copy of the target RNA whose level is measured using at least
one nucleic acid probe that hybridists under at least low, medium,
or high stringency conditions to the target RNA or to the DNA copy,
wherein the nucleic acid probe comprises at least 15 contiguous
nucleotides of an endotoxemia marker polynucleotide. In these
embodiments, the measured level or abundance of the target RNA or
its DNA copy is normalized to the level or abundance of a reference
RNA or a DNA copy of the reference RNA that is present in the same
sample. Suitably, the nucleic acid probe is immobilized on a solid
or semi-solid support. In illustrative examples of this type, the
nucleic acid probe forms part of a spatial array of nucleic acid
probes. In some embodiments, the level of nucleic acid probe that
is bound to the target RNA or to the DNA copy is measured by
hybridization (e.g., using a nucleic acid array). In other
embodiments, the level of nucleic acid probe that is bound to the
target RNA or to the DNA copy is measured by nucleic acid
amplification (e.g., using a polymerase chain reaction (PCR)). In
still other embodiments, the level of nucleic acid probe that is
bound to the target RNA or to the DNA copy is measured by nuclease
protection assay.
[0059] In other embodiments, the expression product or
corresponding expression product is a target polypeptide whose
level is measured using at least one antigen-binding molecule that
is immuno-interactive with the target polypeptide. In these
embodiments, the measured level of the target polypeptide is
normalized to the level of a reference polypeptide that is present
in the same sample. Suitably, the antigen-binding molecule is
immobilized on a solid or semi-solid support. In illustrative
examples of this type, the antigen-binding molecule forms part of a
spatial array of antigen-binding molecule. In some embodiments, the
level of antigen-binding molecule that is bound to the target
polypeptide is measured by immunoassay (e.g., using an ELISA).
[0060] In still other embodiments, the expression product or
corresponding expression product is a target polypeptide whose
level is measured using at least one substrate for the target
polypeptide with which it reacts to produce a reaction product. In
these embodiments, the measured functional activity of the target
polypeptide is normalized to the functional activity of a reference
polypeptide that is present in the same sample.
[0061] In some embodiments, a system is used to perform the
diagnostic methods as broadly described above, which suitably
comprises at least one end station coupled to a base station. The
base station is suitably caused (a) to receive subject data from
the end station via a communications network, wherein the subject
data represents parameter values corresponding to the measured or
normalized level or functional activity of at least one expression
product in the biological sample, and (b) to compare the subject
data with predetermined data representing the measured or
normalized level or functional activity of at least one
corresponding expression product in the reference sample to thereby
determine any difference in the level or functional activity of the
expression product in the biological sample as compared to the
level or functional activity of the corresponding expression
product in the reference sample. Desirably, the base station is
further caused to provide a diagnosis for the presence, absence or
degree of endotoxemia-related conditions. In these embodiments, the
base station may be further caused to transfer an indication of the
diagnosis to the end station via the communications network.
[0062] In another aspect, the invention contemplates use of the
methods broadly described above in the monitoring, treatment and
management of conditions that can lead to endotoxemia, illustrative
examples of which include retained placenta, meningitis,
endometriosis, shock, toxic shock (i.e., a sequelae to tampon use),
gastroenteritis, appendicitis, ulcerative colitis, Crohn's disease,
inflammatory bowel disease, acid gut syndrome, liver failure and
cirrhosis, failure of colostrum transfer in neonates, ischemia (in
any organ), bacteraemia, infections within body cavities such as
the peritoneal, pericardial, thecal, and pleural cavities, burns,
severe wounds, excessive exercise or stress, hemodialysis,
conditions involving intolerable pain (e.g., pancreatitis, kidney
stones), surgical operations, and non-healing lesions. In these
embodiments, the diagnostic methods of the invention are typically
used at a frequency that is effective to monitor the early
development of an endotoxemia-related condition to thereby enable
early therapeutic intervention and treatment of that condition. In
illustrative examples, the diagnostic methods are used at least at
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23 or 24 hour intervals.
[0063] In yet another aspect, the present invention provides
methods for treating, preventing or inhibiting the development of
an endotoxemia-related condition in a subject. These methods
generally comprise detecting aberrant expression of at least one
endotoxemia marker gene in the subject, and administering to the
subject an effective amount of an agent that treats or ameliorates
the symptoms or reverses or inhibits the development of the
endotoxemia-related condition in the subject. Representative
examples of such treatments or agents include but are not limited
to, antibiotics, steroids, intravenous fluids, vasoactives,
palliative support for damaged or distressed organs (e.g. oxygen
for respiratory distress, fluids for hypovolemia) and close
monitoring of vital organs.
[0064] In still another aspect, the present invention provides
isolated polynucleotides, referred to herein as "endotoxemia marker
polynucleotides," which are generally selected from: (a) a
polynucleotide comprising a nucleotide sequence that shares at
least 50% (and at least 51% to at least 99% and all integer
percentages in between) sequence identity with the sequence set
forth in any one of SEQ ID NO: 3, 4, 9, 15, 16, 17, 18, 25, 26, 37,
38, 41, 42, 43, 44, 49, 63, 66, 67, 68, 69, 70, 75, 76, 79, 80, 81,
82, 83, 92, 93, 100, 101, 102, 103, 106, 109, 110, 113, 114, 121,
122, 123, 124, 125, 136, 157, 168, 169, 172, 173, 192, 193, 194,
205, 206, 209, 210, 211, 214, 215, 222, 223, 224, 235, 236, 239,
244, 245, 254, 259, 268, 269, 270, 271, 278, 279, 304, 305, 306,
311, 314, 315 or 320, or a complement thereof; (b) a polynucleotide
comprising a portion of the sequence set forth in any one of SEQ ID
NO: 3, 4, 9, 15, 16, 17, 18, 25, 26, 37, 38, 41, 42, 43, 44, 49,
63, 66, 67, 68, 69, 70, 75, 76, 79, 80, 81, 82, 83, 92, 93, 100,
101, 102, 103, 106, 109, 110, 113, 114, 121, 122, 123, 124, 125,
136, 157, 168, 169, 172, 173, 192, 193, 194, 205, 206, 209, 210,
211, 214, 215, 222, 223, 224, 235, 236, 239, 244, 245, 254, 259,
268, 269, 270, 271, 278, 279, 304, 305, 306, 311, 314, 315 or 320,
or a complement thereof, wherein the portion comprises at least 15
contiguous nucleotides of that sequence or complement; (c) a
polynucleotide that hybridizes to the sequence of (a) or (b) or a
complement thereof, under at least low, medium or high stringency
conditions; and (d) a polynucleotide comprising a portion of any
one of SEQ ID NO: 3, 4, 9, 15, 16, 17, 18, 25, 26, 37, 38, 41, 42,
43, 44, 49, 63, 66, 67, 68, 69, 70, 75, 76, 79, 80, 81, 82, 83, 92,
93, 100, 101, 102, 103, 106, 109, 110, 113, 114, 121, 122, 123,
124, 125, 136, 157, 168, 169, 172, 173, 192, 193, 194, 205, 206,
209, 210, 211, 214, 215, 222, 223, 224, 235, 236, 239, 244, 245,
254, 259, 268, 269, 270, 271, 278, 279, 304, 305, 306, 311, 314,
315 or 320, or a complement thereof, wherein the portion comprises
at least 15 contiguous nucleotides of that sequence or complement
and hybridizes to a sequence of (a), (b) or (c), or a complement
thereof, under at least low, medium or high stringency
conditions.
[0065] In yet another aspect, the present invention provides a
nucleic acid construct comprising a polynucleotide as broadly
described above in operable connection with a regulatory element,
which is operable in a host cell. In certain embodiments, the
construct is in the form of a vector, especially an expression
vector.
[0066] In still another aspect, the present invention provides
isolated host cells containing a nucleic acid construct or vector
as broadly described above. In certain advantageous embodiments,
the host cells are selected from bacterial cells, yeast cells and
insect cells.
[0067] In still another aspect, the present invention provides
probes for interrogating nucleic acid for the presence of a
polynucleotide as broadly described above. These probes generally
comprise a nucleotide sequence that hybridizes under at least low
stringency conditions to a polynucleotide as broadly described
above. In some embodiments, the probes consist essentially of a
nucleic acid sequence which corresponds or is complementary to at
least a portion of a nucleotide sequence encoding the amino acid
sequence set forth in any one of SEQ ID NO: 2, 8, 12, 14, 20, 22,
24, 28, 30, 32, 34, 36, 40, 46, 48, 51, 53, 55, 57, 59, 62, 65, 72,
74, 78, 85, 87, 89, 91, 95, 97, 99, 105, 108, 112, 116, 118, 120,
127, 129, 131, 133, 135, 138, 140, 142, 144, 146, 148, 150, 152,
154, 156, 159, 161, 163, 165, 167, 171, 175, 177, 179, 181, 183,
185, 187, 189, 191, 196, 198, 200, 202, 204, 208, 213, 217, 219,
221, 226, 228, 230, 232, 234, 236, 238, 241, 243, 247, 249, 251,
253, 256, 258, 261, 263, 265, 267, 273, 275, 277, 281, 283, 285,
287, 289, 291, 293, 295, 297, 299, 301, 303, 308, 310, 313, 317,
319, 322, 324 or 326, wherein the portion is at least 15
nucleotides in length. In other embodiments, the probes comprise a
nucleotide sequence which is capable of hybridizing to at least a
portion of a nucleotide sequence encoding the amino acid sequence
set forth in any one of SEQ ID NO: 2, 8, 12, 14, 20, 22, 24, 28,
30, 32, 34, 36, 40, 46, 48, 51, 53, 55, 57, 59, 62, 65, 72, 74, 78,
85, 87, 89, 91, 95, 97, 99, 105, 108, 112, 116, 118, 120, 127, 129,
131, 133, 135, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156,
159, 161, 163, 165, 167, 171, 175, 177, 179, 181, 183, 185, 187,
189, 191, 196, 198, 200, 202, 204, 208, 213, 217, 219, 221, 226,
228, 230, 232, 234, 236, 238, 241, 243, 247, 249, 251, 253, 256,
258, 261, 263, 265, 267, 273, 275, 277, 281, 283, 285, 287, 289,
291, 293, 295, 297, 299, 301, 303, 308, 310, 313, 317, 319, 322,
324 or 326 under at least low, medium or high stringency
conditions, wherein the portion is at least 15 nucleotides in
length. In still other embodiment, the probes comprise a nucleotide
sequence that is capable of hybridizing to at least a portion of
any one of SEQ ID NO: 1, 3, 4, 5, 6, 7, 9, 10, 11, 13, 15, 16, 17,
18, 19, 21, 23, 25, 26, 27, 29, 31, 33, 35, 37, 38, 39, 41, 42, 43,
44, 45, 47, 49, 50, 52, 54, 56, 58, 60, 61, 63, 64, 66, 67, 68, 69,
70, 71, 73, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 86, 88, 90, 92,
93, 94, 96, 98, 100, 101, 102, 103, 104, 106, 107, 109, 110, 111,
113, 114, 115, 117, 119, 121, 122, 123, 124, 125, 126, 128, 130,
132, 134, 136, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155,
157, 158, 160, 162, 164, 166, 168, 169, 170, 172, 173, 174, 176,
178, 180, 182, 184, 186, 188, 190, 192, 193, 194, 195, 197, 199,
201, 203, 205, 206, 207, 209, 210, 211, 212, 214, 215, 216, 218,
220, 222, 223, 224, 225, 227, 229, 231, 233, 235, 236, 237, 239,
240, 242, 244, 245, 246, 248, 250, 252, 254, 255, 257, 259 260,
262, 264, 266, 268, 269, 270, 271, 272, 274, 276, 278, 279, 280,
282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 305,
306, 307, 309, 311, 312, 314, 315, 316, 318, 320 321, 323 or 325
under at least low, medium or high stringency conditions, wherein
the portion is at least 15 nucleotides in length. Representative
probes for detecting the endotoxemia marker polynucleotides
according to the resent invention are set forth in SEQ ID NO:
326-2315 (see Table 2).
[0068] In a related aspect, the invention provides a solid or
semi-solid support comprising at least one nucleic acid probe as
broadly described above immobilized thereon. In some embodiments,
the solid or semi-solid support comprises a spatial array of
nucleic acid probes immobilized thereon.
[0069] In a further aspect, the present invention provides isolated
polypeptides, referred to herein as "endotoxemia marker
polypeptides," which are generally selected from: (i) a polypeptide
comprising an amino acid sequence that shares at least 50% (and at
least 51% to at least 99% and all integer percentages in between)
sequence similarity with a polypeptide expression product of an
endotoxemia marker gene as broadly described above, for example,
especially an endotoxemia marker gene that comprises a nucleotide
sequence that shares at least 50% (and at least 51% to at least 99%
and all integer percentages in between) sequence identity with the
sequence set forth in any one of SEQ ID NO: 3, 4, 9, 15, 16, 17,
18, 25, 26, 37, 38, 41, 42, 43, 44, 49, 63, 66, 67, 68, 69, 70, 75,
76, 79, 80, 81, 82, 83, 92, 93, 100, 101, 102, 103, 106, 109, 110,
113, 114, 121, 122, 123, 124, 125, 136, 157, 168, 169, 172, 173,
192, 193, 194, 205, 206, 209, 210, 211, 214, 215, 222, 223, 224,
235, 236, 239, 244, 245, 254, 259, 268, 269, 270, 271, 278, 279,
304, 305, 306, 311, 314, 315 or 320; (ii) a portion of the
polypeptide according to (i) wherein the portion comprises at least
5 contiguous amino acid residues of that polypeptide; (iii) a
polypeptide comprising an amino acid sequence that shares at least
30% similarity (and at least 31% to at least 99% and all integer
percentages in between) with at least 15 contiguous amino acid
residues of the polypeptide according to (i); and (iv) a
polypeptide comprising an amino acid sequence that is
immuno-interactive with an antigen-binding molecule that is
immuno-interactive with a sequence of (i), (ii) or (iii).
[0070] Still a further aspect of the present invention provides an
antigen-binding molecule that is immuno-interactive with an
endotoxemia marker polypeptide as broadly described above.
[0071] In a related aspect, the invention provides a solid or
semi-solid support comprising at least one antigen-binding molecule
as broadly described above immobilized thereon. In some
embodiments, the solid or semi-solid support comprises a spatial
array of antigen-binding molecules immobilized thereon.
[0072] Still another aspect of the invention provides the use of
one or more endotoxemia marker polynucleotides as broadly described
above, or the use of one or more probes as broadly described above,
or the use of one or more endotoxemia marker polypeptides as
broadly described above, or the use of one or more antigen-binding
molecules as broadly described above, in the manufacture of a kit
for diagnosing the presence of an endotoxemia-related condition in
a subject.
[0073] In still other aspects, the invention is directed to the use
of the diagnostic methods as broadly described above, or one or
more endotoxemia marker polynucleotides as broadly described above,
or the use of one or more probes as broadly described above, or the
use of one or more endotoxemia marker polypeptides as broadly
described above, or the use of one or more antigen-binding
molecules as broadly described above, for diagnosing an
endotoxemia-related condition animals (vertebrates), mammals,
non-human mammals, animals, such as horses involved in load bearing
or athletic activities (e.g., races) and pets (e.g., dogs and
cats).
[0074] The aspects of the invention are directed to animals
(vertebrates), mammals, non-human mammals, animals, such as horses
involved in load bearing or athletic activities (e.g., races) and
pets (e.g., dogs and cats).
BRIEF DESCRIPTION OF THE DRAWINGS
[0075] FIG. 1 is a graphical representation of a receiver operating
curve (ROC) for comparison of gene expression 24 hours
post-induction. The ROC curve generated from these data
demonstrated that 24 hours post-induction, was well separated from
0 hours. The sensitivity and selectivity using two principal
components are 1.00 and 1.00 respectively.
[0076] FIG. 2 is a graphical representation of ROC for comparison
of gene expression 72 hours post-induction. The ROC curve generated
from these data demonstrated that 72 hours post-induction, was well
separated from 0 hours. The sensitivity and selectivity using two
principal components are 0.667 and 1.00 respectively. Using four
principal components this can be improved to 0.883 and 1
respectively (not shown).
DETAILED DESCRIPTION OF THE INVENTION
1. Definitions
[0077] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by those
of ordinary skill in the art to which the invention belongs.
Although any methods and materials similar or equivalent to those
described herein can be used in the practice or testing of the
present invention, preferred methods and materials are described.
For the purposes of the present invention, the following terms are
defined below.
[0078] The articles "a" and "an" are used herein to refer to one or
to more than one (i.e., to at least one) of the grammatical object
of the article. By way of example, "an element" means one element
or more than one element.
[0079] The term "aberrant expression," as used herein to describe
the expression of an endotoxemia marker gene, refers to the
overexpression or underexpression of an endotoxemia marker gene
relative to the level of expression of the endotoxemia marker gene
or variant thereof in cells obtained from a healthy subject or from
a subject lacking endotoxemia, and/or to a higher or lower level of
an endotoxemia marker gene product (e.g., transcript or
polypeptide) in a tissue sample or body fluid obtained from a
healthy subject or from a subject lacking endotoxemia. In
particular, an endotoxemia marker gene is aberrantly expressed if
the level of expression of the endotoxemia marker gene is higher by
at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%, or
even an at least about 100%, 200%, 300%, 400%, 500%, 600%, 700%,
800%, 900% or 1000%, or lower by at least about 10%, 20%, 30% 40%,
50%, 60%, 70%, 80%, 90%, 92%, 94%, 96%, 97%, 98% or 99%, or even an
at least about 99.5%, 99.9%, 99.95%, 99.99%, 99.995% or 99.999%
than the level of expression of the endotoxemia marker gene by
cells obtained from a healthy subject or from a subject without
endotoxemia, and/or relative to the level of expression of the
endotoxemia marker gene in a tissue sample or body fluid obtained
from a healthy subject or from a subject without endotoxemia.
[0080] By "about" is meant a quantity, level, value, number,
frequency, percentage, dimension, size, amount, weight or length
that varies by as much as 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4, 3,
2 or 1% to a reference quantity, level, value, number, frequency,
percentage, dimension, size, amount, weight or length.
[0081] The term "amplicon" refers to a target sequence for
amplification, and/or the amplification products of a target
sequence for amplification. In certain other embodiments an
"amplicon" may include the sequence of probes or primers used in
amplification.
[0082] By "antigen-binding molecule" is meant a molecule that has
binding affinity for a target antigen. It will be understood that
this term extends to immunoglobulins, immunoglobulin fragments and
non-immunoglobulin derived protein frameworks that exhibit
antigen-binding activity.
[0083] As used herein, the term "binds specifically," "specifically
immuno-interactive" and the like when referring to an
antigen-binding molecule refers to a binding reaction which is
determinative of the presence of an antigen in the presence of a
heterogeneous population of proteins and other biologics. Thus,
under designated immunoassay conditions, the specified
antigen-binding molecules bind to a particular antigen and do not
bind in a significant amount to other proteins or antigens present
in the sample. Specific binding to an antigen under such conditions
may require an antigen-binding molecule that is selected for its
specificity for a particular antigen. For example, antigen-binding
molecules can be raised to a selected protein antigen, which bind
to that antigen but not to other proteins present in a sample. A
variety of immunoassay formats may be used to select
antigen-binding molecules specifically immuno-interactive with a
particular protein. For example, solid-phase ELISA immunoassays are
routinely used to select monoclonal antibodies specifically
immuno-interactive with a protein. See Harlow and Lane (1988)
Antibodies, A Laboratory Manual, Cold Spring Harbor Publications,
New York, for a description of immunoassay formats and conditions
that can be used to determine specific immunoreactivity.
[0084] By "biologically active portion" is meant a portion of a
full-length parent peptide or polypeptide which portion retains an
activity of the parent molecule. As used herein, the term
"biologically active portion" includes deletion mutants and
peptides, for example of at least about 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80,
90, 100, 120, 150, 300, 400, 500, 600, 700, 800, 900, 1000
contiguous amino acids, which comprise an activity of a parent
molecule. Portions of this type may be obtained through the
application of standard recombinant nucleic acid techniques or
synthesized using conventional liquid or solid phase synthesis
techniques. For example, reference may be made to solution
synthesis or solid phase synthesis as described, for example, in
Chapter 9 entitled "Peptide Synthesis" by Atherton and Shephard
which is included in a publication entitled "Synthetic Vaccines"
edited by Nicholson and published by Blackwell Scientific
Publications. Alternatively, peptides can be produced by digestion
of a peptide or polypeptide of the invention with proteinases such
as endoLys-C, endoArg-C, endoGlu-C and staphylococcus V8-protease.
The digested fragments can be purified by, for example, high
performance liquid chromatographic (HPLC) techniques. Recombinant
nucleic acid techniques can also be used to produce such
portions.
[0085] The term "biological sample" as used herein refers to a
sample that may be extracted, untreated, treated, diluted or
concentrated from an animal. The biological sample may include a
biological fluid such as whole blood, serum, plasma, saliva, urine,
sweat, ascitic fluid, peritoneal fluid, synovial fluid, amniotic
fluid, cerebrospinal fluid, tissue biopsy, and the like. In certain
embodiments, the biological sample is blood, especially peripheral
blood.
[0086] As used herein, the term "cis-acting sequence," "cis-acting
element" or "cis-regulatory region" or "regulatory region" or
similar term shall be taken to mean any sequence of nucleotides,
which when positioned appropriately relative to an expressible
genetic sequence, is capable of regulating, at least in part, the
expression of the genetic sequence. Those skilled in the art will
be aware that a cis-regulatory region may be capable of activating,
silencing, enhancing, repressing or otherwise altering the level of
expression and/or cell-type-specificity and/or developmental
specificity of a gene sequence at the transcriptional or
post-transcriptional level. In certain embodiments of the present
invention, the cis-acting sequence is an activator sequence that
enhances or stimulates the expression of an expressible genetic
sequence.
[0087] Throughout this specification, unless the context requires
otherwise, the words "comprise," "comprises" and "comprising" will
be understood to imply the inclusion of a stated step or element or
group of steps or elements but not the exclusion of any other step
or element or group of steps or elements.
[0088] By "corresponds to" or "corresponding to" is meant a
polynucleotide (a) having a nucleotide sequence that is
substantially identical or complementary to all or a portion of a
reference polynucleotide sequence or (b) encoding an amino acid
sequence identical to an amino acid sequence in a peptide or
protein. This phrase also includes within its scope a peptide or
polypeptide having an amino acid sequence that is substantially
identical to a sequence of amino acids in a reference peptide or
protein.
[0089] By "effective amount", in the context of treating or
preventing a condition is meant the administration of that amount
of active to an individual in need of such treatment or
prophylaxis, either in a single dose or as part of a series, that
is effective for the prevention of incurring a symptom, holding in
check such symptoms, and/or treating existing symptoms, of that
condition. The effective amount will vary depending upon the health
and physical condition of the individual to be treated, the
taxonomic group of individual to be treated, the formulation of the
composition, the assessment of the medical situation, and other
relevant factors. It is expected that the amount will fall in a
relatively broad range that can be determined through routine
trials.
[0090] The terms "expression" or "gene expression" refer to either
production of RNA message or translation of RNA message into
proteins or polypeptides. Detection of either types of gene
expression in use of any of the methods described herein are part
of the invention.
[0091] By "expression vector" is meant any autonomous genetic
element capable of directing the transcription of a polynucleotide
contained within the vector and suitably the synthesis of a peptide
or polypeptide encoded by the polynucleotide. Such expression
vectors are known to practitioners in the art.
[0092] As used herein, the term "functional activity" generally
refers to the ability of a molecule (e.g., a transcript or
polypeptide) to perform its designated function including a
biological, enzymatic, or therapeutic function. In certain
embodiments, the functional activity of a molecule corresponds to
its specific activity as determined by any suitable assay known in
the art.
[0093] The term "gene" as used herein refers to any and all
discrete coding regions of the cell's genome, as well as associated
non-coding and regulatory regions. The gene is also intended to
mean the open reading frame encoding specific polypeptides,
introns, and adjacent 5' and 3' non-coding nucleotide sequences
involved in the regulation of expression. In this regard, the gene
may further comprise control signals such as promoters, enhancers,
termination and/or polyadenylation signals that are naturally
associated with a given gene, or heterologous control signals. The
DNA sequences may be cDNA or genomic DNA or a fragment thereof. The
gene may be introduced into an appropriate vector for
extrachromosomal maintenance or for integration into the host.
[0094] By "high density polynucleotide arrays" and the like is
meant those arrays that contain at least 400 different features per
cm.sup.2.
[0095] The phrase "high discrimination hybridization conditions"
refers to hybridization conditions in which single base mismatch
may be determined.
[0096] By "housekeeping gene" is meant a gene that is expressed in
virtually all cells since it is fundamental to the any cell's
functions (e.g., essential proteins and RNA molecules).
[0097] "Hybridization" is used herein to denote the pairing of
complementary nucleotide sequences to produce a DNA-DNA hybrid or a
DNA-RNA hybrid. Complementary base sequences are those sequences
that are related by the base-pairing rules. In DNA, A pairs with T
and C pairs with G. In RNA, U pairs with A and C pairs with G. In
this regard, the terms "match" and "mismatch" as used herein refer
to the hybridization potential of paired nucleotides in
complementary nucleic acid strands. Matched nucleotides hybridize
efficiently, such as the classical A-T and G-C base pair mentioned
above. Mismatches are other combinations of nucleotides that do not
hybridize efficiently.
[0098] The phrase "hybridizing specifically to" and the like refer
to the binding, duplexing, or hybridizing of a molecule only to a
particular nucleotide sequence under stringent conditions when that
sequence is present in a complex mixture (e.g., total cellular) DNA
or RNA.
[0099] Reference herein to "immuno-interactive" includes reference
to any interaction, reaction, or other form of association between
molecules and in particular where one of the molecules is, or
mimics, a component of the immune system.
[0100] By "isolated" is meant material that is substantially or
essentially free from components that normally accompany it in its
native state. For example, an "isolated polynucleotide", as used
herein, refers to a polynucleotide, which has been purified from
the sequences which flank it in a naturally-occurring state, e.g.,
a DNA fragment which has been removed from the sequences that are
normally adjacent to the fragment. Alternatively, an "isolated
peptide" or an "isolated polypeptide" and the like, as used herein,
refer to in vitro isolation and/or purification of a peptide or
polypeptide molecule from its natural cellular environment, and
from association with other components of the cell, i.e., it is not
associated with in vivo substances.
[0101] By "marker gene" is meant a gene that imparts a distinct
phenotype to cells expressing the marker gene and thus allows such
transformed cells to be distinguished from cells that do not have
the marker. A selectable marker gene confers a trait for which one
can `select` based on resistance to a selective agent (e.g., a
herbicide, antibiotic, radiation, heat, or other treatment damaging
to untransformed cells). A screenable marker gene (or reporter
gene) confers a trait that one can identify through observation or
testing, i.e., by `screening` (e.g. .beta.-glucuronidase,
luciferase, or other enzyme activity not present in untransformed
cells).
[0102] As used herein, a "naturally-occurring" nucleic acid
molecule refers to a RNA or DNA molecule having a nucleotide
sequence that occurs in nature. For example a naturally-occurring
nucleic acid molecule can encode a protein that occurs in
nature.
[0103] By "obtained from" is meant that a sample such as, for
example, a nucleic acid extract or polypeptide extract is isolated
from, or derived from, a particular source. For instance, the
extract may be isolated directly from a biological fluid or tissue
of the subject.
[0104] The term "oligonucleotide" as used herein refers to a
polymer composed of a multiplicity of nucleotide residues
(deoxyribonucleotides or ribonucleotides, or related structural
variants or synthetic analogues thereof, including nucleotides with
modified or substituted sugar groups and the like) linked via
phosphodiester bonds (or related structural variants or synthetic
analogues thereof). Thus, while the term "oligonucleotide"
typically refers to a nucleotide polymer in which the nucleotide
residues and linkages between them are naturally-occurring, it will
be understood that the term also includes within its scope various
analogues including, but not restricted to, peptide nucleic acids
(PNAs), phosphorothioate, phosphorodithioate, phosphoroselenoate,
phosphorodiselenoate, phosphoroanilothioate, phosphoraniladate,
phosphoroamidate, methyl phosphonates, 2-O-methyl ribonucleic
acids, and the like. The exact size of the molecule can vary
depending on the particular application. Oligonucleotides are a
polynucleotide subset with 200 bases or fewer in length.
Preferably, oligonucleotides are 10 to 60 bases in length and most
preferably 12, 13, 14, 15, 16, 17, 18, 19, or 20 to 40 bases in
length. Oligonucleotides are usually single stranded, e.g., for
probes; although oligonucleotides may be double stranded, e.g., for
use in the construction of a variant nucleic acid sequence.
Oligonucleotides of the invention can be either sense or antisense
oligonucleotides.
[0105] The term "oligonucleotide array" refers to a substrate
having oligonucleotide probes with different known sequences
deposited at discrete known locations associated with its surface.
For example, the substrate can be in the form of a two dimensional
substrate as described in U.S. Pat. No. 5,424,186. Such substrate
may be used to synthesize two-dimensional spatially addressed
oligonucleotide (matrix) arrays. Alternatively, the substrate may
be characterized in that it forms a tubular array in which a two
dimensional planar sheet is rolled into a three-dimensional tubular
configuration. The substrate may also be in the form of a
microsphere or bead connected to the surface of an optic fiber as,
for example, disclosed by Chee et al. in WO 00/39587.
Oligonucleotide arrays have at least two different features and a
density of at least 400 features per cm.sup.2. In certain
embodiments, the arrays can have a density of about 500, at least
one thousand, at least 10 thousand, at least 100 thousand, at least
one million or at least 10 million features per cm.sup.2. For
example, the substrate may be silicon or glass and can have the
thickness of a glass microscope slide or a glass cover slip, or may
be composed of other synthetic polymers. Substrates that are
transparent to light are useful when the method of performing an
assay on the substrate involves optical detection. The term also
refers to a probe array and the substrate to which it is attached
that form part of a wafer.
[0106] The term "operably connected" or "operably linked" as used
herein means placing a structural gene under the regulatory control
of a promoter, which then controls the transcription and optionally
translation of the gene. In the construction of heterologous
promoter/structural gene combinations, it is generally preferred to
position the genetic sequence or promoter at a distance from the
gene transcription start site that is approximately the same as the
distance between that genetic sequence or promoter and the gene it
controls in its natural setting; i.e. the gene from which the
genetic sequence or promoter is derived. As is known in the art,
some variation in this distance can be accommodated without loss of
function. Similarly, the preferred positioning of a regulatory
sequence element with respect to a heterologous gene to be placed
under its control is defined by the positioning of the element in
its natural setting; i.e., the genes from which it is derived.
[0107] The term "pathogen" is used herein in its broadest sense to
refer to an organism or an infectious agent whose infection of
cells of viable animal tissue elicits a disease response.
[0108] The term "polynucleotide" or "nucleic acid" as used herein
designates mRNA, RNA, cRNA, cDNA or DNA. The term typically refers
to polymeric form of nucleotides of at least 10 bases in length,
either ribonucleotides or deoxynucleotides or a modified form of
either type of nucleotide. The term includes single and double
stranded forms of DNA.
[0109] The terms "polynucleotide variant" and "variant" refer to
polynucleotides displaying substantial sequence identity with a
reference polynucleotide sequence or polynucleotides that hybridize
with a reference sequence under stringent conditions that are
defined hereinafter. These terms also encompass polynucleotides in
which one or more nucleotides have been added or deleted, or
replaced with different nucleotides. In this regard, it is well
understood in the art that certain alterations inclusive of
mutations, additions, deletions and substitutions can be made to a
reference polynucleotide whereby the altered polynucleotide retains
a biological function or activity of the reference polynucleotide.
The terms "polynucleotide variant" and "variant" also include
naturally-occurring allelic variants.
[0110] "Polypeptide", "peptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acid residues
and to variants and synthetic analogues of the same. Thus, these
terms apply to amino acid polymers in which one or more amino acid
residues is a synthetic non-naturally-occurring amino acid, such as
a chemical analogue of a corresponding naturally-occurring amino
acid, as well as to naturally-occurring amino acid polymers.
[0111] The term "polypeptide variant" refers to polypeptides which
are distinguished from a reference polypeptide by the addition,
deletion or substitution of at least one amino acid residue. In
certain embodiments, one or more amino acid residues of a reference
polypeptide are replaced by different amino acids. It is well
understood in the art that some amino acids may be changed to
others with broadly similar properties without changing the nature
of the activity of the polypeptide (conservative substitutions) as
described hereinafter.
[0112] By "primer" is meant an oligonucleotide which, when paired
with a strand of DNA, is capable of initiating the synthesis of a
primer extension product in the presence of a suitable polymerizing
agent. The primer is preferably single-stranded for maximum
efficiency in amplification but can alternatively be
double-stranded. A primer must be sufficiently long to prime the
synthesis of extension products in the presence of the
polymerization agent. The length of the primer depends on many
factors, including application, temperature to be employed,
template reaction conditions, other reagents, and source of
primers. For example, depending on the complexity of the target
sequence, the primer may be at least about 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400, 500, to one base
shorter in length than the template sequence at the 3' end of the
primer to allow extension of a nucleic acid chain, though the 5'
end of the primer may extend in length beyond the 3' end of the
template sequence. In certain embodiments, primers can be large
polynucleotides, such as from about 35 nucleotides to several
kilobases or more. Primers can be selected to be "substantially
complementary" to the sequence on the template to which it is
designed to hybridize and serve as a site for the initiation of
synthesis. By "substantially complementary", it is meant that the
primer is sufficiently complementary to hybridize with a target
polynucleotide. Desirably, the primer contains no mismatches with
the template to which it is designed to hybridize but this is not
essential. For example, non-complementary nucleotide residues can
be attached to the 5' end of the primer, with the remainder of the
primer sequence being complementary to the template. Alternatively,
non-complementary nucleotide residues or a stretch of
non-complementary nucleotide residues can be interspersed into a
primer, provided that the primer sequence has sufficient
complementarity with the sequence of the template to hybridize
therewith and thereby form a template for synthesis of the
extension product of the primer.
[0113] "Probe" refers to a molecule that binds to a specific
sequence or sub-sequence or other moiety of another molecule.
Unless otherwise indicated, the term "probe" typically refers to a
polynucleotide probe that binds to another polynucleotide, often
called the "target polynucleotide", through complementary base
pairing. Probes can bind target polynucleotides lacking complete
sequence complementarity with the probe, depending on the
stringency of the hybridization conditions. Probes can be labeled
directly or indirectly and include primers within their scope.
[0114] The term "recombinant polynucleotide" as used herein refers
to a polynucleotide formed in vitro by the manipulation of nucleic
acid into a form not normally found in nature. For example, the
recombinant polynucleotide may be in the form of an expression
vector. Generally, such expression vectors include transcriptional
and translational regulatory nucleic acid operably linked to the
nucleotide sequence.
[0115] By "recombinant polypeptide" is meant a polypeptide made
using recombinant techniques, i.e., through the expression of a
recombinant or synthetic polynucleotide.
[0116] By "regulatory element" or "regulatory sequence" is meant
nucleic acid sequences (e.g., DNA) necessary for expression of an
operably linked coding sequence in a particular host cell. The
regulatory sequences that are suitable for prokaryotic cells for
example, include a promoter, and optionally a cis-acting sequence
such as an operator sequence and a ribosome binding site. Control
sequences that are suitable for eukaryotic cells include promoters,
polyadenylation signals, transcriptional enhancers, translational
enhancers, leader or trailing sequences that modulate mRNA
stability, as well as targeting sequences that target a product
encoded by a transcribed polynucleotide to an intracellular
compartment within a cell or to the extracellular environment.
[0117] The term "sequence identity" as used herein refers to the
extent that sequences are identical on a nucleotide-by-nucleotide
basis or an amino acid-by-amino acid basis over a window of
comparison. Thus, a "percentage of sequence identity" is calculated
by comparing two optimally aligned sequences over the window of
comparison, determining the number of positions at which the
identical nucleic acid base (e.g., A, T, C, G, I) or the identical
amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile,
Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met)
occurs in both sequences to yield the number of matched positions,
dividing the number of matched positions by the total number of
positions in the window of comparison (i.e., the window size), and
multiplying the result by 100 to yield the percentage of sequence
identity. For the purposes of the present invention, "sequence
identity" will be understood to mean the "match percentage"
calculated by the DNASIS computer program (Version 2.5 for windows;
available from Hitachi Software engineering Co., Ltd., South San
Francisco, Calif., USA) using standard defaults as used in the
reference manual accompanying the software.
[0118] "Similarity" refers to the percentage number of amino acids
that are identical or constitute conservative substitutions as
defined in Table A infra. Similarity may be determined using
sequence comparison programs such as GAP (Deveraux et al. 1984,
Nucleic Acids Research 12, 387-395). In this way, sequences of a
similar or substantially different length to those cited herein
might be compared by insertion of gaps into the alignment, such
gaps being determined, for example, by the comparison algorithm
used by GAP.
[0119] Terms used to describe sequence relationships between two or
more polynucleotides or polypeptides include "reference sequence,"
"comparison window," "sequence identity," "percentage of sequence
identity" and "substantial identity". A "reference sequence" is at
least 12 but frequently 15 to 18 and often at least 25 monomer
units, inclusive of nucleotides and amino acid residues, in length.
Because two polynucleotides may each comprise (1) a sequence (i.e.,
only a portion of the complete polynucleotide sequence) that is
similar between the two polynucleotides, and (2) a sequence that is
divergent between the two polynucleotides, sequence comparisons
between two (or more) polynucleotides are typically performed by
comparing sequences of the two polynucleotides over a "comparison
window" to identify and compare local regions of sequence
similarity. A "comparison window" refers to a conceptual segment of
at least 6 contiguous positions, usually about 50 to about 100,
more usually about 100 to about 150 in which a sequence is compared
to a reference sequence of the same number of contiguous positions
after the two sequences are optimally aligned. The comparison
window may comprise additions or deletions (i.e., gaps) of about
20% or less as compared to the reference sequence (which does not
comprise additions or deletions) for optimal alignment of the two
sequences. Optimal alignment of sequences for aligning a comparison
window may be conducted by computerized implementations of
algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin
Genetics Software Package Release 7.0, Genetics Computer Group, 575
Science Drive Madison, Wis., USA) or by inspection and the best
alignment (i.e., resulting in the highest percentage homology over
the comparison window) generated by any of the various methods
selected. Reference also may be made to the BLAST family of
programs as for example disclosed by Altschul et al., 1997, Nucl.
Acids Res. 25:3389. A detailed discussion of sequence analysis can
be found in Unit 19.3 of Ausubel et al., "Current Protocols in
Molecular Biology", John Wiley & Sons Inc, 1994-1998, Chapter
15.
[0120] The terms "subject" or "individual" or "patient", used
interchangeably herein, refer to any subject, particularly a
vertebrate subject, and even more particularly a mammalian subject,
for whom therapy or prophylaxis is desired. Suitable vertebrate
animals that fall within the scope of the invention include, but
are not restricted to, primates, avians, livestock animals (e.g.,
sheep, cows, horses, donkeys, pigs), laboratory test animals (e.g.,
rabbits, mice, rats, guinea pigs, hamsters), companion animals
(e.g., cats, dogs) and captive wild animals (e.g., foxes, deer,
dingoes). A preferred subject is an equine animal in need of
treatment or prophylaxis of endotoxemia. However, it will be
understood that the aforementioned terms do not imply that symptoms
are present.
[0121] The phrase "substantially similar affinities" refers herein
to target sequences having similar strengths of detectable
hybridization to their complementary or substantially complementary
oligonucleotide probes under a chosen set of stringent
conditions.
[0122] The term "template" as used herein refers to a nucleic acid
that is used in the creation of a complementary nucleic acid strand
to the "template" strand. The template may be either RNA and/or
DNA, and the complementary strand may also be RNA and/or DNA. In
certain embodiments, the complementary strand may comprise all or
part of the complementary sequence to the "template," and/or may
include mutations so that it is not an exact, complementary strand
to the "template". Strands that are not exactly complementary to
the template strand may hybridize specifically to the template
strand in detection assays described here, as well as other assays
known in the art, and such complementary strands that can be used
in detection assays are part of the invention.
[0123] The term "transformation" means alteration of the genotype
of an organism, for example a bacterium, yeast, mammal, avian,
reptile, fish or plant, by the introduction of a foreign or
endogenous nucleic acid.
[0124] The term "treat" is meant to include both therapeutic and
prophylactic treatment.
[0125] By "vector" is meant a polynucleotide molecule, suitably a
DNA molecule derived, for example, from a plasmid, bacteriophage,
yeast, virus, mammal, avian, reptile or fish into which a
polynucleotide can be inserted or cloned. A vector preferably
contains one or more unique restriction sites and can be capable of
autonomous replication in a defined host cell including a target
cell or tissue or a progenitor cell or tissue thereof, or be
integrable with the genome of the defined host such that the cloned
sequence is reproducible. Accordingly, the vector can be an
autonomously replicating vector, i.e., a vector that exists as an
extrachromosomal entity, the replication of which is independent of
chromosomal replication, e.g., a linear or closed circular plasmid,
an extrachromosomal element, a minichromosome, or an artificial
chromosome. The vector can contain any means for assuring
self-replication. Alternatively, the vector can be one which, when
introduced into the host cell, is integrated into the genome and
replicated together with the chromosome(s) into which it has been
integrated. A vector system can comprise a single vector or
plasmid, two or more vectors or plasmids, which together contain
the total DNA to be introduced into the genome of the host cell, or
a transposon. The choice of the vector will typically depend on the
compatibility of the vector with the host cell into which the
vector is to be introduced. The vector can also include a selection
marker such as an antibiotic resistance gene that can be used for
selection of suitable transformants. Examples of such resistance
genes are known to those of skill in the art.
[0126] The terms "wild-type" and "normal" are used interchangeably
to refer to the phenotype that is characteristic of most of the
members of the species occurring naturally and contrast for example
with the phenotype of a mutant.
2. Abbreviations
[0127] The following abbreviations are used throughout the
application:
[0128] nt=nucleotide
[0129] nts=nucleotides
[0130] aa=amino acid(s)
[0131] kb=kilobase(s) or kilobase pair(s)
[0132] kDa=kilodalton(s)
[0133] d=day
[0134] h=hour
[0135] s=seconds
3. Markers of Endotoxemia and Uses Therefor
[0136] The present invention concerns the early detection,
diagnosis, or prognosis of endotoxemia or its sequelae (also
referred to herein as "endotoxemia-related conditions"). Markers of
endotoxemia, in the form of RNA molecules of specified sequences,
or polypeptides expressed from these RNA molecules in cells,
especially in blood cells, and more especially in peripheral blood
cells, of subjects with or susceptible to endotoxemia, are
disclosed. These markers are indicators of endotoxemia-related
conditions and, when differentially expressed as compared to their
expression in normal subjects or in subjects lacking
endotoxemia-related conditions, are diagnostic for the presence of
those conditions in tested subjects. Such markers provide
considerable advantages over the prior art in this field. In
certain advantageous embodiments where leukocytes (e.g., peripheral
blood cells) are used for the analysis, it is possible to diagnose
active endotoxemia-related conditions before serum antibodies to
endotoxin, or endotoxaemia-causing agent are detected.
[0137] It will be apparent that the nucleic acid sequences
disclosed herein will find utility in a variety of applications in
detection, diagnosis, prognosis and treatment of
endotoxemia-related conditions. Examples of such applications
within the scope of the present disclosure comprise amplification
of endotoxemia markers using specific primers, detection of
endotoxemia markers by hybridization with oligonucleotide probes,
incorporation of isolated nucleic acids into vectors, expression of
vector-incorporated nucleic acids as RNA and protein, and
development of immunological reagents corresponding to marker
encoded products.
[0138] The identified endotoxemia markers may in turn be used to
design specific oligonucleotide probes and primers. Such probes and
primers may be of any length that would specifically hybridize to
the identified marker gene sequences and may be at least about 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400, 500
nucleotides in length and in the case of probes, up to the full
length of the sequences of the marker genes identified herein.
Probes may also include additional sequence at their 5' and/or 3'
ends so that they extent beyond the target sequence with which they
hybridize.
[0139] When used in combination with nucleic acid amplification
procedures, these probes and primers enable the rapid analysis of
biological samples (e.g., peripheral blood samples) for detecting
marker genes or for detecting or quantifying marker gene
transcripts. Such procedures include any method or technique known
in the art or described herein for duplicating or increasing the
number of copies or amount of a target nucleic acid or its
complement.
[0140] The identified markers may also be used to identify and
isolate full-length gene sequences, including regulatory elements
for gene expression, from genomic DNA libraries, which are suitably
but not exclusively of equine origin. The cDNA sequences identified
in the present disclosure may be used as hybridization probes to
screen genomic DNA libraries by conventional techniques. Once
partial genomic clones have been identified, full-length genes may
be isolated by "chromosomal walking" (also called "overlap
hybridization") using, for example, the method disclosed by
Chinault & Carbon (1979, Gene 5: 111-126). Once a partial
genomic clone has been isolated using a cDNA hybridization probe,
non-repetitive segments at or near the ends of the partial genomic
clone may be used as hybridization probes in further genomic
library screening, ultimately allowing isolation of entire gene
sequences for the endotoxemia markers of interest. It will be
recognized that full-length genes may be obtained using the
full-length or partial cDNA sequences or short expressed sequence
tags (ESTs) described in this disclosure using standard techniques
as disclosed for example by Sambrook, et al. (MOLECULAR CLONING. A
LABORATORY MANUAL (Cold Spring Harbor Press, 1989) and Ausubel et
al., (CURRENT PROTOCOLS 1N MOLECULAR BIOLOGY, John Wiley &
Sons, Inc. 1994). In addition, the disclosed sequences may be used
to identify and isolate full-length cDNA sequences using standard
techniques as disclosed, for example, in the above-referenced
texts. Sequences identified and isolated by such means may be
useful in the detection of the endotoxemia marker genes using the
detection methods described herein, and are part of the
invention.
[0141] One of ordinary skill in the art could select segments from
the identified marker genes for use in the different detection,
diagnostic, or prognostic methods, vector constructs,
antigen-binding molecule production, kit, and/or any of the
embodiments described herein as part of the present invention.
Marker gene sequences that are desirable for use in the invention
are those set fort in SEQ ID NO: 1, 3, 5, 7, 8, 10, 12, 14, 16, 17,
18, 20, 22, 24, 26, 27, 28, 30, 32, 34, 35, 36, 37, 38, 39, 40, 41,
42, 43, 44, 45, 46, 47, 48, 49, 50, 52, 54, 55, 56 or 57 (see Table
1).
4. Nucleic Acid Molecules of the Invention
[0142] As described in the Examples and in Tables 1, the present
disclosure provides 179 markers of endotoxemia, identified by
GeneChip.RTM. analysis of blood obtained from normal horses and
from horses with clinical evidence of an endotoxemia-related
condition. Of the 179 markers identified, 121 comprise coding
regions sequences (see the markers relating to SEQ ID NO: 1, 5, 6,
7, 11, 13, 19, 21, 23, 27, 29, 31, 33, 35, 39, 45, 47, 50, 52, 54,
56, 58, 60, 61, 64, 71, 73, 77, 78, 84, 86, 88, 90, 94, 96, 98,
102, 103, 104, 107, 111, 115, 117, 119, 126, 128, 130, 132, 134,
137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 158, 160, 162,
164, 166, 170, 174, 176, 178, 180, 182, 184, 186, 188, 190, 195,
197, 199, 201, 203, 207, 212, 216, 218, 220, 225, 227, 229, 231,
233, 237, 240, 242, 246, 248, 250, 252, 255, 257, 260, 262, 264,
266, 272, 274, 276, 280, 282, 284, 286, 288, 290, 292, 294, 296,
298, 300, 302, 307, 309, 312, 316, 318, 321, 323 or 325) and 58
comprise 5' and/or 3' untranslated sequences only (see the markers
relating to SEQ ID NO: 3, 4, 9, 15, 16, 17, 18, 25, 26, 37, 38, 41,
42, 43, 44, 49, 63, 66, 67, 68, 69, 70, 75, 76, 79, 80, 81, 82, 83,
92, 93, 100, 101, 102, 103, 106, 109, 110, 113, 114, 121, 122, 123,
124, 125, 136, 157, 168, 169, 172, 173, 192, 193, 194, 205, 206,
209, 210, 211, 214, 215, 222, 223, 224, 235, 236, 239, 244, 245,
254, 259, 268, 269, 270, 271, 278, 279, 304, 305, 306, 311, 314,
315 or 320). These sequences, which are presented in Table 1, are
diagnostic for the presence, stage or degree of an
endotoxemia-related condition (also referred to herein as
"endotoxemia marker polynucleotides"). Sequence analysis has
revealed that the endotoxaemia marker genes can be classified into
subgroups. For example, several endotoxaemia marker genes encode
membrane associated polypeptides involved in the immune response
(e.g., SEQ ID NO. 59, 219, 230, 253, 285, and 295), whereas others
encode cytoplasm associated polypeptides (e.g., SEQ ID NO: 217,
241, 247, 265, 267, 287, and 291), while still others encode
extracellular polypeptides (e.g., SEQ ID NO: 30, 85, 108, 127, 140,
and 226), whereas still others encode nuclear polypeptides (e.g.,
8, 12, 20, 28, 40, 55, 281, 283 and 313) and still others encode
cytoskeleton molecules (e.g., SEQ ID NO: 105 and 213).
[0143] In accordance with the present invention, the sequences of
isolated nucleic acids disclosed herein find utility inter alia as
hybridization probes or amplification primers. These nucleic acids
may be used, for example, in diagnostic evaluation of biological
samples or employed to clone full-length cDNAs or genomic clones
corresponding thereto. In certain embodiments, these probes and
primers represent oligonucleotides, which are of sufficient length
to provide specific hybridization to a RNA or DNA sample extracted
from the biological sample. The sequences typically will be about
10-20 nucleotides, but may be longer. Longer sequences, e.g., of
about 30, 40, 50, 100, 500 and even up to full-length, are
desirable for certain embodiments.
[0144] Nucleic acid molecules having contiguous stretches of about
10, 15, 17, 20, 30, 40, 50, 60, 75 or 100 or 500 nucleotides of a
sequence set forth in any one of SEQ ID NO: 1, 3, 5, 7, 8, 10, 12,
14, 16, 17, 18, 20, 22, 24, 26, 27, 28, 30, 32, 34, 35, 36, 37, 38,
39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 52, 54, 55, 56 or
57 are contemplated. Molecules that are complementary to the above
mentioned sequences and that bind to these sequences under high
stringency conditions are also contemplated. These probes are
useful in a variety of hybridization embodiments, such as Southern
and northern blotting. In some cases, it is contemplated that
probes may be used that hybridize to multiple target sequences
without compromising their ability to effectively diagnose an
endotoxemia-related condition. In general, it is contemplated that
the hybridization probes described herein are useful both as
reagents in solution hybridization, as in PCR, for detection of
expression of corresponding genes, as well as in embodiments
employing a solid phase.
[0145] Various probes and primers may be designed around the
disclosed nucleotide sequences. For example, in certain
embodiments, the sequences used to design probes and primers may
include repetitive stretches of adenine nucleotides (poly-A tails)
normally attached at the ends of the RNA for the identified marker
genes. In other embodiments, probes and primers may be specifically
designed to not include these or other segments from the identified
marker genes, as one of ordinary skilled in the art may deem
certain segments more suitable for use in the detection methods
disclosed. In any event, the choice of primer or probe sequences
for a selected application is within the realm of the ordinary
skilled practitioner. Illustrative probe sequences for detection of
endotoxemia marker genes are presented in Tables 2.
[0146] Primers may be provided in double-stranded or
single-stranded form, although the single-stranded form is
desirable. Probes, while perhaps capable of priming, are designed
to bind to a target DNA or RNA and need not be used in an
amplification process. In certain embodiments, the probes or
primers are labeled with radioactive species .sup.32P, .sup.14C,
.sup.35S, .sup.3H, or other label), with a fluorophore (e.g.,
rhodamine, fluorescein) or with a chemillumiscent label (e.g.,
luciferase).
[0147] The present invention provides substantially full-length
cDNA sequences as well as EST and partial cDNA sequences that are
useful as markers of endotoxemia-related condition. It will be
understood, however, that the present disclosure is not limited to
these disclosed sequences and is intended particularly to encompass
at least isolated nucleic acids that are hybridizable to nucleic
acids comprising the disclosed sequences or that are variants of
these nucleic acids. For example, a nucleic acid of partial
sequence may be used to identify a structurally-related gene or the
full-length genomic or cDNA clone from which it is derived. Methods
for generating cDNA and genomic libraries which may be used as a
target for the above-described probes are known in the art (see,
for example, Sambrook et al., 1989, supra and Ausubel et al., 1994,
supra). All such nucleic acids as well as the specific nucleic acid
molecules disclosed herein are collectively referred to as
"endotoxemia marker polynucleotides." Additionally, the present
invention includes within its scope isolated or purified expression
products of endotoxemia marker polynucleotides (i.e., RNA
transcripts and polypeptides).
[0148] Accordingly, the present invention encompasses isolated or
substantially purified nucleic acid or protein compositions. An
"isolated" or "purified" nucleic acid molecule or protein, or
biologically active portion thereof, is substantially or
essentially free from components that normally accompany or
interact with the nucleic acid molecule or protein as found in its
naturally occurring environment. Thus, an isolated or purified
polynucleotide or polypeptide is substantially free of other
cellular material, or culture medium when produced by recombinant
techniques, or substantially free of chemical precursors or other
chemicals when chemically synthesized. Suitably, an "isolated"
polynucleotide is free of sequences (especially protein encoding
sequences) that naturally flank the polynucleotide (i.e., sequences
located at the 5' and 3' ends of the polynucleotide) in the genomic
DNA of the organism from which the polynucleotide was derived. For
example, in various embodiments, an isolated endotoxaemia marker
polynucleotide can contain less than about 5 kb, 4 kb, 3 kb, 2 kb,
1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally
flank the polynucleotide in genomic DNA of the cell from which the
polynucleotide was derived. A polypeptide that is substantially
free of cellular material includes preparations of protein having
less than about 30%, 20%, 10%, 5%, (by dry weight) of contaminating
protein. When the protein of the invention or biologically active
portion thereof is recombinantly produced, culture medium suitably
represents less than about 30%, 20%, 10%, or 5% (by dry weight) of
chemical precursors or non-protein-of-interest chemicals.
[0149] The present invention also encompasses portions of the
full-length or substantially full-length nucleotide sequences of
the endotoxemia marker genes or their transcripts or DNA copies of
these transcripts. Portions of an endotoxemia marker nucleotide
sequence may encode polypeptide portions or segments that retain
the biological activity of the native polypeptide. Alternatively,
portions of an endotoxemia marker nucleotide sequence that are
useful as hybridization probes generally do not encode amino acid
sequences retaining such biological activity. Thus, portions of an
endotoxemia marker nucleotide sequence may range from at least
about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,
30, 40, 50, 60, 80, 90, 100 nucleotides, or almost up to the
full-length nucleotide sequence encoding the endotoxemia marker
polypeptides of the invention.
[0150] A portion of an endotoxemia marker nucleotide sequence that
encodes a biologically active portion of an endotoxemia marker
polypeptide of the invention may encode at least about 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 300, 400, 500, 600, 700,
800, 900 or 1000, or even at least about 2000, 3000, 4000 or 5000
contiguous amino acid residues, or almost up to the total number of
amino acids present in a full-length endotoxemia marker
polypeptide. Portions of an endotoxemia marker nucleotide sequence
that are useful as hybridization probes or PCR primers generally
need not encode a biologically active portion of an endotoxemia
marker polypeptide.
[0151] Thus, a portion of an endotoxemia marker nucleotide sequence
may encode a biologically active portion of an endotoxemia marker
polypeptide, or it may be a fragment that can be used as a
hybridization probe or PCR primer using standard methods known in
the art. A biologically active portion of an endotoxemia marker
polypeptide can be prepared by isolating a portion of one of the
endotoxemia marker nucleotide sequences of the invention,
expressing the encoded portion of the endotoxemia marker
polypeptide (e.g., by recombinant expression in vitro), and
assessing the activity of the encoded portion of the endotoxemia
marker polypeptide. Nucleic acid molecules that are portions of an
endotoxemia marker nucleotide sequence comprise at least about 15,
16, 17, 18, 19, 20, 25, 30, 50, 75, 100, 150, 200, 250, 300, 350,
400, 450, 500, 550, 600, or 650 nucleotides, or almost up to the
number of nucleotides present in a full-length endotoxemia marker
nucleotide sequence.
[0152] The invention also contemplates variants of the endotoxemia
marker nucleotide sequences. Nucleic acid variants can be
naturally-occurring, such as allelic variants (same locus),
homologues (different locus), and orthologues (different organism)
or can be non naturally-occurring. Naturally occurring variants
such as these can be identified with the use of well-known
molecular biology techniques, as, for example, with polymerase
chain reaction (PCR) and hybridization techniques as known in the
art. Non-naturally occurring variants can be made by mutagenesis
techniques, including those applied to polynucleotides, cells, or
organisms. The variants can contain nucleotide substitutions,
deletions, inversions and insertions. Variation can occur in either
or both the coding and non-coding regions. The variations can
produce both conservative and non-conservative amino acid
substitutions (as compared in the encoded product). For nucleotide
sequences, conservative variants include those sequences that,
because of the degeneracy of the genetic code, encode the amino
acid sequence of one of the endotoxemia marker polypeptides of the
invention. Variant nucleotide sequences also include synthetically
derived nucleotide sequences, such as those generated, for example,
by using site-directed mutagenesis but which still encode an
endotoxemia marker polypeptide of the invention. Generally,
variants of a particular nucleotide sequence of the invention will
have at least about 30%, 40% 50%, 55%, 60%, 65%, 70%, generally at
least about 75%, 80%, 85%, desirably about 90% to 95% or more, and
more suitably about 98% or more sequence identity to that
particular nucleotide sequence as determined by sequence alignment
programs described elsewhere herein using default parameters.
[0153] The endotoxemia marker nucleotide sequences of the invention
can be used to isolate corresponding sequences and alleles from
other organisms, particularly other mammals, especially other
equine species. Methods are readily available in the art for the
hybridization of nucleic acid sequences. Coding sequences from
other organisms may be isolated according to well known techniques
based on their sequence identity with the coding sequences set
forth herein. In these techniques all or part of the known coding
sequence is used as a probe which selectively hybridizes to other
endotoxemia marker coding sequences present in a population of
cloned genomic DNA fragments or cDNA fragments (i.e., genomic or
cDNA libraries) from a chosen organism. Accordingly, the present
invention also contemplates polynucleotides that hybridize to the
endotoxemia marker gene nucleotide sequences, or to their
complements, under stringency conditions described below. As used
herein, the term "hybridizes under low stringency, medium
stringency, high stringency, or very high stringency conditions"
describes conditions for hybridization and washing. Guidance for
performing hybridization reactions can be found in Ausubel et al.,
(1998, supra), Sections 6.3.1-6.3.6. Aqueous and non-aqueous
methods are described in that reference and either can be used.
Reference herein to low stringency conditions include and encompass
from at least about 1% v/v to at least about 15% v/v formamide and
from at least about 1 M to at least about 2 M salt for
hybridization at 42.degree. C., and at least about 1 M to at least
about 2 M salt for washing at 42.degree. C. Low stringency
conditions also may include 1% Bovine Serum Albumin (BSA), 1 mM
EDTA, 0.5 M NaHPO.sub.4 (pH 7.2), 7% SDS for hybridization at
65.degree. C., and (i) 2.times.SSC, 0.1% SDS; or (ii) 0.5% BSA, 1
mM EDTA, 40 mM NaHPO.sub.4 (pH 7.2), 5% SDS for washing at room
temperature. One embodiment of low stringency conditions includes
hybridization in 6.times. sodium chloride/sodium citrate (SSC) at
about 45.degree. C., followed by two washes in 0.2.times.SSC, 0.1%
SDS at least at 50.degree. C. (the temperature of the washes can be
increased to 55.degree. C. for low stringency conditions). Medium
stringency conditions include and encompass from at least about 16%
v/v to at least about 30% v/v formamide and from at least about 0.5
M to at least about 0.9 M salt for hybridization at 42.degree. C.,
and at least about 0.1 M to at least about 0.2 M salt for washing
at 55.degree. C. Medium stringency conditions also may include 1%
Bovine Serum Albumin (BSA), 1 mM EDTA, 0.5 M NaHPO.sub.4 (pH 7.2),
7% SDS for hybridization at 65.degree. C., and (i) 2.times.SSC,
0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO.sub.4 (pH 7.2),
5% SDS for washing at 60-65.degree. C. One embodiment of medium
stringency conditions includes hybridizing in 6.times.SSC at about
45.degree. C., followed by one or more washes in 0.2.times.SSC,
0.1% SDS at 60.degree. C. High stringency conditions include and
encompass from at least about 31% v/v to at least about 50% v/v
formamide and from about 0.01 M to about 0.15 M salt for
hybridization at 42.degree. C., and about 0.01 M to about 0.02 M
salt for washing at 55.degree. C. High stringency conditions also
may include 1% BSA, 1 mM EDTA, 0.5 M NaHPO.sub.4 (pH 7.2), 7% SDS
for hybridization at 65.degree. C., and (i) 0.2.times.SSC, 0.1%
SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO.sub.4 (pH 7.2), 1%
SDS for washing at a temperature in excess of 65.degree. C. One
embodiment of high stringency conditions includes hybridizing in
6.times.SSC at about 45.degree. C., followed by one or more washes
in 0.2.times.SSC, 0.1% SDS at 65.degree. C.
[0154] In certain embodiments, an antigen-binding molecule of the
invention is encoded by a polynucleotide that hybridizes to a
disclosed nucleotide sequence under very high stringency
conditions. One embodiment of very high stringency conditions
includes hybridizing 0.5 M sodium phosphate, 7% SDS at 65.degree.
C., followed by one or more washes at 0.2.times.SSC, 1% SDS at
65.degree. C.
[0155] Other stringency conditions are well known in the art and a
skilled addressee will recognize that various factors can be
manipulated to optimize the specificity of the hybridization.
Optimization of the stringency of the final washes can serve to
ensure a high degree of hybridization. For detailed examples, see
Ausubel et al., supra at pages 2.10.1 to 2.10.16 and Sambrook et
al. (1989, supra) at sections 1.101 to 1.104.
[0156] While stringent washes are typically carried out at
temperatures from about 42.degree. C. to 68.degree. C., one skilled
in the art will appreciate that other temperatures may be suitable
for stringent conditions. Maximum hybridization rate typically
occurs at about 20.degree. C. to 25.degree. C. below the T.sub.m
for formation of a DNA-DNA hybrid. It is well known in the art that
the T.sub.m is the melting temperature, or temperature at which two
complementary polynucleotide sequences dissociate. Methods for
estimating T.sub.m are well known in the art (see Ausubel et al.,
supra at page 2.10.8). In general, the T.sub.m of a perfectly
matched duplex of DNA may be predicted as an approximation by the
formula:
T.sub.m=81.5+16.6 (log.sub.10 M)+0.41 (% G+C)-0.63 (%
formamide)-(600/length)
[0157] wherein: M is the concentration of Na.sup.+, preferably in
the range of 0.01 molar to 0.4 molar; % G+C is the sum of guanosine
and cytosine bases as a percentage of the total number of bases,
within the range between 30% and 75% G+C; % formamide is the
percent formamide concentration by volume; length is the number of
base pairs in the DNA duplex. The T.sub.m of a duplex DNA decreases
by approximately 1.degree. C. with every increase of 1% in the
number of randomly mismatched base pairs. Washing is generally
carried out at T.sub.m-15.degree. C. for high stringency, or
T.sub.m-30.degree. C. for moderate stringency.
[0158] In one example of a hybridization procedure, a membrane
(e.g., a nitrocellulose membrane or a nylon membrane) containing
immobilized DNA is hybridized overnight at 42.degree. C. in a
hybridization buffer (50% deionised formamide, 5.times.SSC,
5.times.Denhardt's solution (0.1% ficoll, 0.1% polyvinylpyrollidone
and 0.1% bovine serum albumin), 0.1% SDS and 200 mg/mL denatured
salmon sperm DNA) containing labeled probe. The membrane is then
subjected to two sequential medium stringency washes (i.e.,
2.times.SSC, 0.1% SDS for 15 min at 45.degree. C., followed by
2.times.SSC, 0.1% SDS for 15 min at 50.degree. C.), followed by two
sequential higher stringency washes (i.e., 0.2.times.SSC, 0.1% SDS
for 12 min at 55.degree. C. followed by 0.2.times.SSC and 0.1% SDS
solution for 12 min at 65-68.degree. C.
5. Polypeptides of the Invention
[0159] The present invention also contemplates full-length
polypeptides encoded by the endotoxemia marker genes of the
invention as well as the biologically active portions of those
polypeptides, which are referred to collectively herein as
"endotoxemia marker polypeptides." Biologically active portions of
full-length endotoxemia marker polypeptides include portions with
immuno-interactive activity of at least about 6, 8, 10, 12, 14, 16,
18, 20, 25, 30, 40, 50, 60 amino acid residues in length. For
example, immuno-interactive fragments contemplated by the present
invention are at least 6 and desirably at least 8 amino acid
residues in length, which can elicit an immune response in an
animal for the production of antigen-binding molecules that are
immuno-interactive with an endotoxemia marker polypeptide of the
invention. Such antigen-binding molecules can be used to screen
other mammals, especially equine mammals, for structurally and/or
functionally related endotoxemia marker polypeptides. Typically,
portions of a full-length endotoxemia marker polypeptide may
participate in an interaction, for example, an intramolecular or an
inter-molecular interaction. An inter-molecular interaction can be
a specific binding interaction or an enzymatic interaction (e.g.,
the interaction can be transient and a covalent bond is formed or
broken). Biologically active portions of a full-length endotoxemia
marker polypeptide include peptides comprising amino acid sequences
sufficiently similar to or derived from the amino acid sequences of
a (putative) full-length endotoxemia marker polypeptide, for
example, the amino acid sequences shown in SEQ ID NO: 2, 4, 6, 9,
11, 13, 15, 19, 21, 23, 25, 29, 31, 33, 51, 53 or 58, which include
less amino acids than a full-length endotoxemia marker polypeptide,
and exhibit at least one activity of that polypeptide. Typically,
biologically active portions comprise a domain or motif with at
least one activity of a full-length endotoxemia marker polypeptide.
A biologically active portion of a full-length endotoxemia marker
polypeptide can be a polypeptide which is, for example, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 300, 400, 500, 600, 700,
800, 900 or 1000, or even at least about 2000 or 3000, or more
amino acid residues in length. Suitably, the portion is a
"biologically-active portion" having no less than about 1%, 10%,
25% 50% of the activity of the full-length polypeptide from which
it is derived.
[0160] The present invention also contemplates variant endotoxemia
marker polypeptides. "Variant" polypeptides include proteins
derived from the native protein by deletion (so-called truncation)
or addition of one or more amino acids to the N-terminal and/or
C-terminal end of the native protein; deletion or addition of one
or more amino acids at one or more sites in the native protein; or
substitution of one or more amino acids at one or more sites in the
native protein. Variant proteins encompassed by the present
invention are biologically active, that is, they continue to
possess the desired biological activity of the native protein. Such
variants may result from, for example, genetic polymorphism or from
human manipulation. Biologically active variants of a native
endotoxemia marker polypeptide of the invention will have at least
40%, 50%, 60%, 70%, generally at least 75%, 80%, 85%, preferably
about 90% to 95% or more, and more preferably about 98% or more
sequence similarity with the amino acid sequence for the native
protein as determined by sequence alignment programs described
elsewhere herein using default parameters. A biologically active
variant of a protein of the invention may differ from that protein
generally by as much 1000, 500, 400, 300, 200, 100, 50 or 20 amino
acid residues or suitably by as few as 1-15 amino acid residues, as
few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2, or even
1 amino acid residue.
[0161] An endotoxemia marker polypeptide of the invention may be
altered in various ways including amino acid substitutions,
deletions, truncations, and insertions. Methods for such
manipulations are generally known in the art. For example, amino
acid sequence variants of an endotoxemia marker protein can be
prepared by mutations in the DNA. Methods for mutagenesis and
nucleotide sequence alterations are well known in the art. See, for
example, Kunkel (1985, Proc. Natl. Acad. Sci. USA 82:488-492),
Kunkel et al (1987, Methods in Enzymol. 154:367-382), U.S. Pat. No.
4,873,192, Watson, J. D. et al. ("Molecular Biology of the Gene",
Fourth Edition, Benjamin/Cummings, Menlo Park, Calif., 1987) and
the references cited therein. Guidance as to appropriate amino acid
substitutions that do not affect biological activity of the protein
of interest may be found in the model of Dayhoff et al. (1978)
Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found.,
Washington, D.C.). Methods for screening gene products of
combinatorial libraries made by point mutations or truncation, and
for screening cDNA libraries for gene products having a selected
property are known in the art. Such methods are adaptable for rapid
screening of the gene libraries generated by combinatorial
mutagenesis of endotoxemia marker polypeptides. Recursive ensemble
mutagenesis (REM), a technique which enhances the frequency of
functional mutants in the libraries, can be used in combination
with the screening assays to identify endotoxemia marker
polypeptide variants (Arkin and Yourvan (1992) Proc. Natl. Acad.
Sci. USA 89:7811-7815; Delgrave et al. (1993) Protein Engineering
6:327-331). Conservative substitutions, such as exchanging one
amino acid with another having similar properties, may be desirable
as discussed in more detail below.
[0162] Variant endotoxemia marker polypeptides may contain
conservative amino acid substitutions at various locations along
their sequence, as compared to the parent endotoxemia marker amino
acid sequence. A "conservative amino acid substitution" is one in
which the amino acid residue is replaced with an amino acid residue
having a similar side chain. Families of amino acid residues having
similar side chains have been defined in the art, which can be
generally sub-classified as follows:
[0163] Acidic: The residue has a negative charge due to loss of H
ion at physiological pH and the residue is attracted by aqueous
solution so as to seek the surface positions in the conformation of
a peptide in which it is contained when the peptide is in aqueous
medium at physiological pH. Amino acids having an acidic side chain
include glutamic acid and aspartic acid.
[0164] Basic: The residue has a positive charge due to association
with H ion at physiological pH or within one or two pH units
thereof (e.g., histidine) and the residue is attracted by aqueous
solution so as to seek the surface positions in the conformation of
a peptide in which it is contained when the peptide is in aqueous
medium at physiological pH. Amino acids having a basic side chain
include arginine, lysine and histidine.
[0165] Charged: The residues are charged at physiological pH and,
therefore, include amino acids having acidic or basic side chains
(i.e., glutamic acid, aspartic acid, arginine, lysine and
histidine).
[0166] Hydrophobic: The residues are not charged at physiological
pH and the residue is repelled by aqueous solution so as to seek
the inner positions in the conformation of a peptide in which it is
contained when the peptide is in aqueous medium. Amino acids having
a hydrophobic side chain include tyrosine, valine, isoleucine,
leucine, methionine, phenylalanine and tryptophan.
[0167] Neutral/polar: The residues are not charged at physiological
pH, but the residue is not sufficiently repelled by aqueous
solutions so that it would seek inner positions in the conformation
of a peptide in which it is contained when the peptide is in
aqueous medium. Amino acids having a neutral/polar side chain
include asparagine, glutamine, cysteine, histidine, serine and
threonine.
[0168] This description also characterizes certain amino acids as
"small" since their side chains are not sufficiently large, even if
polar groups are lacking, to confer hydrophobicity. With the
exception of proline, "small" amino acids are those with four
carbons or less when at least one polar group is on the side chain
and three carbons or less when not. Amino acids having a small side
chain include glycine, serine, alanine and threonine. The
gene-encoded secondary amino acid proline is a special case due to
its known effects on the secondary conformation of peptide chains.
The structure of proline differs from all the other
naturally-occurring amino acids in that its side chain is bonded to
the nitrogen of the .alpha.-amino group, as well as the
.alpha.-carbon. Several amino acid similarity matrices (e.g.,
PAM120 matrix and PAM250 matrix as disclosed for example by Dayhoff
et al. (1978) A model of evolutionary change in proteins. Matrices
for determining distance relationships In M. O. Dayhoff, (ed.),
Atlas of protein sequence and structure, Vol. 5, pp. 345-358,
National Biomedical Research Foundation, Washington D.C.; and by
Gonnet et al., 1992, Science 256(5062): 144301445), however,
include proline in the same group as glycine, serine, alanine and
threonine. Accordingly, for the purposes of the present invention,
proline is classified as a "small" amino acid.
[0169] The degree of attraction or repulsion required for
classification as polar or nonpolar is arbitrary and, therefore,
amino acids specifically contemplated by the invention have been
classified as one or the other. Most amino acids not specifically
named can be classified on the basis of known behavior.
[0170] Amino acid residues can be further sub-classified as cyclic
or noncyclic, and aromatic or nonaromatic, self-explanatory
classifications with respect to the side-chain substituent groups
of the residues, and as small or large. The residue is considered
small if it contains a total of four carbon atoms or less,
inclusive of the carboxylcarbon, provided an additional polar
substituent is present; three or less if not. Small residues are,
of course, always nonaromatic. Dependent on their structural
properties, amino acid residues may fall in two or more classes.
For the naturally-occurring protein amino acids, sub-classification
according to the this scheme is presented in the Table 3.
[0171] Conservative amino acid substitution also includes groupings
based on side chains. For example, a group of amino acids having
aliphatic side chains is glycine, alanine, valine, leucine, and
isoleucine; a group of amino acids having aliphatic-hydroxyl side
chains is serine and threonine; a group of amino acids having
amide-containing side chains is asparagine and glutamine; a group
of amino acids having aromatic side chains is phenylalanine,
tyrosine, and tryptophan; a group of amino acids having basic side
chains is lysine, arginine, and histidine; and a group of amino
acids having sulfur-containing side chains is cysteine and
methionine. For example, it is reasonable to expect that
replacement of a leucine with an isoleucine or valine, an aspartate
with a glutamate, a threonine with a serine, or a similar
replacement of an amino acid with a structurally related amino acid
will not have a major effect on the properties of the resulting
variant polypeptide. Whether an amino acid change results in a
functional endotoxemia marker polypeptide can readily be determined
by assaying its activity. Conservative substitutions are shown in
Table 4 below under the heading of exemplary substitutions. More
preferred substitutions are shown under the heading of preferred
substitutions. Amino acid substitutions falling within the scope of
the invention, are, in general, accomplished by selecting
substitutions that do not differ significantly in their effect on
maintaining (a) the structure of the peptide backbone in the area
of the substitution, (b) the charge or hydrophobicity of the
molecule at the target site, or (c) the bulk of the side chain.
After the substitutions are introduced, the variants are screened
for biological activity.
[0172] Alternatively, similar amino acids for making conservative
substitutions can be grouped into three categories based on the
identity of the side chains. The first group includes glutamic
acid, aspartic acid, arginine, lysine, histidine, which all have
charged side chains; the second group includes glycine, serine,
threonine, cysteine, tyrosine, glutamine, asparagine; and the third
group includes leucine, iso leucine, valine, alanine, pro line,
phenylalanine, tryptophan, methionine, as described in Zubay, G.,
Biochemistry, third edition, Wm.C. Brown Publishers (1993).
[0173] Thus, a predicted non-essential amino acid residue in an
endotoxemia marker polypeptide is typically replaced with another
amino acid residue from the same side chain family. Alternatively,
mutations can be introduced randomly along all or part of an
endotoxemia marker gene coding sequence, such as by saturation
mutagenesis, and the resultant mutants can be screened for an
activity of the parent polypeptide to identify mutants which retain
that activity. Following mutagenesis of the coding sequences, the
encoded peptide can be expressed recombinantly and the activity of
the peptide can be determined.
[0174] Accordingly, the present invention also contemplates
variants of the naturally-occurring endotoxemia marker polypeptide
sequences or their biologically-active fragments, wherein the
variants are distinguished from the naturally-occurring sequence by
the addition, deletion, or substitution of one or more amino acid
residues. In general, variants will display at least about 30, 40,
50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98,
99% similarity to a parent endotoxemia marker polypeptide sequence
as, for example, set forth in any one of SEQ ID NO: 2, 4, 6, 9, 11,
13, 15, 19, 21, 23, 25, 29, 31, 33, 51, 53 or 58. Desirably,
variants will have at least 30, 40, 50, 55, 60, 65, 70, 75, 80, 85,
90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence identity to a
parent endotoxemia marker polypeptide sequence as, for example, set
forth in any one of SEQ ID NO: 2, 4, 6, 9, 11, 13, 15, 19, 21, 23,
25, 29, 31, 33, 51, 53 or 58. Moreover, sequences differing from
the native or parent sequences by the addition, deletion, or
substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300,
500 or more amino acids but which retain the properties of the
parent endotoxemia marker polypeptide are contemplated. endotoxemia
marker polypeptides also include polypeptides that are encoded by
polynucleotides that hybridize under stringency conditions as
defined herein, especially high stringency conditions, to the
endotoxemia marker polynucleotide sequences of the invention, or
the non-coding strand thereof, as described above.
[0175] In one embodiment, variant polypeptides differ from an
endotoxemia marker sequence by at least one but by less than 50,
40, 30, 20, 15, 10, 8, 6, 5, 4, 3 or 2 amino acid residue(s). In
another, variant polypeptides differ from the corresponding
sequence in any one of SEQ ID NO: 2, 4, 6, 9, 11, 13, 15, 19, 21,
23, 25, 29, 31, 33, 51, 53 or 58 by at least 1% but less than 20%,
15%, 10% or 5% of the residues. (If this comparison requires
alignment the sequences should be aligned for maximum similarity.
"Looped" out sequences from deletions or insertions, or mismatches,
are considered differences.) The differences are, suitably,
differences or changes at a non-essential residue or a conservative
substitution.
[0176] A "non-essential" amino acid residue is a residue that can
be altered from the wild-type sequence of an embodiment polypeptide
without abolishing or substantially altering one or more of its
activities. Suitably, the alteration does not substantially alter
one of these activities, for example, the activity is at least 20%,
40%, 60%, 70% or 80% of wild-type. An "essential" amino acid
residue is a residue that, when altered from the wild-type sequence
of an endotoxemia marker polypeptide of the invention, results in
abolition of an activity of the parent molecule such that less than
20% of the wild-type activity is present.
[0177] In other embodiments, a variant polypeptide includes an
amino acid sequence having at least about 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98% or more
similarity to a corresponding sequence of an endotoxemia marker
polypeptide as, for example, set forth in any one of SEQ ID NO: 2,
4, 6, 9, 11, 13, 15, 19, 21, 23, 25, 29, 31, 33, 51, 53 or 58, and
has the activity of that endotoxemia marker polypeptide.
[0178] Endotoxemia marker polypeptides of the invention may be
prepared by any suitable procedure known to those of skill in the
art. For example, the polypeptides may be prepared by a procedure
including the steps of: (a) preparing a chimeric construct
comprising a nucleotide sequence that encodes at least a portion of
an endotoxemia marker polynucleotide and that is operably linked to
a regulatory element; (b) introducing the chimeric construct into a
host cell; (c) culturing the host cell to express the endotoxemia
marker polypeptide; and (d) isolating the endotoxemia marker
polypeptide from the host cell. In illustrative examples, the
nucleotide sequence encodes at least a portion of the sequence set
forth in any one of SEQ ID NO: 2, 4, 6, 9, 11, 13, 15, 19, 21, 23,
25, 29, 31, 33, 51, 53 or 58 or a variant thereof.
[0179] The chimeric construct is typically in the form of an
expression vector, which is suitably selected from self-replicating
extra-chromosomal vectors (e.g., plasmids) and vectors that
integrate into a host genome.
[0180] The regulatory element will generally be appropriate for the
host cell employed for expression of the endotoxemia marker
polynucleotide. Numerous types of expression vectors and regulatory
elements are known in the art for a variety of host cells.
Illustrative elements of this type include, but are not restricted
to, promoter sequences (e.g., constitutive or inducible promoters
which may be naturally occurring or combine elements of more than
one promoter), leader or signal sequences, ribosomal binding sites,
transcriptional start and stop sequences, translational start and
termination sequences, and enhancer or activator sequences.
[0181] In some embodiments, the expression vector comprises a
selectable marker gene to permit the selection of transformed host
cells. Selectable marker genes are well known in the art and will
vary with the host cell employed.
[0182] The expression vector may also include a fusion partner
(typically provided by the expression vector) so that the
endotoxemia marker polypeptide is produced as a fusion polypeptide
with the fusion partner. The main advantage of fusion partners is
that they assist identification and/or purification of the fusion
polypeptide. In order to produce the fusion polypeptide, it is
necessary to ligate the endotoxemia marker polynucleotide into an
expression vector so that the translational reading frames of the
fusion partner and the endotoxemia marker polynucleotide coincide.
Well known examples of fusion partners include, but are not limited
to, glutathione-S-transferase (GST), Fc potion of human IgG,
maltose binding protein (MBP) and hexahistidine (HIS.sub.6), which
are particularly useful for isolation of the fusion polypeptide by
affinity chromatography. In some embodiments, fusion polypeptides
are purified by affinity chromatography using matrices to which the
fusion partners bind such as but not limited to glutathione-,
amylose-, and nickel- or cobalt-conjugated resins. Many such
matrices are available in "kit" form, such as the QIAexpress.TM.
system (Qiagen) useful with (HIS.sub.6) fusion partners and the
Pharmacia GST purification system. Other fusion partners known in
the art are light-emitting proteins such as green fluorescent
protein (GFP) and luciferase, which serve as fluorescent "tags"
that permit the identification and/or isolation of fusion
polypeptides by fluorescence microscopy or by flow cytometry. Flow
cytometric methods such as fluorescence activated cell sorting
(FACS) are particularly useful in this latter application.
[0183] Desirably, the fusion partners also possess protease
cleavage sites, such as for Factor X.sub.a or Thrombin, which
permit the relevant protease to partially digest the fusion
polypeptide and thereby liberate the endotoxemia marker polypeptide
from the fusion construct. The liberated polypeptide can then be
isolated from the fusion partner by subsequent chromatographic
separation.
[0184] Fusion partners also include within their scope "epitope
tags," which are usually short peptide sequences for which a
specific antibody is available. Well known examples of epitope tags
for which specific monoclonal antibodies are readily available
include c-Myc, influenza virus, hemagglutinin and FLAG tags.
[0185] The chimeric constructs of the invention are introduced into
a host by any suitable means including "transduction" and
"transfection", which are art recognized as meaning the
introduction of a nucleic acid, for example, an expression vector,
into a recipient cell by nucleic acid-mediated gene transfer.
"Transformation," however, refers to a process in which a host's
genotype is changed as a result of the cellular uptake of exogenous
DNA or RNA, and, for example, the transformed cell comprises the
expression system of the invention. There are many methods for
introducing chimeric constructs into cells. Typically, the method
employed will depend on the choice of host cell. Technology for
introduction of chimeric constructs into host cells is well known
to those of skill in the art. Four general classes of methods for
delivering nucleic acid molecules into cells have been described:
(1) chemical methods such as calcium phosphate precipitation,
polyethylene glycol (PEG)-mediate precipitation and lipofection;
(2) physical methods such as microinjection, electro-poration,
acceleration methods and vacuum infiltration; (3) vector based
methods such as bacterial and viral vector-mediated transformation;
and (4) receptor-mediated. Transformation techniques that fall
within these and other classes are well known to workers in the
art, and new techniques are continually becoming known. The
particular choice of a transformation technology will be determined
by its efficiency to transform certain host species as well as the
experience and preference of the person practicing the invention
with a particular methodology of choice. It will be apparent to the
skilled person that the particular choice of a transformation
system to introduce a chimeric construct into cells is not
essential to or a limitation of the invention, provided it achieves
an acceptable level of nucleic acid transfer.
[0186] Recombinant endotoxemia marker polypeptides may be produced
by culturing a host cell transformed with a chimeric construct. The
conditions appropriate for expression of the endotoxemia marker
polynucleotide will vary with the choice of expression vector and
the host cell and are easily ascertained by one skilled in the art
through routine experimentation. Suitable host cells for expression
may be prokaryotic or eukaryotic. An illustrative host cell for
expression of a polypeptide of the invention is a bacterium. The
bacterium used may be Escherichia coli. Alternatively, the host
cell may be a yeast cell or an insect cell such as, for example,
SF9 cells that may be utilized with a baculovirus expression
system.
[0187] Recombinant endotoxemia marker polypeptides can be
conveniently prepared using standard protocols as described for
example in Sambrook, et al., (1989, supra), in particular Sections
16 and 17; Ausubel et al., (1994, supra), in particular Chapters 10
and 16; and Coligan et al., CURRENT PROTOCOLS IN PROTEIN SCIENCE
(John Wiley & Sons, Inc. 1995-1997), in particular Chapters 1,
5 and 6. Alternatively, the endotoxemia marker polypeptides may be
synthesized by chemical synthesis, e.g., using solution synthesis
or solid phase synthesis as described, for example, in Chapter 9 of
Atherton and Shephard (supra) and in Roberge et al (1995, Science
269: 202).
6. Antigen-Binding Molecules
[0188] The invention also provides antigen-binding molecules that
are specifically immuno-interactive with an endotoxemia marker
polypeptide of the invention. In one embodiment, the
antigen-binding molecule comprise whole polyclonal antibodies. Such
antibodies may be prepared, for example, by injecting an
endotoxemia marker polypeptide of the invention into a production
species, which may include mice or rabbits, to obtain polyclonal
antisera. Methods of producing polyclonal antibodies are well known
to those skilled in the art. Exemplary protocols which may be used
are described for example in Coligan et al., CURRENT PROTOCOLS IN
IMMUNOLOGY, (John Wiley & Sons, Inc, 1991), and Ausubel et al,
(1994-1998, supra), in particular Section III of Chapter 11.
[0189] In lieu of polyclonal antisera obtained in a production
species, monoclonal antibodies may be produced using the standard
method as described, for example, by Kohler and Milstein (1975,
Nature 256, 495-497), or by more recent modifications thereof as
described, for example, in Coligan et al., (1991, supra) by
immortalizing spleen or other antibody producing cells derived from
a production species which has been inoculated with one or more of
the endotoxemia marker polypeptides of the invention.
[0190] The invention also contemplates as antigen-binding molecules
Fv, Fab, Fab' and F(ab').sub.2 immunoglobulin fragments.
Alternatively, the antigen-binding molecule may comprise a
synthetic stabilized Fv fragment. Exemplary fragments of this type
include single chain Fv fragments (sFv, frequently termed scFv) in
which a peptide linker is used to bridge the N terminus or C
terminus of a VH domain with the C terminus or N-terminus,
respectively, of a VL domain. ScFv lack all constant parts of whole
antibodies and are not able to activate complement. ScFvs may be
prepared, for example, in accordance with methods outlined in
Kreber et al (Kreber et al. 1997, J. Immunol. Methods; 201(1):
35-55). Alternatively, they may be prepared by methods described in
U.S. Pat. No. 5,091,513, European Patent No 239,400 or the articles
by Winter and Milstein (1991, Nature 349:293) and Pluckthun et al
(1996, In Antibody engineering: A practical approach. 203-252). In
another embodiment, the synthetic stabilized Fv fragment comprises
a disulfide stabilized Fv (dsfv) in which cysteine residues are
introduced into the V.sub.H and V.sub.L domains such that in the
fully folded Fv molecule the two residues will form a disulfide
bond between them. Suitable methods of producing dsFv are described
for example in (Glockscuther et al. Biochem. 29: 1363-1367; Reiter
et al. 1994, J. Biol. Chem. 269: 18327-18331; Reiter et al. 1994,
Biochem. 33: 5451-5459; Reiter et al. 1994. Cancer Res. 54:
2714-2718; Webber et al. 1995, Mol. Immunol. 32: 249-258).
[0191] Phage display and combinatorial methods for generating
anti-endotoxemia marker polypeptide antigen-binding molecules are
known in the art (as described in, e.g., Ladner et al. U.S. Pat.
No. 5,223,409; Kang et al. International Publication No. WO
92/18619; Dower et al. International Publication No. WO 91/17271;
Winter et al. International Publication WO 92/20791; Markland et
al. International Publication No. WO 92/15679; Breitling et al.
International Publication WO 93/01288; McCafferty et al.
International Publication No. WO 92/01047; Garrard et al.
International Publication No. WO 92/09690; Ladner et al.
International Publication No. WO 90/02809; Fuchs et al. (1991)
Bio/Technology 9:1370-1372; Hay et al. (1992) Hum Antibod
Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281;
Griffths et al. (1993) EMBO J. 12:725-734; Hawkins et al. (1992) J
Mol Biol 226:889-896; Clackson et al. (1991) Nature 352:624-628;
Gram et al. (1992) PNAS 89:3576-3580; Garrad et al. (1991)
Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res
19:4133-4137; and Barbas et al. (1991) PNAS 88:7978-7982). The
antigen-binding molecules can be used to screen expression
libraries for variant endotoxemia marker polypeptides. They can
also be used to detect and/or isolate the endotoxemia marker
polypeptides of the invention. Thus, the invention also
contemplates the use of antigen-binding molecules to isolate
endotoxemia marker polypeptides using, for example, any suitable
immunoaffinity based method including, but not limited to,
immunochromatography and immunoprecipitation. A suitable method
utilises solid phase adsorption in which anti-endotoxemia marker
polypeptide antigen-binding molecules are attached to a suitable
resin, the resin is contacted with a sample suspected of containing
an endotoxemia marker polypeptide, and the endotoxemia marker
polypeptide, if any, is subsequently eluted from the resin.
Illustrative resins include: Sepharose.RTM. (Pharmacia), Poros.RTM.
resins (Roche Molecular Biochemicals, Indianapolis), Actigel
Superflow.TM. resins (Sterogene Bioseparations Inc., Carlsbad
Calif.), and Dynabeads.TM. (Dynal Inc., Lake Success, N.Y.).
[0192] The antigen-binding molecule can be coupled to a compound,
e.g., a label such as a radioactive nucleus, or imaging agent, e.g.
a radioactive, enzymatic, or other, e.g., imaging agent, e.g., a
NMR contrast agent. Labels which produce detectable radioactive
emissions or fluorescence are preferred. An anti-endotoxemia marker
polypeptide antigen-binding molecule (e.g., monoclonal antibody)
can be used to detect endotoxemia marker polypeptides (e.g., in a
cellular lysate or cell supernatant) in order to evaluate the
abundance and pattern of expression of the protein. In certain
advantageous application in accordance with the present invention,
such antigen-binding molecules can be used to monitor endotoxemia
marker polypeptides levels in biological samples (including whole
cells and fluids) for diagnosing the presence, absence, degree, or
stage of development of endotoxemia. Detection can be facilitated
by coupling (i.e., physically linking) the antibody to a detectable
substance (i.e., antibody labeling). Examples of detectable
substances include various enzymes, prosthetic groups, fluorescent
materials, luminescent materials, bioluminescent materials, and
radioactive materials. Examples of suitable enzymes include
horseradish peroxidase, alkaline phosphatase, p-galactosidase, or
acetylcholinesterase; examples of suitable prosthetic group
complexes include streptavidin/biotin and avidin/biotin; examples
of suitable fluorescent materials include umbelliferone,
fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; examples of bioluminescent materials include luciferase,
luciferin, and aequorin, and examples of suitable radioactive
material include .sup.125I, .sup.131I, .sup.35S or .sup.3H. The
label may be selected from a group including a chromogen, a
catalyst, an enzyme, a fluorophore, a chemiluminescent molecule, a
lanthanide ion such as Europium (Eu.sup.34), a radioisotope and a
direct visual label. In the case of a direct visual label, use may
be made of a colloidal metallic or non-metallic particle, a dye
particle, an enzyme or a substrate, an organic polymer, a latex
particle, a liposome, or other vesicle containing a signal
producing substance and the like.
[0193] A large number of enzymes useful as labels is disclosed in
U.S. Pat. No. 4,366,241, U.S. Pat. No. 4,843,000, and U.S. Pat. No.
4,849,338. Enzyme labels useful in the present invention include
alkaline phosphatase, horseradish peroxidase, luciferase,
.beta.-galactosidase, glucose oxidase, lysozyme, malate
dehydrogenase and the like. The enzyme label may be used alone or
in combination with a second enzyme in solution.
7. Methods of Detecting Aberrant Endotoxemia Marker Gene Expression
or the Presence of Endotoxemia Marker Polynucleotides
[0194] The present invention is predicated in part on the discovery
that: horses with clinical evidence of endotoxemia-related
conditions have aberrant expression of certain genes (referred to
herein as "endotoxemia marker genes") whose transcripts include,
but are not limited to, SEQ ID) NO; 1, 3, 4, 5, 6, 7, 9, 10, 11,
13, 15, 16, 17, 18, 19, 21, 23, 25, 26, 27, 29, 31, 33, 35, 37, 38,
39, 41,42, 43, 44, 45, 47, 49, 50, 52, 54, 56, 58, 60, 61, 63, 64,
66, 67, 68, 69, 70, 71, 73, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,
86, 88, 90, 92, 93, 94, 96, 98, 100, 101, 102, 103, 104, 106, 107,
109, 110, 111, 113, 114, 115, 117, 119, 121, 122, 123, 124, 125,
126, 128, 130, 132, 134, 136, 137, 139, 141, 143, 145, 147, 149,
151, 153, 155, 157, 158, 160, 162, 164, 166, 168, 169, 170, 172,
173, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 193, 194,
195, 197, 199, 201, 203, 205, 206, 207, 209, 210, 211, 212, 214,
215, 216, 218, 220, 222, 223, 224, 225, 227, 229, 231, 233, 235,
236, 237, 239, 240, 242, 244, 245, 246, 248, 250, 252, 254, 255,
257, 259 260, 262, 264, 266, 268, 269, 270, 271, 272, 274, 276,
278, 279, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300,
302, 304, 305, 306, 307, 309, 311, 312, 314, 315, 316, 318, 320
321, 323 or 325, as compared to normal horses or to horses lacking
endotoxemia-related conditions.
[0195] Accordingly, in certain embodiments, the invention features
a method for diagnosing the presence, absence, degree or stage of
an endotoxemia-related condition in a subject, which is typically
of equine origin, by detecting aberrant expression of an
endotoxemia marker gene in a biological sample obtained from the
subject. Accordingly, in order to make such diagnoses, it will be
desirable to qualitatively or quantitatively determine the levels
of endotoxemia marker gene transcripts or the level or functional
activity of endotoxemia marker polypeptides. In some embodiments,
the presence, degree, or stage of development of an
endotoxemia-related condition is diagnosed when an endotoxemia
marker gene product is expressed at a detectably lower level in the
biological sample as compared to the level at which that gene is
expressed in a reference sample obtained from normal subjects or
from subjects lacking that condition. In other embodiments, the
presence, degree, or stage of development of an endotoxemia-related
condition is diagnosed when an endotoxemia marker gene product is
expressed at a detectably higher level in the biological sample as
compared to the level at which that gene is expressed in a
reference sample obtained from normal subjects or from subjects
lacking that condition. Generally, such diagnoses are made when the
level or functional activity of an endotoxemia marker gene product
in the biological sample varies from the level or functional
activity of a corresponding endotoxemia marker gene product in the
reference sample by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,
80%, 90%, 92%, 94%, 96%, 97%, 98% or 99%, or even by at least about
99.5%, 99.9%, 99.95%, 99.99%, 99.995% or 99.999%, or even by at
least about 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or
1000%. The corresponding gene product is generally selected from
the same gene product that is present in the biological sample, a
gene product expressed from a variant gene (e.g., an homologous or
orthologous gene) including an allelic variant, or a splice variant
or protein product thereof. In some embodiments, the method
comprises measuring the level or functional activity of individual
expression products of at least about 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28 29 or 30 endotoxemia marker genes.
[0196] Generally, the biological sample contains blood, especially
peripheral blood, or a fraction or extract thereof. Typically, the
biological sample comprises blood cells such as mature, immature
and developing leukocytes, including lymphocytes, polymorphonuclear
leukocytes, neutrophils, monocytes, reticulocytes, basophils,
coelomocytes, hemocytes, eosinophils, megakaryocytes, macrophages,
dendritic cells natural killer cells, or fraction of such cells
(e.g., a nucleic acid or protein fraction). In specific
embodiments, the biological sample comprises leukocytes including
peripheral blood mononuclear cells (PBMC).
[0197] 7.1 Nucleic Acid-Based Diagnostics
[0198] Nucleic acid used in polynucleotide-based assays can be
isolated from cells contained in the biological sample, according
to standard methodologies (Sambrook, et al., 1989, supra; and
Ausubel et al., 1994, supra). The nucleic acid is typically
fractionated (e.g., poly A.sup.+ RNA) or whole cell RNA. Where RNA
is used as the subject of detection, it may be desired to convert
the RNA to a complementary DNA. In some embodiments, the nucleic
acid is amplified by a template-dependent nucleic acid
amplification technique. A number of template dependent processes
are available to amplify the endotoxemia marker sequences present
in a given template sample. An exemplary nucleic acid amplification
technique is the polymerase chain reaction (referred to as PCR)
which is described in detail in U.S. Pat. Nos. 4,683,195, 4,683,202
and 4,800,159, Ausubel et al. (supra), and in Innis et al., ("PCR
Protocols", Academic Press, Inc., San Diego Calif., 1990). Briefly,
in PCR, two primer sequences are prepared that are complementary to
regions on opposite complementary strands of the marker sequence.
An excess of deoxynucleoside triphosphates are added to a reaction
mixture along with a DNA polymerase, e.g., Taq polymerase. If a
cognate endotoxemia marker sequence is present in a sample, the
primers will bind to the marker and the polymerase will cause the
primers to be extended along the marker sequence by adding on
nucleotides. By raising and lowering the temperature of the
reaction mixture, the extended primers will dissociate from the
marker to form reaction products, excess primers will bind to the
marker and to the reaction products and the process is repeated. A
reverse transcriptase PCR amplification procedure may be performed
in order to quantify the amount of mRNA amplified. Methods of
reverse transcribing RNA into cDNA are well known and described in
Sambrook et al., 1989, supra. Alternative methods for reverse
transcription utilize thermostable, RNA-dependent DNA polymerases.
These methods are described in WO 90/07641. Polymerase chain
reaction methodologies are well known in the art.
[0199] In certain advantageous embodiments, the template-dependent
amplification involves the quantification of transcripts in
real-time. For example, RNA or DNA may be quantified using the
Real-Time PCR technique (Higuchi, 1992, et al., Biotechnology 10:
413-417). By determining the concentration of the amplified
products of the target DNA in PCR reactions that have completed the
same number of cycles and are in their linear ranges, it is
possible to determine the relative concentrations of the specific
target sequence in the original DNA mixture. If the DNA mixtures
are cDNAs synthesized from RNAs isolated from different tissues or
cells, the relative abundance of the specific mRNA from which the
target sequence was derived can be determined for the respective
tissues or cells. This direct proportionality between the
concentration of the PCR products and the relative mRNA abundance
is only true in the linear range of the PCR reaction. The final
concentration of the target DNA in the plateau portion of the curve
is determined by the availability of reagents in the reaction mix
and is independent of the original concentration of target DNA.
[0200] Another method for amplification is the ligase chain
reaction ("LCR"), disclosed in EPO No. 320 308. In LCR, two
complementary probe pairs are prepared, and in the presence of the
target sequence, each pair will bind to opposite complementary
strands of the target such that they abut. In the presence of a
ligase, the two probe pairs will link to form a single unit. By
temperature cycling, as in PCR, bound ligated units dissociate from
the target and then serve as "target sequences" for ligation of
excess probe pairs. U.S. Pat. No. 4,883,750 describes a method
similar to LCR for binding probe pairs to a target sequence.
[0201] Q.beta. Replicase, described in PCT Application No.
PCT/US87/00880, may also be used. In this method, a replicative
sequence of RNA that has a region complementary to that of a target
is added to a sample in the presence of an RNA polymerase. The
polymerase will copy the replicative sequence that can then be
detected.
[0202] An isothermal amplification method, in which restriction
endonucleases and ligases are used to achieve the amplification of
target molecules that contain nucleotide
5'.alpha.-thio-triphosphates in one strand of a restriction site
may also be useful in the amplification of nucleic acids in the
present invention, Walker et al., (1992, Proc. Natl. Acad. Sci.
U.S.A 89: 392-396).
[0203] Strand Displacement Amplification (SDA) is another method of
carrying out isothermal amplification of nucleic acids which
involves multiple rounds of strand displacement and synthesis,
i.e., nick translation. A similar method, called Repair Chain
Reaction (RCR), involves annealing several probes throughout a
region targeted for amplification, followed by a repair reaction in
which only two of the four bases are present. The other two bases
can be added as biotinylated derivatives for easy detection. A
similar approach is used in SDA. Target specific sequences can also
be detected using a cyclic probe reaction (CPR). In CPR, a probe
having 3' and 5' sequences of non-specific DNA and a middle
sequence of specific RNA is hybridized to DNA that is present in a
sample. Upon hybridization, the reaction is treated with RNase H,
and the products of the probe identified as distinctive products
that are released after digestion. The original template is
annealed to another cycling probe and the reaction is repeated.
[0204] Still another amplification method described in GB
Application No. 2 202 328, and in PCT Application No.
PCT/US89/01025, may be used. In the former application, "modified"
primers are used in a PCR-like, template- and enzyme-dependent
synthesis. The primers may be modified by labeling with a capture
moiety (e.g., biotin) and/or a detector moiety (e.g., enzyme). In
the latter application, an excess of labeled probes are added to a
sample. In the presence of the target sequence, the probe binds and
is cleaved catalytically. After cleavage, the target sequence is
released intact to be bound by excess probe. Cleavage of the
labelled probe signals the presence of the target sequence.
[0205] Other nucleic acid amplification procedures include
transcription-based amplification systems (TAS), including nucleic
acid sequence based amplification (NASBA) and 3SR (Kwoh et al.,
1989, Proc. Natl. Acad. Sci. U.S., 86: 1173; Gingeras et al., PCT
Application WO 88/10315). In NASBA, the nucleic acids can be
prepared for amplification by standard phenol/chloroform
extraction, heat denaturation of a clinical sample, treatment with
lysis buffer and minispin columns for isolation of DNA and RNA or
guanidinium chloride extraction of RNA. These amplification
techniques involve annealing a primer which has target specific
sequences. Following polymerization, DNA/RNA hybrids are digested
with RNase H while double stranded DNA molecules are heat denatured
again. In either case the single stranded DNA is made fully double
stranded by addition of second target specific primer, followed by
polymerisation. The double-stranded DNA molecules are then multiply
transcribed by an RNA polymerase such as T7 or SP6. In an
isothermal cyclic reaction, the RNAs are reverse transcribed into
single stranded DNA, which is then converted to double stranded
DNA, and then transcribed once again with an RNA polymerase such as
T7 or SP6. The resulting products, whether truncated or complete,
indicate target specific sequences.
[0206] Vincent and Kong disclose a method termed helicase-dependent
isothermal DNA amplification (HDA) (Vincent and Kong, EMBO Reports,
5(8):795-800, 2004). This method uses DNA helicase to separate DNA
strands and hence does not require thermal cycling. The entire
reaction can be carried out at one temperature and this method
should have broad application to point-of-care DNA diagnostics.
[0207] Davey et al., EPO No. 329 822 disclose a nucleic acid
amplification process involving cyclically synthesizing
single-stranded RNA ("ssRNA"), ssDNA, and double-stranded DNA
(dsDNA), which may be used in accordance with the present
invention. The ssRNA is a template for a first primer
oligonucleotide, which is elongated by reverse transcriptase
(RNA-dependent DNA polymerase). The RNA is then removed from the
resulting DNA:RNA duplex by the action of ribonuclease H(RNase H,
an RNase specific for RNA in duplex with either DNA or RNA). The
resultant ssDNA is a template for a second primer, which also
includes the sequences of an RNA polymerase promoter (exemplified
by T7 RNA polymerase) 5' to its homology to the template. This
primer is then extended by DNA polymerase (exemplified by the large
"Klenow" fragment of E. coli DNA polymerase I), resulting in a
double-stranded DNA ("dsDNA") molecule, having a sequence identical
to that of the original RNA between the primers and having
additionally, at one end, a promoter sequence. This promoter
sequence can be used by the appropriate RNA polymerase to make many
RNA copies of the DNA. These copies can then re-enter the cycle
leading to very swift amplification. With proper choice of enzymes,
this amplification can be done isothermally without addition of
enzymes at each cycle. Because of the cyclical nature of this
process, the starting sequence can be chosen to be in the form of
either DNA or RNA.
[0208] Miller et al. in PCT Application WO 89/06700 disclose a
nucleic acid sequence amplification scheme based on the
hybridization of a promoter/primer sequence to a target
single-stranded DNA ("ssDNA") followed by transcription of many RNA
copies of the sequence. This scheme is not cyclic, i.e., new
templates are not produced from the resultant RNA transcripts.
Other amplification methods include "RACE" and "one-sided PCR"
(Frohman, M. A., In: "PCR Protocols: A Guide to Methods and
Applications", Academic Press, N.Y., 1990; Ohara et al., 1989,
Proc. Natl Acad. Sci. U.S.A., 86: 5673-567).
[0209] Methods based on ligation of two (or more) oligonucleotides
in the presence of nucleic acid having the sequence of the
resulting "di-oligonucleotide", thereby amplifying the
di-oligonucleotide, may also be used for amplifying target nucleic
acid sequences. Wu et al., (1989, Genomics 4: 560).
[0210] Depending on the format, the endotoxemia marker nucleic acid
of interest is identified in the sample directly using a
template-dependent amplification as described, for example, above,
or with a second, known nucleic acid following amplification. Next,
the identified product is detected. In certain applications, the
detection may be performed by visual means (e.g., ethidium bromide
staining of a gel). Alternatively, the detection may involve
indirect identification of the product via chemiluminescence,
radioactive scintigraphy of radiolabel or fluorescent label or even
via a system using electrical or thermal impulse signals (Affymax
Technology; Bellus, 1994, J Macromol. Sci. Pure, Appl. Chem.,
A31(1): 1355-1376).
[0211] In some embodiments, amplification products or "amplicons"
are visualized in order to confirm amplification of the endotoxemia
marker sequences. One typical visualization method involves
staining of a gel with ethidium bromide and visualization under UV
light. Alternatively, if the amplification products are integrally
labeled with radio- or fluorometrically-labelled nucleotides, the
amplification products can then be exposed to x-ray film or
visualized under the appropriate stimulating spectra, following
separation. In some embodiments, visualization is achieved
indirectly. Following separation of amplification products, a
labeled nucleic acid probe is brought into contact with the
amplified endotoxemia marker sequence. The probe is suitably
conjugated to a chromophore but may be radiolabeled. Alternatively,
the probe is conjugated to a binding partner, such as an
antigen-binding molecule, or biotin, and the other member of the
binding pair carries a detectable moiety or reporter molecule. The
techniques involved are well known to those of skill in the art and
can be found in many standard texts on molecular protocols (e.g.,
see Sambrook et al., 1989, supra and Ausubel et al. 1994, supra).
For example, chromophore or radiolabel probes or primers identify
the target during or following amplification.
[0212] In certain embodiments, target nucleic acids are quantified
using blotting techniques, which are well known to those of skill
in the art. Southern blotting involves the use of DNA as a target,
whereas Northern blotting involves the use of RNA as a target. Each
provide different types of information, although cDNA blotting is
analogous, in many aspects, to blotting or RNA species. Briefly, a
probe is used to target a DNA or RNA species that has been
immobilized on a suitable matrix, often a filter of nitrocellulose.
The different species should be spatially separated to facilitate
analysis. This often is accomplished by gel electrophoresis of
nucleic acid species followed by "blotting" on to the filter.
Subsequently, the blotted target is incubated with a probe (usually
labeled) under conditions that promote denaturation and
rehybridisation. Because the probe is designed to base pair with
the target, the probe will bind a portion of the target sequence
under renaturing conditions. Unbound probe is then removed, and
detection is accomplished as described above.
[0213] Following detection/quantification, one may compare the
results seen in a given subject with a control reaction or a
statistically significant reference group of normal subjects or of
subjects lacking an endotoxemia-related condition. In this way, it
is possible to correlate the amount of a endotoxemia marker nucleic
acid detected with the progression or severity of the disease.
[0214] Also contemplated are genotyping methods and allelic
discrimination methods and technologies such as those described by
Kristensen et al. (Biotechniques 30(2): 318-322), including the use
of single nucleotide polymorphism analysis, high performance liquid
chromatography, TaqMang, liquid chromatography, and mass
spectrometry.
[0215] Also contemplated are biochip-based technologies such as
those described by Hacia et al. (1996, Nature Genetics 14: 441-447)
and Shoemaker et al. (1996, Nature Genetics 14: 450-456). Briefly,
these techniques involve quantitative methods for analysing large
numbers of genes rapidly and accurately. By tagging genes with
oligonucleotides or using fixed probe arrays, one can employ
biochip technology to segregate target molecules as high density
arrays and screen these molecules on the basis of hybridization.
See also Pease et al. (1994, Proc. Natl. Acad. Sci. U.S.A. 91:
5022-5026); Fodor et al. (1991, Science 251: 767-773). Briefly,
nucleic acid probes to endotoxemia marker polynucleotides are made
and attached to biochips to be used in screening and diagnostic
methods, as outlined herein. The nucleic acid probes attached to
the biochip are designed to be substantially complementary to
specific expressed endotoxemia marker nucleic acids, i.e., the
target sequence (either the target sequence of the sample or to
other probe sequences, for example in sandwich assays), such that
hybridization of the target sequence and the probes of the present
invention occurs. This complementarity need not be perfect; there
may be any number of base pair mismatches which will interfere with
hybridization between the target sequence and the nucleic acid
probes of the present invention. However, if the number of
mismatches is so great that no hybridization can occur under even
the least stringent of hybridization conditions, the sequence is
not a complementary target sequence. In certain embodiments, more
than one probe per sequence is used, with either overlapping probes
or probes to different sections of the target being used. That is,
two, three, four or more probes, with three being desirable, are
used to build in a redundancy for a particular target. The probes
can be overlapping (i.e. have some sequence in common), or
separate.
[0216] As will be appreciated by those of ordinary skill in the
art, nucleic acids can be attached to or immobilized on a solid
support in a wide variety of ways. By "immobilized" and grammatical
equivalents herein is meant the association or binding between the
nucleic acid probe and the solid support is sufficient to be stable
under the conditions of binding, washing, analysis, and removal as
outlined below. The binding can be covalent or non-covalent. By
"non-covalent binding" and grammatical equivalents herein is meant
one or more of either electrostatic, hydrophilic, and hydrophobic
interactions. Included in non-covalent binding is the covalent
attachment of a molecule, such as, streptavidin to the support and
the non-covalent binding of the biotinylated probe to the
streptavidin. By "covalent binding" and grammatical equivalents
herein is meant that the two moieties, the solid support and the
probe, are attached by at least one bond, including sigma bonds, pi
bonds and coordination bonds. Covalent bonds can be formed directly
between the probe and the solid support or can be formed by a cross
linker or by inclusion of a specific reactive group on either the
solid support or the probe or both molecules. Immobilization may
also involve a combination of covalent and non-covalent
interactions.
[0217] In general, the probes are attached to the biochip in a wide
variety of ways, as will be appreciated by those in the art. As
described herein, the nucleic acids can either be synthesized
first, with subsequent attachment to the biochip, or can be
directly synthesized on the biochip.
[0218] The biochip comprises a suitable solid or semi-solid
substrate or solid support. By "substrate" or "solid support" is
meant any material that can be modified to contain discrete
individual sites appropriate for the attachment or association of
the nucleic acid probes and is amenable to at least one detection
method. As will be appreciated by practitioners in the art, the
number of possible substrates are very large, and include, but are
not limited to, glass and modified or functionalised glass,
plastics (including acrylics, polystyrene and copolymers of styrene
and other materials, polypropylene, polyethylene, polybutylene,
polyurethanes, Teflon.TM., etc.), polysaccharides, nylon or
nitrocellulose, resins, silica or silica-based materials including
silicon and modified silicon, carbon, metals, inorganic glasses,
plastics, etc. In general, the substrates allow optical detection
and do not appreciably fluorescese.
[0219] Generally the substrate is planar, although as will be
appreciated by those of skill in the art, other configurations of
substrates may be used as well. For example, the probes may be
placed on the inside surface of a tube, for flow-through sample
analysis to minimize sample volume. Similarly, the substrate may be
flexible, such as a flexible foam, including closed cell foams made
of particular plastics.
[0220] In certain embodiments, oligonucleotides probes are
synthesized on the substrate, as is known in the art. For example,
photoactivation techniques utilizing photopolymerisation compounds
and techniques can be used. In an illustrative example, the nucleic
acids are synthesized in situ, using well known photolithographic
techniques, such as those described in WO 95/25116; WO 95/35505;
U.S. Pat. Nos. 5,700,637 and 5,445,934; and references cited
within; these methods of attachment form the basis of the
Affymetrix GeneChip.TM. technology.
[0221] In an illustrative biochip analysis, oligonucleotide probes
on the biochip are exposed to or contacted with a nucleic acid
sample suspected of containing one or more endotoxemia
polynucleotides under conditions favoring specific hybridization.
Sample extracts of DNA or RNA, either single or double-stranded,
may be prepared from fluid suspensions of biological materials, or
by grinding biological materials, or following a cell lysis step
which includes, but is not limited to, lysis effected by treatment
with SDS (or other detergents), osmotic shock, guanidinium
isothiocyanate and lysozyme. Suitable DNA, which may be used in the
method of the invention, includes cDNA. Such DNA may be prepared by
any one of a number of commonly used protocols as for example
described in Ausubel, et al., 1994, supra, and Sambrook, et al., et
al., 1989, supra.
[0222] Suitable RNA, which may be used in the method of the
invention, includes messenger RNA, complementary RNA transcribed
from DNA (cRNA) or genomic or subgenomic RNA. Such RNA may be
prepared using standard protocols as for example described in the
relevant sections of Ausubel, et al. 1994, supra and Sambrook, et
al. 1989, supra).
[0223] cDNA may be fragmented, for example, by sonication or by
treatment with restriction endonucleases. Suitably, cDNA is
fragmented such that resultant DNA fragments are of a length
greater than the length of the immobilized oligonucleotide probe(s)
but small enough to allow rapid access thereto under suitable
hybridization conditions. Alternatively, fragments of cDNA may be
selected and amplified using a suitable nucleotide amplification
technique, as described for example above, involving appropriate
random or specific primers.
[0224] Usually the target endotoxemia marker polynucleotides are
detectably labeled so that their hybridization to individual probes
can be determined. The target polynucleotides are typically
detectably labeled with a reporter molecule illustrative examples
of which include chromogens, catalysts, enzymes, fluorochromes,
chemiluminescent molecules, bioluminescent molecules, lanthanide
ions (e.g., Eu.sup.34), a radioisotope and a direct visual label.
In the case of a direct visual label, use may be made of a
colloidal metallic or non-metallic particle, a dye particle, an
enzyme or a substrate, an organic polymer, a latex particle, a
liposome, or other vesicle containing a signal producing substance
and the like. Illustrative labels of this type include large
colloids, for example, metal colloids such as those from gold,
selenium, silver, tin and titanium oxide. In some embodiments in
which an enzyme is used as a direct visual label, biotinylated
bases are incorporated into a target polynucleotide. Hybridization
is detected by incubation with streptavidin-reporter molecules.
[0225] Suitable fluorochromes include, but are not limited to,
fluorescein isothiocyanate (FITC), tetramethylrhodamine
isothiocyanate (TRITC), R-Phycoerythrin (RPE), and Texas Red. Other
exemplary fluorochromes include those discussed by Dower et al.
(International Publication WO 93/06121). Reference also may be made
to the fluorochromes described in U.S. Pat. Nos. 5,573,909 (Singer
et al), 5,326,692 (Brinkley et al). Alternatively, reference may be
made to the fluorochromes described in U.S. Pat. Nos. 5,227,487,
5,274,113, 5,405,975, 5,433,896, 5,442,045, 5,451,663, 5,453,517,
5,459,276, 5,516,864, 5,648,270 and 5,723,218. Commercially
available fluorescent labels include, for example, fluorescein
phosphoramidites such as Fluoreprime.TM. (Pharmacia),
Fluoredite.TM. (Millipore) and FAM (Applied Biosystems
International)
[0226] Radioactive reporter molecules include, for example,
.sup.32P, which can be detected by an X-ray or phosphoimager
techniques.
[0227] The hybrid-forming step can be performed under suitable
conditions for hybridizing oligonucleotide probes to test nucleic
acid including DNA or RNA. In this regard, reference may be made,
for example, to NUCLEIC ACID HYBRIDIZATION, A PRACTICAL APPROACH
(Homes and Higgins, eds.) (IRL press, Washington D.C., 1985). In
general, whether hybridization takes place is influenced by the
length of the oligonucleotide probe and the polynucleotide sequence
under test, the pH, the temperature, the concentration of mono- and
divalent cations, the proportion of G and C nucleotides in the
hybrid-forming region, the viscosity of the medium and the possible
presence of denaturants. Such variables also influence the time
required for hybridization. The preferred conditions will therefore
depend upon the particular application. Such empirical conditions,
however, can be routinely determined without undue
experimentation.
[0228] In certain advantageous embodiments, high discrimination
hybridization conditions are used. For example, reference may be
made to Wallace et al. (1979, Nucl. Acids Res. 6: 3543) who
describe conditions that differentiate the hybridization of 11 to
17 base long oligonucleotide probes that match perfectly and are
completely homologous to a target sequence as compared to similar
oligonucleotide probes that contain a single internal base pair
mismatch. Reference also may be made to Wood et al. (1985, Proc.
Natl. Acid. Sci. USA 82: 1585) who describe conditions for
hybridization of 11 to 20 base long oligonucleotides using 3M
tetramethyl ammonium chloride wherein the melting point of the
hybrid depends only on the length of the oligonucleotide probe,
regardless of its GC content. In addition, Drmanac et al. (supra)
describe hybridization conditions that allow stringent
hybridization of 6-10 nucleotide long oligomers, and similar
conditions may be obtained most readily by using nucleotide
analogues such as locked nucleic acids (Christensen et al., 2001
Biochem J 354: 481-4).
[0229] Generally, a hybridization reaction can be performed in the
presence of a hybridization buffer that optionally includes a
hybridization-optimizing agent, such as an isostabilising agent, a
denaturing agent and/or a renaturation accelerant. Examples of
isostabilising agents include, but are not restricted to, betaines
and lower tetraalkyl ammonium salts. Denaturing agents are
compositions that lower the melting temperature of double stranded
nucleic acid molecules by interfering with hydrogen bonding between
bases in a double stranded nucleic acid or the hydration of nucleic
acid molecules. Denaturing agents include, but are not restricted
to, formamide, formaldehyde, dimethylsulfoxide, tetraethyl acetate,
urea, guanidium isothiocyanate, glycerol and chaotropic salts.
Hybridisation accelerants include heterogeneous nuclear
ribonucleoprotein (hnRP) A1 and cationic detergents such as
cetyltrimethylammonium bromide (CTAB) and dodecyl trimethylammonium
bromide (DTAB), polylysine, spermine, spermidine, single stranded
binding protein (SSB), phage T4 gene 32 protein and a mixture of
ammonium acetate and ethanol. Hybridization buffers may include
target polynucleotides at a concentration between about 0.005 nM
and about 50 nM, preferably between about 0.5 nM and 5 nM, more
preferably between about 1 nM and 2 nM.
[0230] A hybridization mixture containing the target endotoxemia
marker polynucleotides is placed in contact with the array of
probes and incubated at a temperature and for a time appropriate to
permit hybridization between the target sequences in the target
polynucleotides and any complementary probes. Contact can take
place in any suitable container, for example, a dish or a cell
designed to hold the solid support on which the probes are bound.
Generally, incubation will be at temperatures normally used for
hybridization of nucleic acids, for example, between about
20.degree. C. and about 75.degree. C., example, about 25.degree.
C., about 30.degree. C., about 35.degree. C., about 40.degree. C.,
about 45.degree. C., about 50.degree. C., about 55.degree. C.,
about 60.degree. C., or about 65.degree. C. For probes longer than
14 nucleotides, 20.degree. C. to 50.degree. C. is desirable. For
shorter probes, lower temperatures are preferred. A sample of
target polynucleotides is incubated with the probes for a time
sufficient to allow the desired level of hybridization between the
target sequences in the target polynucleotides and any
complementary probes. For example, the hybridization may be carried
out at about 45.degree. C.+/-10.degree. C. in formamide for 1-2
days.
[0231] After the hybrid-forming step, the probes are washed to
remove any unbound nucleic acid with a hybridization buffer, which
can typically comprise a hybridization optimizing agent in the same
range of concentrations as for the hybridization step. This washing
step leaves only bound target polynucleotides. The probes are then
examined to identify which probes have hybridized to a target
polynucleotide.
[0232] The hybridization reactions are then detected to determine
which of the probes has hybridized to a corresponding target
sequence. Depending on the nature of the reporter molecule
associated with a target polynucleotide, a signal may be
instrumentally detected by irradiating a fluorescent label with
light and detecting fluorescence in a fluorimeter; by providing for
an enzyme system to produce a dye which could be detected using a
spectrophotometer; or detection of a dye particle or a colored
colloidal metallic or non metallic particle using a reflectometer;
in the case of using a radioactive label or chemiluminescent
molecule employing a radiation counter or autoradiography.
Accordingly, a detection means may be adapted to detect or scan
light associated with the label which light may include
fluorescent, luminescent, focussed beam or laser light. In such a
case, a charge couple device (CCD) or a photocell can be used to
scan for emission of light from a probe:target polynucleotide
hybrid from each location in the micro-array and record the data
directly in a digital computer. In some cases, electronic detection
of the signal may not be necessary. For example, with enzymatically
generated color spots associated with nucleic acid array format,
visual examination of the array will allow interpretation of the
pattern on the array. In the case of a nucleic acid array, the
detection means is suitably interfaced with pattern recognition
software to convert the pattern of signals from the array into a
plain language genetic profile. In certain embodiments,
oligonucleotide probes specific for different endotoxemia marker
gene products are in the form of a nucleic acid array and detection
of a signal generated from a reporter molecule on the array is
performed using a `chip reader`. A detection system that can be
used by a `chip reader` is described for example by Pirrung et al
(U.S. Pat. No. 5,143,854). The chip reader will typically also
incorporate some signal processing to determine whether the signal
at a particular array position or feature is a true positive or
maybe a spurious signal. Exemplary chip readers are described for
example by Fodor et al (U.S. Pat. No., 5,925,525). Alternatively,
when the array is made using a mixture of individually addressable
kinds of labeled microbeads, the reaction may be detected using
flow cytometry.
[0233] 7.2 Protein-Based Diagnostics
[0234] Consistent with the present invention, the presence of an
aberrant concentration of an endotoxemia marker protein is
indicative of the presence, degree, or stage of development of an
endotoxemia-related condition. Endotoxemia marker protein levels in
biological samples can be assayed using any suitable method known
in the art. For example, when an endotoxemia marker protein is an
enzyme, the protein can be quantified based upon its catalytic
activity or based upon the number of molecules of the protein
contained in a sample. Antibody-based techniques may be employed,
such as, for example, immunohistological and immunohistochemical
methods for measuring the level of a protein of interest in a
tissue sample. For example, specific recognition is provided by a
primary antibody (polyclonal or monoclonal) and a secondary
detection system is used to detect presence (or binding) of the
primary antibody. Detectable labels can be conjugated to the
secondary antibody, such as a fluorescent label, a radiolabel, or
an enzyme (e.g., alkaline phosphatase, horseradish peroxidase)
which produces a quantifiable, e.g., coloured, product. In another
suitable method, the primary antibody itself can be detectably
labeled. As a result, immunohistological labeling of a tissue
section is provided. In some embodiments, a protein extract is
produced from a biological sample (e.g., tissue, cells) for
analysis. Such an extract (e.g., a detergent extract) can be
subjected to western-blot or dot/slot assay of the level of the
protein of interest, using routine immunoblotting methods (Jalkanen
et at., 1985, J. Cell. Biol. 101: 976-985; Jalkanen et al., 1987,
J. Cell. Biol. 105: 3087-3096).
[0235] Other useful antibody-based methods include immunoassays,
such as the enzyme-linked immunosorbent assay (ELISA) and the
radioimmunoassay (RIA). For example, a protein-specific monoclonal
antibody, can be used both as an immunoadsorbent and as an
enzyme-labeled probe to detect and quantify an endotoxemia marker
protein of interest. The amount of such protein present in a sample
can be calculated by reference to the amount present in a standard
preparation using a linear regression computer algorithm (see
Lacobilli et al., 1988, Breast Cancer Research and Treatment 11:
19-30). In other embodiments, two different monoclonal antibodies
to the protein of interest can be employed, one as the
immunoadsorbent and the other as an enzyme-labeled probe.
[0236] Additionally, recent developments in the field of protein
capture arrays permit the simultaneous detection and/or
quantification of a large number of proteins. For example,
low-density protein arrays on filter membranes, such as the
universal protein array system (Ge, 2000 Nucleic Acids Res.
28(2):e3) allow imaging of arrayed antigens using standard ELISA
techniques and a scanning charge-coupled device (CCD) detector.
Immuno-sensor arrays have also been developed that enable the
simultaneous detection of clinical analytes. It is now possible
using protein arrays, to profile protein expression in bodily
fluids, such as in sera of healthy or diseased subjects, as well as
in subjects pre- and post-drug treatment.
[0237] Protein capture arrays typically comprise a plurality of
protein-capture agents each of which defines a spatially distinct
feature of the array. The protein-capture agent can be any molecule
or complex of molecules which has the ability to bind a protein and
immobilize it to the site of the protein-capture agent on the
array. The protein-capture agent may be a protein whose natural
function in a cell is to specifically bind another protein, such as
an antibody or a receptor. Alternatively, the protein-capture agent
may instead be a partially or wholly synthetic or recombinant
protein which specifically binds a protein. Alternatively, the
protein-capture agent may be a protein which has been selected in
vitro from a mutagenized, randomized, or completely random and
synthetic library by its binding affinity to a specific protein or
peptide target. The selection method used may optionally have been
a display method such as ribosome display or phage display, as
known in the art. Alternatively, the protein-capture agent obtained
via in vitro selection may be a DNA or RNA aptamer which
specifically binds a protein target (see, e.g., Potyrailo et al.,
1998 Anal. Chem. 70:3419-3425; Cohen et al., 1998, Proc. Natl.
Acad. Sci. USA 95:14272-14277; Fukuda, et al., 1997 Nucleic Acids
Symp. Ser. 37:237-238; available from SomaLogic). For example,
aptamers are selected from libraries of oligonucleotides by the
Selex.TM. process and their interaction with protein can be
enhanced by covalent attachment, through incorporation of
brominated deoxyuridine and UV-activated crosslinking
(photoaptamers). Aptamers have the advantages of ease of production
by automated oligonucleotide synthesis and the stability and
robustness of DNA; universal fluorescent protein stains can be used
to detect binding. Alternatively, the in vitro selected
protein-capture agent may be a polypeptide (e.g., an antigen) (see,
e.g., Roberts and Szostak, 1997 Proc. Natl. Acad. Sci. USA,
94:12297-12302).
[0238] An alternative to an array of capture molecules is one made
through `molecular imprinting` technology, in which peptides (e.g.,
from the C-terminal regions of proteins) are used as templates to
generate structurally complementary, sequence-specific cavities in
a polymerisable matrix; the cavities can then specifically capture
(denatured) proteins which have the appropriate primary amino acid
sequence (e.g., available from ProteinPrint.TM. and Aspira
Biosystems).
[0239] Exemplary protein capture arrays include arrays comprising
spatially addressed antigen-binding molecules, commonly referred to
as antibody arrays, which can facilitate extensive parallel
analysis of numerous proteins defining a proteome or subproteome.
Antibody arrays have been shown to have the required properties of
specificity and acceptable background, and some are available
commercially (e.g., BD Biosciences, Clontech, BioRad and Sigma).
Various methods for the preparation of antibody arrays have been
reported (see, e.g., Lopez et al., 2003 J. Chromatogr. B 787:19-27;
Cahill, 2000 Trends in Biotechnology 7:47-51; U.S. Pat. App. Pub.
2002/0055186; U.S. Pat. App. Pub. 2003/0003599; PCT publication WO
03/062444; PCT publication WO 03/077851; PCT publication WO
02/59601; PCT publication WO 02/39120; PCT publication WO 01/79849;
PCT publication WO 99/39210). The antigen-binding molecules of such
arrays may recognise at least a subset of proteins expressed by a
cell or population of cells, illustrative examples of which include
growth factor receptors, hormone receptors, neurotransmitter
receptors, catecholamine receptors, amino acid derivative
receptors, cytokine receptors, extracellular matrix receptors,
antibodies, lectins, cytokines, serpins, proteases, kinases,
phosphatases, ras-like GTPases, hydrolases, steroid hormone
receptors, transcription factors, heat-shock transcription factors,
DNA-binding proteins, zinc-finger proteins, leucine-zipper
proteins, homeodomain proteins, intracellular signal transduction
modulators and effectors, apoptosis-related factors, DNA synthesis
factors, DNA repair factors, DNA recombination factors,
cell-surface antigens, hepatitis C virus (HCV) proteases and HIV
proteases.
[0240] Antigen-binding molecules for antibody arrays are made
either by conventional immunization (e.g., polyclonal sera and
hybridomas), or as recombinant fragments, usually expressed in E.
coli, after selection from phage display or ribosome display
libraries (e.g., available from Cambridge Antibody Technology,
Bioinvent, Affitech and Biosite). Alternatively, `combibodies`
comprising non-covalent associations of VH and VL domains, can be
produced in a matrix format created from combinations of
diabody-producing bacterial clones (e.g., available from Domantis).
Exemplary antigen-binding molecules for use as protein-capture
agents include monoclonal antibodies, polyclonal antibodies, Fv,
Fab, Fab' and F(ab').sub.2 immunoglobulin fragments, synthetic
stabilized Fv fragments, e.g., single chain Fv fragments (scFv),
disulfide stabilized Fv fragments (dsFv), single variable region
domains (dAbs) minibodies, combibodies and multivalent antibodies
such as diabodies and multi-scFv, single domains from camelids or
engineered human equivalents.
[0241] Individual spatially distinct protein-capture agents are
typically attached to a support surface, which is generally planar
or contoured. Common physical supports include glass slides,
silicon, microwells, nitrocellulose or PVDF membranes, and magnetic
and other microbeads.
[0242] While microdrops of protein delivered onto planar surfaces
are widely used, related alternative architectures include CD
centrifugation devices based on developments in microfluidics
(e.g., available from Gyros) and specialized chip designs, such as
engineered microchannels in a plate (e.g., The Living Chip.TM.,
available from Biotrove) and tiny 3D posts on a silicon surface
(e.g., available from Zyomyx).
[0243] Particles in suspension can also be used as the basis of
arrays, providing they are coded for identification; systems
include color coding for microbeads (e.g., available from Luminex,
Bio-Rad and Nanomics Biosystems) and semiconductor nanocrystals
(e.g., QDots.TM., available from Quantum Dots), and barcoding for
beads (UltraPlex.TM., available from Smartbeads) and multimetal
microrods (Nanobarcodes.TM. particles, available from Surromed).
Beads can also be assembled into planar arrays on semiconductor
chips (e.g., available from LEAPS technology and BioArray
Solutions). Where particles are used, individual protein-capture
agents are typically attached to an individual particle to provide
the spatial definition or separation of the array. The particles
may then be assayed separately, but in parallel, in a
compartmentalized way, for example in the wells of a microtiter
plate or in separate test tubes.
[0244] In operation, a protein sample, which is optionally
fragmented to form peptide fragments (see, e.g., U.S. Pat. App.
Pub. 2002/0055186), is delivered to a protein-capture array under
conditions suitable for protein or peptide binding, and the array
is washed to remove unbound or non-specifically bound components of
the sample from the array. Next, the presence or amount of protein
or peptide bound to each feature of the array is detected using a
suitable detection system. The amount of protein bound to a feature
of the array may be determined relative to the amount of a second
protein bound to a second feature of the array. In certain
embodiments, the amount of the second protein in the sample is
already known or known to be invariant.
[0245] For analyzing differential expression of proteins between
two cells or cell populations, a protein sample of a first cell or
population of cells is delivered to the array under conditions
suitable for protein binding. In an analogous manner, a protein
sample of a second cell or population of cells to a second array,
is delivered to a second array which is identical to the first
array. Both arrays are then washed to remove unbound or
non-specifically bound components of the sample from the arrays. In
a final step, the amounts of protein remaining bound to the
features of the first array are compared to the amounts of protein
remaining bound to the corresponding features of the second array.
To determine the differential protein expression pattern of the two
cells or populations of cells, the amount of protein bound to
individual features of the first array is subtracted from the
amount of protein bound to the corresponding features of the second
array.
[0246] In an illustrative example, fluorescence labeling can be
used for detecting protein bound to the array. The same
instrumentation as used for reading DNA microarrays is applicable
to protein-capture arrays. For differential display, capture arrays
(e.g. antibody arrays) can be probed with fluorescently labeled
proteins from two different cell states, in which cell lysates are
labeled with different fluorophores (e.g., Cy-3 and Cy-5) and
mixed, such that the color acts as a readout for changes in target
abundance. Fluorescent readout sensitivity can be amplified 10-100
fold by tyramide signal amplification (TSA) (e.g., available from
PerkinElmer Lifesciences). Planar waveguide technology (e.g.,
available from Zeptosens) enables ultrasensitive fluorescence
detection, with the additional advantage of no washing procedures.
High sensitivity can also be achieved with suspension beads and
particles, using phycoerythrin as label (e.g., available from
Luminex) or the properties of semiconductor nanocrystals (e.g.,
available from Quantum Dot). Fluorescence resonance energy transfer
has been adapted to detect binding of unlabelled ligands, which may
be useful on arrays (e.g., available from Affibody). Several
alternative readouts have been developed, including adaptations of
surface plasmon resonance (e.g., available from HTS Biosystems and
Intrinsic Bioprobes), rolling circle DNA amplification (e.g.,
available from Molecular Staging), mass spectrometry (e.g.,
available from Sense Proteomic, Ciphergen, Intrinsic and
Bioprobes), resonance light scattering (e.g., available from
Genicon Sciences) and atomic force microscopy (e.g., available from
BioForce Laboratories). A microfluidics system for automated sample
incubation with arrays on glass slides and washing has been
co-developed by NextGen and Perkin Elmer Life Sciences.
[0247] In certain embodiments, the techniques used for detection of
endotoxemia marker expression products will include internal or
external standards to permit quantitative or semi-quantitative
determination of those products, to thereby enable a valid
comparison of the level or functional activity of these expression
products in a biological sample with the corresponding expression
products in a reference sample or samples. Such standards can be
determined by the skilled practitioner using standard protocols. In
specific examples, absolute values for the level or functional
activity of individual expression products are determined.
[0248] In specific embodiments, the diagnostic method is
implemented using a system as disclosed, for example, in
International Publication No. WO 02/090579 and in copending PCT
Application No. PCT/AU03/01517 filed Nov. 14, 2003, comprising at
least one end station coupled to a base station. The base station
is typically coupled to one or more databases comprising
predetermined data from a number of individuals representing the
level or functional activity of endotoxemia marker expression
products, together with indications of the actual status of the
individuals (e.g., presence, absence, degree, or stage of
development of an endotoxemia-related condition) when the
predetermined data was collected. In operation, the base station is
adapted to receive from the end station, typically via a
communications network, subject data representing a measured or
normalized level or functional activity of at least one expression
product in a biological sample obtained from a test subject and to
compare the subject data to the predetermined data stored in the
database(s). Comparing the subject and predetermined data allows
the base station to determine the status of the subject in
accordance with the results of the comparison. Thus, the base
station attempts to identify individuals having similar parameter
values to the test subject and once the status has been determined
on the basis of that identification, the base station provides an
indication of the diagnosis to the end station.
[0249] 7.3 Kits
[0250] All the essential materials and reagents required for
detecting and quantifying endotoxemia maker gene expression
products may be assembled together in a kit. The kits may also
optionally include appropriate reagents for detection of labels,
positive and negative controls, washing solutions, blotting
membranes, microtiter plates dilution buffers and the like. For
example, a nucleic acid-based detection kit may include (i) an
endotoxemia marker polynucleotide (which may be used as a positive
control), (ii) a primer or probe that specifically hybridizes to an
endotoxemia marker polynucleotide. Also included may be enzymes
suitable for amplifying nucleic acids including various polymerases
(Reverse Transcriptase, Taq, Sequenase.TM. DNA ligase etc.
depending on the nucleic acid amplification technique employed),
deoxynucleotides and buffers to provide the necessary reaction
mixture for amplification. Such kits also generally will comprise,
in suitable means, distinct containers for each individual reagent
and enzyme as well as for each primer or probe. Alternatively, a
protein-based detection kit may include (i) an endotoxemia marker
polypeptide (which may be used as a positive control), (ii) an
antigen-binding molecule that is immuno-interactive with an
endotoxemia marker polynucleotide. The kit can also feature various
devices and reagents for performing one of the assays described
herein; and/or printed instructions for using the kit to quantify
the expression of an endotoxemia marker gene.
8. Methods of Treatment or Prophylaxis
[0251] The present invention also extends to the management of
endotoxaemia-related conditions, or prevention of further
progression of endotoxaemia-related conditions, or assessment of
the efficacy of therapies in subjects following positive diagnosis
for the presence, or stage of endotoxaemia-related conditions in
the subjects. Generally, the management of endotoxaemia-related
conditions is highly intensive and can include identification and
amelioration of the underlying cause and aggressive use of
therapeutic compounds such as, vasoactive compounds, antibiotics,
steroids, antibodies to endotoxin, and anti tumour necrosis factor
agents. In addition, palliative therapies.sup.1 aimed at restoring
and protecting organ function can be used such as intravenous
fluids and oxygen. .sup.1 Cohen J & Glauser M P. Lancet 338:
736-739 (1991).
[0252] Typically, the therapeutic agents will be administered in
pharmaceutical (or veterinary) compositions together with a
pharmaceutically acceptable carrier and in an effective amount to
achieve their intended purpose. The dose of active compounds
administered to a subject should be sufficient to achieve a
beneficial response in the subject over time such as a reduction
in, or relief from, the symptoms of endotoxaemia. The quantity of
the pharmaceutically active compounds(s) to be administered may
depend on the subject to be treated inclusive of the age, sex,
weight and general health condition thereof. In this regard,
precise amounts of the active compound(s) for administration will
depend on the judgement of the practitioner. In determining the
effective amount of the active compound(s) to be administered in
the treatment or prevention of endotoxaemia, the medical
practitioner or veterinarian may evaluate severity of any symptom
associated with the presence of endotoxaemia including tachycardia,
fever, chills, vomiting, diarrhea, skin rash, headaches, confusion,
muscle aches, seizures. In any event, those of skill in the art may
readily determine suitable dosages of the therapeutic agents and
suitable treatment regimens without undue experimentation.
[0253] The therapeutic agents may by administered in concert with
adjunctive (palliative) therapies to increase oxygen supply to
major organs, increase blood flow to major organs and/or to reduce
the inflammatory response. Illustrative examples of such adjunctive
therapies include non steroidal-anti inflammatory drugs (NSAIDs),
intravenous saline and oxygen.
[0254] In order that the invention may be readily understood and
put into practical effect, particular preferred embodiments will
now be described by way of the following non-limiting examples.
EXAMPLES
Example 1
Identification of Specific Diagnostic Genes for
Endotoxaemia-Related Conditions
Experimental Disease Trial Design
[0255] A clinical trial was performed on three blocks of fours
horses each. The first block consisted of four horses that were
dosed orally with 12.5 mg/kg of oligofructose.sup.2 as part of the
trial procedure described by Pollitt.sup.3 which is specifically
designed to induce endotoxaemia and subsequent acute laminitis. The
second block consisted of four horses that underwent the same trial
procedure but were dosed with normal saline (0.9%) solution
(controls). The same four horses in the second block then underwent
the trial procedure (following a period of recovery) for a second
time but were dosed with oligofructose (block three). All horses
were stalled under the same conditions for the duration of the
procedure (120 hours). .sup.2 Raftilose.RTM., Orafti Active Food
Ingredients, Aanndorenstraat, B-3300 Tienen, Belgium..sup.3 van Eps
A & Pollitt CC. Equine Vet J. 36(3):255-60 (2004).
[0256] Endotoxaemia-related conditions in horses (including
laminitis) can be induced experimentally and one of the more
reliable methods of induction is by carbohydrate overload through
oral dosing with oligofructose.
[0257] Blood samples were collected at four time points--Hour 0
prior to dosing and at hours 24, 48, and 72 hours after dosing. The
sample at Hour 0 acted as a control for each horse.
[0258] The following tests and observations were undertaken at all
of the above time points:
[0259] (i) physical examination, rectal temperature, digital pulse,
hoof temperature, heart and respiratory rate, faecal pH, hoof
shifting; and
[0260] (ii) haematology and biochemistry.
[0261] Blood samples from each of the animals on Hours 0, 24, 48
and 72 of the trial were analysed using GeneChips.TM. (method of
use is described below in detail in "Generation of Gene Expression
Data") containing thousands of genes expressed in white blood cells
of horses. Analysis of these data (see "Identification of
Diagnostic Marker Genes" below) reveals a number of specific genes
that differ in expression between animals before and after
experimental induction of endotoxaemia and laminitis from Hour 24
following dosing. It is possible to design an assay that measures
the RNA level in the sample from the expression of at least one and
desirably at least two endotoxaemia marker genes, representative
transcript sequences of which are set forth in SEQ ID NO: 1, 3, 4,
5, 6, 7, 9, 10, 11, 13, 15, 16, 17, 18, 19, 21, 23, 25, 26, 27, 29,
31, 33, 35, 37, 38, 39, 41, 42, 43, 44, 45, 47, 49, 50, 52, 54, 56,
58, 60, 61, 63, 64, 66, 67, 68, 69, 70, 71, 73, 75, 76, 77, 78, 79,
80, 81, 82, 83, 84, 86, 88, 90, 92, 93, 94, 96, 98, 100, 101, 102,
103, 104, 106, 107, 109, 110, 111, 113, 114, 115, 117, 119, 121,
122, 123, 124, 125, 126, 128, 130, 132, 134, 136, 137, 139, 141,
143, 145, 147, 149, 151, 153, 155, 157, 158, 160, 162, 164, 166,
168, 169, 170, 172, 173, 174, 176, 178, 180, 182, 184, 186, 188,
190, 192, 193, 194, 195, 197, 199, 201, 203, 205, 206, 207, 209,
210, 211, 212, 214, 215, 216, 218, 220, 222, 223, 224, 225, 227,
229, 231, 233, 235, 236, 237, 239, 240, 242, 244, 245, 246, 248,
250, 252, 254, 255, 257, 259 260, 262, 264, 266, 268, 269, 270,
271, 272, 274, 276, 278, 279, 280, 282, 284, 286, 288, 290, 292,
294, 296, 298, 300, 302, 304, 305, 306, 307, 309, 311, 312, 314,
315, 316, 318, 320 321, 323 or 325.
Materials and Methods
Blood Collection
[0262] Blood is collected from a horse (in a non-agitated state)
for the purpose of extraction of high quality RNA or protein.
Suitable blood collection tubes for the collection, preservation,
transport and isolation of RNA include PAXgene.TM. tubes
(PreAnalytix Inc., Valencia, Calif., USA). Alternatively, blood can
be collected into tubes containing solutions designed for the
preservation of nucleic acids (available from Roche, Ambion,
Invitrogen and ABI). For the determination of protein levels, 50 mL
of blood is prevented from clotting by collection into a tube
containing 4 mL of 4% sodium citrate. White blood cells and plasma
are isolated and stored frozen for later analysis and detection of
specific proteins. PAXgene tubes can be kept at room temperature
prior to RNA extraction. Clinical signs are recorded in a standard
format.
Total RNA Extraction
[0263] A kit available from Qiagen Inc (Valencia, Calif., USA) has
the reagents and instructions for the isolation of total RNA from
2.5 mL blood collected in the PAXgene Blood RNA Tube. Isolation
begins with a centrifugation step to pellet nucleic acids in the
PAXgene blood RNA tube. The pellet is washed and resuspended and
incubated in optimized buffers together with Proteinase K to bring
about protein digestion. An additional centrifugation is carried
out to remove residual cell debris and the supernatant is
transferred to a fresh microcentrifuge tube. Ethanol is added to
adjust binding conditions, and the lysate is applied to the PAXgene
RNA spin column. During brief centrifugation, RNA is selectively
bound to the silica-gel membrane as contaminants pass through.
Remaining contaminants are removed in three efficient wash steps
and RNA is then eluted in Buffer BR5.
[0264] Determination of RNA quantity and quality is necessary prior
to proceeding and can be achieved using an Agilent Bioanalyzer and
Absorbance 260/280 ratio using a spectrophotometer.
Generation of Gene Expression Data
Choice of Method
[0265] Measurement of specific RNA levels in a tissue sample can be
achieved using a variety of technologies. Two common and readily
available technologies that are well known in the art are: [0266]
GeneChip.RTM. analysis using Affymetrix technology. [0267]
Real-Time Polymerase Chain Reaction (TaqMan.TM. from Applied
Biosystems for example).
[0268] GeneChips.RTM. quantitate RNA by detection of labeled cRNA
hybridized to short oligonucleotides built on a silicon substrate.
Details on the technology and methodology can be found at
www.affymetrix.com.
[0269] Real-Time Polymerase Chain Reaction (RT-PCR) quantitates RNA
using two PCR primers, a labeled probe and a thermostable DNA
polymerase. As PCR product is generated a dye is released into
solution and detected. Internal controls such as 18S RNA probes are
often used to determine starting levels of total RNA in the sample.
Each gene and the internal control are run separately. Details on
the technology and methods can be found at www.appliedbiosytems.com
or www.qiagen.com or www.biorad.com. Applied Biosystems offer a
service whereby the customer provides DNA sequence information and
payment and is supplied in return all of the reagents required to
perform RT-PCR analysis on individual genes.
[0270] GeneChip.RTM. analysis has the advantage of being able to
analyze thousands of genes at a time. However it is expensive and
takes over 3 days to perform a single assay. RT-PCR generally only
analyses one gene at a time, but is inexpensive and can be
completed within a single day.
[0271] RT-PCR is the method of choice for gene expression analysis
if the number of specific genes to be analyzed is less than 20.
GeneChip.RTM. or other gene expression analysis technologies (such
as Illumina Bead Arrays) are the method of choice when many genes
need to be analyzed simultaneously.
[0272] The methodology for GeneChip.RTM. data generation and
analysis and Real Time PCR is presented below in brief.
GeneChip.RTM. Data Generation
cDNA & cRNA Generation
[0273] The following method for cDNA and cRNA generation from total
RNA has been adapted from the protocol provided and recommended by
Affymetrix (www.affymetrix.com).
[0274] The steps are: [0275] A total of 3 .mu.g of total RNA is
used as a template to generate double stranded cDNA. [0276] cRNA is
generated and labeled using biotinylated Uracil (dUTP). [0277]
biotin-labeled cRNA is cleaned and the quantity determined using a
spectrophotometer and MOPS gel analysis. [0278] labeled cRNA is
fragmented to 300 bp in size. [0279] RNA quantity is determined on
an Agilent "Lab-on-a-Chip" system (Agilent Technologies).
Hybridization, Washing & Staining
[0280] The steps are: [0281] A hybridization cocktail is prepared
containing 0.05 .mu.g/.mu.L of labeled and fragmented cRNA,
spike-in positive hybridization controls, and the Affymetrix
oligonucleotides B2, bioB, bioC, bioD and cre. [0282] The final
volume (80 .mu.L) of the hybridization cocktail is added to the
GeneChip.RTM. cartridge. [0283] The cartridge is placed in a
hybridization oven at constant rotation for 16 hours. [0284] The
fluid is removed from the GeneChip.RTM. and stored. [0285] The
GeneChip.RTM. is placed in the fluidics station. [0286] The
experimental conditions for each GeneChip.RTM. are recorded as an
.EXP file. [0287] All washing and staining procedures are carried
out by the Affymetrix fluidics station with an attendant providing
the appropriate solutions. [0288] The GeneChip.RTM. is washed,
stained with steptavidin-phycoerythin dye and then washed again
using low salt solutions. [0289] After the wash protocols are
completed, the dye on the probe array is `excited` by laser and the
image captured by a CCD camera using an Affymetrix Scanner
(manufactured by Agilent).
Scanning & Data File Generation
[0290] The scanner and MAS 5 software generates an image file from
a single GeneChip.RTM. called a .DAT file (see figure
overleaf).
[0291] The .DAT file is then pre-processed prior to any statistical
analysis.
[0292] Data pre-processing steps (prior to any statistical
analysis) include: [0293] .DAT File Quality Control (QC). [0294]
.CEL File Generation. [0295] Scaling and Normalization.
.DAT File Quality Control
[0296] The .DAT file is an image. The image is inspected manually
for artifacts (e.g. high/low intensity spots, scratches, high
regional or overall background). (The B2 oligonucleotide
hybridization performance is easily identified by an alternating
pattern of intensities creating a border and array name.) The MAS 5
software used the B2 oligonucleotide border to align a grid over
the image so that each square of oligonucleotides was centered and
identified.
[0297] The other spiked hybridization controls (bioB, bioC, bioD
and cre) are used to evaluate sample hybridization efficiency by
reading "present" gene detection calls with increasing signal
values, reflecting their relative concentrations. (If the .DAT file
is of suitable quality it is converted to an intensity data file
(.CEL file) by Affymetrix MAS 5 software).
.CEL File Generation
[0298] The .CEL files generated by the MAS 5 software from .DAT
files contain calculated raw intensities for the probe sets. Gene
expression data is obtained by subtracting a calculated background
from each cell value. To eliminate negative intensity values, a
noise correction fraction based from a local noise value from the
standard deviation of the lowest 2% of the background is
applied.
[0299] All .CEL files generated from the GeneChips.RTM. are
subjected to specific quality metrics parameters.
[0300] Some metrics are routinely recommended by Affymetrix and can
be determined from Affymetrix internal controls provided as part of
the GeneChip.RTM.. Other metrics are based on experience and the
processing of many GeneChips.RTM..
Analysis of GeneChip.RTM. Data
[0301] Two illustrative approaches to normalising data may be used:
[0302] Affymetrix MAS 5 Algorithm. [0303] Robust Multi-chip
Analysis (RMA) algorithm of Irizarry (Irizarray et al., 2002,
Biostatistics (in print)).
[0304] Those of skill in the art will recognise that many other
approaches might be adopted, without materially affecting the
invention.
Affymetrix AS 5 Algorithm
[0305] .CEL files are used by Affymetrix MAS 5 software to
normalize or scale the data. Scaled data from one chip are compared
to similarly scaled data from other chips.
[0306] Affymetrix MAS 5 normalization is achieved by applying the
default "Global Scaling" option of the MAS 5 algorithm to the .CEL
files. This procedure subtracts a robust estimate of the center of
the distribution of probe values, and divides by a robust estimate
of the probe variability. This produces a set of chips with common
location and scale at the probe level.
[0307] Gene expression indices are generated by a robust averaging
procedure on all the probe pairs for a given gene. The results are
constrained to be non-negative.
[0308] Given that scaling takes place at the level of the probe,
rather than at the level of the gene, it is possible that even
after normalization there may be chip-to-chip differences in
overall gene expression level. Following standard MAS5
normalization, values for each gene were de-trended with respect to
median chip intensity. That is, values for each gene were regressed
on the median chip intensity, and residuals were calculated. These
residuals were taken as the de-trended estimates of expression for
each gene
[0309] Median chip intensity was calculated using the Affymetrix
MAS5 algorithm, but with a scale factor fixed at one.
RMA Algorithm
[0310] This algorithm quantifies the expression of a set of chips,
rather than of a single chip. It estimates background intensities
using a robust statistical model applied to perfect match probe
data. It does not make use of mis-match probe data. Following
implicit background correction, chips are processed using Quantile
Quantile normalization (Rizarray et al., 2002, Biostatistics (in
print)).
DNA Extraction
[0311] A kit available from Qiagen Inc (Valencia, Calif., USA) has
the reagents and instructions for the isolation of total DNA from
8.5 mL blood collected in the PAXgene Blood DNA Tube. Isolation
begins with the addition of additional lysis solution followed by a
centrifugation step. The pellet is washed and resuspended and
incubated in optimized buffers together with Proteinase K to bring
about protein digestion. DNA is precipitated using alcohol and an
additional centrifugation is carried out to pellet the nucleic
acid. Remaining contaminants are removed in a wash step and the DNA
is then resuspended in Buffer BG4.
[0312] Determination of DNA quantity and quality is necessary prior
to proceeding and can be achieved using a spectrophotometer or
agarose gel electrophoresis.
Genotyping Analysis
[0313] Many methods are available to genotype DNA. A review of
allelic discrimination methods can be found in Kristensen et al.
(Biotechniques 30(2): 318-322 (2001). An illustrative method for
genotyping using allele-specific PCR is described here.
Primer Design
[0314] Upstream and downstream PCR primers specific for particular
alleles can be designed using freely available computer programs,
such as Primer3
(http://frodo.wi.mit.edu/primer3/primer3_code.html). Alternatively
the DNA sequences of the various alleles can be aligned using a
program such as ClustalW (http://www.ebi.ac.uk/clustalw/) and
specific primers designed to areas where DNA sequence differences
exist but retaining enough specificity to ensure amplification of
the correct amplicon. Preferably a PCR amplicon is designed to have
a restriction enzyme site in one allele but not the other. Primers
are generally 18-25 base pairs in length with similar melting
temperatures.
PCR Amplification
[0315] The composition of PCR reactions has been described
elsewhere (Clinical Applications of PCR, Dennis Lo (Editor),
Blackwell Publishing, 1998). Briefly, a reaction contains primers,
DNA, buffers and a thermostable polymerase enzyme. The reaction is
cycled (up to 50 times) through temperature steps of denaturation,
hybridization and DNA extension on a thermocycler such as the MJ
Research Thermocycler model PTC-96V.
DNA Analysis
[0316] PCR products can be analyzed using a variety of methods
including size differentiation using mass spectrometry, capillary
gel electrophoresis and agarose gel electrophoresis. If the PCR
amplicons have been designed to contain differential restriction
enzyme sites, the DNA in the PCR reaction is purified using
DNA-binding columns or precipitation and re-suspended in water, and
then restricted using the appropriate restriction enzyme. The
restricted DNA can then be run on an agarose gel where DNA is
separated by size using electric current. Various alleles of a gene
will have different sizes depending on whether they contain
restriction sites. Thus, homozygotes and heterozygotes can be
determined.
Real-Time PCR Data Generation
[0317] Background information for conducting Real-time PCR may be
obtained, for example, at
http://dorakmt.tripod.com/genetics/realtime.html and in a review by
Bustin SA (2000, J Mol Endocrinol 25:169-193).
TaqMan.TM. Primer and Probe Design Guidelines
[0318] 1. The Primer Express.TM. (ABI) software designs primers
with a melting temperature (Tm) of 58-60.degree. C., and probes
with a Tm value of 10.degree. C. higher. The Tm of both primers
should be equal.
[0319] 2. Primers should be 15-30 bases in length.
[0320] 3. The G+C content should ideally be 30-80%. If a higher G+C
content is unavoidable, the use of high annealing and melting
temperatures, cosolvents such as glycerol, DMSO, or 7-deaza-dGTP
may be necessary.
[0321] 4. The run of an identical nucleotide should be avoided.
This is especially true for G, where runs of four or more Gs is not
allowed.
[0322] 5. The total number of Gs and Cs in the last five
nucleotides at the 3' end of the primer should not exceed two (the
newer version of the software has an option to do this
automatically). This helps to introduce relative instability to the
3' end of primers to reduce non-specific priming. The primer
conditions are the same for SYBR Green assays.
[0323] 6. Maximum amplicon size should not exceed 400 bp (ideally
50-150 bases). Smaller amplicons give more consistent results
because PCR is more efficient and more tolerant of reaction
conditions (the short length requirement has nothing to do with the
efficiency of 5' nuclease activity).
[0324] 7. The probes should not have runs of identical nucleotides
(especially four or more consecutive Gs), G+C content should be
30-80%, there should be more Cs than Gs, and not a G at the 5' end.
The higher number of Cs produces a higher ARn. The choice of probe
should be made first.
[0325] 8. To avoid false-positive results due to amplification of
contaminating genomic DNA in the cDNA preparation, it is preferable
to have primers spanning exon-exon junctions. This way, genomic DNA
will not be amplified (the PDAR kit for human GAPDH amplification
has such primers),
[0326] 9. If a TaqMan.TM. probe is designed for allelic
discrimination, the mismatching nucleotide (the polymorphic site)
should be in the middle of the probe rather than at the ends,
[0327] 10. Use primers that contain dA nucleotides near the 3' ends
so that any primer-dimer generated is efficiently degraded by
AmpErase.TM. UNG (mentioned in p. 9 of the manual for EZ RT-PCR
kit; P/N 402877). If primers cannot be selected with dA nucleotides
near the ends, the use of primers with 3' terminal dU-nucleotides
should be considered.
[0328] (See also the general principles of PCR Primer Design by
InVitroGen.)
General Method
[0329] 1. Reverse transcription of total RNA to cDNA should be done
with random hexamers (not with oligo-dT). If oligo-dT has to be
used long mRNA transcripts or amplicons greater than two kilobases
upstream should be avoided, and 18S RNA cannot be used as
normalizer,
[0330] 2. Multiplex PCR will only work properly if the control
primers are limiting (ABI control reagents do not have their
primers limited),
[0331] 3. The range of target cDNA used is 10 ng to 1 .mu.g. If DNA
is used (mainly for allelic discrimination studies), the optimum
amount is 100 ng to 1 .mu.g,
[0332] 4. It is ideal to treat each RNA preparation with RNAse free
DNAse to avoid genomic DNA contamination. Even the best RNA
extraction methods yield some genomic DNA. Of course, it is ideal
to have primers not amplifying genomic DNA at all but sometimes
this may not be possible,
[0333] 5. For optimal results, the reagents (before the preparation
of the PCR mix) and the PCR mixture itself (before loading) should
be vortexed and mixed well. Otherwise there may be shifting Rn
value during the early (0-5) cycles of PCR. It is also important to
add probe to the buffer component and allow it to equilibrate at
room temperature prior to reagent mix formulation.
TaqMan.TM. Primers and Probes
[0334] The TaqMan.TM. probes ordered from ABI at midi-scale arrive
already resuspended at 100 .mu.M. If a 1/20 dilution is made, this
gives a 5 .mu.M solution. This stock solution should be aliquoted,
frozen and kept in the dark. Using 1 .mu.L of this in a 50 .mu.L
reaction gives the recommended 100 nM final concentration.
[0335] The primers arrive lyophilized with the amount given on the
tube in pmols (such as 150.000 pmol which is equal to 150 nmol). If
X nmol of primer is resuspended in X .mu.L of H.sub.2O, the
resulting solution is 1 mM. It is best to freeze this stock
solution in aliquots. When the 1 mM stock solution is diluted
1/100, the resulting working solution will be 10 .mu.M. To get the
recommended 50-900 nM final primer concentration in 50 .mu.L
reaction volume, 0.25-4.50 .mu.L should be used per reaction (2.5
.mu.L for 500 nM final concentration).
[0336] The PDAR primers and probes are supplied as a mix in one
tube. They have to be used 2.5 .mu.L in a 50 .mu.L reaction
volume.
Setting Up One-step TaqMan.TM. Reaction
[0337] One-step real-time PCR uses RNA (as opposed to cDNA) as a
template. This is the preferred method if the RNA solution has a
low concentration but only if singleplex reactions are run. The
disadvantage is that RNA carryover prevention enzyme AmpErase
cannot be used in one-step reaction format. In this method, both
reverse transcriptase and real-time PCR take place in the same
tube. The downstream PCR primer also acts as the primer for reverse
transcriptase (random hexamers or oligo-dT cannot be used for
reverse transcription in one-step RT-PCR). One-step reaction
requires higher dNTP concentration (greater than or equal to 300 mM
vs 200 mM) as it combines two reactions needing dNTPs in one. A
typical reaction mix for one-step PCR by Gold RT-PCR kit is as
follows:
TABLE-US-00001 Reagents Volume H.sub.2O + RNA: 20.5 .mu.L [24 .mu.L
if PDAR is used] 10X TaqMan buffer: 5.0 .mu.L MgCl.sub.2 (25 mM):
11.0 .mu.L dATP (10 mM): 1.5 .mu.L [for final concentration of 300
.mu.M] dCTP (10 mM): 1.5 .mu.L [for final concentration of 300
.mu.M] dGTP (10 mM): 1.5 .mu.L [for final concentration of 300
.mu.M] dUTP (20 mM): 1.5 .mu.L [for final concentration of 600
.mu.M] Primer F (10 .mu.M) *: 2.5 .mu.L [for final concentration of
500 nM] Primer R (10 .mu.M) *: 2.5 .mu.L [for final concentration
of 500 nM] TaqMan Probe *: 1.0 .mu.L [for final concentration of
100 nM] AmpliTaq Gold: 0.25 .mu.L [can be increased for higher
efficiency] Reverse Transcriptase: 0.25 .mu.L RNAse inhibitor: 1.00
.mu.L * If a PDAR is used, 2.5 .mu.L of primer + probe mix
used.
[0338] Ideally 10 pg-100 ng RNA should be used in this reaction.
Note that decreasing the amount of template from 100 ng to 50 ng
will increase the C.sub.T value by 1. To decrease a C.sub.T value
by 3, the initial amount of template should be increased 8-fold.
ABI claims that 2 picograms of RNA can be detected by this system
and the maximum amount of RNA that can be used is 1 microgram. For
routine analysis, 10 pg-100 ng RNA and 100 pg-1 .mu.g genomic DNA
can be used.
Cycling Parameters for One-Step PCR
[0339] Reverse transcription (by MuLV) 48.degree. C. for 30
min.
[0340] AmpliTaq activation 95.degree. C. for 10 min.
[0341] PCR: denaturation 95.degree. C. for 15 sec and
annealing/extension 60.degree. C. for 1 min (repeated 40 times) (On
ABI 7700, minimum holding time is 15 seconds.)
[0342] The recently introduced EZ One-stept.TM. RT-PCR kit allows
the use of UNG as the incubation time for reverse transcription is
60.degree. C. thanks to the use of a thermostable reverse
transcriptase. This temperature also a better option to avoid
primer dimers and non-specific bindings at 48.degree. C.
Operating the ABI 7700
[0343] Make sure the following before starting a run:
[0344] 1. Cycle parameters are correct for the run.
[0345] 2. Choice of spectral compensation is correct (off for
singleplex, on for multiplex reactions).
[0346] 3. Choice of "Number of PCR Stages" is correct in the
Analysis Options box (Analysis/Options). This may have to be
manually assigned after a run if the data is absent in the
amplification plot but visible in the plate view, and the X-axis of
the amplification is displaying a range of 0-1 cycles.
[0347] 4. No Template Control is labeled as such (for accurate ARn
calculations).
[0348] 5. The choice of dye component should be made correctly
before data analysis.
[0349] 6. You must save the run before it starts by giving it a
name (not leaving as untitled). Also at the end of the run, first
save the data before starting to analyze.
[0350] 7. The ABI software requires extreme caution. Do not attempt
to stop a run after clicking on the Run button. You will have
problems and if you need to switch off and on the machine, you have
to wait for at least an hour to restart the run.
[0351] When analyzing the data, remember that the default setting
for baseline is 3-15. If any C.sub.T value is <15, the baseline
should be changed accordingly (the baseline stop value should be
1-2 smaller than the smallest C.sub.T value). For a useful
discussion of this matter, see the ABI Tutorial on Setting
Baselines and Thresholds. (Interestingly, this issue is best
discussed in the manual for TaqMan.TM. Human Endogenous Control
Plate.)
[0352] If the results do not make sense, check the raw spectra for
a possible CDC camera saturation during the run. Saturation of CDC
camera may be prevented by using optical caps rather than optical
adhesive cover. It is also more likely to happen when SYBR Green I
is used, when multiplexing and when a high concentration of probe
is used.
Interpretation of Results
[0353] At the end of each reaction, the recorded fluorescence
intensity is used for the following calculations:
[0354] Rn.sup.+ is the Rn value of a reaction containing all
components, Rn.sup.- is the Rn value of an unreacted sample
(baseline value or the value detected in NTC). ARn is the
difference between Rn.sup.+ and Rn.sup.-. It is an indicator of the
magnitude of the signal generated by the PCR.
[0355] There are three illustrative methods to quantitate the
amount of template:
[0356] 1. Absolute standard method: In this method, a known amount
of standard such as in vitro translated RNA (cRNA) is used.
[0357] 2. Relative standard: Known amounts of the target nucleic
acid are included in the assay design in each run,
[0358] 3. Comparative C.sub.T method: This method uses no known
amount of standard but compares the relative amount of the target
sequence to any of the reference values chosen and the result is
given as relative to the reference value (such as the expression
level of resting lymphocytes or a standard cell line).
The Comparative CT Method (.DELTA..DELTA.CT) for Relative
Quantitation of Gene Expression
[0359] This method enables relative quantitation of template and
increases sample throughput by eliminating the need for standard
curves when looking at expression levels relative to an active
reference control (normalizer). For this method to be successful,
the dynamic range of both the target and reference should be
similar. A sensitive method to control this is to look at how
.DELTA.C.sub.T (the difference between the two CT values of two
PCRs for the same initial template amount) varies with template
dilution. If the efficiencies of the two amplicons are
approximately equal, the plot of log input amount versus
.DELTA.C.sub.T will have a nearly horizontal line (a slope of
<0.10). This means that both PCRs perform equally efficiently
across the range of initial template amounts. If the plot shows
unequal efficiency, the standard curve method should be used for
quantitation of gene expression. The dynamic range should be
determined for both (1) minimum and maximum concentrations of the
targets for which the results are accurate and (2) minimum and
maximum ratios of two gene quantities for which the results are
accurate. In conventional competitive RT-PCR, the dynamic range is
limited to a target-to-competitor ratio of about 10:1 to 1:10 (the
best accuracy is obtained for 1:1 ratio). The real-time PCR is able
to achieve a much wider dynamic range.
[0360] Running the target and endogenous control amplifications in
separate tubes and using the standard curve method requires the
least amount of optimization and validation. The advantage of using
the comparative C.sub.T method is that the need for a standard
curve is eliminated (more wells are available for samples). It also
eliminates the adverse effect of any dilution errors made in
creating the standard curve samples.
[0361] As long as the target and normalizer have similar dynamic
ranges, the comparative C.sub.T method (.DELTA..DELTA.C.sub.T
method) is the most practical method. It is expected that the
normalizer will have a higher expression level than the target
(thus, a smaller C.sub.T value). The calculations for the
quantitation start with getting the difference (.DELTA.C.sub.T)
between the C.sub.T values of the target and the normalizer:
.DELTA.C.sub.T=C.sub.T (target)-C.sub.T (normalizer)
[0362] This value is calculated for each sample to be quantitated
(unless, the target is expressed at a higher level than the
normalizer, this should be a positive value. It is no harm if it is
negative). One of these samples should be chosen as the reference
(baseline) for each comparison to be made. The comparative
.DELTA..DELTA.C.sub.T calculation involves finding the difference
between each sample's .DELTA.C.sub.T and the baseline's
.DELTA.C.sub.T. If the baseline value is representing the minimum
level of expression, the .DELTA..DELTA.C.sub.T values are expected
to be negative (because the .DELTA.C.sub.T for the baseline sample
will be the largest as it will have the greatest C.sub.T value). If
the expression is increased in some samples and decreased in
others, the .DELTA..DELTA.C.sub.T values will be a mixture of
negative and positive ones. The last step in quantitation is to
transform these values to absolute values. The formula for this
is:
comparative expression level=2.sup.-.DELTA.CT
[0363] For expressions increased compared to the baseline level
this will be something like 2.sup.3=8 times increase, and for
decreased expression it will be something like 2.sup.-3=1/8 of the
reference level. Microsoft Excel can be used to do these
calculations by simply entering the C.sub.T values (there is an
online ABI tutorial at
http://www.appliedbiosystems.com/support/tutorials/7700 amp/ on the
use of spread sheet programs to produce amplification plots; the
TaqMan.TM. Human Endogenous Control Plate protocol also contains
detailed instructions on using MS Excel for real-time PCR data
analysis).
[0364] The other (absolute) quantification methods are outlined in
the ABI User Bulletins
(http://docs.appliedbiosystems.com/search.taf?_UserReference=A86583271898-
50A13A0C598 E). The Bulletins #2 and #5 are most useful for the
general understanding of real-time PCR and quantification.
[0365] Recommendations on Procedures:
[0366] 1. Use positive-displacement pipettes to avoid inaccuracies
in pipetting,
[0367] 2. The sensitivity of real-time PCR allows detection of the
target in 2 pg of total RNA. The number of copies of total RNA used
in the reaction should ideally be enough to give a signal by 25-30
cycles (preferably less than 100 ng). The amount used should be
decreased or increased to achieve this.
[0368] 3. The optimal concentrations of the reagents are as
follows: [0369] i. Magnesium chloride concentration should be
between 4 and 7 mM. It is optimized as 5.5 mM for the
primers/probes designed using the Primer Express software. [0370]
ii. Concentrations of dNTPs should be balanced with the exception
of dUTP (if used). Substitution of dUTP for dTTP for control of PCR
product carryover requires twice dUTP that of other dNTPs. While
the optimal range for dNTPs is 500 .mu.M to 1 mM (for one-step
RT-PCR), for a typical TaqMan reaction (PCR only), 200 .mu.M of
each dNTP (400 .mu.M of dUTP) is used. [0371] iii. Typically 0.25
.mu.L (1.25 U) AmpliTaq DNA Polymerase (5.0 U/.mu.L) is added into
each 50 .mu.L reaction. This is the minimum requirement. If
necessary, optimization can be done by increasing this amount by
0.25 U increments. [0372] iv. The optimal probe concentration is
50-200 nM, and the primer concentration is 100-900 nM. Ideally,
each primer pair should be optimized at three different
temperatures (58, 60 and 62.degree. C. for TaqMan primers) and at
each combination of three concentrations (50, 300, 900 nM). This
means setting up three different sets (for three temperatures) with
nine reactions in each (50/50 mM, 50/300 mM, 50/900, 300/50,
300/300, 300/900, 900/50, 900/300, 900/900 mM) using a fixed amount
of target template. If necessary, a second round of optimization
may improve the results. Optimal performance is achieved by
selecting the primer concentrations that provide the lowest C.sub.T
and highest .DELTA.Rn. Similarly, the probe concentration should be
optimized for 25-225 nM.
[0373] 4. If AmpliTaq Gold DNA Polymerase is being used, there has
to be a 9-12 min pre-PCR heat step at 92-95.degree. C. to activate
it. If AmpliTaq Gold DNA Polymerase is used, there is no need to
set up the reaction on ice. A typical TaqMan reaction consists of 2
min at 50.degree. C. for UNG (see below) incubation, 10 min at
95.degree. C. for Polymerase activation, and 40 cycles of 15 sec at
95.degree. C. (denaturation) and 1 min at 60.degree. C. (annealing
and extension). A typical reverse transcription cycle (for cDNA
synthesis), which should precede the TaqMan reaction if the
starting material is total RNA, consists of 10 min at 25.degree. C.
(primer incubation), 30 min at 48.degree. C. (reverse transcription
with conventional reverse transcriptase) and 5 min at 95.degree. C.
(reverse transcriptase inactivation).
[0374] 5. AmpErase uracil-N-glycosylase (UNG) is added in the
reaction to prevent the reamplification of carry-over PCR products
by removing any uracil incorporated into amplicons. This is why
dUTP is used rather than dTTP in PCR reaction. UNG does not
function above 55.degree. C. and does not cut single-stranded DNA
with terminal dU nucleotides. UNG-containing master mix should not
be used with one-step RT-PCR unless rTth DNA polymerase is being
used for reverse transcription and PCR (TaqMan EZ RT-PCR kit).
[0375] 6. It is necessary to include at least three No
Amplification Controls (NAC) as well as three No Template Controls
(NTC) in each reaction plate (to achieve a 99.7% confidence level
in the definition of +/-thresholds for the target amplification,
six replicates of NTCs must be run). NAC former contains sample and
no enzyme. It is necessary to rule out the presence of fluorescence
contaminants in the sample or in the heat block of the thermal
cycler (these would cause false positives). If the absolute
fluorescence of the NAC is greater than that of the NTC after PCR,
fluorescent contaminants may be present in the sample or in the
heating block of the thermal cycler.
[0376] The dynamic range of a primer/probe system and its
normalizer should be examined if the .DELTA..DELTA.C.sub.T method
is going to be used for relative quantitation. This is done by
running (in triplicate) reactions of five RNA concentrations (for
example, 0, 80 pg/.mu.L, 400 pg/.mu.L, 2 ng/.mu.L and 50 ng/.mu.L).
The resulting plot of log of the initial amount vs C.sub.T values
(standard curve) should be a (near) straight line for both the
target and normalizer real-time RT-PCRs for the same range of total
RNA concentrations.
[0377] 8. The passive reference is a dye (ROX) included in the
reaction (present in the TaqMan universal PCR master mix). It does
not participate in the 5' nuclease reaction. It provides an
internal reference for background fluorescence emission. This is
used to normalize the reporter-dye signal. This normalization is
for non-PCR-related fluorescence fluctuations occurring
well-to-well (concentration or volume differences) or over time and
different from the normalization for the amount of cDNA or
efficiency of the PCR. Normalization is achieved by dividing the
emission intensity of reporter dye by the emission intensity of the
passive reference. This gives the ratio defined as Rn.
[0378] 9. If multiplexing is done, the more abundant of the targets
will use up all the ingredients of the reaction before the other
target gets a chance to amplify. To avoid this, the primer
concentrations for the more abundant target should be limited.
[0379] 10. TaqMan Universal PCR master mix should be stored at 2 to
8.degree. C. (not at -20.degree. C.).
[0380] 11. The GAPDH probe supplied with the TaqMan Gold RT-PCR kit
is labeled with a JOE reporter dye, the same probe provided within
the Pre-Developed TaqMan.TM. Assay Reagents (PDAR) kit is labeled
with VIC. Primers for these human GAPDH assays are designed not to
amplify genomic DNA.
[0381] 12. The carryover prevention enzyme, AmpErase UNG, cannot be
used with one-step RT-PCR which requires incubation at 48.degree.
C. but may be used with the EZ RT-PCR kit.
[0382] 13. One-step RT-PCR can only be used for singleplex
reactions, and the only choice for reverse transcription is the
downstream primer (not random hexamers or oligo-dT).
[0383] It is ideal to run duplicates to control pipetting errors
but this inevitably increases the cost.
[0384] 15. If multiplexing, the spectral compensation option (in
Advanced Options) should be checked before the run.
[0385] 16. Normalization for the fluorescent fluctuation by using a
passive reference (ROX) in the reaction and for the amount of
cDNA/PCR efficiency by using an endogenous control (such as GAPDH,
active reference) are different processes.
[0386] 17. ABI 7700 can be used not only for quantitative RT-PCR
but also end-point PCR. The latter includes presence/absence assays
or allelic discrimination assays (such as SNP typing).
[0387] 18. Shifting Rn values during the early cycles (cycle 0-5)
of PCR means initial disequilibrium of the reaction components and
does not affect the final results as long as the lower value of
baseline range is reset.
[0388] 19. If an abnormal amplification plot has been noted
(C.sub.T value<15 cycles with amplification signal detected in
early cycles), the upper value of the baseline range should be
lowered and the samples should be diluted to increase the C.sub.T
value (a high C.sub.T value may also be due to contamination).
[0389] 20. A small .DELTA.Rn value (or greater than expected
C.sub.T value) indicates either poor PCR efficiency or low copy
number of the target.
[0390] 21. A standard deviation>0.16 for C.sub.T value indicates
inaccurate pipetting.
[0391] 22. SYBR Green entry in the Pure Dye Setup should be
abbreviated as "SYBR" in capitals. Any other abbreviation or lower
case letters will cause problems.
[0392] 23. The SDS software for ABI 7700 have conflicts with the
Macintosh Operating System version 8.1. The data should not be
analyzed on such computers.
[0393] 24. The ABI 7700 should not be deactivated for extended
periods of time. If it has ever been shutdown, it should be allowed
to warm up for at least one hour before a run. Leaving the
instrument on all times is recommended and is beneficial for the
laser. If the machine has been switched on just before a run, an
error box stating a firmware version conflict may appear. If this
happens, choose the "Auto Download" option.
[0394] 25. The ABI 7700 is only one of the real-time PCR systems
available, others include systems from BioRad, Cepheid, Corbett
Research, Roche and Stratagene.
Example 2
Identification of Diagnostic Marker Genes and Priority Ranking of
Genes
[0395] For experimental groups, differences in gene expression
between animals before and after experimental induction of
endotoxaemia were analysed using the empirical Bayes approach of
Lonnstedt and Speed (Lonnstedt and Speed, 2002, Statistica Sinica
12: 31-46).
[0396] The objectives were to: (a) identify changes in gene
expression during the acute endotoxaemic phase of disease, and (b)
evaluate the diagnostic potential of these changes, for detecting
enodtoxaemia.
[0397] Comparison between dosed and control horses involved some
information which is within horses (i.e. some information is
available from the longitudinal comparison of horses which were
used both as controls and as treated animals), and some information
which is between horses (involving cross-sectional comparisons
between horses which were dosed and horses which were not). In
addition, some planed samples were not available. The result is an
unbalanced, non-orthogonal mixed effects study.
[0398] Gene expression data were generated, and quality metrics
were generated for each chip. Only chips providing high quality
data and passing all quality metrics were used in subsequent
analyses. The chips were then processed using the RMA (Robust
Multichip Analysis) algorithm as implemented in the R Bioconductor
project. Following calculation of expression measures, the
distribution of the chips was compared using Box and Whisker plots,
kernel density estimates and MA plots. Outliers were removed from
further analyses.
[0399] Results obtained were corroborated using Microarray Analysis
Software 5.0 (provided by Affymetrix) and a list of "housekeeping"
genes to scale the data. Housekeeping genes were determined a
priori by identifying those genes that vary the least in gene
expression across healthy horses of various breed, age, sex, and
geographical location, and across horses with various diseases.
[0400] Positive horses at each time point were compared with all
horses at time zero, and negative horses at the time point
concerned. For example, horses which were positive at 24 hours were
compared with all horses at day 0, and negative horses at 24 hours.
Two approaches were used for each comparison: univariate
comparisons made gene at a time, and multivariate comparisons using
the entire gene set. For the univariate comparisons, the analysis
of each gene was made on a linear mixed model, in which horse was a
random effect and time (time 0 vs current time) and status (control
or induced) were fixed effects. Individual p values were adjusted
using the Holm step down procedure (Holm, S. 1979, Scandinavian
Journal of Statistics 6: 65-70) to provide strong control of the
Family-Wise Error Rate (FWER). For multivariate analyses, a
composite strategy was employed involving, reduced space linear
discriminate analysis, support vector machines and classification
tree techniques. Genes that showed statistically significant
differences before and after experimental induction of endotoxaemia
were tabulated for each day post dosing.
[0401] A list of genes ranked by p value for comparisons made
between hours 0 and 24, 48 and 72 post-dosing is shown in Table 5.
This analysis is based on two-group comparisons (Hour 0 versus
hours 24, 48, and 72) with p Values adjusted using Holm's and the
FDR method. Results are based on the full outcome from the
empirical Bayes method.
[0402] Using linear mixed models, and at 24 hours, 159 genes were
statistically significant when Holm's correction was applied and
995 genes following FDR adjustment. Using classification and
regression trees, 829 of the 3105 genes on the GeneChip.TM.
separated the groups perfectly with a p value of 0.002.
[0403] Using linear mixed models, and at 72 hours, no genes were
statistically significant when Holm's correction was applied and 62
genes following FDR adjustment. Using classification and regression
trees, 125 of the 3105 genes on the GeneChip.TM. separated the
groups perfectly with a p value of 0.001.
[0404] Using linear mixed models, and at 120 hours, no genes were
statistically significant when either Holm's correction or FDR
adjustment were applied. Using classification and regression trees,
7 of the 3105 genes on the GeneChip.TM. separated the groups
perfectly with a p value of 0.019.
[0405] The genes listed in Table 5 are ranked in order of their t
statistic or value--which may be interpreted as a signal-to-noise
ratio. The tabulation also displays the log 2 fold change (M
value), and the adjusted p values. Genes with a negative t value
(and hence a negative M value) are down regulated. Genes with
positive t and M values are up-regulated. The priority ranking of
significant genes (p<0.05) is based on increasing t value for
the first time point (24 hours) followed by ranking on increasing t
value at 72 hours. Note, some genes are significant for both 24 and
72 hours, others are significant for either 24 or 72 hours.
Example 3
Demonstration of Diagnostic Potential to Determine Endotoxaemia
[0406] In addition, the diagnostic potential of the entire set of
genes was assessed using discriminant analysis (Venables and
Ripley, 2002, Modern Applied Statistics in S. Springer) on the
principal component scores (Jolliffe, I. T. Principal components
analysis, Springer-Verlag, 1986) calculated from gene expression.
The entire process was cross-validated. Sensitivity and specificity
were calculated for a uniform prior. This may be interpreted as a
form of shrinkage regularization, where the estimates are shrunken
to lie in a reduced space.
[0407] Cross-validated discriminant function scores were used to
estimate a receiver operator curve. The receiver operator curve was
calculated by moving a critical threshold along the axis of the
discriminant function scores. Both raw empirical ROCs were
calculated, and smoothed ROCs using Lloyd's method (Lloyd, C. J.
1998, Journal of the American Statistical Association 93:
1356-1364). Curves were calculated for the comparison of clinically
normal and clinically affected animals. Separate curves were
calculated, using gene expression at each day post-inoculation. The
area under the receiver operator curve was calculated by the
trapezoidal rule, applied to both the empirical ROC and the
smoothed ROC.
[0408] The ROC curve provides a useful summary of the diagnostic
potential of an assay. A perfect diagnostic assay has an ROC curve
which is a horizontal line passing through the point with
sensitivity and specificity both equal to one. The area under the
ROC curve for such a perfect diagnostic is 1. A useless diagnostic
assay has a ROC curve which is given by a 45 degree line through
the origin. The area for such an uninformative diagnostic is
0.5.
[0409] The ROC curves for the analysis based on comparisons between
time point 0 and time points 24 hours and 72 hours are presented
FIGS. 1-2, respectively. The diagnostic capability is very
high.
Example 4
Predictive Gene Sets
[0410] Although about 180 genes have been identified as having
diagnostic potential, a much fewer number are generally required
for acceptable diagnostic performance.
[0411] Table 6 shows the cross-validated classification success,
sensitivity and specificity obtained from a linear discriminant
analysis, based on two genes selected from the set of potential
diagnostic genes. The pairs presented are those producing the
highest prediction success, many other pairs of genes produce
acceptable classification success. The identification of alternate
pairs of genes would be readily apparent to those skilled in the
art. Techniques for identifying pairs include (but are not limited
to) forward variable selection (Venables W. N. and Ripley B. D.
Modern Applied Statistics in S 4.sup.th Edition 2002. Springer),
best subsets selection, backwards elimination (Venables W. N. and
Ripley B. D., 2002, supra), stepwise selection (Venables W. N. and
Ripley B. D., 2002, supra) and stochastic variable elimination
(Figueirodo M. A. Adeaptive Sparseness for Supervised
Learning).
[0412] Table 7 shows the cross-validated classification success
obtained from a linear discriminant analysis based on three genes
selected from the diagnostic set. Only twenty sets of three genes
are presented. It will be readily apparent to those of skill in the
art that other suitable diagnostic selections based on three
endotoxemia marker genes can be made.
[0413] Table 8 shows the cross-validated classification success
obtained from a linear discriminant analysis based on four genes
selected from the diagnostic set. Only twenty sets of four genes
are presented. It will be readily apparent to practitioners in the
art that other suitable diagnostic selections based on four
endotoxemia marker genes can be made.
[0414] Table 9 shows the cross-validated classification success
obtained from a linear discriminant analysis based on five genes
selected from the diagnostic set. Only twenty sets of five genes
are presented. It will be readily apparent to practitioners in the
art that other suitable diagnostic selections based on five
endotoxemia marker genes can be made.
[0415] Table 10 shows the cross-validated classification success
obtained from a linear discriminant analysis based on six genes
selected from the diagnostic set. Only twenty sets of six genes are
presented. It will be readily apparent to practitioners in the art
that other suitable diagnostic selections based on six endotoxemia
marker genes can be made.
[0416] Table 11 shows the cross-validated classification success
obtained from a linear discriminant analysis based on seven genes
selected from the diagnostic set. Only twenty sets of seven genes
are presented. It will be readily apparent to practitioners in the
art that other suitable diagnostic selections based on seven
endotoxemia marker genes can be made.
[0417] Table 12 shows the cross-validated classification success
obtained from a linear discriminant analysis based on eight genes
selected from the diagnostic set. Only twenty sets of eight genes
are presented. It will be readily apparent to practitioners in the
art that other suitable diagnostic selections based on eight
endotoxemia marker genes can be made.
[0418] Table 13 shows the cross-validated classification success
obtained from a linear discriminant analysis based on nine genes
selected from the diagnostic set. Only twenty sets of nine genes
are presented. It will be readily apparent to practitioners in the
art that other suitable diagnostic selections based on nine
endotoxemia marker genes can be made.
[0419] Table 14 shows the cross-validated classification success
obtained from a linear discriminant analysis based on ten genes
selected from the diagnostic set. Only twenty sets of ten genes are
presented. It will be readily apparent to practitioners in the art
that other suitable diagnostic selections based on ten endotoxemia
marker genes can be made.
[0420] Table 15 shows the cross-validated classification success
obtained from a linear discriminant analysis based on 12 genes
selected from the diagnostic set. Only 20 sets of twenty genes are
presented. It will be readily apparent to practitioners in the art
that other suitable diagnostic selections based on twenty
endotoxemia marker genes can be made.
[0421] Table 16 shows the cross-validated classification success
obtained from a linear discriminant analysis based on 13 genes
selected from the diagnostic set. Only 20 sets of twenty genes are
presented. It will be readily apparent to practitioners in the art
that other suitable diagnostic selections based on twenty
endotoxemia marker genes can be made.
[0422] Further numbers of genes introduced noise (and subsequently
lower specificity and sensitivity) through observational overload
compared to the number of variables.
[0423] The genes listed in Table 5 are ranked in order of their t
statistic--which may be interpreted as a signal-to-noise ratio. The
tabulation also displays the log 2 fold change (M value), and the
adjusted p values. Genes with a negative t value (and hence a
negative M value) are down regulated.
Example 5
Demonstration of Specificity
[0424] The specificity of the endotoxemia signature was examined by
training a classifier on the trial data only and running the
classifier over a large gene expression dataset of over 850
GeneChips.RTM.. Gene expression results in the database were
obtained from samples from horses with various diseases and
conditions including; clinical, induced acute and chronic EPM,
herpes virus infection, degenerative osteoarthritis, stress,
Rhodococcus infection, endotoxemia, laminitis, gastric ulcer
syndrome, animals in athletic training and clinically normal
animals.
[0425] Three classifiers were generated. All were based on the
comparison of positives at 24 hours with all horses at time zero,
and negative horses at 24 hours. The first used all the genes on
the GeneChip.TM.. The second used only those genes that were
statistically significant (Holm's adjusted p value<0.05). The
third was based on all of the genes except for 45 that had been
identified as being involved in at least one other gene signature
for disease. The latter was the most specific. It was able to
identify all eight endotoxemic horses in the database. It also
identified five other horses, one with severe gastritis, one with
botulism, another with Wobbler syndrome and two others with an
unknown diagnosis.
[0426] Using this method and a gene signature of 159 genes, a
specificity of 99% for endotoxemia was obtained from a population
sample size of over 850.
Example 7
Gene Ontology
[0427] Gene sequences were compared against the GenBank database
using the BLAST algorithm (Altschul, S. F., Gish, W., Miller, W.,
Myers, E. W. & Lipman, D. J. (1990) "Basic local alignment
search tool." J. Mol. Biol. 215:403-410), and gene homology and
gene ontology searches were performed in order to group genes based
on function, metabolic processes or cellular component. Table 17
lists and groups the genes based on these criteria. See also Table
1, which contains sequence information for each gene.
[0428] The disclosure of every patent, patent application, and
publication cited herein is hereby incorporated herein by reference
in its entirety.
[0429] The citation of any reference herein should not be construed
as an admission that such reference is available as "Prior Art" to
the instant application.
[0430] Throughout the specification the aim has been to describe
the preferred embodiments of the invention without limiting the
invention to any one embodiment or specific collection of features.
Those of skill in the art will therefore appreciate that, in light
of the instant disclosure, various modifications and changes can be
made in the particular embodiments exemplified without departing
from the scope of the present invention. All such modifications and
changes are intended to be included within the scope of the
appended claims.
TABLE-US-00002 Lengthy table referenced here
US20090264305A1-20091022-T00001 Please refer to the end of the
specification for access instructions.
TABLE-US-LTS-00001 LENGTHY TABLES The patent application contains a
lengthy table section. A copy of the table is available in
electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20090264305A1).
An electronic copy of the table will also be available from the
USPTO upon request and payment of the fee set forth in 37 CFR
1.19(b)(3).
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20090264305A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20090264305A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References