U.S. patent application number 11/053027 was filed with the patent office on 2005-06-30 for diagnosis and management of infection caused by chlamydia.
This patent application is currently assigned to Vanderbuilt University. Invention is credited to Mitchell, William M., Stratton, Charles W..
Application Number | 20050143386 11/053027 |
Document ID | / |
Family ID | 34437824 |
Filed Date | 2005-06-30 |
United States Patent
Application |
20050143386 |
Kind Code |
A1 |
Mitchell, William M. ; et
al. |
June 30, 2005 |
Diagnosis and management of infection caused by Chlamydia
Abstract
The present invention provides a method of treating coronary
artery disease in a patient in need thereof by administering to the
patient an antichlamydial amount of a rifamycin for a duration to
treat said coronary artery disease.
Inventors: |
Mitchell, William M.;
(Nashville, TN) ; Stratton, Charles W.;
(Nashville, TN) |
Correspondence
Address: |
CLARK & ELBING LLP
101 FEDERAL STREET
BOSTON
MA
02110
US
|
Assignee: |
Vanderbuilt University
Nashville
TN
|
Family ID: |
34437824 |
Appl. No.: |
11/053027 |
Filed: |
February 8, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11053027 |
Feb 8, 2005 |
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10100759 |
Mar 19, 2002 |
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6884784 |
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10100759 |
Mar 19, 2002 |
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09073661 |
May 6, 1998 |
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6579854 |
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09073661 |
May 6, 1998 |
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09025521 |
Feb 18, 1998 |
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09025521 |
Feb 18, 1998 |
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08911593 |
Aug 14, 1997 |
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09073661 |
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09025176 |
Feb 18, 1998 |
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6258532 |
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09073661 |
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09025174 |
Feb 18, 1998 |
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6562582 |
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60045739 |
May 6, 1997 |
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60045779 |
May 6, 1997 |
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60045780 |
May 6, 1997 |
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60045784 |
May 6, 1997 |
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60045787 |
May 6, 1997 |
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60045689 |
May 6, 1997 |
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Current U.S.
Class: |
514/252.13 ;
514/171; 514/253.08; 514/28; 514/312; 514/570 |
Current CPC
Class: |
A61K 31/573 20130101;
A61K 31/455 20130101; A61K 31/192 20130101; A61K 31/496 20130101;
A61K 31/7052 20130101; A61K 31/7052 20130101; A61K 31/192 20130101;
Y10S 514/824 20130101; A61K 2300/00 20130101; A61K 2300/00
20130101; A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 31/455
20130101; A61K 45/06 20130101; A61K 31/496 20130101; A61K 2300/00
20130101; A61K 31/573 20130101 |
Class at
Publication: |
514/252.13 ;
514/253.08; 514/312; 514/171; 514/028; 514/570 |
International
Class: |
A61K 031/7052; A61K
031/573; A61K 031/496; A61K 031/4709; A61K 031/192 |
Claims
We claim:
1. A method of treating coronary artery disease in a patient in
need thereof, said method comprising the step of administering to
the patient an antichlamydial amount of a rifamycin for a duration
to treat said coronary artery disease.
2. The method claim 1, wherein said rifamycin is rifampin.
3. The method of claim 1, further comprising the step of
administering to the patient an immunosuppressant or an
anti-inflammatory agent.
4. The method of claim 3, wherein said anti-inflammatory agent is
prednisone, cortisone, hydrocortisone, or naproxen.
5. The method claim 1, further comprising the step of administering
to the patient an azalide or a macrolide.
6. The method of claim 5, wherein said azalide is azithromycin.
7. The method of claim 1, further comprising the step of
administering to the patient a quinolone or a fluoroquinolone.
8. The method of claim 7, wherein said fluoroquinolone is ofloxacin
or levofloxacin.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of Ser. No.
10/100,759, filed Mar. 19, 2002, which is a continuation
application of and claims priority to U.S. Ser. No. 09/073,661,
filed May 6, 1998, which is a continuation-in-part of U.S. Ser. No.
09/025,521, filed Feb. 18, 1998, which is a continuation-in-part of
U.S. Ser. No. 08/911,593, filed Aug. 14, 1997. U.S. Ser. No.
09/073,661 is also a continuation-in-part of U.S. Ser. No.
09/025,176 and U.S. Ser. No. 09/025,174, each filed Feb. 18, 1998,
and claims benefit of U.S. Provisional Application Nos. 60/045,739,
60/045,779, 60/045,780, 60/045,784, 60/045,787, and 60/045,689,
each filed May 6, 1997. Each of the foregoing applications is
incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] Chlamydiae are obligate intracellular microorganisms which
parasitize eukaryotic cells and are ubiquitous throughout the
animal kingdom. Members of the chlamydial genus are considered
bacteria with a unique biphasic developmental cycle having distinct
morphological and functional forms. This developmental growth cycle
alternates between 1) intracellular life forms, of which two are
currently recognized, a metabolically-active, replicating organism
known as the reticulate body (RB) and a persistent, non-replicating
organism known as the cryptic phase; and 2) an extracellular life
form that is an infectious, metabolically-inactive form known as
the elementary body (EB).
[0003] EBs are small (300-400 nm) infectious, spore-like forms
which are metabolically inactive, non-replicating, and found most
often in the acellular milieu. EBs are resistant to a variety of
physical insults such as enzyme degradation, sonication and osmotic
pressure. This physical stability is thought to be a result of
extensive disulfide cross-linking of the cysteine-rich major outer
membrane protein (MOMP) (Bavoil et al., Infection and Immunity,
44:479-485 (1984); Hackstadt et al., Journal of Bacteriology,
161:25-31 (1985); Hatch et al., Journal of Bacteriology,
165:379-385 (1986); Peeling et al., Infection and Immunity,
57:3338-3344 (1989); J. C. A. Bardwell, Molecular Microbiology,
14:199-205 (1994); and T. P. Hatch, Journal of Bacteriology,
178:1-5 (1993)). Under oxidizing conditions in the acellular milieu
of the host, the outer membrane of EBs is relatively impermeable as
well as resistant to inactivation. EBs are thus well suited to
survive long enough outside of their hosts to be transmitted to a
new host in the form of a droplet nuclei (Theunissen et al.,
Applied Environmental Microbiology, 59:2589-2593 (1993)) or a
fomite (Fasley et al., The Journal of Infectious Diseases,
168:493-496 (1993)).
[0004] Infection by members of the genus Chlamydiae induces a
significant inflammatory response at the cellular level. For
example, genital lesions produced by Chlamydia trachomatis
frequently elicit a vigorous influx of lymphocytes, macrophages,
and plasma cells, suggesting the development of humoral and
cellular immunity. Yet, clinically, the initial infection is
frequently varied in symptomatology and may even be asymptomatic.
Once fully established, the Chlamydia are difficult to eradicate,
with frequent relapse following antibiotic therapy. Evidence also
indicates that the Chlamydia may become dormant and are then shed
in quantities too few to reliably detect by culture.
[0005] Chlamydia pneumoniae (hereinafter "C. pneumoniae") is the
most recent addition to the genus Chlamydiae and is isolated from
humans and currently is recognized as causing approximately 10
percent of community acquired cases of pneumonia (Grayston et al.,
J. Inf. Dis. 161:618-625 (1990)). This newly recognized pathogen
commonly infects the upper and lower respiratory tract and is now
recognized as ubiquitous in humans. C. pneumoniae is well-accepted
as a human pathogen that may be difficult to eradicate by standard
antibiotic therapy (Hammerschlag et al., Clin. Infect. Dis.
14:178-182 (1992)). C. pneumoniae is known to persist as a silent
or mildly symptomatic pathogen, resulting in a chronic, persistent
infection (J. Schacter, In: Baun AL, eg. Microbiology of Chlamydia,
Boca Raton, Fla. CRC Press, 1988, pp. 153-165).
[0006] The current therapy for suspected/confirmed C. pneumoniae
infection is with a short course (e.g., 2-3 weeks) of a single
antibiotic. C. pneumoniae is susceptible in vitro to tetracyline,
erythromycin, clarithromycin, and fluoroquinolones such as
ofloxacin and sparfloxacin (Kuo et al., Antimicrob Agents Chemother
32:257-258 (1988); Welsh et al., Antimicrob Agents Chemother
36:291-294 (1992); Chirgwin et al., Antimicrob Agents Chemother
33:1634-1635 (1989); Hammerschlag et al., Antimicrob Agents
Chemother 36:682-683 (1992); Hammerschlag et al., Antimicrob Agents
Chemother 36:1573-1574); M. R. Hammerschlag, Antimicrob Agents
Chemother 38:1873-1878 (1994); M. R. Hammerschlag, Infect. Med. pp.
64-71 (1994)). Despite this demonstration of in vitro
susceptibility, C. pneumoniae infections may relapse following
antibiotic therapy with these agents. In vitro studies on the
persistence of Chlamydiae despite specific and appropriate
antibiotic therapy have suggested that the presence of antibiotics
promotes the formation of an intracellular, non-replicative state
(Beatty et al., Microbiol. Rev. 58:686-699 (1994)), typically
referred to as the latent or cryptic phase. This change can be
thought of as a stringent response and is seen also with nutrient
starvation and exposure to .gamma.-interferon. Removal of the
stressful influence allows the organism to resume replication.
Thus, in this way, the organism can escape current antibiotic
therapy used in clinical practice.
[0007] In view of the chronic and persistent nature of chlamydial
infections, there is a need for reliable, accurate methods for
diagnosis of pathogenic infection as well as therapeutic approaches
to manage the infection. Due to the highly infective nature of
Chlamydia EBs and their ability to reinfect cells, there is also a
need for antichlamydial therapy which totally eradicates this
pathogen, thereby preventing the long term sequelae of such chronic
infections.
SUMMARY OF THE INVENTION
[0008] The present invention provides a unique approach for the
diagnosis and management of infection by Chlamydia species,
particularly C. pneumoniae. The invention is based upon the
discovery that a combination of agents directed toward many of the
various stages of the chlamydial life cycle can successfully manage
infection and ultimately prevent reinfection/reactivation of the
pathogen. Accordingly, one embodiment of the invention pertains to
methods of treating infection by a Chlamydia species, comprising
administering to an individual in need thereof a combination of
antichlamydial agents, comprising at least two agents, each of
which is targeted against a different phase of the chlamydial life
cycle. For example, the method can be carried out using agents
chosen from among the following groups: a) at least one agent
targeted against the elementary body phase of the chlamydial life
cycle; b) at least one agent targeted against the replicating phase
of the chlamydial life cycle; and c) at least one agent targeted
against a cryptic phase of the chlamydial life cycle. The
chlamydial pathogen can be eliminated more rapidly when a
combination comprising agents targeted against each phase of the
chlamydial life cycle is administered.
[0009] The invention also pertains to novel combinations of
antichlamydial agents and to novel pharmaceutical compositions
including at least two antichlamydial agents, each of which is
targeted against a different phase of the chlamydial life cycle.
For example, the agents can be selected from the group consisting
of: a) at least one agent targeted against the elementary body
phase of the chlamydial life cycle; b) at least one agent targeted
against the replicating phase of the chlamydial life cycle; and c)
at least one agent targeted against a cryptic phase of the
chlamydial life cycle. These compositions and combinations of
agents can further comprise one or a combination of adjunct
compounds, including anti-inflammatory agents, immunosuppressive
agents and anti-porphyrial agents. Use of the combination of
antichlamydial agents or compositions thereof for the manufacture
of a medicament for the management of Chlamydia infection is also
described. In a particular embodiment, the agents can be assembled
individually, admixed or instructionally assembled.
[0010] The invention also pertains to a novel therapy comprising a
specific agent targeted against the elementary body phase of the
chlamydial life cycle which, if used for a sufficient period of
time, allows active infection to be completed without the creation
of infectious EBs.
[0011] In order to facilitate patient compliance during a course of
therapy, the invention provides a means for packaging therapeutic
agents, described herein, for the management of Chlamydia
infection. For example, a pack can comprise at least two different
agents, each of which is targeted against a different phase of the
chlamydial life cycle. These agents can be selected from the group
consisting of: a) at least one agent targeted against the
elementary body phase of the chlamydial life cycle; b) at least one
agent targeted against the replicating phase of the chlamydial life
cycle; and c) at least one agent targeted against a cryptic phase
of the chlamydial life cycle. Optional adjunct compounds, as
mentioned previously, can likewise be present in the pack. A
preferred pack will comprise a plurality of agents that are
targeted at two, but preferably to all, of the stages of the
chlamydial life cycle. The pack can provide a unit dosage of the
agents or can comprise a plurality of unit dosages, and may be
labeled with information, such as the mode and order of
administration (e.g., separate, simultaneous or sequential) of each
component contained therein.
[0012] The invention also encompasses a method for evaluating the
infection status of an individual and/or the progress of therapy in
an individual undergoing therapy for infection caused by Chlamydia.
The method comprises quantifying antibody titer or other measure to
the pathogen and comparing the measure to antibody measure
quantified at a time earlier in the therapy, whereby the difference
between the measures is indicative of the progress of the therapy.
The invention also pertains to a method for monitoring the course
of therapy for treating infection by Chlamydia, comprising
determining presence or absence of Chlamydia in an infected
individual at time intervals during course of therapy. In a
particular embodiment, this is determined by PCR assay for pathogen
DNA or antigen capture assay for pathogen.
[0013] Detection of the presence of Chlamydia in a sample of
biological material taken from an individual thought to be infected
therewith is important in determining the course of therapy and the
agents to be used. This can be achieved by detecting the presence
of DNA encoding MOMP of Chlamydia or other chlamydial genes in the
individual. In one aspect of the invention, diseases associated
with Chlamydia infection, such as inflammatory diseases, autoimmune
diseases and diseases in which the individual is immunocompromised,
can be treated by managing (i.e., significantly reducing infection
or eradicating) the Chlamydia infection using the novel approach
described herein. Both clinical and serological improvements/
resolutions in patient status have been demonstrated.
[0014] The invention also pertains to a susceptibility test for
identifying agent(s) capable of significantly reducing/eliminating
chlamydial infection. The method comprises preparing tissue culture
from cell lines; inoculating these cells with Chlamydia in the
absence of cycloheximide; allowing the Chlamydia to infect these
cells for several days; adding agent(s) to be tested, which
agent(s) is/are replaced as needed for the duration of incubation;
isolating chlamydial nucleic acid from the cells; and assessing the
presence or absence of chlamydial DNA using a suitable nucleotide
amplification assay, such as PCR. Preferably the presence or
absence of signal for amplified DNA encoding MOMP of Chlamydia or
other chlamydial protein is determined. Absence of a signal
indicates a reduction in the degree of infection below that which
is detectable by nucleic acid amplification techniques and strongly
suggests eradication of the microorganism. The susceptibility tests
described herein are particularly useful as a drug screening tool
for assessing the activity of single agents or combinations of
agents against Chlamydia infection.
[0015] The unique and novel aspect of the susceptabilty test
described herewithin is that it measures the presence or absence of
chlamydial DNA and thus can detect cryptic forms and/or elementary
bodies both of which are viable, yet are not replicating.
[0016] In one embodiment, a suitable nucleotide assay for
identifying agents effective against a cryptic form of chlamydia
comprises, in the presence of agent(s) to be tested, is performed
by subjecting cultured cells to protease/reducing agent (e.g.,
dithiotreitol (DTT)) and protease digestion or guanidine
isothiocyanate (also known as guanidine thiocyanate) for a
prescribed period of time; extracting DNA from the treated
solution; exposing DNA to appropriate polymerase, dNTPs and primers
for DNA amplification of MOMP or other protein of the Chlamydia
species; and determining the presence or absence of amplified DNA
by visualizing the ethidium bromide treated DNA product by gel
electrophoresis, for example. In particular embodiments, the
Chlamydia species is C. pneumoniae and the appropriate primers are
CHLMOMPDB2 and CHLMOMPCB2.
[0017] The invention further relates to a method of identifying
cells containing a cryptic form of a Chlamydia species by a nucleic
acid amplification technique (e.g., PCR) comprising subjecting
cultured cells to protease digestion; stopping protease activity;
exposing cells to appropriate heat-stable DNA polymerase, dNTPs and
labeled primers (e.g., 3'-biotin labeled, 5'-biotin labeled) for
amplification of DNA encoding MOMP of the Chlamydia species;
washing the cells; exposing the cells to a reporter molecule (e.g.,
strepavidin-conjugated signal enzyme); exposing the cells to an
appropriate substrate for the reporter molecule (e.g., conjugated
enzyme); and visualizing the amplified DNA encoding MOMP by
visualizing the product of the reaction.
[0018] A method of identifying cells containing a cryptic form of
Chlamydia comprises treating cultured cells, thought to be infected
with Chlamydia, with a disulfide reducing agent; subjecting
cultured cells to protease digestion; exposing cells to appropriate
polymerase, dNTPs and primers for DNA amplification of nucleic acid
encoding a chlamydial protein; exposing the cells to a reporter
molecule enzyme; exposing the cells to an appropriate substrate for
the reporter enzyme; and determining the presence of a cryptic form
of Chlamydia by visualizing the amplified DNA encoding a chlamydial
protein. Preferably the amplification technique is PCR and the
primers are CHLMOMPDB2 and CHLMOMPCB2 of Chlamydia pneumoniae.
[0019] A similar method can be used as an assay for identifying an
agent which is effective against a cryptic form of Chlamydia.
Accordingly, the method comprises treating cultured cells grown in
the absence of cycloheximide, thought to be infected with
Chlamydia, with a disulfide reducing agent; allowing the chlamydia
to replicate; adding a test agent; subjecting cultured cells to
protease digestion; exposing cells to appropriate polymerase, dNTPs
and primers for DNA amplification of a chlamydial protein; exposing
the cells to a reporter molecule enzyme; exposing the cells to an
appropriate substrate for the reporter enzyme; and determining the
presence of a cryptic form of Chlamydia by visualizing the
amplified DNA encoding a chlamydial protein, such as MOMP.
[0020] Also described is a method of detecting chlamydial
elementary bodies in a sample comprising contacting the sample with
a disulfide reducing agent before using a DNA amplification
technique to detect chlamydial DNA in the sample.
[0021] The present invention pertains to methods for clearing
biological material infected with Chlamydia to produce
Chlamydia-free cell lines and animals, and to methods of
maintaining biological material, e.g, cell lines and animals, such
that they remain Chlamydia-free. According to the method, a
biological material is cleared from Chlamydia infection by
contacting the biological material with at least two agents but
preferably three agents, each of which is targeted against a
different phase of the chlamydial life cycle, until the biological
material no longer tests positive for Chlamydia. The agents can be
selected from the group consisting of a) agents targeted against a
cryptic phase of the chlamydial life cycle; b) agents targeted
against the elementary body phase of the chlamydial life cycle; and
c) agents targeted against the replicating phase of the chlamydial
life cycle. In one embodiment, the agent targeted against the
elementary body phase is a disulfide reducing agent. In another
embodiment, the agent targeted against a cryptic phase is a
nitroaromatic compound, such as nitroimidazoles, nitrofluans,
analogs, derivatives and combinations thereof.
[0022] Biological material that has been cleared of Chlamydia
infection, according to the methods of this invention, are also
described. The biological material can be a continuous cell line
such as HeLa-CF, HL-CF, H-292-CF, HuEVEC-CF and McCoy-CF; wherein
"CF" is a shorthand annotation for "Chlamydia-free". Alternatively,
the biological material can be an animal, such as a mouse, rabbit
or other animal model, which is negative for Chlamydia.
[0023] The invention also pertains to methods of maintaining a
Chlamydia-free status in animals and cell lines which have been
cleared of Chlamydia infection by the methods of this invention, or
have never been infected, such as their Chlamydia-free offspring or
progeny. Cells or animals can be maintained as Chlamydia-free by
maintaining them on antibiotics and/or treating their nutrients and
environment to ensure that they are Chlamydia-free. Particularly, a
source of nutrients to be administered to Chlamydia-free cells or
animals can be treated to inactivate or remove any chlamydial
elementary bodies therefrom. This can be accomplished by exposing
the nutrients to gamma irradiation for a period of time and level
of exposure adequate to inactivate the elementary bodies. In
addition to, or alternatively, a source of nutrients can be passed
through a filtration system to physically remove the chlamydial
elementary bodies therefrom. Optionally, the source of nutrients
can be first treated with a disulfide reducing agent, such as
dithiothreitol, before the filtration step is performed. The filter
should be of adequate size such that objects larger than 0.5
microns are prevented from passing through.
[0024] The invention further pertains to a diagnostic kit or pack
comprising an assembly of materials selected from the group
consisting of antibiotics, reagents, Chlamydia-free cell lines, and
combinations thereof, or other materials that would be necessary to
perform any of the methods described herein.
[0025] The invention further relates to a method of detecting
viable Chlamydia in a biological material suspected of being
contaminated therewith, comprising culturing Chlamydia-free cells
or animals in the presence of biological material and then
determining the presence or absence of viable Chlamydia in the
culture.
[0026] The invention also pertains to a method for differentiating
porphyria caused by Chlamydia species from porphyria caused by a
genetic disorder. The method comprises measuring peripheral red
blood cell enzymes and/or performing a fecal and/or urinary
porphyrin screen, wherein if the peripheral red blood cell enzymes
are normal or elevated and fecal/urinary screen are elevated in one
or more components of the heme pathway, the porphyria is not caused
by a genetic disorder and may be caused by Chlamydia. The invention
relates to a method for diagnosing secondary porphyria caused by
Chlamydia in an individual having symptoms associated therewith,
comprising determining the presence or amount of obligatory enzymes
in heme biosynthesis in red blood cells of the individual and
determining the presence of Chlamydia in the individual. The
invention further relates to a method for differentiating secondary
porphyria caused by Chlamydia from that caused by a genetic
disorder in an individual, comprising treating infection by
Chlamydia at many stages of its life cycle and then assessing
whether porphyrins have been reduced, wherein a decrease in the
porphyrin levels is indicative that the porphyria is secondary and
caused by Chlamydia.
[0027] The subject invention also pertains to a method for treating
porphyria caused by Chlamydia in an individual in need thereof,
comprising reducing the levels of active stage, latent stage and
elementary bodies of the pathogen from the individual and
administering one or more compounds which reduce adverse effects
associated with secondary porphyria. In one embodiment, the method
additionally comprises administering a compound which reduces the
adverse effects of porphyrins associated with porphyria. In a
particular embodiment, the compound is cimetidine. This method can
also be valuably combined with additional steps, including at least
one of administering antioxidants; orally administering activated
charcoal; administering a high carbohydrate diet regimen;
administering hydroxychloroquine; administering benzodiazepine
drugs; performing hemodialysis; performing plasmaphoresis; and
adminstering chelating agents; and administering intravenous
hematin.
[0028] The invention also pertains to a method of detecting
elevated porphyrin levels in an individual by testing that
individual for antibodies to porphyrins. The invention also
pertains to diagnosing deficiency by detecting antibodies to B-12.
Monoclonal and polyclonal antibodies to prophyrins and/or Vitamin
B12 can be produced.
[0029] The invention further pertains to a method which can be
automated using a computerized system, for example, to formulate a
drug therapy for management of infection caused by Chlamydia. The
method comprises determining targets within the chlamydial life
cycle, for each determined target; identifying agents that are
active against the target; and combining at least a subset of the
identified agents to provide a combination therapy for management
of infection caused by Chlamydia, the agents in said subset
individually being active against different targets in the life
cycle of Chlamydia. The targets include identifying phases of the
chlamydial life cycle and for each identified life cycle phase,
determining at least one vulnerable aspect of the organism during
that life cycle phase, each said determined vulnerable aspect
defining a target within the chlamydial life cycle. Agents
identified by the method are then tested using the susceptibility
testing procedure described herein and initial dosages for
combination agents are set based on pharmacokenetics and
pharmacodynamics for the agents prescribed individually, said
setting initial dosage including modifying the combination dosage
according to results of the susceptibility testing and in vivo
efficacy.
[0030] The invention also relates to a method of purifying a blood
sample, comprising subjecting the blood sample to hemodialysis or
plasmaphoresis; in particular, the plasmaphoresis is carried out
using a plasmaphoresis apparatus utilizing a sulfone-containing
filter or a charcoal-containing filter. The blood sample can be
obtained from a blood bank or repository.
BRIEF DESCRIPTION OF THE DRAWINGS
[0031] FIGS. 1A and 1B show a sequence alignment of various
Chlamydia MOMPs.
[0032] FIG. 2 shows the expressed thioredoxin fusion protein
containing a polyhistidine affinity chromatography site, an
enterokinase cleavage site, and the full length MOMP protein with
an alanine insertion after aa1. Amino to carboxyl reads left to
right. Total amino acid content in the expressed protein is 530
residues.
[0033] FIG. 3 illustrates the constant and variable domain (VD) of
various Chlamydia species.
[0034] FIG. 4 illustrates the peptide amino acid sequences employed
for the construction of peptide based ELISAs with species
specificity for VD1.
[0035] FIG. 5 illustrates the peptides for VD2 which are used
similarly to the VD1 sequences.
DETAILED DESCRIPTION OF THE INVENTION
[0036] This invention describes specific antichlamydial agents that
are used singly or in combination to eliminate or interfere with
more than one of the distinct phases of the life cycle of Chlamydia
species. These chlamydial phases include the intracellular
metabolizing/replicating phase; the intracellular "cryptic" phases;
and the extracellular EB phase. Current concepts of susceptibility
testing for chlamydiae and antimicrobial therapy for their
associated infections address only one phase, the replicating
phase. Unless multiple phases of the life cycle are addressed by
antichlamydial therapy, the pathogen is likely to escape the
desired effects of the antimicrobial agent(s) used and cause
recurrent infection after reactivation from latency. For the
purposes of this invention, "cryptic phase" embraces any
non-replicating, intracellular form, of which there are a number of
distinct stages, including but not limited to intracellular EBs,
EBs transforming into RBs and vice versa, miniature RBs,
non-replicating RBs and the like.
[0037] Diagnostic and therapeutic methods for the management of
Chlamydia infections are described in detail below. For the
purposes of this invention, "management of Chlamydia infection" is
defined as a substantial reduction in the presence of all
phases/forms of Chlamydia in the infected host by treating the host
in such a way as to minimize the sequellae of the infection.
Chlamydia infections can thus be managed by a unique approach
referred to herein as "combination therapy" which is defined for
the purpose of this application as the administration of multiple
agents which together are targeted at least two but preferably many
of the multiple phases of the chlamydial life cycle, each agent
taken separately, simultaneously or sequentially over the course of
therapy. When used alone, these agents are unable to eliminate or
manage chlamydial infection. The diagnostic methods and combination
therapies described below are generally applicable for infection
caused by any Chlamydia species, including but not limited to C.
pneumoniae, C. trachomatis, C. psittaci and C. pecorum. Infections
in which the causative agent is C. pneumoniae are emphasized.
[0038] Antichlamydial agents, which have been identified as
effective against Chlamydia by the susceptibility testing methods
described herein, can be used singly to affect Chlamydia in a
single stage of its life cycle or as part of a combination therapy
to manage Chlamydia infection. For example, compounds identified as
anti-cryptic phase drugs, anti-EB phase drugs, anti-DNA-dependent
RNA polymerase drugs and nicotinic acid cogener drugs can be used
alone or in combination to eliminate, reduce or prevent one or more
of the distinct phases of the chlamydial life cycle. Certain of
these compounds have not heretofore been shown to have
antichlamydial activity.
DIAGNOSIS OF CHLAMYDIA INFECTION
[0039] The invention pertains to methods for diagnosing the
presence of Chlamydia in a biological material, as well as to the
use of these methods to evaluate the serological status of an
individual undergoing antichlamydial combination therapy. For
purposes of this application, "biological material" includes, but
is not limited to, bodily secretions, bodily fluids and tissue
specimens. Examples of bodily secretions include cervical
secretions, trachial-bronchial secretions and pharyngeal
secretions. Suitable bodily fluids include blood, sweat, tears,
cerebral spinal system fluid, serum, sputum, ear wax, urine,
synovial fluid and saliva. Animals, cells and tissue specimens such
as from a variety of biopsies are embraced by this term.
[0040] In one embodiment, peptide-based assays are disclosed for
the detection of one or more immunoglobulins, such as IgG, IgM, IgA
and IgE, against antigenic determinants within the full length
recombinant MOMPs of various Chlamydia species. Detection of IgG
and/or IgM against antigenic determinants-within the full length
recombinant MOMP of C. pneumoniae is preferred. IgA determinations
are useful in the analysis of humoral responses to Chlamydia in
secretions from mucosal surfaces (e.g., lung, GI tract,
gerontourinary tract, etc.). Similarly, IgE determinations are
useful in the analysis of allergic manifestations of disease. Table
1 below provides the GenBank Accession numbers of various MOMPs for
Chlamydia species.
1 TABLE 1 GenBank Accession Species Strain ID No. C. trachomatis A
CTL/A M33636 C. trachomatis A CTL/A M58938 M33535 C. trachomatis A
CTL/A J03813 C. trachomatis B CTL/B M33636 C. trachomatis C CTL/L
M17343 M19128 C. trachomatis D CTL/D A27838 C. trachomatis E CTL/E
X52557 C. trachomatis F CTL/F X52080 M30501 C. trachomatis H CTL/H
X16007 C. trachomatis L1 CTL/L1 M36533 C. trachomatis L2 CTL/L2
M14738 M19126 C. trachomatis L3 CTL/L3 X55700 C. trachomatis Mouse
Pneumo CTL/MP X60678 C. pecorum Ovine CPC/OP Z18756 Polylarthritis
C. psittaci Strain 6BC CPS/6B X56980 C. psittaci Feline CPS/F
X61096 C. trachomatis Da CTL/DA X62921 S45921 C. pneumoniae TWAR
CPN/HU1 M64064 M34922 M64063 C. pneumoniae Horse CPN/EQ2 L04982 (?
C. pecorum) C. pneumoniae TWAR CPN/MS not assigned C. psittaci
Horse CPS/EQ1 L04982
[0041] For example, a biological material, such as a sample of
tissue and/or fluid, can be obtained from an individual and a
suitable assay can be used to assess the presence or amount of
chlamydial nucleic acids or proteins encoded thereby. Suitable
assays include immunological methods such as enzyme-linked
immunosorbent assays (ELISA), including luminescence assays (e.g.,
fluorescence and chemiluminescence), radioimmunoassay, and
immunohistology. Generally, a sample and antibody are combined
under conditions suitable for the formation of an antibody-protein
complex and the formation of antibody-protein complex is assessed
(directly or indirectly). In all of the diagnostic methods
described herein, the antibodies can be directly labeled with an
enzyme, fluorophore, radioisotope or luminescer. Alternatively,
antibodies can be covalently linked with a specific scavenger such
as biotin. Subsequent detection is by binding avidin or strepavidin
labeled with an indicator enzyme, flurophore, radioisotope, or
luminescer. In this regard, the step of detection would be by
enzyme reaction, fluorescence, radioactivity or luminescence
emission, respectively.
[0042] The antibody can be a polyclonal or monoclonal antibody,
such as anti-human monoclonal IgG or anti-human monoclonal IgM.
Examples of useful antibodies include mouse anti-human monoclonal
IgG that is not cross reactive to other immunoglobulins (Pharmagen;
Clone G18-145, Catalog No. 34162D); mouse anti-human monoclonal IgM
with no cross reactivity to other immunoglobulins (Pharmagen; Clone
G20-127, Catalog No. 34152D). Peptide-based immunoassays can be
developed which are Chlamydia specific or provide species
specificity, but not necessarily strain specificity within a
species, using monoclonal or polyclonal antibodies that are not
cross-reactive to antigenic determinants on MOMP of a chlamydial
species not of interest.
[0043] Recombinant-based immunological assays have been developed
to quantitate the presence of immunoglobulins against the Chlamydia
species. Full length recombinant Chlamydia MOMP can be synthesized
using an appropriate expression system, such as in E. coli or
Baculovirus. The expressed protein thus serves as the antigen for
suitable immunological methods, as discussed above. Protein-based
immunological techniques can be designed that are species- and
strain-specific for various Chlamydia.
[0044] Diagnosis of chlamydial infection can now be made with an
improved IgM/IgG C. pneumoniae method of quantitation using ELISA
techniques, Western blot confirmation of ELISA specificity and the
detection of the MOMP gene of C. pneumoniae in serum using specific
amplification primers that allow isolation of the entire gene for
analysis of expected strain-specific differences.
[0045] Any known techniques for nucleic acid (e.g., DNA and RNA)
amplification can be used with the assays described herein.
Preferred amplification techniques are the polymerase chain
reaction (PCR) methodologies which comprise solution PCR and in
situ PCR, to detect the presence or absence of unique genes of
Chlamydia. Species-specific assays for detecting Chlamydia can be
designed based upon the primers selected. Examples of suitable PCR
amplification primers are illustrated below in Table 2. Examples of
preferred primers are illustrated in Table 3.
2TABLE 2 Initial and Terminal Nucleotide Sequences of Chlamydial
MOMP Genes in which entire sequence is known GenBank Accession No.
ID Initial Fifty Nucleotides SEQ ID NO. M64064/M34922/M64063 CPNHU1
ATGAAAAAACTCTTAAAGTCGGCGTT- ATTATCCGCCGCATTTGCTGGTTC 1 None
CPNHU2.sup.a ATGAAAAAACTCTTAAAGTCGGCGTTATTATCCGCCGCATTTGCTGGTTC 2
L04982 CPNEQ1 ATGAAAAAACTCTTGAAGTCGGCATTATTGTTTGCCGCTACGGGTTCCGC 3
L04982 CPNEQ2 ATGAAAAAACTCTTAAAGTCGGCGTTATTATCCGCCGCATTTGCTGGTTC 4
X56980 CPS/6B ATGAAAAAACTCTTGAAATCGGCATTATTGTTTGCCGCTAC- GGGTTCCGC
5 M36703 CPS/AB1 ATGAAAAAACTCTTGAAATCGGCATTATTGT-
TTGCCGCTACGGGTTCCGC 6 L39020 CPS/AB2
ATGAAAAAACTCTTGAAATCGGCATTATTGTTTGCCGCTACGGGTTCCGC 7 L25436
CPS/AV/C ATGAAAAAACTCTTGAAATCGGCATTATTATTTGCCGCTACGGGTTCCGC 8
X61096 CPS/F ATGAAAAAACTCTTAAAATCGGCATTATTATTTGCCGCTGCGGGTTCCG- C 9
M33636/N58938/J03813 CTL/A ATGAAAAAACTCTTGAAATCGGTATTA-
GTATTTGCCGCTTTGAGTTCTGC 10 M17343/M19128 CTL/C
ATGAAAAAACTCTTGAAATCGGTATTAGTATTTGCCGCTTTGAGTTCTGC 11 X62921/S45921
CTL/DA ATGAAAAAACTCTTGAAATCGGTATTAGTATTTGCCGCTTTGAGTTCTGC 12 X52557
CTL/E ATGAAAAAACTCTTGAAATCGGTATTAGTATTTGCCGCTTT- GAGTTCTGC 13
X52080/M30501 CTL/F ATGAAAAAACTCTTGAAATCGGTAT-
TAGTATTTGCCGCTTTGAGTTCTGC 14 X16007 CTL/H
ATGAAAAAACTCTTGAAATCGGTATTAGTATTTGCCGCTTTGAGTTCTGC 15 M36533 CTL/L1
ATGAAAAAACTCTTGAAATCGGTATTAGTGTTTGCCGCTTTGAGTTCTGC 16 M14738/M19126
CTL/L2 ATGAAAAAACTCTTGAAATCGGTATTAGTGTTTGCCGCTTTG- AGTTCTGC 17
X55700 CTL/L3 ATGAAAAAACTCTTGAAATCGGTATTAGTGTT- TGCCGCTTTGAGTTCTGC
18 X60678 CTL/MP ATGAAAAAACTCTTGAAATCGGTATTAGCATTTGCCGTTTTGGGTTCTGC
19 Chlamydial Species Strain ID Terminal Fifty Nucleotides SEQ ID
NO. C. pneumoniae TWAR CPNHU1 GTTTAATTAACGAGAGAGCTGCTCA-
CGTATCTGGTCAGTTCAGATTCTAA 20 C. pneumoniae MS CPNHU2
GTTTAATTAACGAGAGAGCTGCTCACGTATCTGGTCAGTTCAGATTCTAA 21 C. psittaci
Horse CPNEQ1 CAACGTTAATCGACGCTGACAAATGGTCAATCACTGGTGAAGCACGCTTA 22
C. pneumoniae Horse CPNEQ2 GTTTAATTAACGAGAGAGCTGCTCACA-
TATCTGGTCAGTTCAGATTCTAA 23 C. psittaci SBE CPS/6B
AACGTTAATCGACGCTGACAAATGGTCAATCACTGGTGAAGCACGCTTAA 24 C. psittaci
Ewe CPS/AB1 AACGTTAATCGACGCTGACAAATGGTCAATCACTGGTGAAGCACGCTTAA 25
abortion C. psittaci Bovine CPS/AB2
GCTTAATCAATGAAAGAGCCGCTCACATGAATGCTCAATTCAGATTCTAA 26 abortion C.
psittaci Avian CPS/AV/C GCTTAATCAATGAAAGAGCTGCTCACATGAATG-
CTCAATTCAGATTCTAA 27 C. psittaci Feline CPS/F
GCTTAATCGACGAAAGAGCTGCTCACATTAATGCTCAATTCAGATTCTAA 28 C.
trachomatis Hu/A CTL/A
CGCAGTTACAGTTGAGACTCGCTTGATCGATGAGAGAGCAGCTCACGTAA 29 C.
trachomatis Hu/C CTL/C GCTTGATCGATGAGAGAGCAGGTCACGT-
AAATGCACAATTCCGGTTCTAA 30 C. trachomatis Hu/Da CTL/DA
GCTTGATCGATGAGAGAGCAGCTCACGTAAATGCACAATTCCGCTTCTAA 31 C.
trachomatis HU/E CTL/E
CGCTTGATCGATGAGAGACTGCTCACGTAAATGCACAATTCCGCTTCTAA 32 C.
trachomatis Hu/F CTL/F GCTTGATCGATGAGAGAGCTGCTCACGT-
AAATGCACAATTCCGCTTCTAA 33 C. trachomatis Hu/H CTL/H
GCTTGATCGATGAGAGAGCAGCTCACGTAAATGCACAATTCCGCTTCTAA 34 C.
trachomatis Hu/L1 CTL/L1
GCTTGATCGATGAGAGAGCTGCTCACGTAAATGCACAATTCCGCTTCT- AA 35 C.
trachomatis Hu/L2 CTL/L2 GCTTGATCGATGAGAGAGCTGCTC-
ACGTAAATGCACAATTCCGCTTCTAA 36 C. trachomatis Hu/L3 CTL/L3
GCTTGATCGATGAGAGAGCAGCTCACGTAAATGCACAATTCCGCTTCTAA 37 C.
trachomatis Mouse CTL/MP
GCTTGATCGATGAAAGAGCAGCTCACGTAAATGCTCAGTTCCGTTTCT- AA 38
.sup.aSequence from a cerebral spinal fluid of a patient with
multiple sclerosis isolated by the inventors. Sequence is identical
to TWAR C. pneumoniae with exception of a C/T mutation at NT 54 and
a G/A mutation at NT 126. .sup.bTerminator condon underlined
[0046]
3TABLE 3 Primers for PCR Amplification of Entire MOMP Gene.sup.a
Chlamydia Plus Strand Primer Species Strain ID Sequence
T.sub.m.sup.b SEQ ID NO. C. pneunioniae TWAR CHLMOMP ATGAAAAAAC
TCTTAAAGTC GGCGTTATTA 61.4.degree. 105 DB2 TCCGCCGC C. trachomatis
L2 CTMOMP ATGAAAAAAC TCTTGAAATC GGTATTAGTG 61.2.degree. 106 L2DB
TTTGCCGCTT TGAG C. psittaci Feline PSOMP ATGAAAAAAC TCTTAAAATC
GGCATTATTA 62.1.degree. 107 FPN-D TTTGCCGCTG CGGG C. psittaci 6BC
PSOMP ATGAAAAAAC TCTTGAAATC GGCATTATTG 63.0.degree. 108 6BC-b
TTTGCCGCTA CGGG C. trachomatis Mouse CTMU ATGAAAAAAC TCTTGAAATC
GGTATTAGCA 63.5.degree. 109 MOMP-D TTTGCCGTTT TGGGTTCTGC Chlamydia
Minus Strand Primer Species Strain ID Sequence T.sub.m.sup.b SEQ ID
NO. C. pneumoniae TWAR CHLMOMP TTAGAATCTG AACTGACCAG ATACGTGAGC
64.4.degree. 110 CB2 AGCTCTCTCG C. trachomatis L2 CTMOMP TTAGAAGCGG
AATTGTGCAT TTACGTGAGC 61.5.degree. 111 L2CB AGCTC C. psittaci
Feline PSOMP TTAGAATCTG AATTGAGCAT TAATGTGAGC 62.2.degree. 112
FPN_C AGCTCTTTCG TCG C. psittaci 6BC PSOMP TTAGAATCTG AATTGACCAT
TCATGTGAGC 63.4.degree. 113 GBC_C AGCTCTTTCA TTGATTAAGC G C.
trachomatis Mouse CTMU TTAGAAACGG AACTGAGCAT TTACGTGAGC
63.2.degree. 114 MOMP_C TGCTCTTTCA TC .sup.aAll primers amplify
under identical amplification conditions: 94.degree. C. for 1 min.,
58.degree. C. for 2 min., 74.degree. C. for 3 min., for 35 cycles
with 72.degree. C. for 10 min. extension of last cycle.
.sup.bMelting temperature in degrees Celsius of a nucleic acid
isomer based on the equation of Mermur and Doty (J. Mol. Biol. 5:
109-118, 1962) where T.sub.m = 81.5 + 16.6 log.sub.10
(Na.sup.+/K.sup.+) + 41 (GC) - 600/L where (Na.sup.+/K.sup.+) in a
molar cation concentration, GC in the mole fraction of GC and L is
the sequence fragment length. (Na.sup.+/K.sup.+) used for
computation was 0.05 M.
[0047] Ligase chain reaction can also be carried out by the methods
of this invention; primers/probes therefor can be constructed using
ordinary skill. Amplification of the entire MOMP gene is useful for
mutational analysis and the production of recombinant MOMP. Shorter
primers can be used for specific amplification of most of the MOMP
genome with a modification of amplification protocol. For example,
a 22 bp negative strand primer of the sequence 5'-CAGATACGTG
AGCAGCTCTC TC-3' (CPNMOMPC; SEQ ID NO. 39) with a computed
T.sub.m=55.degree. C. plus a 25 bp positive strand primer of the
sequence 5'-CTCTTAAAGT CGGCGTTATT ATCCG-3' (CPNMOMPD; SEQ ID NO.
40) with a computed T.sub.m=53.9.degree. C. can be used as a primer
pair by adjusting the hybridization step in the amplification
protocol (Table 2) from 58.degree. C. to 50.degree. C. Similarly,
smaller regions of MOMP can be amplified by a large variety of
primer pairs for diagnostic purposes although the utility of strain
identification is reduced and amplification may be blocked if one
or both primer pairs hybridize to a region that has been mutated.
Extensive experience with the full length MOMP PCR amplification
indicates that mutational events within the CHLMOMPDB2 and
CHLMOMPCB2 hybridization sites are rare or non-existent.
[0048] The nucleic acid amplification techniques described above
can be used to evaluate the course of antichlamydial therapy. The
continued absence of detectable chlamydial DNA encoding MOMP as a
function of antichlamydial therapy is indicative of clinical
management of the chlamydial infection. Serological improvement can
be based upon the current serological criteria for eradication of
chronic Chlamydia reported below in Table 4.
4TABLE 4 Serological Criteria for Eradication of Chronic Chlamydia
pneumoniae Infection IgM .ltoreq.1:25 IgG Stable titer 1:100 PCR
Negative
[0049] Preferred PCR techniques are discussed in detail below in
the Example Section. In general, solution PCR is carried out on a
biological material by first pre-incubating the material in an
appropriate reducing agent that is capable of reducing the
disulfide bonds which maintain the integrity of the MOMP and other
surface proteins of the chiamydial elementary bodies, thereby
compromising the outer protective shell of the EBs and allowing
protease penetration. Suitable disulfide reducing agents include,
but are not limited to, dithiothreitol, succimer, glutathione,
DL-penicillamine, D-penicillamine disulfide, 2,2'-dimercaptoadipic
acid, 2,3-dimercapto-1-propone-sulfide acid. Appropriate
concentrations of these reducing agents can be readily determined
by the skilled artisan without undue experimentation using a 10
.mu.M concentration of dithiothreitol (the preferred reducing
agent) as a guideline. Failure to include a reducing agent in the
initial step may prevent DNA of EBs from being isolated in the
subsequent step. Data presented in Example 1 shows the effects of
various reducing agents on the susceptibility of EBs to proteinase
K digestion. The in vitro data shows that dithiothreitol is most
effective at opening EBs for protease digestion.
[0050] Once the outer shell of the EBs has been released, the
pre-incubated material is subjected to protein digestion using a
protease (e.g., proteinase K), or functionally equivalent enzyme.
The DNA is extracted and subjected to a nucleic acid amplification
technique, e.g., PCR. The entire gene or portion thereof containing
unique antigenic determinant(s) encoding MOMP or other suitable
gene can then be amplified using appropriate primers flanking the
gene to be amplified. For example, the gene or portion thereof can
be the gene encoding MOMP, OMP-B, GRO-ES, GRO-EL, DNAK, 16S RNA,
23S RNA, the gene encoding ribonuclease-P, a 76 kd attachment
protein or a KDO-transferase gene. In an alternative method,
guanidine thiocyanate, at preferably a concentration of 4M, or
functionally equivalent reducing denaturant may be substituted for
the disulfide reduction/protease steps.
[0051] The amplified DNA is then separated and identified by
standard electrophoretic techniques. DNA bands are identified using
ethidium bromide staining and UV light detection. PCR primers can
be designed to selectively amplify DNA encoding MOMP of a
particular Chlamydia species, such as the MOMP of C. pneumoniae, C.
pecorum, C. trachomatis, C. psittaci (See FIG. 1). Primers that are
from about 15-mer to about 40-mer can be designed for this
purpose.
[0052] For in situ PCR, the amplification primers are designed with
a reporter molecule conjugated to the 5'-terminus. Suitable
reporter molecules are well known and can be used herein. However,
biotin-labeled primers are preferred. For the MOMP gene, the
primers CHLMOMPDB2 and CHLMOMPCB2 have been engineered with a
biotin at the 5'-terminus. For in situ PCR, using biotin labels
incorporated at the 5'-terminus of the amplification primers, each
DNA chain amplification results in each double strand DNA
containing 2 molecules of biotin. Alternatively, other specific DNA
sequences can be used, although the above-described sequence is the
preferred embodiment since the large product produced (1.2 kb)
prevents diffusion that may be encountered with smaller DNA
amplifications. Similarly, other detection labels can be
incorporated (i.e., fluorescein, for example) at the 5'-end or
digoxigenin-dUTP (replacement for dTPP) can be incorporated within
the amplified DNA. Alternatively to labeling the product, specific
hybridization probes to constant regions of the amplified DNA can
be used to identify an amplified product. This latter method has
particular utility for the construction of automated laboratory
equipment for solution-based PCR. For example, strepavidin-coated
ELISA plates can be used to capture one or both strands of a biotin
5'-labeled DNA with detection by fluorescence of a fluorescein or
other incorporated fluorophore detection probe.
CLEARING AND MAINTAINING CHLAMYDIA-FREE ORGANISMS
[0053] The present invention provides a unique approach for
creating and maintaining animals and cell lines which are free of
Chlamydia infection. Also described herein are methods for creating
nutrients and culture media that are suitable for use with animals
and cell lines that have been cleared of Chlamydia infection.
[0054] Attempts to culture isolates of C. pneumoniae from blood and
cerebrospinal fluid (CSF) have resulted in the discovery that the
continuous cell lines routinely used to cultivate C. pneumoniae are
cryptically infected with C. pneumoniae. These include not only in
house stocks of HeLa, HL, H-292, HuEVEC and McCoy cells, but also
stocks obtained from the American Type Culture Collection (ATCC),
The University of Washington Research Foundation for HL cells, as
well as a commercial supplier (Bartells) of H-292 and McCoy cells
for the clinical culture of Chlamydia. The presence of a cryptic
form of C. pneumoniae in these cells has been repeatedly
demonstrated by solution PCR amplifying the MOMP. In situ PCR in
HeLa cells against the MOMP demonstrates the MOMP genes to be
present in 100% of cells. Nevertheless, fluoro-scenated mAb to LPS
in McCoy cells does not yield any indication of Chlamydia (i.e.,
reactive against all Chlamydia) while fluoroscenated mAb to C.
pneumoniae MOMP yields a generalized fluorescence throughout the
cytoplasm that can be confused with non-specific autofluorescence.
Infection with Chlamydia trachomatis (Bartells supply) yields the
typical inclusion body staining with the LPS mAb (i.e., cross
reactive with all species of Chlamydia) with no change in
cytoplasmic signal with anti-MOMP mAb against C. pneumoniae. These
findings (solution PCR, in situ PCR, mAb reactivity) were
interpreted as consistent with a cryptic (non-replicating)
infection by C. pneumoniae of cells commonly used to culture the
organism. Further, virtually all untreated rabbits and mice tested
to date have PCR signals for the C. pneumoniae MOMP gene.
[0055] This creates a currently unrecognized problem of major
significance for those clinical labs providing C. pneumoniae
culture services as well as investigators who now do not know
whether their results in animals or in cell culture will be
affected by cryptic chlamydial contamination. Clinical and research
laboratories currently have no way to determine whether an organism
is, in fact, Chlamydia-free.
[0056] This invention pertains to a method for clearing cells and
animals of C. pneumoniae and keeping them clear. Clearing them
entails contacting the infected organism with agents used singly or
in combination to eliminate or interfere with more than one of the
distinct phases of the life cycle of Chlamydia species. Keeping
them clear entails either maintaining them on antibiotics and/or
treating their nutrients and environment to ensure they are
Chlamydia-free. In a preferred embodiment, maintenance conditions
comprise a combination of isoniazid (INH) (1 .mu.g/ml),
metronidazole (1 .mu.g/ml), and dithiothreitol (10 .mu.M) in the
culture medium. Media changes are accomplished every 3 days or
twice per week. The cells can be removed from the protective
solution between 1 and 7 days before they are to be used for
culture or other purpose.
[0057] These techniques have now made it possible to create a
variety of Chlamydia-free (CF) organisms, including continuous cell
lines called HeLa-CF, HL-CF, H-292-CF, HuEVEC-CF, McCoy-CF, African
green monkey and other cell lines that are capable of supporting
chiamydial growth. Various CF strains of mice, rabbits and other
animal models for research use can be produced.
[0058] Because Chlamydia is highly infectious, organisms which have
been cleared of extracellular, replicating and cryptic infections
must be protected from exposure to viable EBs if the organisms are
to remain clear. The inventors have discovered that many of the
nutrients and other materials used to maintain continuous cell
lines are contaminated with viable Chlamydia EBs. For example,
every lot of fetal calf serum has tested positive for the Chlamydia
MOMP gene by PCR. Since extensive digestion is required for
isolation of the DNA, we have concluded it is bound in EBs. C.
pneumoniae can also be cultured directly from fetal calf serum.
Thus, it is necessary to inactivate EBs in these materials, such as
culture media and nutrients, used to maintain the Chlamydia-free
status of the organism. Collectively these materials are referred
to herein as "maintenance materials". In one embodiment, nutrients
and culture media are subjected to gamma irradiation to inactivate
Chlamydia therein. Preferably, the material should be irradiated
for a period of time sufficient to expose the material to at least
10,000 rads of gamma radiation. It is important for the material to
be contained in vessels that do not absorb high energy radiation.
The preferred vessel is plastic. In another embodiment, the
maintenance materials are treated with a disulfide reducing agent
(e.g.,dithiothreitol (10 .mu.M) for about 30 minutes) and then the
treated maintenance materials are passed through a standard
submicron (e.g., about 0.45 microns) filtration system. The
reducing agent causes any EBs to expand to the size where a 0.45
micron filter will block their passage. Examples of suitable
disulfide reducing agents include, but are not limited to,
dithiothreitol, succimer, glutathione, DL-penicillamine,
D-penicillamine disulfide, 2,2'-dimercaptoadipic acid,
2,3-dimercapto-1-propone-sulfide acid. In yet another embodiment,
maintenance materials are treated with a disulfide reducing agent,
preferably dithiothreitol (e.g., about 10 .mu.M concentration),
before the materials are passed through a filtration system to
remove Chlamydia therefrom.
[0059] In order to insure that research tools, such as cell lines
and animals, remain Chlamydia-free, an assay has been designed to
evaluate whether an organism is Chlamydia-free. The method
comprises obtaining a sample of cells or tissue culture; optionally
culturing the cells in the presence or absence of cycloheximide;
and determining the presence or absence of Chlamydia nucleic acid
by a suitable amplification technique, such as PCR. The absence of
nucleic acid amplification signal is indicative that the status of
the organism is Chlamydia-free.
SUSCEPTIBILITY TESTING FOR EVALUATING ACTIVE AGENTS AGAINST VARIOUS
FORMS OF CHLAMYDIA
[0060] This invention pertains to novel approaches for the
susceptibility testing of Chlamydia species that are necessitated
by the complex life cycle of the chlamydial pathogen as well as by
its diverse, extensive, and heretofore unappreciated ability to
cause chronic, cryptic and persistent systemic infections that are
refractory to short duration therapy with conventional single
agents. The inventors have discovered that successful management or
eradication of chronic/systemic chlamydial infections can be
predicted by using the described unique methods for in vitro and in
vivo susceptibility testing.
[0061] The invention is based upon the discovery that current
susceptibility testing methods for Chlamydiae do not accurately
predict the ability of antimicrobial agents to successfully and
totally eradicate chronic chlamydial infections. This is because
the current susceptibility testing methods measure only replication
of chlamydia and ignores the well-known "cryptic phase" in which
intracellular Chlamydiae are not actively replicating. Moreover, it
has also been discovered that the so-called "cryptic phase" of
Chlamydiae includes multiple and different sub-phases. The
following are some of the phases of the chlamydial life cycle in
which the intracellular Chlamydiae are not replicating: an initial
intranuclear phase in which elementary bodies (EBs) transition to
reticulate bodies (RBs), an intracytoplasmic phase in which there
is a transition of the RB phenotype to the EB phenotype, an
intracytoplasmic phase with a nonreplicating, but metabolizing RB,
and intracellular/extracellular EB phases, including endocytotic
and exocytotic phases, in which there is neither replication nor
metabolism. In order to assess the cumulative and long term effect
of antimicrobial therapy on these multiple life phases, unique in
vitro and in vivo susceptibility test methods have been developed
and are described herein.
[0062] The term "susceptibility" as used herein is intended to mean
a physiological response of an organism to an environmental or
chemical stimuli. The desired physiological response to stimuli is
one which adversely affects the pathogen's viability to replicate
or reside within the host cell and, ideally, would result in the
reduction or complete elimination (i.e., death) of that
pathogen.
[0063] A. In Vitro Methodology
[0064] One aspect of the invention pertains to methods for
evaluating the susceptibility of the distinct phases and stages of
the life cycle of Chlamydia, particularly the cryptic phase to a
particular agent(s), since prior techniques have failed,
heretofore, to appreciate the need for drugs that can clear
infected cells of cryptic Chlamydia. A preferred drug screening
method which accomplished this objective utilizes tissue culture
cells which are maintained, in the absence of cycloheximide in
order to encourage cryptic infection. Cryptic infection is uncommon
in cells used in standard cell culture susceptibility techniques
because Chlamydia in cycloheximide-paralyzed cells need not compete
with the host cell for metabolites and hence are encouraged to
replicate.
[0065] The in vitro method uses standard tissue culture cells, but
without the addition of cycloheximide. Moreover, the chlamydiae are
allowed to replicate for several days prior to the addition of one
or more test agents. A "test agent" can be any compound or
combination of compounds to be evaluated as an antichlamydial agent
for its ability to significantly reduce the presence of Chlamydia
in living cells. For example, a test agent can include, but is not
limited to, antibiotics, antimicrobial agents, antiparasitic
agents, antimalarial agent, disulfide reducing agents and
antimycobacterial agents. Antimicrobial agent(s) (test agent) is
then added to the replicating cells. The antimicrobial
agents/growth medium are periodically replaced for the duration of
the incubation time, which is preferably weeks rather than days.
The test agent(s) is/are replaced when needed for the duration of
the incubation time (days to weeks) to ensure that the test agent
is present and has not been otherwise degraded. Finally, the end
point after the prolonged incubation time is the complete absence
of chlamydial DNA, as determined by a nucleic acid amplification
technique, such as the polymerase chain reaction (PCR) methodology.
Standard nucleic acid amplification techniques (such as PCR) are
used to ascertain the presence or absence of signal for chlamydial
DNA encoding MOMP or another unique Chlamydia gene to determine
whether the test agent or combination of agents is/are effective in
reducing Chlamydia infection. The loss of signal (i.e., below the
detectable level of the nucleic acid amplification technique) in
cells with antibiotic(s) versus its presence in controls is an
indication of efficacy of the agent or combination of agents
against Chlamydia.
[0066] Accordingly, the susceptibility test of this invention can
be used to identify an agent or agents which are targeted against
any particular species of Chlamydia and can be used to identify
agent(s) targeted against the cryptic form of the pathogen, i.e.,
is capable of inhibiting or eliminating the cryptic form of the
pathogen. In one embodiment, this is done by performing the
susceptibility test while placing the cells under stringent
environmental conditions known to induce Chlamydia to enter a
cryptic phase. Agents that are effective against Chlamydia, as
ascertained by the susceptibility testing protocols described
herein, can be used as part of a therapy for the management of
Chlamydia infections. Suitable therapeutic protocols are described
in detail below, with a particular focus on targeting agents toward
specific stages of the chlamydial life cycle.
[0067] The methods described herein are unique because they
evaluate the activity of antimicrobial agents in the absence of
cycloheximide which provides a more clinically relevant
intracellular milieu. For example, any normally operating,
energy-dependent host cell membrane pumps which might move
antimicrobial agents in or out of the cell are inactivated by the
use of cycloheximide. The methods described herein are unique
because they utilize culture medium which has previously been
inactivated. The methods are also unique because they measure the
effect of a prolonged duration of exposure to the antimicrobial
agent(s) after the intracellular infection by chlamydiae has become
established. Finally, the method is unique because it measures the
presence/absence of chlamydial DNA as the endpoint, for example by
measuring PCR signal. By using complete eradication of chlamydial
DNA as an endpoint, the susceptibility test confirms that all
phases of Chlamydiae have been eradicated as opposed to there
having been merely a temporary halt in replication.
[0068] When a nucleic acid amplification methodology, such as PCR,
is used to evaluate assay endpoint, the nucleic acid assay (e.g.,
PCR) method can be enhanced by the unique application of a reducing
agent, such as dithiothreitol (DTT), in order to perturb the coat
of chlamydial EBs and hence allow exposure of the DNA by the action
of a protein digestive compound, such as proteinase K. In other
words, the reducing agent permits the EB coating to rupture. By
using an assay for DNA in which EBs are specifically uncoated, the
susceptibility test endpoint assesses the presence or absence of
EBs as well as the presence or absence of both replicating and
nonreplicating RBs. Thus, this approach for chlamydial
susceptibility testing allows quantitative antimicrobial
susceptibility assays of single and combination agents in which the
cumulative effect of the agent(s) on the complete eradication of
all life phases is measured. Examples of results obtained with this
in vitro method are described below.
[0069] In one embodiment, a suitable nucleic acid assay for
identifying agents effective against the cryptic form of Chlamydia
comprises, in the presence of agent(s) to be tested, subjecting
cultured cells to reducing agent (e.g., dithiotreitol) and protease
digestion or guanidine isothiocyanate (also known as guanidine
thiocyanate) for a prescribed period of time; extracting DNA from
the treated solution; exposing DNA to appropriate polymerase, dNTPs
and primers for DNA amplification of MOMP or other protein of the
Chlamydia species; and determining the presence or absence of
amplified DNA by visualizing the ethidium bromide treated DNA
product by gel electrophoresis, for example, or alternatively by
Southern Blot. In particular embodiments, the Chlamydia species is
C. pneumoniae and the appropriate primers are CHLMOMPDB2 and
CHLMOMPCB2.
[0070] The invention further relates to a method of identifying
cells containing a non-EB cryptic form of a Chlamydia species by a
nucleic acid amplification technique (e.g., PCR) comprising
subjecting cultured cells to protease digestion; stopping protease
activity; exposing cells to appropriate heat-stable DNA polymerase,
dNTPs and labeled primers (e.g., 3'-biotin labeled, 5'-biotin
labeled) for amplification of DNA encoding MOMP of the Chlamydia
species; washing the cells; exposing the cells to a reporter
molecule (e.g., strepavidin-conjugated signal enzyme); exposing the
cells to an appropriate substrate for the reporter molecule (e.g.,
conjugated enzyme); and visualizing the amplified DNA encoding MOMP
by visualizing the product of the reaction.
[0071] The invention pertains to a-method of identifying cells
containing a cryptic form of Chlamydia. The method comprises
treating cultured cells, thought to be infected with Chlamydia,
with a disulfide reducing agent; subjecting cultured cells to
protease digestion; exposing cells to appropriate polymerase, dNTPs
and primers for DNA amplification of nucleic acid encoding of a
chlamydial protein; exposing the cells to a reporter molecule
enzyme; exposing the cells to an appropriate substrate for the
reporter enzyme; and determining the presence of a cryptic form of
Chlamydia by visualizing the amplified DNA encoding a chlamydial
protein. Preferably, the amplification technique is PCR and the
primers are CHLMOMPDB2 and CHLMOMPCB2 of Chlamydia pneumoniae.
[0072] A similar method can be used as an assay for identifying an
agent which is effective against a cryptic form of Chlamydia.
Accordingly, the method comprises treating cultured cells grown in
the absence of cycloheximide, thought to be infected with
Chlamydia, with a disulfide reducing agent; allowing the Chlamydia
to replicate; adding a test agent; subjecting cultured cells to
protease digestion; exposing cells to appropriate polymerase, dNTPs
and primers for DNA amplification of a gene encoding chlamydial
protein; exposing the cells to a reporter molecule enzyme; exposing
the cells to an appropriate substrate for the reporter enzyme; and
determining the presence of cryptic form of Chlamydia by
visualizing the amplified DNA encoding a chlamydial protein, such
as MOMP.
[0073] B. In Vivo Methodology
[0074] In another aspect of the invention, the susceptibility test
can be used to evaluate the status of a human or animal undergoing
therapy for the management of Chlamydia infection. For example, a
biological material is isolated from the human or animal to undergo
combination therapy. The biological material is treated such that
the Chlamydia is isolated therefrom. This chlamydial isolate is
allowed to infect Chlamydia free cells. These infected cells are
then exposed to the combination of agents being used in the
individual undergoing combination therapy. Alternatively, the
individual's serum containing the antimicrobial agents can be added
to the infected cells as a "serum bactericidal test" for
intracellular chlamydial infection. The presence of chlamydial DNA
is then measured.
[0075] The in vivo method uses the murine model although other
animals such as rats or rabbits can be used. In this method, mice
(or any other animal) are inoculated intranasally with
2.times.10.sup.5 chlamydial EBs per ml. The inventors have
confirmed the work of Yang and colleagues (J. Infect. Dis.,
171:736-738 (1995)) in which intranasal inoculation of chlamydial
EBs results in systemic dissemination and, in particular, causes
infection of the spleen. The inventors have discovered that this
systemic dissemination also results in the presence of EBs in the
blood of the mice. Therefore, infectivity can be measured by blood
culture or by serum/whole blood PCR for chlamydial DNA. Systemic
infection is also confirmed and monitored by the presence of
elevated IgM and IgG antibody titers. After the systemic murine
infection has been established, antimicrobial agents are given to
the mice. This is most easily done by adding the antibiotics to the
drinking water. The effect of antichlamydial therapy is monitored
by serum/whole blood PCR. When the serum/PCR assay suggests
eradication of chlamydiae from the bloodstream, the mice are
sacrificed and PCR for chlamydial DNA is done on lung, heart,
liver, and spleen homogenates. This method is unique because it
measures the complete eradication of all life forms of chlamydiae
in known murine target organs for chlamydial infection. This in
vivo susceptibility method has revealed, for example, that
antimicrobial therapy with the triple agents, INH, metronidazole
and penicillamine, can completely eradicate C. pneumoniae from
infected mice in four months. Moreover, following complete
eradication of chlamydiae, multiple attempts to reinfect these
cured mice via intranasal inoculation have proven unsuccessful.
This suggests that effective management and complete eradication
results in the development of protective immunity, and that
effective management is therefore a way to create effective
immunity.
[0076] Performing PCR for chlamydial DNA on homogenates of other
organ systems can be used to determine the effectiveness of
particular antibiotic combinations in eradicating chlamydial
infection in those organ systems. Establishment of prior chlamydial
infection of those systems can be done by either biopsy or
antibody-enhanced radiological imaging. Alternatively, prior
infection can be determined statistically by performing PCR for
chlamydial DNA on homogenates of the same organ systems in a
similarly inoculated but untreated control population.
Organ-specific susceptibility is determined by comparing rates of
positive PCR assays in the control and treated populations.
[0077] An alternative or complementary method of determining the
presence of cryptic chlamydial infections in an animal or cell
culture is to expose the culture to chlamydia-stimulating
compounds. Such compounds include (but are not limited to)
cycloheximide, corticosteriods (such as prednisone) and other
compounds which are known to stimulate reactivation of cryptic
intracellular infections, and disulfide reducing agents (such as
dithiotreitol) and other chemicals which cause EBs to turn into
RBs. Once the cryptic forms have entered a more active phase, they
can be detected using standard detection techniques such as visual
detection of inclusion bodies, immunochemical detection of
chlamydial antigen, or reverse transcriptase-PCR.
ANTICHLAMYDIAL THERAPY DIRECTED TOWARD THE INITIAL STAGE OF
CHLAMYDIA INFECTION
[0078] A number of effective agents that are specifically directed
toward the initial phase of chlamydial infection (i.e., transition
of the chlamydial EB to an RB) have been identified. This "cryptic"
growth phase, unlike that of the replicating chlamydial
microorganism, which uses host cell energy, involves electrons and
electron transfer proteins, as well as nitroreductases. Based upon
this, it has been discovered that the initial phase of Chlamydia
infection is susceptible to the antimicrobial effects of
nitroimidazoles, nitrofurans and other agents directed against
anaerobic metabolism in bacteria.
[0079] Nitroimidazoles and nitrofurans are synthetic antimicrobial
agents that are grouped together because both are nitro (NO.sub.2-)
containing ringed structures and have similar antimicrobial
effects. These effects require degradation of the agent within the
microbial cell such that electrophilic radicals are formed. These
reactive electophilic intermediates then damage nucleophilic
protein sites including ribosomes, DNA and RNA. Nitroimidazoles and
nitrofurans currently are not considered to possess antimicrobial
activity against members of the Chlamydia species. This lack of
antimicrobial activity, however, is due to the fact that
conventional susceptibility testing methods only test for effect on
the replicating form of Chlamydia species.
[0080] Examples of suitable nitroimidazoles include, but are not
limited to, metronidazole, tinidazole, bamnidazole, benznidazole,
flunidazole, ipronidazole, misonidazole, moxnidazole, ronidazole,
sulnidazole, and their metabolites, analogs and derivatives
thereof. Metronidazole is most preferred. Examples of nitrofurans
that can be used include, but are not limited to, nitrofurantoin,
nitrofurazone, nifurtimox, nifuratel, nifuradene, nifurdazil,
nifurpirinol, nifuratrone, furazolidone, and their metabolites,
analogs and derivatives thereof. Nitrofurantoin is preferred within
the class of nitrofurans.
[0081] Throughout this application and for purposes of this
invention, "metabolites" are intended to embrace products of
cellular metabolism of a drug in the host (e.g., human or animal)
including, but not limited to, the activated forms of prodrugs. The
terms "analogs" and "derivatives" are intended to embrace isomers,
optically active compounds and any chemical or physical
modification of an agent, such that the modification results in an
agent having similar or increased, but not significantly decreased,
effectiveness against Chlamydia, compared to the effectiveness of
the parent agent from which the analog or derivative is obtained.
This comparison can be ascertained using the susceptibility tests
described herein.
[0082] Cells to be treated can already be cryptically infected or
they can be subjected to stringent metabolic or environmental
conditions which cause or induce the replicating phase to enter the
cryptic phase. Such stringent conditions can include changing
environmental/culturing conditions in the instance where the
infected cells are exposed to .gamma.-interferon; or by exposing
cells to conventional antimicrobial agents (such as macrolides and
tetracyclines) which induce this cryptic phase of chlamydial
infection in human host cells.
NOVEL ANTICHLAMYDIAL THERAPY DIRECTED TOWARD THE REPLICATING AND
CRYPTIC STATIONARY PHASES OF CHLAMYDIA INFECTION
[0083] A unique class of antichlamydial agents that is effective
against the replicating and cryptic stationary phases of Chlamydia
(and possibly against some other stages of the cryptic phase) have
been identified using the susceptibility tests described herein.
This novel class of agents comprises ethambutol and isonicotinic
acid congeners which include isoniazid (INH), isonicotinic acid
(also known as niacin), nicotinic acid, pyrazinamide, ethionamide,
and aconiazide; where INH is most preferred. Although these are
currently considered effective only for mycobacterial infections,
due in part to currently available susceptibility testing
methodologies, it has been discovered that these agents, in
combination with other antibiotics, are particularly effective
against Chlamydia. It is believed that the isonicotinic acid
congeners target the constitutive production of catalase and
peroxidase, which is a characteristic of microorganisms, such as
mycobacteria, that infect monocytes and macrophages. Chlamydia can
also successfully infect monocytes and macrophages.
[0084] Using INH to eradicate Chlamydia from macrophages and
monocytes subsequently assists these cells in their role of
fighting infection. However, these agents appear to be less
effective, in vitro, against the cryptic phase. Thus, ethambutol,
INH and other isonicotinic acid congeners ideally should be used in
combination with agents that target other phases of the chlamydial
life cycle. These isonicotinic acid congeners are nevertheless
excellent agents for the long term therapy of chronic/systemic
chlamydial infection generally, and in particular to chlamydial
infection of endothelial and smooth muscle cells in human blood
vessels.
[0085] INH and its congeners can be used to clear infection from
monocytes and/or macrophages. When monocytes and macrophages are
infected by Chlamydia, they become debilitated and cannot properly
or effectively fight infection. It is believed that, if the
chlamydial infection, per se, is cleared from these cells, then the
monocytes and macrophages can resume their critical roles fighting
chlamydial or other infection(s). Thus, patient responsiveness to
combination therapy can be optimized by the inclusion of
isonicotinic acid congeners. Accordingly, one aspect of the
invention provides a specific method for reempowering monocytes or
macrophages that have been compromised by a Chlamydia infection
and, in turn, comprise treating the infection in other sites. Such
compromised macrophages or monocytes can be activated by treating
the chlamydial infection by contacting the infected macrophages
and/or monocytes with an antichlamydial agent.
THERAPY DIRECTED TOWARD ELEMENTARY BODIES OF CHLAMYDIA
[0086] As discussed above, it has been discovered that adverse
conditions, such as limited nutrients, antimicrobial agents, and
the host immune response, produce a stringent response in
Chlamydia. Such adverse conditions are known to induce stringent
responses in other microorganisms (C. W. Stratton, In: Antibiotics
in Laboratory Medicine, Fourth Edition. Lorian V (ed) Williams
& Wilkins, Baltimore, pp 579-603 (1996)) and not surprisingly
induce a stringent response in Chlamydia. This stringent response
in Chlamydia alters the morphological state of the intracellular
microorganism and creates dormant forms, including the
intracellular EB, which then can cryptically persist until its
developmental cycle is reactivated. Conversely, the host cell may
lyse and allow the EBs to reach the extracellular milieu. Thus, it
is necessary to utilize a combination of agents directed toward the
various life stages of Chlamydia and, in particular, against the
elementary body for successful management of infection.
[0087] During the unique chlamydial life cycle, it is known that
metabolically-inactive spore-like EBs are released into the
extracellular milieu. Although these released EBs are infectious,
they may not immediately infect nearby susceptible host cells until
appropriate conditions for EB infectivity are present. The result
of this delay in infection is the extracellular accumulation of
metabolically-inactive, yet infectious, EBs. This produces a second
type of chlamydial persistence referred to herein as EB
"tissue/blood load". This term is similar in concept to HIV load
and is defined herein as the number of infectious EBs that reside
in the extracellular milieu. Direct microscopic visualization
techniques, tissue cell cultures, and polymerase chain reaction
test methods have demonstrated that infectious EBs are frequently
found in the blood of apparently healthy humans and animals. This
phenomenon is clearly of great clinical importance in chlamydial
infections as these metabolically-inactive EBs escape the action of
current antichlamydial therapy which is directed only against the
replicating intracellular forms term, anti-replicating phase
therapy for chlamydial infections has been shown to result in
intracellular infection relapse. Thus, the duration and nature of
antichlamydial therapy required for management of chlamydial
infections is, in part, dictated by the extracellular load of EBs.
For purposes of this invention, short term therapy can be
approximately two to three weeks; long term therapy in contrast is
for multiple months.
[0088] As described in previous sections, it is also believed that
persistence of chiamydial infections, in part, may be due to the
presence of cryptic forms of Chlamydia within the cells. This
cryptic intracellular chlamydial form apparently can be activated
by certain host factors such as cortisone (Yang et al., Infection
and Immunity, 39:655-658 (1983); and Malinverni et al., The Journal
of Infectious Diseases, 172:593-594 (1995)). Antichlamydial therapy
for chronic Chlamydia infections must be continued until any
intracellular EBs or other intracellular cryptic forms have been
activated and extracellular EBs have infected host cells. This
reactivation/reinfection by chlamydial EBs clearly is undesirable
as it prolongs the therapy of chlamydial infections, as well as
increases the opportunity for antimicrobial resistance to
occur.
[0089] Physiochemical agents have been identified that can
inactivate chlamydial EBs in their respective hosts by reducing
disulfide bonds which maintain the integrity of the outer membrane
proteins of the EBs. For Chlamydia, disruption of the outer
membrane proteins of EBs thereby initiates the transition of the EB
form to the RB form. When this occurs in the acellular milieu where
there is no available energy source, the nascent RB perishes or
falls victim to the immune system. Thus, disulfide reducing agents
that can interfere with this process are suitable as compounds for
eliminating EBs.
[0090] One such class of disulfide reducing agents are
thiol-disulfide exchange agents. Examples of these include, but are
not limited to, 2,3-dimercaptosuccinic acid (DMSA; also referred to
herein as "succimer"); D,L,-.beta.,.beta.-dimethylcysteine (also
known as penicillamine); .beta.-lactam agents (e.g., penicillins,
penicillin G, ampicillin and amoxicillin, which produce
penicillamine as a degradation product), cycloserine,
dithiotreitol, mercaptoethylamine (e.g., mesna, cysteiamine,
dimercaptol), N-acetylcysteine, tiopronin, and glutathione. A
particularly effective extracellular antichlamydial agent within
this class is DMSA which is a chelating agent having four ionizable
hydrogens and two highly charged carboxyl groups which prevent its
relative passage through human cell membranes. DMSA thus remains in
the extracellular fluid where it can readily encounter
extracellular EBs. The two thiol (sulfhydryl) groups on the
succimer molecule (DMSA) are able to reduce disulfide bonds in the
MOMP of EBs located in the extracellular milieu.
[0091] Penicillamine can also be used as a disulfide reducing agent
to eliminate chlamydial EBs. However, the use of penicillamine may
cause undesirable side effects. Thus, as an alternative, those
P-lactam agents which are metabolized or otherwise converted to
penicillamine-like agents in vivo (i.e., these agents possess a
reducing group) can be orally administered to the human or animal
as a means of providing a controlled release of derivative
penicillamine, by non-enzymatic acid hydrolysis of the penicillin,
under physiologic conditions. Clavulonic acid is not required for
this hydrolysis or for using .beta.-lactam agents to create
penicillamine in vivo.
CURRENTLY RECOGNIZED AGENTS ACTIVE AGAINST CHLAMYDIA
REPLICATION
[0092] As chlamydial RBs transform into EBs, they begin to utilize
active transcription of chlamydial DNA and translation of the
resulting mRNA. As such, these forms of Chlamydia are susceptible
to currently used antimicrobial agents. The antichlamydial
effectiveness of these agents can be significantly improved by
using them in combination with other agents directed at different
stages of Chlamydia life cycle, as discussed herein.
[0093] Classes of suitable antimicrobial agents include, but are
not limited to, rifamycins (also known as ansamacrolides),
quinolones, fluoroquinolones, chloramphenicol,
sulfonamides/sulfides, azalides, cycloserine, macrolides and
tetracyclines. Examples of these agents which are members of these
classes, as well as those which are preferred, are illustrated
below in Table 5.
5TABLE 5 Agents Effective Against the Replicating Phase of
Chlamydia Drug Class Examples Preferred Quinolones/ Ofloxacin
Levofloxacin Fluoroquinolones Levofloxacin Trovafloxacin
Sparfloxacin Norfloxacin Lomefloxacin Cinoxacin Enoxacin Nalidixic
Acid Fleroxacin Ciprofloxacin Sulfonamides Sulfamethoxazole
Sulfamethoxazole/ Trimethoprim Azalides Azithromycin Azithromycin
Macrolides Erythromycin Clarithromycin Clarithromycin Lincosamides
Lincomycin Clindamycin Tetracyclines Tetracycline Minocycline
Doxycycline Minocycline Methacycline Oxytetracyline Rifamycins
Rifampin Rifampin (Ansamacrolides) Rifabutin
[0094] All members of the Chlamydia species, including C.
pneumoniae, are considered to be inhibited, and some killed, by the
use of a single agent selected from currently used antimicrobial
agents such as those described above. However, using the new
susceptability test, the inventors have found complete eradication
of Chlamydia cannot be achieved by the use of any one of these
agents alone because none are efficacious against all phases of the
Chlamydia life cycle and appear to induce a stringent response in
Chlamydia causing the replicating phase to transform into cryptic
forms. This results in a persistent infection in vivo or in vitro
that can be demonstrated by PCR techniques which assess the
presence or absence of chlamydial DNA. Nevertheless, one or more of
these currently used agents, or a new agent directed against the
replicating phase of Chlamydia, should be included as one of the
chlamydial agents in a combination therapy in order to slow or halt
the transition of the EB to the RB as well as to inhibit chlamydial
replication.
METHODOLOGY FOR SELECTING POTENTIAL AGENT COMBINATIONS
[0095] In attempting to manage or eradicate a systemic infection,
it is critical to target multiple phases in the life cycle of
Chlamydia, otherwise viable Chlamydia in the untargeted phases will
remain after therapy and result in continued, chronic infection.
This fundamental insight is at the core of this invention.
[0096] A preferred method for selecting an appropriate combination
of agents that satisfies the requirements of this strategy
comprises a plurality of steps as follows:
[0097] 1. Identify the phases of the chlamydial life cycle. For
example, the following phases are currently known:
[0098] a. Elementary Body ("EB")--Extracellular or Intracellular.
Intracellular EBs may represent a type of "cryptic phase".
[0099] b. EB to Reticulate Body ("RB") transition phase.
[0100] c. Stationary RB phase. This is what is traditionally
thought of as the "cryptic phase".
[0101] d. Replicating RB phase.
[0102] e. RB to EB transition phase (also called
"condensation").
[0103] 2. Evaluate the relative importance of targeting each
particular phase in eradicating reservoirs of Chlamydia from the
host organism. For example, the life-cycle stages listed in step 1
can be prioritized based on the following assumptions:
[0104] a. In the host, extracellular and intracellular EBs
represent a very important reservoir of infectious agents that
result in chronic and persistent infection.
[0105] b. Most intracellular RBs in chronic infections are
non-replicating. The 3-4 day reproduction cycle seen in
cycloheximide-treated eukaryotic cells is an artifact of an
atypical, cell culture environment designed primarily to propagate
Chlamydia.
[0106] c. The transition phases represent only a small portion of
Chlamydia in chronic infections.
[0107] 3. Identify "targets" for each phase of the selected life
cycle phases. A target is an attribute of Chlamydia which is
vulnerable during a particular life cycle phase. For example, the
disulfide bonds in MOMP are a target during the EB phase.
[0108] 4. Identify agents with known or theoretical mechanism(s) of
action against those targets.
[0109] 5. Estimate whether those agents would be merely inhibitory
or, preferably, cidal, through an understanding of their mechanism
of action.
[0110] 6. Confirm the estimate by using the following
approaches:
[0111] a. In the case of anti-EB agents, treat EBs with the agent,
then attempt to infect cells with the treated EBs. If the cells do
not become infected, the agent is EB-cidal.
[0112] b. In the case of other agents, use the susceptibility tests
disclosed elsewhere herein, to determine whether the agent, either
alone or in combination with other agents, is chlamydicidal.
[0113] 7. Select a combination of agents that, through their
individual effects, provide activity against targets for the most
important phases within the chlamydial life cycle. Preferably, a
combination should target as many phases of the life cycle as
possible, seeking to maximize the total of the relative important
scores of the phases targeted while minimizing the number of drugs
involved.
[0114] 8. Test the combination using the susceptibility testing
procedures described elsewhere. This step is necessary because the
selected combination may or may not be chlamydicidal for various
reasons such as intracellular penetration and/or efflux.
[0115] 9. Set initial dosages based on clinical standards which
consider the pharmacokenetics and pharmacodynamics for the drugs
prescribed individually; modifications, if needed, are based on
results of susceptibility testing and in vivo efficacy.
[0116] Table 6 provides an example of how the foregoing methodology
can be used. The preferred embodiment includes agents which:
[0117] a) Target disulfide bonds in the EB and condensation
phases;
[0118] b) Target non-oxidative metabolism in the stationary/cryptic
phase;
[0119] c) Target constitutive production of peroxidases and
catalyses in the stationary and replicative phases;
[0120] d) In the latter two cases, work through physio-chemical
disruption of the organism through free radicals, which are very
difficult for organism to develop resistance to; and
[0121] e) Optionally adds an agent to target DNA-dependent RNA
polymerase in the EB->RB phase.
[0122] The foregoing methodology for selecting combination
therapies can be automated (e.g., by a computer system) at one or a
combination of the steps described above. This methodology is
applicable even after greater understanding of the chlamydial life
cycle leads to a re-prioritization or even sub-division of the
life-cycle phases, new theoretical targets within Chlamydia are
identified, or new drugs are developed which attack currently known
or new targets within Chlamydia. For example, the phases of the
life cycle could be further sub-classified based on the type of
host cell the phase is in. Thus, stationary phase RBs in
macrophages could be considered a separate phase than stationary
phase RBs in hepatocytes. This allows the methodology to be used to
design a single or multi-tissue specific combination of agents.
6TABLE 6 Example of using Theoretical Effect on Various Targets
within the Chlamydial Life Cycle to Pick a Combination Therapy
Constitutive production of DNA- Ribosomes Rel- Potentially
peroxidases dependent Folic involved in ative vulnerable Disulfide
Non-oxidative and RNA acid protein Im- attributes of bonds
metabolism catalyses Topoisomerases polymerase pathway synthesis
por- Chlamydia: Theoretical Targets tance Phase in EB
(Extracellular X 8 Chla- or Intracellular) mydial EB->RB p p X 6
Life Transition Cycle Stationary X X p p p p 8 Phase RB ("Cryptic
phase") Replicating RB p X X p X X 7 RB->EB X p p 6 Transition
("Condensation") Pharma- Non-Novel Quinolones, Rifamycins Sulfon-
Azalides, ceutical Classes Fluroquinolones amides Macrolides, Com-
Lincosamides, pounds Tetracyclines Novel Disulfide Agents that
Agents Classes reducing agents strip activated electrons from by
carrier proteins peroxidases and become and free-radicals catalyses
to become free-radicals Examples: Thiol-disulfide Nitroimidazoles
Isonicotinic reducing agents & Nitrofurans acid cogeners Drugs
of Penicillamine Metronidazole INH Levofloxacin Rifampin Sulfa-
Azythromycin Choice (based (from or Trovafloxavin methoxizole/
Clarithromycin on effectiveness) Amoxicillan) Nitrofurantoin
trimethoprim Minocycline Preferred Penicillamine Metronidazole INH
.+-.Rifampin Embodiment (Amoxicillan) X = known target relevant to
that life phase; p = possible target relevant to that life
phase
DISEASES ASSOCIATED WITH CHLAMYDIAL INFECTION
[0123] An association has been discovered between chronic Chlamydia
infection of body fluids and/or tissues with several disease
syndromes of previously unknown etiology in humans which respond to
unique antichlamydial regimens described herein. To date, these
diseases include Multiple Sclerosis (MS), Rheumatoid Arthritis
(RA), Inflammatory Bowel Disease (IBD), Interstitial Cystitis (IC),
Fibromyalgia (FM), Autonomic nervous dysfunction (AND,
neural-mediated hypotension); Pyoderma Gangrenosum (PG), Chronic
Fatigue (CF) and Chronic Fatigue Syndrome (CFS). Other diseases are
under investigation. Correlation between Chlamydia infection and
these diseases has only recently been established as a result of
the diagnostic methodologies and combination therapies described
herein.
[0124] Based on this evidence, published evidence of an association
between atherosclerosis and Chlamydia (Grupta et al., Circulation,
96:404-407 (1997)), and an understanding of the impact Chlamydia
infections have on infected cells and the immune systems, the
inventors have discovered a connection between Chlamydia and a
broad set of inflammatory, autoimmune, and immune deficiency
diseases. Thus, the invention describes methods for diagnosing
and/or treating disease associated with Chlamydia infection, such
as autoimmune diseases, inflammatory diseases and diseases that
occur in immunocompromised individuals by diagnosing and/or
treating the Chlamydia infection in an individual in need thereof,
using any of the assays or therapies described herein. Progress of
the treatment can be evaluated serologically, to determine the
presence or absence of Chlamydia using for example the diagnostic
methods provided herein, and this value can be compared to
serological values taken earlier in the therapy. Physical
improvement in the conditions and symptoms typically associated
with the disease to be treated should also be evaluated. Based upon
these evaluating factors, the physician can maintain or modify the
antichlamydial therapy accordingly. For example, the physician may
change an agent due to adverse side-effects caused by the agent,
ineffectiveness of the agent, or for other reason. When antibody
titers rise during treatment then alternate compounds should be
substituted in order to achieve the lower antibody titers that
demonstrate specific susceptibility of the Chlamydia to the new
regimen. A replacement or substitution of one agent with another
agent that is effective against the same life stage of Chlamydia is
desirable.
[0125] The therapies described herein can thus be used for the
treatment of acute and chronic immune and autoimmune diseases when
patients are demonstrated to have a Chlamydia load by the
diagnostic procedures described herein which diseases include, but
are not limited to, chronic hepatitis, systemic lupus
erythematosus, arthritis, thyroidosis, scleroderma, diabetes
mellitus, Graves' disease, Beschet's disease and graft versus host
disease (graft rejection). The therapies of this invention can also
be used to, treat any disorders in which a chlamydial species is a
factor or co-factor.
[0126] Thus, the present invention can be used to treat a range of
disorders in addition to the above immune and autoimmune diseases
when demonstrated to be associated with chlamydial infection by the
diagnostic procedures described herein; for example, various
infections, many of which produce inflammation as primary or
secondary symptoms, including, but not limited to, sepsis syndrome,
cachexia, circulatory collapse and shock resulting from acute or
chronic bacterial infection, acute and chronic parasitic and/or
infectious diseases from bacterial, viral or fungal sources, such
as a HIV, AIDS (including symptoms of cachexia, autoimmune
disorders, AIDS dementia complex and infections) can be treated, as
well as Wegners Granulomatosis.
[0127] Among the various inflammatory diseases, there are certain
features of the inflammatory process that are generally agreed to
be characteristic. These include fenestration of the
microvasculature, leakage of the elements of blood into the
interstitial spaces, and migration of leukocytes into the inflamed
tissue. On a macroscopic level, this is usually accompanied by the
familiar clinical signs of erythema, edema, tenderness
(hyperalgesia), and pain. Inflammatory diseases, such as chronic
inflammatory pathologies and vascular inflammatory pathologies,
including chronic inflammatory pathologies such as aneurysms,
hemorrhoids, sarcoidosis, chronic inflammatory bowel disease,
ulcerative colitis, and Crohn's disease and vascular inflammatory
pathologies, such as, but not limited to, disseminated
intravascular coagulation, atherosclerosis, and Kawasaki's
pathology are also suitable for treatment by methods described
herein. The invention can also be used to treat inflammatory
diseases such as coronary artery disease, hypertension, stroke,
asthma, chronic hepatitis, multiple sclerosis, peripheral
neuropathy, chronic or recurrent sore throat, laryngitis,
tracheobronchitis, chronic vascular headaches (including migraines,
cluster headaches and tension headaches) and pneumonia when
demonstrated to be pathogenically related to Chlamydia
infection.
[0128] Treatable disorders when associated with Chlamydia infection
also include, but are not limited to, neurodegenerative diseases,
including, but not limited to, demyelinating diseases, such as
multiple sclerosis and acute transverse myelitis; extrapyramidal
and cerebellar disorders, such as lesions of the corticospinal
system; disorders of the basal ganglia or cerebellar disorders;
hyperkinetic movement disorders such as Huntington's Chorea and
senile chorea; drug-induced movement disorders, such as those
induced by drugs which block CNS dopamine receptors; hypokinetic
movement disorders, such as Parkinson's disease; Progressive
supranucleo palsy; Cerebellar and Spinocerebellar Disorders, such
as astructural lesions of the cerebellum; spinocerebellar
degenerations (spinal ataxia, Friedreich's ataxia, cerebellar
cortical degenerations, multiple systems degenerations (Mencel,
Dejerine-Thomas, Shi-Drager, and Machado Joseph)); and systemic
disorders (Refsum's disease, abetalipoprotemia, ataxia,
telangiectasia, and mitochondrial multi-system disorder);
demyelinating core disorders, such as multiple sclerosis, acute
transverse myelitis; disorders of the motor unit, such as
neurogenic muscular atrophies (anterior horn cell degeneration,
such as amyotrophic lateral sclerosis, infantile spinal muscular
atrophy and juvenile spinal muscular atrophy); Alzheimer's disease;
Down's Syndrome in middle age; Diffuse Lewy body disease; Senile
Dementia of Lewy body type; Wernicke-Korsakoff syndrome; chronic
alcoholism; Creutzfeldt-Jakob disease; Subacute sclerosing
panencephalitis, Hallerrorden-Spatz disease; and Dementia
pugilistica, or any subset thereof.
[0129] It is also recognized that malignant pathologies involving
tumors or other malignancies, such as, but not limited to leukemias
(acute, chronic myelocytic, chronic lymphocytic and/or
myelodyspastic syndrome); lymphomas (Hodgkin's and non-Hodgkin's
lymphomas, such as malignant lymphomas (Burkitt's lymphoma or
Mycosis fungoides)); carcinomas (such as colon carcinoma) and
metastases thereof; cancer-related angiogenesis; infantile
hemangiomas; alcohol-induced hepatitis. Ocular neovascularization,
psoriasis, duodenal ulcers, angiogenesis of the female reproductive
tract, can also be treated when demonstrated by the diagnostic
procedures described herein to be associated with Chlamydial
infection.
[0130] An immunocompromised individual is generally defined as a
person who exhibits an attenuated or reduced ability to mount a
normal cellular or humoral defense to challenge by infectious
agents, e.g., viruses, bacterial, fungi and protozoa. Persons
considered immunocompromised include malnourished patients,
patients undergoing surgery and bone narrow transplants, patients
undergoing chemotherapy or radiotherapy, neutropenic patients,
HIV-infected patients, trauma patients, burn patients, patients
with chronic or resistant infections such as those resulting from
myeloodysplastic syndrome, and the elderly, all of who may have
weakened immune systems. A protein malnourished individual is
generally defined as a person who has a serum albumin level of less
than about 3.2 grams per deciliter (g/dl) and/or unintentional
weight loss greater than 10% of usual body weight.
[0131] The course of therapy, serological results and clinical
improvements from compassionate antichlamydial therapy in patients
diagnosed with the diseases indicated were observed and are
reported in Example 5. The data provides evidence to establish that
treatment of Chlamydia infection results in the serological and
physical improvement of a disease state in the patient undergoing
combination therapy. These observations were consistent among a
variety of different diseases which fall within a generalized
disease class.
OTHER DISEASES OF UNKNOWN ETIOLOGY WITH NEW EVIDENCE FOR A
CHLAMYDIA PNEUMONIAE ETIOLOGY
[0132] Both C. trachomatis and C. psittaci exhibit a protean
disease complex dependent on different serovars. One known basis
for this diversity to date is the amino acid sequence of the MOMP.
FIG. 1 shows a sequence alignment of various Chlamydia MOMPs. Note
that the size and sequence are relatively homologous except for the
four variable regions that are responsible for the serovar
(serotype) basis of classification. Further, it has been discovered
that C. pneumoniae infects blood vessel endothelial cells from
which EBs are released in the blood stream. In addition,
macrophages are known targets for C. pneumoniae and may serve as
reservoirs and provide an additional mechanism of transmission. C.
pneumoniae is thus able to spread throughout the human body,
establishing infection in multiple sites and in multiple organ
systems. Infected sites may exist for an extended period without
inducing symptoms that are noticed by the patient or by an
examining physician. Sequence variability of MOMPs or other
chlamydial antigens may provide a basis for organ specificity while
other chlamydial proteins, such as the 60K and 70K heat shock
proteins or LPS, may influence immune response.
[0133] C. psittaci and C. pecorum are known to cause a host of
infections in economically significant animals. Thus, the teachings
of this invention are relevant to animals. Throughout this
application and for purposes of this invention, "patient" is
intended to embrace both humans and animals. Virtually all rabbits
and mice tested to date have PCR signals for C. pneumoniae. They
can be used as appropriate animal models for treatment using
specific combination antibiotics to improve therapy. (Banks et al.,
Ameri. J. of Obstetrics and Gynecology 138(7Pt2):952-956 (1980));
(Moazed et al., Am. J. Pathol. 148(2):667-676 (1996)); (Masson et
al., Antimicrob. Agents Chemother. 39(9):1959-1964 (1995)); (Patton
et al., Antimicrob. Agents Chemother. 37(1):8-13 (1993)); (Stephens
et al., Infect. Immun. 35(2):680-684 (1982)); and (Fong et al., J.
Clin. Microbiol. 35(1):48-52 (1997)).
[0134] Coupled with these developments are the recently developed
rabbit models of coronary artery disease, where rabbits exposed to
C. pneumoniae subsequently develop arterial plaques similar to
humans (Fong et al., J. Clin. Microbiol. 35:48-52 (1997)). Most
recently, a study at St. George's Hospital in London found that
roughly 3/4 of 213 heart attach victims have significant levels of
antibodies to C. pneumoniae antibody and that those that have such
antibodies achieve significantly lower rates of further adverse
cardiac events when treated with antibiotics (Gupta et al.,
Circulation 95:404-407 (1997)). Taken together, these three pieces
of evidence (the bacteria found in diseased tissue, inoculation
with the bacteria causes diseases, and treating for the bacteria
mitigates disease) make a case for a causal connection.
ADJUNCT AGENTS USED IN CONJUNCTION WITH THE COMBINATION THERAPY
[0135] In addition to the combination therapies discussed above,
other compounds can be co-administered to an individual undergoing
antichlamydial therapy for the management of chronic/systemic
infection. For example, it may be desirable to include one or a
combination of anti-inflammatory agents and/or immunosuppressive
agents to amelioriate side-effects that may arise in response to a
particular antichlamydial agent, e.g., Herxheimer reactions.
Initial loading with an anti-inflammatory steroid can be introduced
to minimize side-effects of the antichlamydial therapy in those
patients in which clinical judgment suggests the possibility of
serious inflammatory sequelae.
[0136] Suitable anti-inflammatory agents (steroidal and
nonsteroidal agents) include, but are not limited to, Prednisone,
Cortisone, Hydrocortisone and Naproxin. Preferably the
anti-inflammatory agent is a steroidal agent, such as Prednisone.
The amount and frequency of administration of these adjunct
compounds will depend upon patient health, age, clinical status and
other factors readily apparent to the medical professional.
[0137] Vitamin C (2 gms bid) has also been introduced based on the
report that Vitamin C (ascorbic acid) at moderate intracellular
concentrations stimulates replication of C. trachomatis (Wang et
al., J. Clin. Micro. 30:2551-2554 (1992)) as well as its potential
effect on biofilm charge and infectivity of the bacterium and
specifically the EB (Hancock, R. E. W., Annual Review in
Microbiology, 38:237-264 (1984)).
[0138] Additionally, probenicid can optionally be added to the
therapy as an enhancer. Probenecid is known to increase plasma
levels of penicillins by blocking the uricosuric and renal tubular
secretion of these drugs.
DIAGNOSIS AND TREATMENT OF SECONDARY PORPHYRIA
[0139] Chlamydia is a parasite of normal energy production in
infected eukaryotic cells. As a result, host cells have
insufficient energy available for their normal functioning. The
energy shortage also causes the host cell mitochondria to attempt
to synthesize certain critical enzymes involved in energy
production in order to increase energy production. Because
Chlamydia also prevents this synthesis from completing, these
enzyme's precursors, called porphyrins, build up in cell and often
escape into the intracellular milieu. Porphyrins readily form
free-radicals, that, in turn, damage cells. Thus, there is an
obligate secondary porphyria that accompanies many chlamydial
infections. Therapy for this secondary porphyria, which is adjunct
to anti-chlamydial therapy, involves at least three strategies: a)
supplement the cellular energy supply to mitigate cell malfunction
and the formation of porphyrins; b) reduce the levels of systemic
porphyrins; and c) mitigate the harmful effects of the
porphyrins.
[0140] The pathogenesis of chronic/systemic chlamydial infection is
unique in that the intracellular infection by this parasite results
in a number of heretofore unrecognized concomitant and obligatory
metabolic/ autoimmune disorders including secondary porphyria with
associated autoantibodies against the porphyrins. Cross reaction
with Vitamin B 12 can result in a subclinical autoimmune-mediated
Vitamin B 12 deficiency. These associated disorders often require
diagnosis and preventive and/or specific adjunctive therapy.
[0141] The first of these concomitant disorders is a porphyria
which is a direct result of the chlamydial infection of host cells.
This form of porphyria is a secondary porphyria as it is not the
result of a genetic deficiency of the enzymes involved in the
biosynthesis of heme. Based upon the discovery of this secondary
form of porphyria, a unique approach for the diagnosis and
treatment of obligatory and secondary disorders caused by Chlamydia
infections has been developed. The adjunctive therapy described
herein can be used in combination with the appropriate
antimicrobial therapy required for eradication of the pathogen.
This adjunctive therapy for secondary porphyria is particularly
important for long-term antimicrobial therapy of chronic/systemic
infections as such therapy often evokes symptoms of secondary
porphyria.
[0142] The discussion below outlines the believed mechanism by
which Chlamydiae induce these secondary metabolic disorders. The
phrase "chlamydial-induced porphyria" is defined herein as an
obligatory and secondary metabolic disorder which is the direct
result of a chlamydial infection and which may find clinically
relevant phenotypic expression requiring interventional
therapy.
[0143] Chlamydiae are prokaryocytes that develop in eukaryotic
cells and utilize part of the host cell metabolism (Becker, Y.,
Microbiological Reviews, 42:247-306 (1978); McClairty, G.,
Microbiology, 2:157-164(1994)). The transition of elementary bodies
(EBs) to reticulate bodies (RBs) for Chlamydia species requires the
presence of functioning mitochondria in the infected cell as well
as the production by the host cell of nucleoside triphosphates
which are needed for chlamydial biosynthesis of nucleic acids
(Becker, Y., Microbiological Reviews, 42:247-306 (1978); McClairty,
G., Microbiology, 2:157-164(1994); Ormsbee, R. A. and Weiss, E.,
Science, 2:1077 (1963); Weiss, E., Jour. of Bacteriology,
90:243-253 (1965); Weiss, E. and Kiesow, L. A., Bacteriology
Proceedings, 85 (1966); Weiss, E. and Wilson, N. N., Jour. of
Bacteriology, 97:719 (1969); Hatch et al., Jour. of Bacteriology,
150:662-670 (1985)). Chlamydiae are known to possess fragments of
the glycolytic, pentose phosphate, and citric acid pathways and.
appear to be capable of converting glucose-6-phosphate (but not
glucose) to pyruvate and pentose (Ormsbee, R. A. and Weiss, E.,
Science, 2:1077 (1963); Weiss, E. and Kiesow, L. A., Bacteriology
Proceedings, 85 (1966)). However, Chlamydiae seem to lack enzymes
needed for the net generation of adenosine triphosphate
(ATP)(Weiss, E., Jour. of Bacteriology, 90:243-253 (1965)). Thus,
chlamydial development is dependent on 5 active mitochondrial and
nuclear function of the host cell. For this reason, Chlamydiae are
considered obligatory intracellular parasites (McClairty, G.,
Microbiology, 2:157-164(1994)). Chlamydial dependence on host cell
energy must necessarily deplete the host cell's existing energy
output at the net expense of depriving host cell biosynthetic
pathways.
[0144] The requirement of an exogenous source of ATP and the
presence of a specific ATP transport system in Chlamydiae have
provided supporting evidence for the energy parasite concept (Hatch
et al., Jour. of Bacteriology, 150:662-670 (1985)). This ATP
transport system is an ATP-adenosine diphosphate (ADP) exchange
mechanism (Peeling et al., Infect. and Immun., 57:3334-3344 (1989))
similar to that found in mitochondria (Penefsky, H. S. and Cross,
R. L., Adv. Enzym. and Rel. Areas in Molec. Bio., 64:173-214
(1991)). Moreover, electron microscopic studies have shown that
replicating Chlamydiae are always found in close proximity to
mitochondria. Therefore, it has been suggested that Chlamydiae
behave in the reverse manner of mitochondria in that mitochondria
import ADP from the host cell cytoplasm and export ATP, while
Chlamydiae import ATP and export ADP (Becker, Y., Microbiological
Reviews, 42:247-306 (1978)).
[0145] The production of ATP within the mitochondria is powered by
a mechanism called chemiosmotic coupling (Kalckar, H. M., Annu.
Review of Biochem., 60:1-37 (1991); Lehninger, A. L., The
Mitochondrion: Molecular Basis of Structure and Function, The
Benjamin Company, Incorporated, New York; Slater, E. C., Europ.
Journ. of Biochem., 166:489-504 (1987); Babcock, G. T. and
Wickstrom, M., Nature, 356:301-309 (1992); Senior, A. E.,
Physiology Review, 68:177-231 (1988); Pedersen, P. I. and Carafoli,
E., Trends in Biochem. Sci., 12:145-150 (1987); Pedersen, P. I. and
Carafoli, E., Trends in Biochem. Sci., 12:145-150 (1987)). The
citric acid cycle drives oxidation of NADH or FADH2, which, in
turn, releases a hydride ion (H-), which is quickly converted to a
proton (H+) and two high-energy electrons (2 e-). As the
high-energy electron pair is transferred to each of these three
multiprotein complexes, the protons produced pass freely from the
mitochondria matrix to the intermembrane space via channels in
complexes I, III and IV. Thus, the transfer of electrons from NADH
down the electron transport chain causes protons to be pumped out
of the mitochondrial matrix and into the intermembrane space. These
protons then reenter the matrix through a specific channel in
complex V. This proton gradient across the inner membrane results
in the proton motive force which drives ATP synthesis.
[0146] Chlamydial ATPase in essence is competing for protons with
host cell mitochondrial ATPase. This, of course, reduces the ATP
produced by the mitochondria. A net reduction of ATP in the host
cell mitochondria results in a concomitant lowering of the electron
transfer in the host cell mitochondria because electron transfer
and ATP synthesis are obligatorily coupled; neither reaction occurs
without the other. The establishment of a large electrochemical
proton gradient across the inner mitochondrial membrane halts
normal electron transport and can even cause a reverse electron
flow in some sections of the host cell respiratory chain. The
reduction of electron transfer in the host cell mitochondria, in
turn, lowers the translocation and reduction of extramatrix
mitochondrial ferric iron to intramatrix ferrous iron. This energy
depletion, in turn, interferes with the biosynthesis of heme.
A. BIOSYNTHESIS OF HEME
[0147] Heme is a Fe2+ complex in which the ferrous ion is held
within the organic ligand, tetrapyrrolic macrocycle. The
heme-containing tetrapyrrolic macrocyclic pigments are known as
porphyrinogens and play a major role in cellular biochemistry. A
number of critical cellular functions such as electron transport,
reduction of oxygen, and hydroxylation are mediated by a family of
heme-based cytochromes including catalase, peroxidase and
superoxide dismutase. Moreover, the oxygen-carrying properties of
hemoglobin and myoglobin are based on heme. Many cellular enzymes
such as cytochrome P-450 and tryprophan pyrolase contain heme.
[0148] The biosynthesis of heme (Battersby et al., Nature, 285:17-
(1980); Batterspy, A. R., Proceedings of the Royal Society of
London, 225:1-26 (1985)) is an energy-dependent process which is
adversely affected by depletion of host cell energy. The metabolic
consequence of the interruption of heme biosynthesis is porphyria
(Ellefson, R. D., Mayo Clinic Proceedings, 57:454-458 (1982);
Hindmarsh, J. T., Clin. Chem., 32:1255-1263 (1986); Meola, T. and
Lim, H. W., Bullous Diseases, 11:583-596 (1993); Moore, M. R.,
Int'l. Journ. of Biochem., 10:1353-1368 (1993)). Heme synthesis is
a series of irreversible biochemical reactions of which some occur
in the cell mitochondria and some in the cytoplasm. The
intramitochondrial reactions are mainly oxidation-reduction while
those in the cytosol are condensation and decarboxylation.
[0149] Porphyrinogens, porphyrins and porphyria are all related to
heme synthesis. The biosynthesis of heme occurs in all human cells
and involves a relatively small number of starting materials that
are condensed to form porphyrinogens; the porphyrins are formed
from the porphyrinogens by non-enzymatic oxidation. As
porphyrinogens progress through the heme biosynthesis pathway, the
numbers of carboxyl side groups on the corresponding porphyrins
decreases, as does the water solubility of the compounds.
[0150] The porphyrias are consequences of any impairment of the
formation of porphyrinogens or in their transformation to heme.
Porphyrins are formed from porphyrinogens by non-enzymatic
oxidation. Each of the various genetic porphyrias is linked to an
enzyme deficiency in the heme biosynthesis pathway. As a
consequence of the enzyme defects, there is increased activity of
the initial and rate-controlling enzyme of this biosynthesis
pathway that results in overproduction and increased excretion of
porphyrinogen precursors and porphyrinogens. The steps of heme
biosynthesis are laid out in Table 7.
7TABLE 7 Simplified outline of enzymes and precursors in the
Biosynthesis of Heme Other Enzyme precursor Inhibitor Result.sup.b
glycine and succinyl coenzyme A .DELTA.-ALA synthase pyridoxal 5'-
heme delta-aminolevulinic phosphate acid (.DELTA.-ALA) .DELTA.-ALA
dehydratase* lead porphobilinogen and heme (PBG) PBG deaminase*
tetrapyrrole hydoxymethylbilane uroporphyrinogen-
uroporphyrinogen-III.sup.a III cosynthase* uroporphyrinogen
7,6,5-carboxyl decarboxylase* porphyrinogen-III coproporphyrinogen
coproporphyrinogen- oxidase III protoporphyrinogen
protoporphyrinogen oxidase protoporphyrinogen protoporphyrin
oxidase ferrochelatase ferrous ion heme .sup.aIn the absence of
this step, the symmetric uroporphyrinogen-I is formed .sup.bBecomes
precursor of the next step *Present in circulating red cells
[0151] When porphyrinogens accumulate due to enzymatic defects in
the heme biosynthesis pathway, they are oxidized to
photosensitizing porphyrins. Porphyrins are classified as
photodynamic agents because they generally require
superoxide/oxygen/electrons to exert their damaging biologic
effects. Porphyrins may be converted from ground state to excited
state molecules after absorption of radiation. Excited state
porphyrins transfer energy to oxygen molecules and produce reactive
oxygen species such as singlet oxygen, superoxide anion, super
oxide radical, hydroxyl radical and hydrogen peroxide. Reactive
oxygen species have been noted to disrupt membrane lipids,
cytochrome P-450 and DNA structure. If these reactive oxygen
species are released into the extracellular space, as seen in acute
porphyria, autooxidation of surrounding tissue may result. Thus,
the accumulation of porphyrinogens/porphyrins in human tissues and
body fluids produces a condition of chronic system overload of
oxidative stress with long term effects particularly noted for
neural, hepatic and renal tissue.
B. CHLAMYDIA AND SECONDARY PORPHYRIA
[0152] As mentioned, ferric/ferrous translocation is a critical
step in the biosynthesis of heme as it catalyses the oxidative
entry of coproporphyrinogen into the mitochondria matrix as
protoporphyrin; Chlamydia interfere with this step by reducing
electron transfer in the host cell. When coproporhyrinogen is
unable to return to the mitochondrial matrix, it accumulates first
in the cytosol and then in the extracellular milieu. Within the
mitochondrial matrix, the final steps in the biosynthesis of heme
are halted. Because the accumulation of heme within the
mitochondrial matrix normally exerts a negative feedback on heme
biosynthesis, the reduction of heme caused by the inability of
coproporphyrinogen to return to the mitochondrial matrix results in
the increased production of heme precursors such as .DELTA.-ALA and
PBG, the first and second products in heme biosynthesis. Thus,
porphyrin precursors such as .DELTA.-ALA and PBG begin to
accumulate in the mitochondrial matrix, then in the cytosol, and
then in the extracellular milieu.
[0153] Depletion of host cell energy by the intracellular infection
with Chlamydia species causes additional energy-related
complications. As fewer electrons are available to move through the
electron transport chain of the host cell mitochondrial matrix
membrane, the citric acid cycle produces more succinyl-CoA which,
in turn, promotes increased synthesis of .DELTA.-ALA. The net
result is an increased amount of heme precursors which become
porphyrins. The presence of porphyrins in the mitochondrial matrix
damages the cell as these molecules are unstable and form free
radicals. The high energy electrons generated by these free
radicals is "captured" by ubiquinone and cytochrome c which are
present in the mitochondrial matrix membrane. This, of course,
effectively uncouples electron transport from ATP synthesis and
"short circuits" the proton-motice force: ATP synthesis is then
reduced. Less ATP, in turn, means increased porphyrins and a
destructive cycle is begun.
[0154] The clinical result of the intracellular and extracellular
accumulation of porphyrins, if extensive, is a tissue/organ
specific porphyria which produces many of the classical
manifestations of hereditary porphyria. As the chlamydial-infected
host cells lyse, as happens in the normal life cycle of Chlamydia,
the intracellular porphyrins are released and result in a secondary
porphyria. Moreover, when the chlamydial infection involves hepatic
cells, the use of any pharmacologic agents that are metabolized by
cytochrome P-450 in the liver will increase the need for cytochrome
P-450, which is a heme-based enzyme. Hence, the biosynthesis of
heme in the liver becomes increased. When hepatic cells are
infected with Chlamydia species, the decreased energy in the host
cell does not allow heme biosynthesis to go to completion and
porphyrins in the liver/entero-hepatic circulation are increased.
It also has been noted that any host cell infected with Chlamydia
species has an increased amount of intracellular porphyrins that
are released when antimicrobial agents kill the microorganism.
[0155] Although a number of investigators have reported enigmatic
porphyria in patients who had no evidence of abnormal enzymes in
the heme biosynthesis pathway (Yeung Laiwah et al., Lancet,
i:790-792 (1983); Mustajoki, P. and Tenhunen, R., Europ. Journ. of
Clin. Invest., 15:281-284 (1985)), the intrinsic secondary,
obligatory porphyria caused by chlamydial infection disclosed
herein has neither been described nor hypothesized in the medical
literature. This obligatory secondary porphyria clearly is of
paramount importance in dealing with chronic systemic chlamydial
infections as are seen with intravascular infections caused by
Chlamydia pneumoniae.
[0156] The diagnosis of chlamydial-associated secondary porphyria
is important because of the well known neuropsychiatiric
manifestations of porphyrias (Gibson et al., Journal of Pathology
and Bacteriology, 71:495-509 (1956); Bonkowsky et al., Seminars in
Liver Diseases, 2:108-124 (1982); Brennan et al., International
Journal of Biochemistry, 833-835 (1980); Burgoyne et al.,
Psychotherapy and Psychosomatics, 64:121-131 (1995)). Moreover,
chronic exposure to excess porphyrins has been associated with
cancer (Kordac V., Neoplasma, 19:135-139 (1972); Lithner et al.,
Acta Medica Scandanavia, 215:271-274 (1984)). Of particular
interest is that infection with Chlamydia pneumoniae has been
associated with lung cancer (Cerutti PA., Science, 227:375-381
(1985)).
[0157] The diagnosis of genetic porphyria in patients with systemic
chlamydial infections is important as these patients may
precipitate a severe porphyric attack when they receive
antimicrobial agents to treat their infection. Thus, in order to
control the severe porphyria, these patients may require
intravenous hematin and/or plasmapheresis in addition to the oral
anti-porphyric agents. In contrast, the diagnosis of
chlamydial-associated secondary porphyria may be difficult as the
porphyria may be minimal and tissue-specific. The measurement of 24
hour urine porphyrins is not sensitive enough in every case of
chlamydial infection to detect the secondary porphyria caused by
chlamydial infection.
[0158] In view of the foregoing discussion of the etiology of
porphyria, one aspect of the invention pertains to methods for
differentiating porphyria caused by Chlamydia from that caused by a
latent genetic disorder in an individual. The method comprises
treating infection by Chlamydia at all stages of its life cycle,
using the therapies described in detail elsewhere in this
disclosure, and then assessing whether symptoms of porphyria have
been reduced. A reduction in the symptoms of porphyria (e.g.,
biochemical, enzymatic or physical manifestation) are indicative
that the porphyria is a secondary porphyria caused by
Chlamydia.
[0159] The diagnosis of genetic porphyria is most easily done
during an acute porphyric attack as there are porphyrinogen
precursors and porphyrins in the blood, urine and stool (Kauppinen
et al., British Journal of Cancer, 57:117-120(1988)). The diagnosis
of secondary porphyria is not as easy to do as there may not be an
abnormal amount of porphyrinogen precursors and porphyrins in the
blood, urine, or stool. However, several early enzymes in the
pathway for heme biosynthesis can be readily measured in peripheral
red blood cell (Percy et al., South African Forensic Medicine
Journal, 52:219-222 (1977); Welland et al., Metabolism, 13:232-250
(1964); McColl et al., Journal of Medical Genetics, 19:271-276
(1982)). Specific hereditary porphyrias that can be diagnosed with
the measurement of low levels of peripheral red blood cell enzymes
are acute intermittent porphyria, congenital erythropoietic
porphyria, .DELTA.-aminolevulinic acid dehydratase deficiency
porphyria, and porphyria cutanea tarda. Therefore, elevated
porphyrin levels in patients who do not have low levels of these
enzymes is suggestive of a non-genetic porphyria, such as
chlamydially induced secondary porphyria. For example, in one
embodiment, porphyria caused by Chlamydia in an individual having
symptoms associated therewith can be diagnosed by determining the
presence and/or amount of obligatory enzymes in heme biosynthesis
in red blood cells of the individual. The presence or amount of the
obligatory enzyme is compared to a normal patient who does not have
porphyria or to an earlier test result in the patient to determine
the patient's porphyria symptoms and/or whether therapy is
effective. For example, the presence of ALA synthase and/or PBF
deaminase or any of the other known enzymes involved in heme
biosynthesis (see Table 7), in abnormal levels (i.e., significant
deviation from normal levels in healthy patients who do not have
genetic porphyria) is indicative of secondary porphyria.
[0160] The diagnosis of chlamydial-associated secondary porphyria
may be difficult as the porphyria may be minimal and
tissue-specific. The measurement of 24 hour urine or stool
porphyrins may not be sensitive enough in many cases of chlamydial
infection to detect the secondary porphyria. Here, the diagnosis
depends on the fact that if excess porphyrins are reaching the
circulation, the precursor red blood cells will absorb these and
make heme. Thus, the enzymes for heme biosynthesis in the
differentiated red blood cell become elevated and remain elevated
for the life of the red cell. This allows the diagnosis of episodic
low-level secondary porphyria as is seen with chlamydial
infections. Thus, elevated heme synthesis levels can be used to
diagnose intracellular porphyria. See Example 7.
[0161] As discussed above, some patients having a Chlamydia-induced
porphyria do not have abnormal levels of heme precursors. For those
patients it may be appropriate to determine the presence of
Chlamydia as well as porphyrins in the individual. The presence of
both the pathogen and porphyrins (e.g., determined by ELISA assay
described below) is indicative of secondary chlamydial porphyria,
rather than a genetic based porphyria. A proper diagnosis can thus
determine the therapeutic regimen needed to treat infection and
symptoms of secondary porphyria.
[0162] The inventors have discovered the existence of antibodies to
the various metabolites of heme biosynthesis, as well as Vitamin B
12 (cobalamin), which is molecularly similar to these metabolites,
in patients with active systemic infection with C. pneumoniae. The
antibodies are primarily IgM; this is similar to the antibody
responses to the MOMP of C. pneumoniae in severely symptomatic
patients. Example 8 illustrates titers in symptomatic patients with
systemic C. pneumoniae infections. The presence of antibodies to
Vitamin B 12 may have functional significance by decreasing the
amount of bioavilable Vitamin B 12. Thus, a Chlamydia infection may
cause a previously unrecognized secondary Vitamin B 12 deficiency.
Administration (e.g., intramuscular) of large quantities of
VitaminB 12 (1000 to 5000 .mu.g) (e.g., parenteral cobalamin
therapy) creates large amounts of Vitamin B 12 available for
binding to the native receptors of antibodies with an affinity for
Vitamin B 12, thereby saturating these anti-Vitamin B 12 antibodies
and increasing the amount of bioavailable circulating Vitamin
B12.
[0163] The previously unknown fact that the body produces
antibodies to porphyrins makes it possible to diagnose the presence
of porphyrins in a patient or animal by determining the presence of
anti-porphyrin antibodies. The inventors have developed a method in
which IgM and IgG antibodies to porphyrins can be measured with an
ELISA method. This has been shown to be a much more accurate method
to determine the chronic presence of porphyrins.
[0164] Porphyrins can also be used to create monoclonal and
polyclonal antibodies using standard methods known to any one
skilled in the field. These antibodies can be used in a variety of
diagnostic assays and anti-porphyrin therapeutic strategies.
[0165] Treatment of Chlamydia infection may exacerbate secondary
porphyria by increasing the metabolism of cryptic Chlamydia or by
accelerating the death of infected cells with elevated
intracellular porphyrin levels.
[0166] Once secondary porphyria is diagnosed, chlamydial infection
and symptoms associated with porphyria can be treated. The
following therapeutic regimen is aimed at controlling the
chlamydial-associated secondary/obligatory porphyria, symptoms of
which can actually increase during antimicrobial therapy of the
chlamydial infection. This porphyric reaction to antimicrobial
therapy should be recognized as such and differentiated from the
expected cytokine-mediated immune response precipitated by antigen
dump during anti-chlamydial therapy. These obligatory and secondary
chlamydial metabolic disorders are treated by specific diets and a
combination of pharmacological agents, each directed at different
aspects of the metabolic disorders. For example, chlamydial-induced
porphyria can be treated with a specific antiporphyric diet and a
combination of antiporphyric agents, each directed at different
aspects of porphyrins/porphyria. For purposes of this invention,
the term "antiporphyric agent(s)" is intended to embrace any of the
therapies described herein for management of porphyria. In addition
to the antiporphyric diet and antiporphyric agents, the patient may
require intravenous glucose and hematin, renal dialysis, and/or
plasmaphoresis, particularly for those patients having both genetic
porphyria and secondary porphyria induced by a chlamydial
infection. Suitable diets and antiporphyric agents are described in
detail below.
C. THERAPIES TO ENHANCE CELLULAR FUNCTION
[0167] Glucose is an important source of cellular energy. Glucose
levels can be enhanced by diet and through vitamin supplements as
described below.
[0168] A high carbohydrate diet should be maintained to promote
production of glucose (Pierach et al., Journal of the American
Medical Association, 257:60-61 (1987)). Approximately 70% of the
caloric intake should be in the form of complex carbohydrates such
as bread, potato, rice and pasta. The remaining 30% of the daily
diet should comprise protein and fat, which should ideally be in
the form of fish or chicken. Red meats, including beef, dark
turkey, tuna and salmon, contain tryptophan. Increased levels of
tryptophan in the liver inhibit the activity of phosphoenol
pyruvate carboxykinase with consequent disruption of
gluconeogenesis. This accounts for the abnormal glucose tolerance
seen in porphyria. Increased plasmic concentrations of tryptophan
also enhances tryptophan transport into the brain. The
concentration of tryptophan in the brain is the rate-limiting
factor for the synthesis of the neurotransmitter
5-hydroxytryptamine (5-HT, serotonin). Serotonin is synthesized by
the endothelium of brain capillaries for circulating tryptophan.
Thus, increased concentrations of tryptophan in the brain would be
expected to enhance production of serotonin and its metabolic,
5-hydroxyindole-acetic acid (5HIAA). Acute increases in serotonin
turnover in the brain are followed by vascular and metabolic
changes which include decreases in glucose consumption,
disturbances in EEG tracings, and decreases in the postischemic
neurological score. In addition, while serotonin increases brain
perfusion on a single injection, repetitive administration
initially opens the blood-brain barrier and subsequently induces
vasoconstriction. It is likely that any transient opening of the
blood-brain barrier by serotonin could allow circulating substrates
such as ALA and PBG, if present, to enter the central nervous
system. As would be expected from the location of serotonin
receptors and from the barrier function of the endothelium of
cerebral arteries, the constricting effect of serotonin is
amplified in cerebral arteries where endothelium is damage or
removed. Damaged endothelial cells, as would be expected with
chlamydial infection, would no longer have operational catabolic
processes for serotonin. This would be particularly true in the
event of depleted ATP as caused by chlamydial infection. This means
that increased concentrations of serotonin will reach the smooth
muscle layer of the cerebral vessels and cause more constriction.
Finally, serotonin is also stored in blood platelets. Because blood
platelets do not adhere and aggregate under normal conditions, they
do not release serotonin when the vessel lumen is intact. However,
if the vessel lumen is altered by chlamydial infection, platelet
deposition and release of serotonin can occur.
[0169] Another adverse effect of increased serotonin levels due to
porphyria is seen with nervous tissues. Sympathetic nerve endings
store serotonin taken up from the circulation. These serotonergic
neurons form plexuses around brain vessels where they are likely to
liberate their serotonin contents when subjected to cellular lysis
from any cause including ischemia, free-radical ionizing damage to
cell membranes, and/or chlamydial infection.
[0170] In rats, elevated circulating tryptophan has been shown to
produce structural alteration of brain astrocytes, oligodendroglia,
and neurons, as well as degeneration of Purkinje cells and wasting
of axons. Similar neurohistological alterations have been reported
in patients with acute porphyria. Elevated tryptophan levels in
plasma and brain have been associated with human encepholopathy.
Finally, serotonin is also recognized as an active neurotransmitter
in the gastrointestinal tract. The pharmacologic effects of
serotonin in the central nervous system and gastrointestinal tract
resemble the neurological manifestations of acute porphyric
attacks. In fact, administration of either tryptophan or serotonin
to humans have been reported to cause severe abdominal pain,
psychomotor disturbances, nausea, and dysuria; all of which are
symptoms of acute porphyria.
[0171] Sucrose and fructose should be avoided (Bottomly et al.,
American Journal of Clinical Pathology, 76:133-139 (1981)) because
the ingestion of large amounts of fructose trigger hepatic
gluconeogenesis which then decreases the available glucose which is
derived from glycogen breakdown within the liver. It is recommended
that sport drinks which contain glucose be consumed.
[0172] It is recommended that a patient suffering from porphyria
avoid milk products. Milk products contain lactose and lactoferrin,
and have been empirically shown to make symptoms of porphyria
worse.
[0173] Multivitamins containing the B complex vitamins should be
administered daily (e.g., one or multiple times), preferably in
excess of RDA, to enhance glucose availability. Hepatic breakdown
of glycogen with generation of glucose is assisted by taking these
multivitamins that contain the B complex vitamins. Pyridoxine
minimizes the porphyrin related porphyrial neuropathy. B complex
vitamins include folic acid (e.g., 400 .mu.g per dosage; 1200 .mu.g
daily maximum); vitamin B-1 (thiamin; e.g., 10 mg per dosage; 30 mg
daily maximum); B-2 (riboflavin; e.g., 10 mg per dosage; 30 mg
daily maximum); B-5 (panothenate; e.g., 100 mg per dosage; 300 mg
daily maximum); B-6 (pyridoxine; e.g., 100 mg per dosage; 300 mg
daily maximum) or pyridoxal-5-phosphate (e.g., 25 mg per dosage;
100 mg daily maximum) and B-12 (e.g., 500 .mu.g per dosage; 10,000
.mu.g daily maximum). The preferred method of administration is
oral for the majority of these vitamins (twice daily), except for
B-12 for which sublingual administration (three-times daily) is
preferred. It has been discovered that one important effect of this
secondary porphyria in some patients is the production of IgM and
IgG antibodies against coproporphyrinogen-III. These antibodies
cross-react with Vitamin B12 (cobalamin) and can thus cause a
deficiency. Vitamin B12 supplementation (e.g., parenteral cobalamin
therapy) can remedy the deficiency.
D. REDUCING PORPHYRIN LEVELS
[0174] Dietary and pharmaceutical methods can be used to reduce
systemic porphyrin levels (both water-soluble and fat-soluble).
[0175] Plenty of oral fluids in the form of bicarbonated water or
"sports drinks" (i.e., water with glucose and salts) should be
incorporated into the regimen. This flushes water-soluble
porphyrins from the patient's system. Drinking seltzer water is the
easiest way to achieve this goal. The color of the urine should
always be almost clear instead of yellow. It is noted that
dehydration concentrates prophyrins and makes patients more
symptomatic.
[0176] Activated charcoal can be daily administered in an amount
sufficient to absorb fat-soluble porphyrins from the enterohepatic
circulation. Treatment with activated oral charcoal, which is
nonabsorbable and binds porphyrins in the gastrointestinal tract
and hence interrupts their enterohepatic circulation, has been
associated with a decrease of plasma and skin porphyrin levels.
Charcoal should be taken between meals and without any other oral
drugs or the charcoal will absorb the food or drugs rather than the
porphyrins. For those who have difficulty taking the charcoal due
to other medications being taken during the day, the charcoal can
be taken all at one time before bed. Taking between 2 and 20 grams,
preferably at least 6 grams (24.times.250 mg capsules) of activated
charcoal per day (Perlroth et al., Metabolism, 17:571-581 (1968))
is recommended. Much more charcoal can be safely taken; up to 20
grams six times a day for nine months has been taken without any
side effects.
[0177] For severe porphyria, chelating and other agents may be
administered, singularly or in combination, to reduce levels of
porphyrins in the blood. Examples of chelating agents include but
are not limited to Kemet (succimer; from about 10 mg/kg to about 30
mg/kg); ethylene diamine tetracetic acid (EDTA); BAL (dimercaprol;
e.g., 5 mg/kg maximum tolerated dosage every four hours), edetate
calcium disodium (e.g., from about 1000 mg/m.sup.2 to about 5000
mg/m.sup.2 per day; can be used in combination with BAL);
deferoxamine mesylate (e.g., from about 500 mg to about 6000 mg per
day); trientine hydrochloride (e.g., from about 500 mg to about 3 g
per day); panhematin (e.g., from about 1 mg/kg to about 6 mg/kg per
day), penacillamine. Intravenous hematin may also be administered.
Quinine derivatives, such as but limited to hydroxychloroquine,
chloroquine-and quinacrine, should-be administered to the patient
daily at a dosage of from about 100 mg to about 400 mg per day,
preferably about 200 mg once or twice per day with a maximum daily
dose of 1 g. Hydrochloroquine is most preferred. The mechanism of
action of hydroxychloroquine is thought to involve the formation of
a water-soluble drug-porphyria complex which is removed from the
liver and excreted in the urine (Tschudy et al., Metabolism,
13:396-406 (1964); Primstone et al., The New England Journal of
Medicine, 316:390-393 (1987)).
[0178] To reduce severe porphyric attacks during therapy for
chronic Chlamydia infections, the use of hemodialysis,
plasmapheresis, chelating agents and/or intravenous hematin may be
needed. Any one of these or a combination thereof can be used to
treat the patient and is well within the knowledge of the skilled
artisan how to carry out these adjunct therapies.
E. MITIGATING THE EFFECTS OF PORPHYRINS
[0179] Antioxidants at high dosages (preferably taken twice per
day) help to mitigate the effects of free radicals produced by
porphyrins. Examples of suitable antioxidants include but are not
limited to Vitamin C (e.g., 1 gram per dosage; 10 g daily maximum);
Vitamin E (e.g., 400 units per dosage; 3000 daily maximum);
L-Carnitine (e.g., 500 mg per dosage; 3 g daily maximum); coenzyme
Q-10 (uniquinone (e.g., 30 mg per dosage; 200 mg daily maximum);
biotin (e.g., 5 mg per dosage; 20 mg daily maximum); lipoic acid
(e.g., 400 mg per dosage; 1 g daily maximum); selenium (e.g., 100
.mu.g per dosage; 300 .mu.g daily maximum); gultamine (e.g., from 2
to about 4 g per dosage); glucosamine (e.g., from about 750 to
about 1000 mg per dosage); and chondroitin sulfate (e.g., from
about 250 to about 500 mg per dosage).
[0180] The above-mentioned therapeutic diets can be combined with
traditional or currently recognized drug therapies for porphyria.
In one embodiment, benzodiazapine drugs, such as but not limited to
valium, klonafin, flurazepam hydrochloride (e.g., Dalmanc3, Roche)
and alprazolam (e.g., Xanax), can be administered. Preferably,
sedatives, such as alprazolam (e.g., Xanax; 0.5 mg per dosage for 3
to 4 times daily), can be prescribed for panic attacks and
flurazepam hydrochloride (e.g., Dalmane3, Roche or Restoril3 (e.g.,
30 mg per dosage)) can be prescribed for sleeping. The rationale is
based upon the presence of peripheral benzodiazepine receptors in
high quantities in phagocytic cells known to produce high levels of
radical oxygen species. A protective role against hydrogen peroxide
has been demonstrated for peripheral benzodiazipine receptors. This
suggests that these receptors may prevent mitochondria from radical
damages and thereby regulate apoptosis in the hematopoietic system.
Benzodiazepines have also been shown to interfere with the
intracellular circulation of heme and porphyrinogens (Scholnick et
al., Journal of Investigative Dermatology, 1973, 61:226-232). This
is likely to decrease porphyrins and their adverse effects. The
specific benzodiazipine will depend on the porphyrin-related
symptoms.
[0181] Cimetidine can also be administered separately or in
combination with benzodiazepine drugs. Cimetidine has been shown to
effectively scavenge hydroxyl radicals although it is an
ineffective scavenger for superoxide anion and hydrogen peroxide.
Cimetidine appears to be able to bind and inactivate iron, which
further emphasizes its antioxidant capacity. Cimetidine also is an
effective scavenger for hypochlorous acid and monochloramine, which
are cytotoxic oxidants arising from inflammatory cells, such as
neutrophils. Cimetidine thus would be expected to be useful for the
therapy of free-radical-mediated oxidative damage caused by
chlamydial porphyria. Recent studies in Japan have found that
cimetadine is effective for treating porphyria. The recommended
amount of cimetadine is about 400 mg once or twice per day.
[0182] The complexity of the chiamydial life cycle, the host
response to infection as well as to therapy, the high frequency of
untoward side effects of antimicrobial therapy, the obligatory
metabolic disorders, and the need for prolonged therapy make
patient education, monitoring and support a necessary and key
factor in the successful eradication of chronic/systemic chlamydial
infections. When the presence of chlamydial in the blood is
detected by culture and/or PCR and the IgM and IgG antibody titers
are elevated, a presumptive diagnosis of chronic/systemic
chlamydial infection is made. The potential for secondary effects
such as porphyria should then be screened. For example, this can be
evaluated by performing one or a combination of the following
tests: 1) complete blood count (CBC); 2) Liver function tests; 3)
Uric acid; 4) Serum iron studies; 5) IgM and IgG antibodies to
coproporpyrinogen-III and Vitamin B12; and, 6) ALA dehydratase and
PBG deaminase. Urine and stool samples should also be tested for
presence of porphyrins, preferably using 24 hour samples. In a
preferred embodiment of the therapeutic regimen, the patient is
placed on the antiporphyric regimen, preferably for at least two
weeks before any antibiotics are started. Following this, a
reducing agent is started. These include amoxicillin (500 mg every
12 hours), penicillamine (250 mg every 12 hours), and cycloserine
(250 mg every 12 hours). The patient is closely monitored for at
least two weeks on this regimen to determine if any side effects
occur. This regimen is continued for the entire course of therapy
and is critical as it decreases the EB load. After the patient has
adjusted to the amoxicillin or penicillamine, a combination of
antimicrobial agents is added. The patient is closely monitored to
determine tolerance to the antimicrobial agents.
[0183] Vitamins, antioxidants and other antiporphyric agents can be
incorporated, in the amounts described herein, into nutraceuticals,
medical foods, dietary supplements and dietary nutritional
formulations including beverages and foods such as nutritional bar,
for the management of non-genetic, secondary porphyria caused by a
Chlamydia infection. Alternatively, a combination of vitamins and
antioxidants can be co-packed in a pack or kit as described
elsewhere herein and/or co-formulated into a composition in amounts
suitable for administration to an individual having non-genetic,
secondary prophyria.
MODES OF ADMINISTRATION
[0184] Based upon the ability of the-combination therapy of this
invention to improve both the serological and physical status of a
patient undergoing treatment, pharmaceutical compositions or
preparations can be made comprising at least two different agents
chosen from the following groups: a) at least one agent targeted
against elementary body phase of chlamydial life cycle (e.g.,
disulfide reducing agents); b) at least one agent targeted against
replicating phase of chlamydial life cycle (e.g., antimycobacterial
agents); and c) at least one agent targeted against cryptic phase
of chlamydial life cycle (e.g., anaerobic bactericidal agents). As
discussed in greater detail below, the agents can be formulated in
a physiologically acceptable vehicle in a form which will be
dependent upon the method in which it is administered.
[0185] In another aspect, the invention pertains to a combination
of agents comprising at least two agents, each of which is targeted
against a different phase of the chlamydial life cycle, as
previously discussed. The combination of antichlamydial agents can
be used in the management of chlamydial infection or prophylaxis
thereof to prevent recurrent infection. The combination of agents
can be in the form of an admixture, as a pack (discussed in detail
below) or individually, and/or by virtue of the instruction to
produce such a combination. It should be understood that
combination therapy can comprise multiple agents that are effective
within a particular phase of the chlamydial life cycle. The
combination of antichlamydial agents can further comprise
immunosuppressants, anti-inflammatory agents, vitamin C and
combinations thereof.
[0186] In a preferred embodiment, if only one antichlamydial agent
is elected to be used in an asymptomatic patient to reduce/prevent
chronic infection, this agent is a reducing agent, such as
penicillamine.
[0187] The novel therapeutic methods described herein can be used
to ameliorate conditions/symptoms associated with the disease
states described above, when the disease is onset or aggravated by
infection by Chlamydia. The agents of this invention can be
administered to animals including, but not limited to, fish,
amphibians, reptiles, avians and mammals including humans.
Compounds and agents described herein can be administered to an
individual using standard methods and modes which are typically
routine for the disease state.
[0188] Combination(s) of antichlamydial agents of this invention
can be used for the manufacture of a medicament for simultaneous,
separate or sequential use in managing chlamydial infection or
prophylaxis thereof. The agents can also be used for the
manufacture of a medicament for therapy of a disease associated
with chlamydia infection, such as autoimmune disease, inflammatory
disease, immunodeficiency disease.
[0189] The agents can be administered subcutaneously,
intravenously, parenterally, intraperitoneally, intradermally,
intramuscularly, topically, enteral (e.g., orally), sublingually,
rectally, nasally, buccally, vaginally, by inhalation spray, by
drug pump or via an implanted reservoir in dosage formulations
containing conventional non-toxic, physiologically acceptable
carriers or vehicles. The preferred method of administration is by
oral delivery. The form in which it is administered (e.g., syrup,
elixir, capsule, tablet, solution, foams, emulsion, gel, sol) will
depend in part on the route by which it is administered. For
example, for mucosal (e.g., oral mucosa, rectal, intestinal mucosa,
bronchial mucosa) administration, via nose drops, aerosols,
inhalants, nebulizers, eye drops or suppositories can be used. The
compounds and agents of this invention can be administered together
with other biologically active agents.
[0190] In a specific embodiment, it may be desirable to administer
the agents of the invention locally to a localized area in need of
treatment; this may be achieved by, for example, and not by way of
limitation, local infusion during surgery, topical application
(e.g., for skin conditions such as psoriasis), transdermal patches,
by injection, by means of a catheter, by means of a suppository, or
by means of an implant, said implant being of a porous, non-porous,
or gelatinous material, including membranes, such as sialastic
membranes or fibers. For example, the agent can be injected into
the joints.
[0191] In a specific embodiment when it is desirable to direct the
drug to the central nervous system, techniques which can
opportunistically open the blood brain barrier for a time adequate
to deliver the drug there through can be used. For example, a
composition of 5% mannitose and water can be used. In another
embodiment, the agents can be delivered to a fetus through the
placenta since many of the agents are small enough to pass through
the placental barrier.
[0192] The present invention also provides pharmaceutical
compositions. Such compositions comprise a therapeutically (or
prophylactically) effective amount of the agent, and a
pharmaceutically acceptable carrier or excipient. Such a carrier
includes but is not limited to saline, buffered saline, dextrose,
water, glycerol, ethanol, and combinations thereof. The carrier and
composition can be sterile. The formulation should suit the mode of
administration.
[0193] Suitable pharmaceutically acceptable carriers include but
are not limited to water, salt solutions (e.g., NaCl), alcohols,
gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols,
gelatin, carbohydrates such as lactose, amylose or starch,
magnesium stearate, talc, silicic acid, viscous paraffin, perfume
oil, fatty acid esters, hydroxymethylcellulose, polyvinyl
pyrolidone, etc. The pharmaceutical preparations can be sterilized
and if desired, mixed with auxiliary agents, e.g., lubricants,
preservatives, stabilizers, wetting agents, emulsifiers, salts for
influencing osmotic pressure, buffers, coloring, flavoring and/or
aromatic substances and the like which do not deleteriously react
with the active compounds.
[0194] The composition, if desired, can also contain minor amounts
of wetting or emulsifying agents, or pH buffering agents. The
composition can be a liquid solution, suspension, emulsion, tablet,
pill, capsule, sustained release formulation, or powder. The
composition can be formulated as a suppository, with traditional
binders and carriers such as triglycerides. Oral formulation can
include standard carriers such as pharmaceutical grades of
mannitol, lactose, starch, magnesium stearate, polyvinyl
pyrollidone, sodium saccharine, cellulose, magnesium carbonate,
etc.
[0195] The composition can be formulated in accordance with the
routine procedures as a pharmaceutical composition adapted for
intravenous administration to human beings. Typically, compositions
for intravenous administration are solutions in sterile isotonic
aqueous buffer. Where necessary, the composition may also include a
solubilizing agent and a local anesthetic to ease pain at the site
of the injection. Generally, the ingredients are supplied either
separately or mixed together in unit dosage form, for example, as a
dry lyophilized powder or water free concentrate in a hermetically
sealed container such as an ampoule or sachette indicating the
quantity of active agent. Where the composition is to be
administered by infusion, it can be dispensed with an infusion
bottle containing sterile pharmaceutical grade water, saline or
dextrose/water. Where the composition is administered by injection,
an ampoule of sterile water for injection or saline can be provided
so that the ingredients may be mixed prior to administration.
[0196] For topical application, there are employed as nonsprayable
forms, viscous to semi-solid or solid forms comprising a carrier
compatible with topical application and having a dynamic viscosity
preferably greater than water. Suitable formulations include but
are not limited to solutions, suspensions, emulsions, creams,
ointments, powders, enemas, lotions, sols, liniments, salves,
aerosols, etc., which are, if desired, sterilized or mixed with
auxiliary agents, e.g., preservatives, stabilizers, wetting agents,
buffers or salts for influencing osmotic pressure, etc. The drug
may be incorporated into a cosmetic formulation. For topical
application, also suitable are sprayable aerosol preparations
wherein the active ingredient, preferably in combination with a
solid or liquid inert carrier material, is packaged in a squeeze
bottle or in admixture with a pressurized volatile, normally
gaseous propellant, e.g., pressurized air.
[0197] Agents described herein can be formulated as neutral or salt
forms. Pharmaceutically acceptable salts include those formed with
free amino groups such as those derived from hydrochloric,
phosphoric, acetic, oxalic, tartaric acids, etc., and those formed
with free carboxyl groups such as those derived from sodium,
potassium, ammonium, calcium, ferric hydroxides, isopropylamine,
triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
[0198] The amount of agents which will be effective in the
treatment of a particular disorder or condition will depend on the
nature of the disorder or condition, and can be determined by
standard clinical techniques. In addition, in vitro or in vivo
assays may optionally be employed to help identify optimal dosage
ranges. The precise dose to be employed in the formulation will
also depend on the route of administration, and the seriousness of
the disease or disorder, and should be decided according to the
judgment of the practitioner and each patient's circumstances.
Effective doses may be extrapolated from dose-response curves
derived from in vitro or animal model test systems.
[0199] The invention also provides a pharmaceutical pack or kit
comprising one or more containers filled with one or more of the
ingredients of the pharmaceutical compositions and/or adjunct
therapies of the invention. Optionally associated with such
container(s) can be a notice in the form prescribed by a
governmental agency regulating the manufacture, use or sale of
pharmaceuticals or biological products, which notice reflects
approval by the agency of manufacture, use of sale for human
administration. The pack or kit can be labeled with information
regarding mode of administration, sequence of drug administration
(e.g., separately, sequentially or concurrently), or the like. The
pack or kit may also include means for reminding the patient to
take the therapy. The pack or kit can be a single unit dosage of
the combination therapy or it can be a plurality of unit dosages.
In particular, the agents can be separated, mixed together in any
combination, present in a single vial or tablet. Agents assembled
in a blister pack or other dispensing means is preferred. For the
purpose of this invention, unit dosage is intended to mean a dosage
that is dependent on the individual pharmacodynamics of each agent
and administered in FDA approved dosages in standard time
courses.
DIAGNOSTIC REAGENTS
[0200] The invention also provides a diagnostic reagent pack or kit
comprising one or more containers filled with one or more of the
ingredients used in the assays of the invention. Optionally
associated with such container(s) can be a notice in the form
prescribed by a governmental agency regulating the manufacture, use
or sale of diagnostic products, which notice reflects approval by
the agency of manufacture, use of sale for human administration.
The pack or kit can be labeled with information regarding mode of
administration, sequence of execution (e.g., separately,
sequentially or concurrently), or the like. The pack or kit can be
a single unit assay or it can be a plurality of unit assays. In
particular, the agents can be separated, mixed together in any
combination, present in a single vial or tablet. For the purpose of
this invention, unit assays is intended to mean materials
sufficient to perform only a single assay.
[0201] The invention will be further illustrated by the following
non-limiting examples of diagnostic and therapeutic methods. All
percentages are by weight unless otherwise specified.
EXAMPLES
Example 1
POLYMERASE CHAIN REACTION (PCR) FOR THE FULL LENGTH MOMP GENE OF C.
PNEUMONIAE AND OTHER SPECIES OF CHLAMYDIA (DIAGNOSTIC)
[0202] a. Solution PCR
[0203] Serum, blood or tissue samples were pre-incubated in the
presence of 10 .mu.M dithiothreitol at room temperature for 2 hours
to reduce the disulfide bonds and facilitate release of the outer
shell of the elementary bodies. CSF and other body fluids are also
suitable for use as described. Other suitable reducing agents for
use in this step include, but are not limited to, succimer and
glutathione (e.g., including, but not limited to, glutathione
esters, other analogs and deriviatives). The failure to include a
reducing agent initially may result in a negative PCR signal
following the protease digestion step. Appropriate concentrations
of these reducing agents can be readily determined by the skilled
artisan without undue experimentation using the 10 .mu.M
concentration of dithiothreitol as a guideline. Alternatively,
guanidine isothiocyanate may be substituted for the disulfide
reduction/protease step. Table 8 shows the effect of various
reducing agents on susceptibility of EBs to proteinase K digestion
in order to allow DNA extraction for PCR amplification.
8TABLE 8 Effect of various reducing agents on susceptibility of EBs
to proteinase K digestion in order to allow DNA extraction for PCR
amplification. PCR Reducing Agent Concentration Signal.sup.a
Reducing Agent Concentration PCR Signal.sup.a Dithiothreitol 10 mM
+ 2,3-Dimercapto-1- 10 mM - 1 mM + Propone-sulfide acid 1 mM - 100
.mu.M + 100 .mu.M + 10 .mu.M + 10 .mu.M - 1 .mu.M + 1 .mu.M -
Succimer 10 mM - Meso-2,2'-Dimercapto 10 mM + 1 mM + adipic acid 1
mM + 100 .mu.M + 100 .mu.M + 10 .mu.M + 10 .mu.M + 1 .mu.M - 1
.mu.M - DL-Penicillamine 10 mM - Glutathione 10 mM - 1 mM - 1 mM
wk+ 100 .mu.M + 100 .mu.M - 10 .mu.M - 10 .mu.M +/- 1 .mu.M - 1
.mu.M +/- D-Penicillamine disulfide 10 mM + Control 0 - 1 mM - 100
.mu.M - 10 .mu.M - 1 .mu.M - .sup.aAll assays performed on control
serum #1154, which on repeated assay without reducing agents,
yields a negative PCR signal for the 1.2 kB MOMP gene of C.
pneumoniae. Analysis on agarose gel with ethidium bromide
visualization under UV light.
[0204] Serum, blood, or tissue samples are lysed overnight at
37.degree. C. in the presence of SDS which inhibits DNAses and
proteinase K which digests protein (i.e., 2.times.cell lysis
buffer: 1% SDS, 0.2 M NaCl, 10 mM EDTA, 20 mM Tris-KCl, pH 7.5 plus
proteinase K to a final concentration of 20 mg/ml). Following
digestion, the lysate is extracted.times.1 with phenol followed by
chloroform extraction.times.2. DNA is precipitated from the final
aqueous phase by the addition of {fraction (1/10)} volume Na
acetate (3 M) and 2-2.5 volume of cold ethanol. The DNA is pelleted
by centrifugation and the DNA is resuspended in 10-20 .mu.l water
with PCR amplification performed in the same microtube. The entire
gene of MOMP (1.2 kb) is amplified using the CHLMOMPDB2 coding
strand primer (5'-ATGAAAAAAC TCTTAAAGTC GGCGTTATTA TCCGCCGC; SEQ ID
NO. 41) and the CHLMOMPCB2 complimentary strand primer
(5'-TTAGAATCTG AACTGACCAG ATACGTGAGC AGCTCTCTCG; SEQ ID NO. 42).
Alternatively, shortened primers can be used by making suitable
modifications in the primer: DNA hybridization temperature for PCR
detection only. The appropriate primer selection, however, may
result in the absence of signal if an unknown strain with mutations
in one or both primer binding regions is present. The frequency of
positive signals using the preferred primers which amplify the full
length MOMP gene suggests that mutations in these regions of C.
pneumoniae is rare. Standard conditions for this gene product in a
50-.mu.l volume is 35 cycles with 1 second ramp times between steps
of 94.degree. C. for 1 minute, 58.degree. C. for 2 minutes and
74.degree. C. for 3 minutes. The PCR reaction used 0.1 mM of each
primer in Deep Vent buffer with 200 mM of each dNTP, and 1.0 U Deep
Vent DNA polymerase. Amplified DNA is separated and identified by
electrophoresis in 1.2% agarose or 6% polyacrylamide gels run in
the TBE buffer (88 mM Tris-borate, 89 mM boric acid, 2 mM EDTA) at
120 volts for 1 hour. DNA bands are identified by ethidium bromide
staining and UV light detection. Product specificity has been
verified by restriction enzyme analysis of cleavage products as
well as DNA sequence analysis. Negative controls consist of
amplification of lysis buffer extracts. Extreme care must be
exercised to screen all components of the cell lysis and
amplification buffer components to exclude contaminant MOMP DNA
which are common contaminants in such lab and molecular biology
grade chemicals.
[0205] b. In situ PCR
[0206] This procedure identifies individual cells containing RB and
cryptic forms of C. pneumoniae. Cultured cells are adhered to glass
slides with formalin, or formalin fixed tissue sections embedded in
paraffin are adhered to glass slides and subjected to protease
digestion (i.e., pepsin, trypsin, chymotrypsin, or other specific
proteases). Each digestion time and pH (i.e., pepsin at pH 2.5 or
trypsin at pH 7-8, etc.) with a standard concentration of each
protease must be evaluated for each tissue type for optimal
digestion times. Protease activity is stopped by washing and/or pH
change and the target cells exposed to Taq polymerase, dNTPs, and
primers. For the MOMP gene the primers CHLMOMPDB2 and CHLMOMPCB2
have been engineered with a biotin at the 5'-terminus. For in situ
PCR, using biotin labels incorporated at the 5'-terminus of the
amplification primers, each DNA chain amplification results in each
double strand DNA containing 2 molecules of biotin. Standard
conditions of amplification are identical to solution PCR described
above. Following the end of the PCR cycle, the slides are washed
and exposed to strepavidin-.beta.-galactosidase (or other
strepavidin conjugated signal enzyme). Visualization of the
amplified MOMP gene is accomplished by bound enzyme hydrolysis of
soluble substrate yielding an insoluble product which can then be
visualized by standard light microscopy.
[0207] Alternatively, other specific DNA sequences, including
subsections of the full MOMP gene (e.g., subsections including gene
sequences for the peptides in FIG. 4) can be used, although the
above-described sequence is the preferred embodiment since the
large product produced (1.2 kb) prevents diffusion that may be
encountered with smaller DNA amplifications. Similarly, other
detection labels can be incorporated (i.e., fluorescein, for
example) at the 5'-end or dixoxigenin & UTP can be incorporated
within the amplified DNA. Alternatively to labeling the product,
specific hybridization probes to constant regions of the amplified
DNA can be used to identify an amplified product. This latter
method has particular utility for the construction of automated
laboratory equipment for solution-based PCR. For example,
strepavidin-coated ELISA plates can be used to capture one or both
strands of a biotin 5'-labeled DNA with detection by fluorescence
of a fluorescen or other incorporated fluorophore detection
probe.
Example 2
ENZYME LINKED IMMUNO SORBENT ASSAY (ELISA; DIAGNOSTIC)
[0208] a. Recombinant MOMP-Based ELISA
[0209] The full length MOMP gene of C. pneumoniae was directionally
cloned into the pET expression plasmid at the NCOI and NOTI
restriction sites using primers to introduce these unique
restriction sites into the MOMP ends. Primer sequences are as
follows:
9 CPOMPDNCO (Coding strand): (SEQ ID NO. 43) 5'-AGCTTACCAT
GGCTAAAAAA CTCTTAAAGT CGGCGTTATT ATCCG-3' CPOMP_CNOT (complimentary
strand): (SEQ ID NO. 44) 5'-ATATGCGGCC GCTCATAGAA TCTGAACTGA
CCAGATACG-3'
[0210] The construction of the MOMP insert into the pET expression
vector (Novagen, Inc.) yields, on transformation of permissive E.
coli, an amino terminal thioredoxin fusion domain, a polyhistidine
for Ni.sup.+-affinity chromatography, a solubility sequence of
approximately 5 kD, and an endopeptidase cleavage site which yields
a full length MOMP with a modified amino terminal (as illustrated
in FIG. 2) containing an alanine insert between the amino terminal
methionine and the adjacent lysine. Either the full length
expressed recombinant fusion protein or the modified MOMP following
endopeptidase cleavage can be used as the antigen for a Chlamydia
species ELISA. Other expression systems in E. coli or Baculovirus
can be used for synthesis of the MOMP protein as the antigen in
ELISA. The process is performed by non-covalent attachment of 50 ng
recombinant MOMP in each well (rows 1-11) of a 96 well microtiter
plate (Immulon 4) in carbonate buffer at pH 9.5 with an overnight
incubation at 4.degree. C. The plate is washed with PBS, 0.15%
Tween20.times.3 and is then blocked with PBS, 1% BSA, 0.15% Tween,
20 at 300 ml per well for 1 hour at RT and then washed.times.3 with
PBS, 0.15% Tween20. Serum is-serially diluted in PBS in triplicate
in a separate plate and 50 .mu.l of each well transferred to
corresponding wells of a MOMP ligand plate, and the following
sequence is followed: incubate at 37.degree. C. for 1 hour using a
parafilm or other suitable cover to prevent non-uniform
evaporation. Wash with PBS, 1% FCS, 0.05% NaN3.times.5. Incubate
each well with a predetermined dilution of biotin conjugated
anti-human monoclonal IgG or monoclonal IgM. Incubate at 37.degree.
C. for 1 hour with cover. Wash (.times.3) with PBS, 1% FCS, 0.05%
NaN3. Follow with 50 .mu.l strepavidin-alkaline phosphatase
conjugate (1:200 in PBS, 1% BSA, 0.15% Tween20) for 1 hour at
37.degree. C. with cover. Wash.times.3 with PBS, 1% CS (calf
serum), 0.05% NaN3. Color is developed with p-nitrophenyl phosphate
in glycine buffer at pH 9.6. The color yield is measured on a
microtiter colorimeter using a 405 nm filter. The end point titer
is the highest dilution of serum or secretion yielding a color
yield>3 SD over background (n=8). Analysis is simplified by
computer-generated end point antibody titer or other antibody level
measure identification and/or quantity of specific antibody (IgG,
IgM, or total Ig) in the test sample using appropriate controls.
Other strepavidin or avidin enzyme conjugates can be substituted
such as strepavidin peroxidase or strepavidin-galactosidase with an
approximate substitute yielding a detectable color for
quantitation.
[0211] b. Peptide-Based ELISA
[0212] The recombinant MOMP-based ELISA described above provides a
sensitive method for the quantitation of immunoglobulins against
the Chlamydia genus in serum, plasma, CSF, and other body fluids.
In order to provide ELISA assays that are species- and potentially
strain-specific for the various Chlamydia, two regions in the MOMP
have been identified which show minimal amino acid sequence
homologies and which are predicted by computer analysis
(Intelligenetics) to be excellent antigenic domains by virtue of
hydrophilicity and mobility on the solvent-accessible surface of
MOMP. FIG. 3 illustrates the constant and variable domain (VD) of
the various chlamydial species. The identified species-specific
antigenic domains are located in VD1 and VD2. FIG. 4 illustrates
the peptide amino acid sequences employed for the construction of
peptide based ELISAs with species specificity for VD1. FIG. 5
illustrates the peptides for VD2 which are used similarly to the
VD1 sequences. ELISA methodology parallels that described above for
the recombinant MOMP-based ELISA. In addition, a highly antigenic
domain (FIG. 6) common to all Chlamydia has been identified and was
developed as an alternative genus-specific ELISA for the Chlamydia.
Immunization of rabbits has verified the antigenicity of each
peptide to each peptide (Table 9). Monoclonal antibodies have
further verified the specificities and antigenicity of each peptide
(Table 8) as predicted by computer analysis of the
nucleotide-generated amino acid sequence of each species-specific
MOMP.
10TABLE 9 Antigenic Responses To Peptides From 4 Species of
Chlamydia Identified By Hydrophilicity And Peptide Movement As
Highly Antigenic Chlamydia Titer.sup.a Species Peptide.sup.b Pre
Post C. pneumoniae 90-105 100 >3200 C. trachomatis L2 91-106 800
>3200 C. psittaci 92-106 400 >3200 C. trachomatis (mouse)
89-105 0 >3200 C. pneumoniae 158-171 25 >3200 C. trachomatis
L2 159-175 200 >3200 C. psittaci 160-172 100 >3200 C.
trachomatis (mouse) 158-171 800 >3200 C. pneumoniae 342-354 200
>3200 C. trachomatis L2 342-354 100 >3200 C. psittaci
ND.sup.c C. trachomatis (mouse) ND.sup.c .sup.aReciprocal titer
.sup.bImmunogenic peptide and ELISA antigen of specific amino acid
sequence against the indicated pre-immunization and
post-immunization rabbit serum .sup.cND, not done
[0213] Table 10 illustrates reciprocal titers of a polyclonal and
monoclonal antibody against C. trachomatis cross-reactive against
C. pneumoniae peptide encompassing amino acids 342-354 and a
recombinant full length MOMP from C. pneumoniae.
11TABLE 10 Reciprocal titers of a polyclonal and a monoclonal
antibody against C. trachomatis cross-reactive against C.
pneumoniae peptide encompassing amino acids 342-354 and a
recombinant full length MOMP from C. pneumonia. Titer.sup.a Antigen
Polyclonal Ab.sup.b Monoclonal Ab.sup.c CPN Momp.sup.d 400 0 CPN
90-105.sup.e 50 0 CPN 158-171.sup.f 50 0 CPN 342-354.sup.g >3200
1600 .sup.aReciprocal titer .sup.bPolyclonal goat Ab from Chemicon
Inc. against MOMP of C. trachomatis .sup.cMonoclonal Ab (ICN, Inc.)
against MOMP of C. trachomatis .sup.dC. pneumoniae recombinant MOMP
.sup.eAmino acid peptide 90-105 of C. pneumoniae .sup.fAmino acid
peptide 158-171 of C. pneumoniae .sup.gAmino acid peptide 342-354
of C. pneumoniae
Example 3
DETECTION ASSAY METHODS (DIAGNOSTIC)
[0214] a. Immunoglobulin (Ig) assay
[0215] C. pneumoniae EBs were grown in primary human umbilical vein
endothelial cells (HuEVEC; early passage), HeLa 199, or a suitable
alternative in the presence of 1 .mu.g/ml cycloheximide at
35.degree. C. under 5% CO.sub.2. Permissive cells were lysed by
sonication at 3 days, thereby liberating EBs. The latter were
harvested from infection flasks, sonicated, and cellular debris
were removed after sonication by a low speed centrifugation
(.about.600.times.g) for 5 minutes. EBs were pelleted by high speed
centrifugation (30,000.times.g) for 30 minutes at 4.degree. C. The
EB pellet was washed with PBS .times.1 and was reconstituted in 2
ml PBS per four 25-cm.sup.2 culture flask and sonicated at maximum
power for 20 seconds and a 0.5 cycle time using a Braun-Sonic U
sonicator. EB protein concentration was determined by the Bradford
method and the sonicated infectious EB suspension was rendered
non-infectious by the addition of 37% formaldehyde to a final 10%
formaldehyde concentration with constant agitation during addition.
Formalin-treated EBs were added to 96-well plates at 50 .mu.l per
well containing 50 ng EB (total of 5 .mu.g/plate) and air dried.
The plate was washed with PBS-0.15% Tween20 .times.3 and was then
blocked with PBS-1% BSA-0.15% Tween20 at 300 .mu.l per well for 1
hour at room temperature and then washed .times.3 with PBS-0.15%
Tween20. Serum was serially diluted in PBS in duplicate in a
separate plate and 50 .mu.l of each well transferred to
corresponding wells of a MOMP ligand plate and the following
sequence was followed: incubate at 37.degree. C. for 1 hour using a
parafilm cover; wash with PBS-l% FCS-0.05% NaN3 .times.5; incubate
each well with a predetermined dilution of biotin-conjugated,
antihuman monoclonal IgG or monoclonal IgM; incubate at 37.degree.
C. for 1 hour with cover; wash (.times.3) with PBS, 1% FCS, 0.05%
NaN3; follow with 50 .mu.l strepavidin-alkaline phosphatase
conjugate (1:200 in PBS-1% BSA-0.15% Tween20) for 1 hour at
37.degree. C. with cover; and wash .times.3 with PBS, 1% CS, 0.05%
NaN3. Color was developed with p-nitrophenyl phosphate in glycine
buffer at pH 9.6. The color yield was measured on a Flow microtiter
colorimeter using a 405 nm filter. End point titer was the highest
dilution of serum or secretion yielding a color yield>3 SD over
background (n=8).
[0216] b. Western Blot
[0217] Western blots were prepared by SDS-PAGE of C. pneumoniae EBs
(non-formalin fixed) harvested from infected HuEVEC or HeLa cell
lysates, electrophoresed under standard SDS-PAGE conditions, and
transferred to nitrocellulose achieved with an active diffusion
transfer. Albumin-blocked strips were prepared from nitrocellulose
sheets and incubated 1 hour with 1.2 ml of a 1:40 dilution of test
serum. Detection was achieved with an alkaline
phosphatase-conjugated, mouse anti-human antibody, and developed
with 5-bromo-4-chloro-3'-indolyphosphate p-toluidine/nitro-blue
tetrazolium chloride (BCIP/NBT, Pierce Chemical Company).
Polyclonal animal anti-human antibodies can alternatively be
used.
[0218] c. Antigen Capture Assay for Chlamydial MOMP
[0219] The peptides described in FIGS. 3-5 were conjugated via
disulfide bonding to keyhole limpet hemocyanin (KLH) by standard
methods (Bernatowicz et al., Anal. Biochem. 155(1):95-102 (1986)).
The peptide conjugates in alum were used to generate polyclonal
and/or monoclonal antibodies to the species-specific domains of
MOMP which is used as a capture antibody in 96 well microtiter
plates. Final configuration can follow a number of alternative
routes to yield quantitation of MOMP in body fluids. The favored
configuration utilizes biotin labeled recombinant MOMP in a
competition assay with strepavidin/alkaline phosphatase generated
color development based on the quantity of biotinylated recombinant
MOMP displaced by unlabeled MOMP in body fluids.
Example 4
IN VITRO ANTIMICROBIAL SUSCEPTIBILITY TESTING FOR C. PNEUMONIAE
[0220] Tissue culture cells containing cryptic phase C. pneumoniae
(H-292, HeLa, HEL, HuEVEC, McCoy, etc.) are plated at subconfluency
in a 96-well microtiter plate (flasks or plates or other
configurations can be alternatively used) and cultured in the
presence of various antibiotics (singly and in combination) with
the medium changed daily. Analysis of chlamydiacidal activity is
carried out by assessing loss of solution PCR signal, or relative
activity can be quantified by dilution titer of the starting
material using the absence of PCR signal as the endpoint titer
(i.e., last dilution to yield specific PCR signal).
[0221] Two week exposure of single agents including the
fluoroquinolone, ofloxacin, and the macrolide, clarithromycin, at 1
.mu.g/ml failed to clear HeLa cells in culture of a detectable PCR
signal for the MOMP gene of Chlamydia pneumoniae. In contrast,
triple agents consisting of isoniazid (INH), metronidazole, and
penicillamine (1 .mu.g/ml each) resulted in no detectable PCR
signal (Table 11). None of these agents, effective in the triple
combination, is currently recognized as an anti-chlamydial
agent.
[0222] Table 12 provides the results of an expanded study of
antimicrobial susceptibilities at two different concentrations of
antimicrobial agents, used alone and in combination, when exposed
to the antimicrobial agents for two weeks. In addition to the
agents already mentioned, minocycline, doxycycline, rifampin and
sulfamethoxizole/trimethoprim, at all concentrations tested, failed
to clear the PCR signal for chlamydial MOMP. Only the triple
combination of isoniazid, metronidazole and penicillamine cleared
the PCR signal in two weeks. The triple combination was effective
at both low and high concentrations. Table 12 also demonstrates the
effect of a 4 week exposure with the same expanded series of
antimicrobial agents alone and in combination. A number of triple
combinations of antimicrobial agents resulted in cell cultures in
which the PCR signal for the chlamydial MOMP gene could not be
detected at four weeks. The most effective combinations to have the
greatest impact on the important life cycle phases are those
predicted according to the methodology described in the section
above entitled "Methodology for Selecting Potential Agent
Combinations".
12TABLE 11 Susceptibility to Antibiotics for Cryptic C. pneumoniae
Cultured in HeLa Cells.sup.a Antibiotic Conc (.mu.g/ml) PCR.sup.b
Ofloxacin 1 positive Clarithromycin 1 positive INH 1 positive
Metronidazole 1 positive Penicillamine 1/1 positive INH +
Metronidazole + 1/1/4 negative Penicillamine Control 0 positive
.sup.aCultured in the presence of the indicated antibiotic(s), but
with no cycloheximide. Media changes at 48-72 hours. .sup.bAnalysis
following 2 weeks exposure to antimicrobioal agents.
[0223]
13TABLE 12 Susceptibility to Antibiotics by PCR for Cryptic
Chlamydia pneumoni.oe butted.. Cultured in HeLa Cells.sup.1 Phase
of the Chlamydial Life Cycle EB (Extracellular EB->RB Stationary
Phase RB RB->EB Transition Concentration PCR PCR or
Intracellular) Transition ("Cryptic phase") Replicating RB
("Condensation") (.mu.g/ml) 2 week 4 week Comment 0 + + Control
Minocycline 0.25 + + One drug Minocycline 1 + + One drug
Doxycycline 0.25 + + One drug Doxycycline 1 + + One drug
TMP/SMZ.sup.2 25 + + One drug TMP/SMZ.sup.2 100 + + One drug
Clarithromycin 0.25.sup.1 + + One drug Ofloxacin 0.25 + + One drug
Metronidazole 0.25 + + One drug Rifampin 0.25 + + One drug
Isoniazide Isoniazide 1 + + Misses EB phase Metronidazole
TMP/SMZ.sup.1 25/0.25 + + Misses EB phase Metronidazole Ofloxacin
0.25/0.25 + + Misses EB phase Rifampin Metronidazole 0.25/0.25 + +
Misses EB phase penicillamine Rifampin Metronidazole penicillamine
4/.25/.25 + + Misses replicating phase Rifampin Metronidazole
Ofloxacin .25/.25/.25 + + Misses EB phase penicillamine
Metronidazole Doxycyclin penicillamine 1/.25/.25 + + Concentration
too low penicillamine Metronidazole Doxycyclin penicillamine 4/1/1
+ - Covers key phases penicillamine Metronidazole Minocycline
penicillamine 1/.25/.25 + - Covers key phases penicillamine
Metronidazole Minocycline penicillamine 4/1/1 + - Original report
of 4 week positive was a typo penicillamine Metronidazole TMP/SMZ
penicillamine 4/1/100 + - Covers key phases penicillamine
Metronidazole Clarithromycin penicillamine 1/.25/.25 + - Covers key
phases penicillamine Isoniazid Isoniazid penicillamine 1/.25/.25 -
- Covers key phases Metronidazole penicillamine Isoniazid Isoniazid
penicillamine 4/1/1 - - Covers key phases Metronidazole
.sup.1Cultured in the presence of the indicated antibiotics, but
with no cycloheximide. Media changes at 48-72 hours. + = positive;
- = negative .sup.2TMP/SMX = trimethoprim/sulfamethoxazole
Example 5
RESPONSE TO ANTIBIOTIC THERAPY
[0224] Table 13(a) illustrates typical responses to combination
antibiotic therapy in a variety of patients with diagnostic
evidence of an active infection by C. pneumoniae. Unlike typical
immune responses to infection with infectious agents, most of the
included patients have not only detectable IgM titers against the
chlamydial genus but in many cases very high IgM titers. With
specific therapy over time the IgM titers generally fall, with a
rise in IgG titer (as expected). Current methods of detecting
antibodies against C. pneumoniae (Indirect immunofluoresence, MIF)
are incapable of accurately identifying high IgM titers against C.
pneumoniae. Moreover, current procedures are genus specific and not
species specific as are peptide-based ELISAs.
[0225] With clearing of the pathogen, the IgG titers fall.
Concomitant with combination antibiotic therapy, there is generally
an improvement of patient symptoms associated with the specific
diagnosis indicative of evidence of an active chlamydial
infection.
[0226] Table 13(b) describes the course of therapy for a number of
individuals treated with a combination of agents and their clinical
outcomes.
[0227] Table 13(c) describes the detailed case histories of the
patients undergoing combination therapy, as reported in Table
13(b).
[0228] Table 13(d) provides a listing of drugs and standard dosages
for those used herein.
14TABLE 13a Serological and PCR Responses to Combination Antibiotic
Therapy Titer Patient Diagnosis.sup.a IgM IgG Time on Therapy PCR
Status PH FM 800 800 6 months + 3200 1600 + 800 200 wk+
Asymptomatic BL MS 2000 500 + Dramatic 400 3200 9 months wk+
Improvement MM CFS/AND 3200 800 + Improvement; Relapse 400 1600 1
month + (non-compliant) PM CFS 2000 25 6 months + 400 800 wk+
Asymptomatic AM IBD 800 0 6 months wk+ 90% Improvement 3200 400 +
FO MS 800 3200 st+ 800 800 10 months + Improvement in 400 800 wk+
speech and bowel 400 800 + continence WM CF 25 25 Pre-illness serum
wk+ 1000 25 <--Antibiotics start st+ 50 800 + 50 1600 wk+ 50 400
- Asymptomatic HM CF 2000 100 + 3200 3200 6 months + 200 800 wk+
Asymptomatic CN CFS 3200 800 + 800 800 8 months wk+ 75% Improvement
AN MS/CFS 400 400 wk+ Improved Strength 200 3200 st+ Fatigue
decrease JS CFS 2000 2000 st+ (severe) 2000 2000 5 months + 200 800
- Asymptomatic AG IBD 3200 400 + . . . 800 400 9 months +
Improvement 800 800 + in joint Sx 800 400 - AT CF 3200 3200 + 1600
1600 9 months + 1600 1600 + 800 800 + Asymptomatic 400 400 + LH RA
3200 1600 wk+ 800 400 6 months wk+ Improvement 200 50 + HS MS 2000
400 + 3200 800 5 months + 50 200 - Improvement ST CFS/FM >1000
100 wk+ 1000 100 7 months wk+ 400 100 + 800 3200 + 100 100 +
Asymptomatic RV CF 1000 100 + 400 1600 10 months + 400 400 -
Asymptomatic .sup.aCF = Chronic Fatigue <6 months, CFS = Chronic
Fatigue Syndrome, FM = Fibromyalgia, IBD = Inflammatory Bowel
Disease, MS = Multiple Sclerosis, AND = Autonomic nervous
dysfunction (neural-mediated hypotension), RA = Rheumatoid
Arthritis IgM >> IgG: immune tolerance to the antigen; IgG
>> IgM: successful immune control of the antigen
[0229]
15TABLE 13b Treatment Regimens Treatment Regimen Phase of
Chlamydial Life Cycle EB (Extra-or EB->RB Stationary Replicating
RB->EB Duration Patient Sex Diag Intracellular) Transition Phase
RB RB Transition Enhancer (months) Comments BL M MS Rifampin Flagyl
Floxin 2 Flagyl Bactrim, 5 Levaquin -- -- -- -- -- 3 Took a break,
had relapse Flagyl Bactrim, 2 Levaquin Penicillimine Flagyl
Bactrim, Penicillimine 7 Levaquin Penicillimine Rifampin INH INH
Penicillimine Probenicid 3 MC M MS Rifampin INH INH 9 Flagyl
Levaquin 6 probably not compliant Minocycline -- -- -- -- -- --
Discontinued JM M MS Flagyl Floxin 7 Bactrim Minocycline
Amoxicillin Levaquin Amoxicillin 4 Bactrim Amoxicillin Levaquin
Amoxicillin Probenicid 3 Bactrim LL F MS Flagyl Levaquin 15
Minocycline Penicillimine Levaquin Penicillimine Probenicid 1
Minocycline AN F MS Tenitizole Floxin ? She was given a copy of the
protocol, but ran her own therapy FO M MS Prednizone 0.25 Phased in
over several days to mitigate effect of therapy Flagyl Biaxin 2
Biaxin 1 Stoped flagyl due to persistence of side effects Kemet
Biaxin Kemet 0.5 Kemet Flagyl Biaxin Kemet 6 Began phasing Flagyl
back in over a month Kemet Flagyl Biaxin Kemet 1 Began 2 week
switchover to Amoxicillin Amoxicillin Amoxicillin Amoxicillin
Flagyl Biaxin Amoxicillin 2 Amoxicillin Flagyl Biaxin Amoxicillin
Probenicid 6 Began 6 week phase in of probenicid JC F MS
Amoxicillin Amoxicillin 1 Phased in over 7 months . . . Amoxicillin
Amoxicillin Probenicid 1 Amoxicillin Bactrim Amoxicillin Probenicid
1 Amoxicillin INH Bactrim Amoxicillin Probenicid 7 FW M MS
Penicillimine Flagyl Doxicycline Penicillimine 7 Penicillimine INH
INH Penicillimine Probenicid 5 Bactrim -- -- -- -- -- -- Stopped
treatment LH F RA Penicillimine Flagyl Minocycline Penicillimine 11
Penicillimine Flagyl Minocycline Penicillimine Probenicid 3 -- --
-- -- -- -- 3 PCR negative, symptom free, but titer @ 1:800.
Decided to stop. Penicillimine Flagyl Minocycline Penicillimine
Probenicid 2 After symptoms flared, PCR went positive, and titer to
1:1600, restarted therapy XX F IC Amoxicillin INH INH Amixicillan
Probenicid 4 Symptoms gone after 4 Bactrim months of treatment NC F
PG Amox INH INH Amoxicillin 7 Continued improvement Bactrim CH M PG
Amoxicillin INH IHN Amoxicillin 3 Levaquin Amoxicillin INH IHN
Amoxicillin 2 Bactrim -- -- -- -- -- -- Discontinued after all
ulcers cleared up except for those in poorly blood-supplied leg RI
M PG Missing patient chart PL M PG Amoxicillin INH IHN Amoxicillin
2 Non-compliant because Bactrim could not afford medicines -- -- --
-- -- -- 1 Amoxicillin INH IHN Amoxicillin 0.5 Would often only
take what Bactrim he had left. -- -- -- -- -- -- 2 Off for 2
months, then flared Amoxicillin INH IHN Amoxicillin 1 No subsequent
follow-up Zithromax TW M PG Flagyl Minocycline 4 Amoxicillin INH
INH Amoxicillin 2 Levaquin -- -- -- -- -- 1 Amoxicillin Levaquin 4
No improvement -- -- -- -- -- Moved to topical antibiotics AM M UC
Flagyl Biaxin 11 Amoxicillan Flagyl Biaxin Amoxicillan 2 INH INH
Amoxicillan Flagyl Bactrim Amoxicillan Probenicid 5 Now doing very
well INH INH AG F UC Flagyl Doxycycline 6 -- -- -- -- -- --
Discontinued after symptoms resolved. DM F IBD Flagyl Doxycycline 7
Cupramine.sup.1 Flagyl Doxycycline Cupramine Probenicid 5 -- -- --
-- -- -- Discontinued after doing well clinically; wanted to start
a family. RP F UC Flagyl Biaxin 5 -- -- -- -- -- -- Discontinued
after impvt AB F CD Flagyl Doxycycline 7 -- -- -- -- -- --
Non-compliant EU F UC Flagyl Doxycycline 9 -- -- -- -- -- -- 1
Stopped Amoxicillan Flagyl Doxycycline Amoxicillan Probenicid 2
Restarted after symptoms flared. Now doing well again RR CD Flagyl
Doxycycline 2 Colectomy 2 months prior Amoxicillan Flagyl
Doxycycline Amoxicillan Probenicid 6 Now doing well; no evidence of
active disease .sup.1125 mg BID
[0230]
16TABLE 13c Detailed Case Histories Patient Diag Test data.sup.1
Case History BL MS Row 2 First symptoms began with numbness of the
left arm and leg which rapidly progressed to a partial
Brown-Sequard syndrome (i.e.-cord myelitis) with an associated
urinary retention. Despite therapy with corticosteroids, and Beta
interferon he rapidly progressed over the next three months with an
EDSS = 8.0 (triplegic plus speech and swallowing impairments). A
positive CSF PCR and culture for C. pneumoni.ae butted. led to
treatment with combination antibiotics. The patient improved on all
spheres of neurologic function over the following six months. His
EDSS score 9 months later was 3.0 with return to work and routine
athletic activities (e.g.-jogging). His neurological status remains
stable and he continues on an anti-chlamydial combination regimen.
MC MS This patient had a ten year history of MS with evidence of
progressive ataxia and weakness in the legs. Over 5 months his EDSS
score worsened from 7.0 to 8.0. His CSF was positive by PCR for C.
pneumoni.ae butted. and he was placed on combination antibiotics.
Over the next six months he gradually improved in his balance,
coordination and lower extremity strength. His most recent EDSS
score was 6.5. JM MS Initially seen with rapidly progressive
paraparesis secondary to MS. He failed to response to
corticosteroids on two successive occasions. Five months later, his
EDSS score was 7.5. Following a positive C. pneumoni.ae butted. PCR
he was placed on combination antibiotics. He has gradually gained
strength in his lower extremities and five months later was able to
walk with a walker (EDSS = 6.5) while being maintained on
combination antibiotics. LL MS Patient with a long history (14
years) of secondary progressive MS with recent progressive bulbar
symptoms, axtaxia, and paraplegia (EDSS = 8.5). PCR for the MOMP
gene of C. pneumoni.ae butted. in the CSF was positive. She was
placed on combination antibiotics with no further progression of
symptoms for the last six months. AN MS Row 10 Long history of MS
and wheel chair bound for approximately ten years. She has received
continuous physical therapy to retain leg muscle tone. Following
approximately 6 months of combination antibiotics, she was able to
stand unaided and take several unaided steps. She reports a
significant decrease in fatigue and cognitive dysfunction. She
remains on combination antibiotics and other supportive
medications. FO MS Row 6 Wheel chair bound with a long history of
MS with a 2-3 year progression of severe dysarthri.ae butted. and
incontinence. On combination antibiotics (14 months) he has had
improvement of speech and incontinence. Speech, ability to open
mouth for dentist, stamina all improved. Can stand better on his
own mid- transfer. He remains wheel chair bound. JC MS Diagnosis of
MS with development of a foot drop approximately one year prior to
therapy requiring the use of a cane in walking. Approximately four
months after initiation of combination antibiotic therapy, patient
reports reversal of foot drop and no longer requires a cane. She
continues on antibiotic therapy. FW MS Male executive in late 50s
with a 15 year history of MS. Used a cane for a rolling, unstable
gait. Easily fatigued. After 12 months of combination antibiotics,
was able to walk without cane or excessive fatigue, although his
gait can still wander. Can easily make it across the parking lot,
which had previously been a challenge. Stopped antibiotics even
though was still PCR positive; plans to restart therapy if he has
another flare-up. LH RA Row 14 Patient LH had an active case of RA
which was moderately debilitating. Following two months of
combination antibiotic therapy, her RA is in complete remission. XX
IC She responded to combination antibiotics with complete remission
of symptoms after one month. Cessation of antibiotics resulted in a
return of IC symptoms. NC PG PCR+ 61 year old male who had had
lesions for several years. Large ulcerated lesions on feet that
resolved on combination antibiotic therapy. Only residual
hypertrophic scars remain. CH PG PCR+ 75-year-old male diabetic
with multiple, large, severe lesions on both legs, abdomen, and
arms. Lesions first formed in 1993. Severity of process required
chronic nursing home care at an estimated cost of $300-400 per day.
All lesions above the knee have resolved on combination antibiotic
therapy: lesions only remain on right lower leg, where inadequate
blood supply offers poor prognosis. The patient no longer requires
nursing home care. RI PG PCR+ Original severe PG lesions on legs
required bilateral amputation. Lesions now occurring on arms.
Treatment with combination antibiotics has resulted in resolution
of lesions although not complete to date. [No update - chart
missing] PL PG PCR+ 18 year old female with history of leg ulcers.
Multiple PG lesions completely healed on combination antibiotic
therapy. Patient then lost his job and could not afford to maintain
drug regimen. Upon re-flaring of ulcers, re-started therapy and
ulcers improved again. TW PG Severe PG, initiated after a chemical
burn in 1991, but with PCR negative serology for C. pneumoni.ae
butted.. Patient did not initially respond to combination
antibiotic therapy. A positive biopsy culture for C. pneumoni.ae
butted. resulted in the recent re-institution of combination
antibiotics. However, after no improvement, patient went off
therapy. AM IBD Row 5 This is a 35 year old male who first
presented as a prostititis ten years ago at the age of 25. This
progressed to acute ulcerative colitis, involving the entire colon,
which was associated with severe arthritis, iritis, and weight
loss. Diagnosis was biopsy confirmed. Control required high doses
of corticosteroids and azacol. Attempts to reduce steroids resulted
in partial control of symptoms. Six months prior to initiation of
combination antibiotic therapy, patient was experiencing frequency
(20-25 times per day), frank bleeding, and mucus in the stool.
Patient on combination antibiotics for one year. Following
significant stress, patient had significant increase in symptoms.
Alteration of antibiotic combination has resulted in normal bowel
habits with no mucus and minimal blood. Associated neuropsychiatric
manifestations of cognitive dysfunction and depression have
resolved. Steroids have been discontinued. AG IBD Row 12 This is a
27 year old white female with a two month history of fulminate,
progressive ulcerative colitis which had not responded to the usual
medical therapy. A total abdominal colectomy with ileostomy and
rectal pouch was done. The microscopic appearance confirmed
ulcerative colitis. Following the colectomy, the patient
experienced neurologic symptoms, fatigue, myalgias, arthralgias,
and an acneoform skin rash. Serology was performed for C.
pneumoni.ae butted. and was positive with an IgM of 1:3200, IgG
1:400 and PCR positive. Therapy with combination antibiotics was
initiated. After six months of antimicrobial therapy, her serology
was IgM 1:800, IgG 1:400, and PCR positive. The neurologic
symptoms, fatigue, myalgias, arthralgias, and acneoform rash
resolved completely. There was no further evidence of inflammatory
bowel disease, and the ileostomy was successfully anastomised to
the rectal stump. The patient has felt more energetic. Serology
after 1 year was PCR negative. DM IBD This 37 year old female had a
six year history of inflammatory bowel disease (uncertain CD or UC)
associated with painless rectal bleeding, arthritis, myalgias, skin
ulceration, abdominal cramping/diarrhea, and rectal fistulas. She
had increasing fatigue which caused her to frequently miss work as
a minor executive. On combination antibiotic therapy, all symptoms
resolved but recurred with cessation of antibiotics while on
vacation. Reinstitution of combination antibiotics resulted in a
second remission of symptoms. Prior to combination antibiotic
therapy, she had not gone longer than 3 months without an anal
manifestation of IBD. She has been symptom free of IBD for over a
year. RP IBD Patient presented with proctocolectomy and ileostomy
due to UC. Following a flu-like illness in 1993, she became
fatigued and anemic with blood-tinged diarrhea. Examination of her
ileostomy pouch revealed inflammation and ulcerations. Upper GI
series/small bowel series revealed no abnormalities and no cause of
her anemia was diagnosed. On combination antibiotics her ileostomy
activity was more regular and less spastic. She claimed to feel
better with higher energy levels and ceased antibiotic therapy. Six
months post- antibiotic therapy she remained asymptomatic other
than a moderate anemia. AB IBD Patient with long history of CD
involving small bowel, large bowel, and anus. She had been treated
with a small bowel resection and fissurectomy. She continued to
suffer from numerous rectal fistulas. On combination antibiotics
she experience some symptomatic improvement but failed to
completely resolve her IBD symptoms. She discontinued antibiotics
due to a probable chronic Herxheimer reaction. Currently she is
lost to follow-up. EU IBD Colitis with inflamed distal sigmoid
colon and proctitis associated with frequent loose stools with
significant mucus. Following six weeks of combination antibiotic
therapy with a significant reduction in symptoms. Shortly after
cessation of antibiotics her symptoms return. Reinstitution of
antibiotics resulted in a second remission of the majority of her
symptoms with resolution of her proctitis on visual exam. NM CFS
Vanderbilt University initial patient that resulted in our first
association of C. pneumoni.ae butted., initially complained of the
insidious onset of debilitating fatigue. This was associated with a
severe cognitive dysfunction that disrupted his ability to function
as the supervisor of a clinical diagnostic laboratory. Despite six
months of intensive diagnostic efforts by the Infectious Disease
Clinic at Vanderbilt no definitive or presumptive diagnosis could
be made. A subsequent high antibody titer against C. pneumoni.ae
butted. led to standard anti-chlamydial antibiotic therapy over a
three month period with gradual disappearance of fatigue and
cognition symptoms. On cessation of a fluroquinolone antibiotic,
symptoms returned within two weeks. He was placed on combination
antibiotics with complete reversal of symptoms after six months. He
remains asymptomatic. JS CFS Row 11 Academic physician with a
greater than 10 year history of CFS. Cognition problems resulted in
his grounding himself as a private pilot. Initial treatment with
combination antibiotics results in an apparent Herxheimer reaction
with resolution over a two week period with gradual improvement in
symptoms. After three months therapy, he piloted a light aircraft
under instruments from Florida to North Carolina. He remains on
combination antibiotics for over a year and is asymptomatic. PM CFS
Row 4 Physician with long-standing CFS. Treated with combination
antibiotics with gradual resolution of symptoms. During course of
treatment developed cardiac myopathy. Currently asymptomatic from
CFS. Cardiac myopathy resolved over six month period on combination
antibiotics. MM CFS Row 2 CFS and AD. Resolution of postural
tachycardia over 1 month combination antibiotic therapy. Partial
reversal of fatigue during this period. Patient non- compliant
after one month and lost to follow-up. PH FM Row 1 Three year
history of debilitating FM following the stress of being a stalking
victim. Patient relatively asymptomatic after nine months
combination antibiotic therapy. CN CFS Row 9 Five year history of
severe CFS with debilitating cognitive dysfunction and depression.
Gradual improvement on combination antibiotics for approximately
nine months. Estimated 75% of normal function. PG CFS Ten year
history CFS with cognitive dysfunction. Complete response to
combination antibiotics over a course of one year. AT CF Row 13
Moderate fatigue and cognitive dysfunction following acute
infectious illness. Depression was major problem. During one year
course of combination antibiotics fatigue and cognitive dysfunction
largely reversed. During mid- course of therapy patient developed
acute anxiety attacks relieved by anti- porphyrin therapy. WM CF
Row 7 CF following acute stress. Pre-illness serum negative for
anti-Chlamydia pneumoni.ae butted. antibodies which peaked six
weeks following stress. Pre-illness PCR was weak positive that
became strongly positive. On combination antibiotic therapy at 3
months became asymptomatic. Cessation of antibiotics resulted in
symptomatic relapse. Currently asymptomatic with low serum
antibodies and negative PCR. HM CF Row 8 Medical student with short
history of CF and cognitive dysfunction affecting studies.
Combination antibiotics over a multi-month course resulted in
complete reversal of symptoms. ST CFS Row 17 Mother of Patient AT.
Three year history of CFS with FM. Combination antibiotic therapy
has resulted in partial reversal of symptoms allowing her to retain
a job in jeopardy. Estimated 80-90% normal function currently. RV
CF History of fatigue although non-incapacitating. Combination
antibiotic therapy has resulted in 100% return to normal function.
EB CFS Teen-ager with long history of CFS resulting in home-bound
schooling. On combination antibiotic therapy returned to school and
recently graduated. Recovery has not been complete probably
secondary to non-compliance in therapy. .sup.1Refers to row in
Table 13b which has the ELISA and PCR histories for these
patients.
[0231]
17TABLE 13d Drugs and Standard Dosages Unit Daily Drug Generic
dosage dosage Cupramine Penicillimine 250 mg 2.times. Amoxicillin
500 mg 2.times. Flagyl Metronidazole 500 mg 2.times. INH 300 mg
1.times. Rifampin 300 mg 2.times. Floxin Ofloxacin 400 mg 2.times.
Levaquin 500 mg 1.times. Bactrim SMZ/TMP Double 2.times. Strength
Biaxin Clarythromycin 500 mg 2.times. Minocycline 100 mg 2.times.
Doxycycline 100 mg 2.times. Probenicid 500 mg 2.times.
Example 6
EXAMPLE OF CLEARING MICE
[0232] A set of mice were tested for infection with C. pneumoniae.
Of 10 mice tested, 8 (80%) were PCR positive for C. pneumoniae. The
mice were then placed on triple-antibiotic therapy: Amoxicillin,
Metronidazole and INH at 50 .mu.g/ml each in their water. Based on
their water comsumption of 6.8 to 7 ml per day, the mice were
effectively receiving approximately 350 .mu.g of each drug each
day.
[0233] The mice were tested again, by PCR, on the first generation
of pups once they were old enough. They still tested positive by
PCR. The mouse colony was then maintained on the combination
therapy in water for several months. Approximately seven months
after the start of this study, probenicid was added to the water as
well. Roughly 2 to 3 weeks after the probenicid was added, the then
third or fourth generation of mice was again tested. All 22 mice
tested were then PCR negative.
Example 7
DETERMINATION OF SECONDARY PORPHYRIA
[0234] Patents with systemic infections caused by C. pneumoniae
were evaluated for secondary porphyria. The presence of enzymes
(i.e., .DELTA.-ALA synthase and PBG deaminase) for heme
biosynthesis were determined using known methods. Elevated fecal
and urinary prophyrins were measured at 24 hours. The results are
reported in Table 14.
18TABLE 14 Examples of Secondary Porphyria in Patients with
Systemic infections caused by C. pneumoni.ae butted..sup.a Enzymes
of Heme biosynthesis.sup.b Patient ALA PBG Elevated Fecal
Porphyrins (24 hr) Elevated Urinary Porphyrins (24 hr) ID synthase
deaminase Porphyrin Level Normal Porphyrin Level Normal KRH 6.0
11.7 Protoporphyrin 913 <500 Coproporphryn 115 <60 Dicarboxyl
porphyrin 596 <150 (tetracarboxyl) KB 1.8 7.8 None Coproporphryn
III 248 <45 Isocoproporphryn 142 <10 MB Not done Not done
Tetracarboxyl 287 <200 Not done Coproporphryn 177 <150
Coproporyn III 396 <200 Tricarboxly porphryn III 71 <50
Uroporphryn III SE 6.6 9.7 Isocoproporphyrin 446 <200
Coproporphyrin 89 <60 Protoporphyrin 3512 <1500
Semi-protoporphyrin 2951 <1500 Total dicarboxyl porphyrins 3390
<1500 PE 6.4 10.0 None Porphyrobelinigen 2.5 <1.5 TE 5.9 9.4
Protoporphyrin 2633 <1500 None RH 7.2 9.7 Corproporphyrin I 913
<500 None Corproporphyrin III 596 <150 Protoporphyrin 2884
<1500 Semiprotoporphyrin 2305 <1500 Total dicaboxyl
porphyrins 3706 <1500 NH 7.9 11.5 Uroporphyrin I 241 <120
Pentacarboxyl porphyrin 4 <3 Uroporphyrin III 125 <50
Semirprotoporphyrin 3470 <1500 GK Not done Not done
Coproporphyrin III 175 <50 Not done Total dicarboxyl porphyrins
1635 <1500 AL Not done Not done Uroporphyrin I 237 <50 Not
done Coproporphyrin I 601 <500 Coproporphyrin III 476 <150
Protoporphyrin 1865 <1500 JW 6.7 11.5 Hectacarboxyl porphyrin I
13 <10 None Hectacarboxyl porphyrin III 18 <10 Dicarboxyl
porphyrin 107 <100 HW 7.2 11.2 Isohexycarboxyl porphyrin 19
<10 None Coproporphyrin I 573 <500 Semiprotoporphyrin 1712
<1500 Dicarboxyl porphyrin 2769 <1500 .sup.bReported as
mmol/sec/l. ALA PGB synathase deaminase High levels >4.0 >7.0
Low levels <3.5 <6.0 .sup.aAll assays performed at the Mayo
Clinic, which established the normal reference values. Levels above
normal justify a diagnosis of porphyria.
Example 8
PRESENCE OF AUTOANTIBODIES TO PORPHYRIN RING STRUCTURES
[0235] Patients with systemic infections caused by C. pneumoniae
were tested for the presence of antibodies to porphyrin ring
structures (i.e., vitamin B12, coproporphyrinogen--III,
protoporphyrin, porphyrobelinigen and .sup.a-ALA). IgM and IgG
antibody titers were determined using an ELISA assay, for which the
protocol is described below.
[0236] ELISA Assay Protocol
[0237] 1. Plate Preparation: Coproporphyrin III is used as an
example but the procedure is also preformed by coating plates with
one of the following other ring structures at the same
concentration: vitamin B12, protoporphyrin IX, porphobilinogen and
.DELTA.-aminolevulinic acid. Add 50 ng. of Coproporphyrin III in 50
.mu.l of carbonate coating buffer to each well in columns 1-11 of a
96 well Immulon 4 microtiter plate. Cover with plastic wrap and
incubate overnight at 4.degree. C.
[0238] 2. Block step: Wash plates three times with Tween 20 wash
buffer. (0.1M Tris; 0.15% Tween 20; 0.05% NaN.sub.3) Block plates
by adding 200 .mu.l of Tris block buffer (Tris 0.1M; 1% bovine
serum albumin; 0.15% Tween 20) to each well of columns 1-11. Leave
the wells of column 12 dry. Wrap plates with plastic wrap and
incubate at room temperature for 1 hour.
[0239] 3. Sample preparation: While plates are blocking prepare
samples and controls. Dilute patient's sera 1:10 in block and
vortex well.
[0240] 4. Plate preparation: Wash plates with Tween 20 wash buffer
three times and add 50 .mu.l block in each well in each row except
row A and every column except column 12. Leave row A and column 12
empty.
[0241] 5. Plate configuration: Place 100 .mu.l of patient dilutions
in duplicate in row A. Prepare plates in duplicate and label one
plate for IgG and one for IgM detection. Use the Cetus Propet
apparatus to twofold serially dilute (1:10 to 1:1280) the samples
in column 1-10. The following loading configuration is used for
patient samples and controls:
[0242] Sample 1--columns 1 and 2
[0243] Sample 2--columns 3 and 4
[0244] Sample 3--columns 5 and 6
[0245] Sample 4--columns 7 and 8
[0246] High positive control--column 9
[0247] Low positive or negative control--column 10
[0248] Block only--column 11
[0249] Column 12 dry--air blank
[0250] Wrap with plastic wrap and incubate at 37.degree. C. for 1
hour.
[0251] 6. Detection antibodies: Prepare five minutes before
incubation is up separate 1:2000 dilutions in Tris block buffer of
mouse monoclonal biotin labeled anti-human IgG and IgM. Wash the
plates in FCS wash (0.1M Tris; 0.05% NaN.sub.3; 0.15% Tween 20; 1%
FCS) four times and place 50 .mu.l of the anti-human IgG dilution
in each well of columns 1-11 of the plates labeled IgG. Repeat
using anti-human IgM in plates labeled IgM. Wrap with plastic wrap
and incubate for one hour at 37.degree. C.
[0252] 7. Ligand: Prepare five minutes before the incubation is up
a 1:1000 dilution of streptavidin-alkaline phosphatase conjugate in
Tris block buffer. Wash the plates with FCS wash four times and
place 50 .mu.l of the streptavidin dilution in each well of columns
1-11. Wrap with plastic wrap and incubate one hour at 37.degree.
C.
[0253] 8. Prepare P-Nitrophenlphosphate (PNPP) 30 minutes before
incubation is complete by dissolving Immunopure PNPP tablets in
diethanolamine substrate buffer. Prepare one tablet in five ml
1.times. DEA substrate buffer for each plate.
[0254] 9. When incubation is complete wash plates in FCS wash four
times and add 50 .mu.l of PNPP to each well of columns 1-11. Wrap
with plastic film and allow color to develop by incubating one hour
at room temperature. It is best to protect from light during the
incubation period. At the end of the incubation period stop the
color development by adding 50 .mu.l of 3N NaOH to each well of
columns 1-11.
[0255] 10. Read the plates at a wavelength 404 nM using a Titertek
plate reader.
[0256] The results are reported below in Table 15.
19TABLE 15 Examples of Antibody Titers.sup.a to Porphyrin Ring
Structures in Patients with Systemic infections caused by C.
pneumoni.ae butted. Patient B12 Copro III Protoporphyrin
Porphyrobelinigen -ALA ID IgM IgG IgM IgG IgM IgG IgM IgG IgM IgG
KRH 1:640 1:160 1:640 1:160 1:1280 1:640 1:1280 1:80 1:640 1:640 KB
1:640 1:80 1:320 1:40 1:1280 1:1280 1:160 1:40 1:160 1:320 MB 1:160
1:160 1:160 1:80 1:160 1:60 1:160 1:160 1:320 1:640 SE 1:1280 1:160
1:1280 1:80 1:1280 1:1280 1:640 1:640 1:640 1:1280 AEM 1:1280 1:320
1:1280 1:160 -- -- -- -- -- -- GK 1:640 1:20 1:320 1:20 1:1280 1:80
1:1280 1:40 1:1280 1:40 AL 1:1280 1:20 1:1280 1:10 1:1280 1:80
1:1280 1:40 1:1280 1:40 PE -- -- 1:640 1:20 1:640 1:640 1:320 1:20
1:320 1:640 RH -- -- 1:160 1:80 1:40 1:640 1:160 1:160 1:40 1:320
NH -- -- 1:320 1:160 1:320 1:1280 1:640 1:320 1:160 1:320 JW -- --
1:320 1:80 1:640 1:640 1:160 1:80 1:320 1:320 SW-H -- -- 1:640 1:40
1:640 1:320 1:640 1:40 1:320 1:160 Cord 1 0 0 1:10 1:80 0 1:80 --
-- 1:10 1:10 Cord 2 0 1:20 1:10 1:80 1:10 1:160 0 1:80 0 1:20 Cord
3 0 1:20 1:20 1:80 0 1:20 0 1:10 0 0 .sup.aAntibodies are
quantitated in an ELISA format
EQUIVALENTS
[0257] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
spirit and scope of the invention as defined by the appended
claims. Those skilled in the art will recognize or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
specifically herein. Such equivalents are intended to be
encompassed in the scope of the claims.
Sequence CWU 1
1
* * * * *