U.S. patent number 3,857,393 [Application Number 05/328,048] was granted by the patent office on 1974-12-31 for apparatus for use in the augmentation of the production of antibodies in animals and humans and the collection thereof.
This patent grant is currently assigned to Bio-Response, Inc.. Invention is credited to Samuel Rose.
United States Patent |
3,857,393 |
Rose |
December 31, 1974 |
APPARATUS FOR USE IN THE AUGMENTATION OF THE PRODUCTION OF
ANTIBODIES IN ANIMALS AND HUMANS AND THE COLLECTION THEREOF
Abstract
It has been found that very large production of antibodies can
be achieved by removing specific feedback regulatory antibodies by
means of a lymphoresis performed under special conditions in a
patient or subject (e.g., an animal or human) with induced
anatomical and physiological changes.
Inventors: |
Rose; Samuel (La Jolla,
CA) |
Assignee: |
Bio-Response, Inc. (New York,
NY)
|
Family
ID: |
26834338 |
Appl.
No.: |
05/328,048 |
Filed: |
January 30, 1973 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
|
136476 |
Apr 22, 1971 |
3719182 |
|
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Current U.S.
Class: |
604/8 |
Current CPC
Class: |
C07K
16/06 (20130101); C07K 16/00 (20130101); A61M
1/362 (20140204); A61M 1/3679 (20130101) |
Current International
Class: |
C07K
16/00 (20060101); C07K 16/06 (20060101); A61M
1/36 (20060101); A61m 001/03 () |
Field of
Search: |
;128/214R,214A,214Z,1R
;424/12,85 |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Truluck; Dalton L.
Attorney, Agent or Firm: Kenyon & Kenyon Reilly Carr
& Chapin
Parent Case Text
This is a continuation of application Ser. No. 136,476, filed on
Apr. 22, 1971, now U.S. Pat. No. 3,719,182.
Claims
I claim:
1. Apparatus for the augmentation of production of a specific
antibody from a patient upom whom there has been performed a
thoracic duct fistula comprising:
a. means adapted to be inserted into the fistula for removing lymph
including lymph cells and lymph fluid from the thoracic duct, the
lymph fluid including the specific antibody;
b. means connected to the lymph removing means for separating lymph
cells from lymph fluid of the lymph removed from the thoracic duct,
the separated lymph cells lacking specific anitbody;
c. means connected to the separating means for intravascularly
returning the separated lymph cells to the patient; and
d. means connected to the separating means for removing the
specific antibody from the lymph fluid of the lymph from which the
lymph cells have been separated.
2. Apparatus in accordance with claim 1 in which the means
connected to the lymph removing means for separating lymph cells
from the lymph fluid of the lymph removed from the thoracic duct
comprises a centrifuge separator.
3. Apparatus in accordance with claim 1 and further comprising
means connected to the means for removing the specific antibody
from the lymph fluid for returning the lymph fluid from the
removing means to the patient.
4. Apparatus in accordance with claim 1 and further comprising
means for administering to the patient an antigen to which the
specific antibody corresponds.
5. Apparatus in accordance with claim 1 and further comprising
means for providing fluid to the patient as a replacement for the
lymph fluid removed with the lymph for the thoracic duct.
6. Apparatus in accordance with claim 1 and further comprising
means connected to the separating means for washing the separated
lymph cells.
7. Apparatus in accordance with claim 1 in which the means for
removing the specific antibody from the lymph fluid of the lymph
from which the lymph cells have been separated comprises
immuno-adsorbent apparatus, the immuno-adsorbent apparatus
including an immuno-adsorbent which adsorbs the specific
antibody.
8. Apparatus in accordance with claim 7 and further comprising
means for feeding immuno-adsorbent to the immuno-adsorbent
apparatus.
9. Apparatus in accordance with claim 7 and further comprising
removing means for the immuno-adsorbent with the specific antibody
attached thereto from the immuno-adsorbent apparatus.
10. Apparatus in accordance with claim 7 in which the
immuno-adsorbent is selected from the group consisting of
insolubilized horse anti-Fc, heterologous horse anti-Fc,
insolubilized sheep anti-Fc, heterologous sheep anti-Fc,
insolubilized rabbit albumin, horse anti-gamma-globulin,
heterologous anti-Fc, and heterologous anti-gamma-globulin.
11. Apparatus in accordance with claim 7 and further comprising
means connected to the immuno-adsorbent apparatus for transferring
lymph fluid free of the specific antibody from the immuno-adsorbent
apparatus to the patient.
12. Apparatus in accordance with claim 11 and further comprising
means for filtering the lymph fluid free of the specific antibody
prior to being returned to the patient.
Description
The subject is first given antigen administration then, preferably,
but not mandatorily, is splenectomized. A thoracic duct fistula is
next performed. The central venous system pressure is then
preferably raised so that it is above the atmospheric pressure of
the thoracic duct. In this manner, substantially all the lymph
fluid is allowed to flow out of the thoracic duct from the fistula
(through an indwelling catheter) for a prolonged period of time.
The lymph is separated into cells and lymph fluid. The cells are
returned to the subject intravenously. The subject must be given
replacement fluid, which can be of several kinds, but all lacking
the specific antibody.
By virtue of the above procedure, the plasma and extra-cellular
fluid of the subject is continuously depleted of feedback antibody
because the antibody is continuously removed prior to its reaching
the blood stream. Because of this lack of antibody, in the presence
of antigen administration, it is found that the antibody production
in the lymphoid tissue and therefore its content in the lymph fluid
is enormous and ever-increases. The tremendous increase in antibody
production appears to be several orders of magnitude greater than
other modes of antibody production and therefore appears to have
very substantial utility in the fields of biology, chemistry and
veterinary and clinical medicine. BACKGROUND OF THE INVENTION
1. Introduction
Antibodies are protein in nature and fall into the category of
immuno-globulins. Most of the antibodies are found in
gamma-globulin. Antibodies are synthesized by the lymphoid tissue
of animals and man as the immunological response to challenges,
that is foreign bodies, introduced into the animal or man. These
challenges are called antigens. Antibodies react specifically with
antigens in various ways to hasten elimination of antigens and
reduce the possibility of infection.
The production and collection of antibodies from animals is, at
present, a very costly procedure.
2. Utility of Antibodies
Antibodies have a wide variety of known uses. First, antibodies are
used as prophylactic therapy for animals and humans who have been
exposed (or who have been thought to be exposed or are in danger of
being exposed) to various infections and other antigens. This type
of therapy applies to normal individuals or animals and those with
immunological deficiences induced by disease, congenital anamolies,
iatrogenically induced or accidentally induced (e.g., through
radiation overexposure, etc.) or other immunological deficiencies.
The injection of antibodies is utilized as part of, or as a whole
regimen of, the therapy of various infectious and disease states in
animals and man and applies to individuals with normal and actual
or potential immunological deficient states.
Antibodies are used as part or whole of the therapy to suppress the
active development or expression of an immune state. For example,
antibodies to RH antigen are given to RH-negative women to suppress
their becoming actively immunized to RH antigen thereby decreasing
the chance of ill effect to their progeny. It is possible that
antibodies could be utilized to prevent humans from rejecting organ
and tissue transplants. Allogenically or heterogenically produced
antibodies (intact or altered) should protect grafts or hosts from
immunological damage. Antibodies (intact or altered) against tissue
and other antigens can be used to prevent the development of
autoimmune disease or to treat such autoimmune disease after it has
been established.
Antibodies are used as specific immuno-chemical reagents to study,
identify, localize, isolate, inactivate and measure the amount of
various compounds (such as specific components of normal or altered
plasma proteins, hormones, enzymes, normal and malignant tissue,
and cellular antigens etc.). Thus, antibodies against
transplantation antigens are used for tissue typing and labeled
antibodies against tumor antigens could be used for detection and
diagnosis of cancer.
Prior Art Method for Production of Antibodies
Prior to the process of this invention the art of collecting
antibodies from immunized animals or humans fell into two different
categories. First, one could periodically remove whole blood and,
secondly, one could periodically remove whole blood, separate the
blood into cells and fluid and return the cells. In both cases only
a small proportion, approximately one-quarter of the antibody
contained in the blood stream and one-eighth of the antibody
contained in the body, is removed.
These techniques for production of antibody are very costly because
of the small proportion of antibody present in the body which is
removed. It is known that plasmaphoresis, or periodic removal of
whole blood and return of the cells of the subject (animal or
human), causes increased antibody production in the host. However,
because only a small proportion of antibody present in the blood is
removed, the amount of increase in antibody production is limited
until this loss is corrected. The procedure also has other
substantial drawbacks. The procedure requires the handling of blood
which has a high cellular content compared to lymph. The procedure
can only be performed intermittently rather than continuously.
Also, the procedure for plasmaphoresis removes the antibody only
after the antibody has exerted some of its feedback control and
therefore the augmentation of the production of the antibody is not
high.
In short, the removal of antibody by plasmaphoresis has limitations
in that only a small proportion of the total antibody in the body
is removed, the antibody cannot be removed continuously, is only
removed after the antibody has had a chance to circulate in the
bloodstream and after it has already exerted some feedback action.
A high antibody production and collection is therefore not
achieved.
Other procedures, such as the serial transfer of spleen cells from
an immunized animal into non-immunized irradiated syngenic
recipients and tissue culture procedures, demonstrate the
magnification of the immune response in the absence of feedback
antibody. These two procedures are not presently applicable to the
production and collection of large amounts of antibody. Serial
transfer of spleen cells shows that augmentation of antibody
production can occur, but the antibody producing cells are
distributed in all the recipients making the collection of the
antibody difficult and inefficient. Present techniques of tissue
culture of lymphoid tissue do not sustain prolonged antibody
production in high amounts and therefore this technique is not yet
applicable to the production and collection of large amounts of
antibody.
The present invention is directed towards a process whereby
tremendous production and collection of antibodies is provided by
means of a continuous process which utilizes standardized or
available apparatus.
Rationale for the Present Invention
Antibodies are synthesized in the lymph nodes, spleen and other
lymphoid masses of the body of the animal or human. The flow of
antibodies from their sites of production to the venous circulation
are shown diagrammatically in FIG. 1 under conditions of normal
physiology and anatomy. Antibodies are secreted directly from the
spleen 20 into the venous system 22, via venous channels 24, and
without first flowing into the lymphatic vessels. Secondly, the
antibodies leave the major lymphoid masses 26 by their efferent
ducts 28. Data are available to support the lymphatic routes of
antibody exit from the lymphoid tissues. The efferent ducts 28 from
most but not all lymphoid masses join to make up the thoracic duct
30 which enters the major vein in the neck. Thirdly, the antibodies
leave the minor lymphoid masses 32 via efferent lymph ducts 34. The
efferent ducts 34 open via alternate lymphatico-venous channels 36
directly into the venous circulation system 22. The efferent ducts
34, from the minor lymphoid masses 32, also open via
lymphatico-lymph anastomotic channels 40 directly into the thoracic
duct 30, or into vessels which communicate with the thoracic
duct.
Thus, it will be seen that, under conditions of normal physiology
and anatomy, if a thoracic duct fistula is made, antibodies which
are synthesized in the spleen and some lymphoid masses can reach
the blood stream directly from the spleen 20 and from lymphoid
masses 32 via channels 34 and 36.
Under natural conditions, antibody is eliminated either slowly, as
by degradation, or quickly when the organism is invaded by antigens
which complex the antibody. Under appropriate experimental
conditions, antibody can be eliminated quickly by physical means.
Apart from the influence of the presence of antigen-antibody
complexes, the natural and experimental methods of eliminating
antibody would be expected to have a similar quantitative and
qualitative effect in augmenting the immune response. A moderate
augmentation of the immune response and antibody production occurs
if a small proportion of the total antibody in the body is removed
by plasmaphoresis. However, unless a high proportion or all of the
newly synthesized and augmented antibody production is removed
prior to its reaching the bloodstream, a new state of equilibrium
with relatively steady but increased antibody production and steady
blood level of the antibody is established.
It is the rationale of the present invention that only by removing
the newly synthesized and augmented antibody production as it is
made, and prior to its reaching the bloodstream, can a continuing
out-of-equilibrium state, with respect to antibody, be established.
This continuing out-of-equilibrium state will result in a prolonged
low blood titre and a continuing increase in antibody production.
Since the out of equilibrium state is the cause for the increasing
antibody production and results from the induced antibody loss,
such circumstances lead to a very large production of antibodies,
which can be collected from the subject rather readily.
To take the rationale further, if the central venous pressure is
raised and the thoracic duct is converted into a fistula so that
the pressure in the thoracic duct is low, a differential pressure
between the thoracic duct and veins is established. Under these
circumstances, all production of lymph flows into the thoracic duct
fistula. Now, if further a splenectomy is caused so that no
antibodies can flow directly from the spleen into the bloodstream,
then virtually all production of lymph (and with it, all production
of antibodies) flows into the thoracic duct fistula, and does not
enter the bloodstream at all.
If then the lymph fluid is not returned to the subject, it is found
that increasing production of antibodies many, many times that of
usual antibody production is produced in the lymphoid masses and
flows out through the fistula in the thoracic duct, and is
collected therefrom.
A similar phenomena will occur if the lymph fluid after removal of
antibody is sent directly back to the blood stream of the
subject.
The foregoing rationale is the basis for the present invention and
can be summarized in the following manner.
SUMMARY OF THE INVENTION
The present invention relates to a process for the continuous
prolonged fluid lymphoresis from an immunized animal or human. The
lymph is separated into cells and lymph fluid. The cells are
returned intravascularly together with replacement fluid which can
be of several kinds but all lacking specific antibody, e.g., plasma
or serum from non-immunized animals, or lymph (from the same
animal) from which specific antibody or gamma-globulin has been
removed.
The lymphoresis is normally carried out in the animal or human with
certain induced anatomical and physiological changes and utilizes
available or standardized apparatus. The process results in an
increased production of antibody, in the human or animal, which
increased production rises as the process continues.
The lymphoresis procedure consists of establishing a thoracic duct
fistula, preferably under conditions such that the lymph fluid is
caused to flow only in the thoracic duct, and after collecting the
lymph fluid, processing it in such a manner as to separate the
lymph cells from the lymph fluid. The lymph cells are then returned
intravenously to the subject and the cell-free lymph fluid is
collected and either (1) not returned to the subject or (2) may be
processed on-stream to extract from it, the specific antibody or
gamma-globulin in which case, the anti-body free or gamma-globulin
free lymph fluid is preferably returned to the bloodstream of the
subject.
The animal or human's health is maintained by appropriate
replacement therapy of serum, proteins, hormones, etc.
The body of the animal or human is thus depleted of feedback
antibody and responds by producing antibody in enormous quantities
since the immunological system of the animal or human senses that
it is lacking in the antibody that its nature has come to demand in
the presence of antigen. In this fashion, antibody is produced in
tremendous quantities from very few animals and, in turn, can be
collected at a very, very small fraction of the cost of present day
methods now employed.
DESCRIPTION OF THE FIGURES
FIG. 1 is a diagrammatic view of the sources of antibody
production, namely spleen and lymphoid masses, and of the normal
flow of the antibodies from the source to the venous system of the
animal or human;
FIG. 2 is a diagrammatic view of the same system which has been
surgically and medically altered in accordance with the purpose of
the invention;
FIG. 3 is a schematic presentation of one embodiment of the process
of the invention showing the separation of lymph into cells and
lymph fluid. The cells of the lymph are returned free of lymph
fluid. The lymph fluid is not returned to the subject who receives
instead plasma or serum from non-immunized subjects. In addition,
the subject receives antigen and serum or albumin or dextran or
other osmotically active materials, the foregoing items (except
antigen) being given to increase the venous pressure. The venous
pressure can also be elevated by surgical procedures which increase
the resistance of the venous return to the heart;
FIG. 4 is a schematic presentation of another embodiment of the
process in which antibody is removed from the lymph by an
immuno-adsorbent. The cells contained in the lymph are returned to
the subject as is the antibody-free and cell-free lymph. In
addition, the subject receives antigen and albumin or serum or
dextran or other osmotically active materials, the foregoing items
(except antigen) being given to increase the venous pressure;
FIG. 5 is a schematic presentation of a third embodiment of the
process. The lymph is separated into cells and fluid. The cells are
returned to the subject. Gamma-globulin is removed from the lymph
by horse (or heterologous) Anti Fc. The subject is given pooled
gamma-globulin prepared from pooled serum of plasma. In addition,
the subject receives antigen plus albumin (or serum or dextran or
other osmotically active materials); and
FIGS. 6 through 14 illustrate, graphically, the augmented
production of antibody resulting from the various embodiments of
the present invention.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
The following description relates to the presently preferred
embodiment of my process wherein maximization of the immune
response (i.e., antibody production) occurs. Maximum augmentation
requires (a) antigen administration prior to and during fistula
procedure; (b) splenectomy of the subject prior to fistula; (c)
raised central venous pressure during fistula; (d) thoracic duct
fistula procedure; and (e) separation of cells and lymph fluid with
return of cells without lymph fluid followed by the separation of
antibodies or gamma-globulin from the lymph fluid and return of the
lymph fluid (minus the antibodies or gamma-globulin) to the subject
in a continuous manner, or alternatively to this procedure, wasting
the lymph fluid (with respect to the subject) and providing
adequate replacement therapy to the subject. Antibodies are then
collected from the wasted lymph fluid.
However, it is to be understood that many variations in the above
procedure are possible which, while they do not result in
maximization of the immune response, do result in greatly increased
production of antibody from the subject. Thus, antigen
administration to a subject over several weeks followed by
lymphoresis through a thoracic duct fistula (but without
splenectomy and raised central venous pressure), removal of
antibodies from the lymph fluid, and the return of the
antibody-free lymph fluid to the subject (or in the alternative,
wasting of the lymph fluid but providing adequate replacement
therapy) has also been found to result in highly augmented antibody
production.
Specific examples of these variations will be described at a later
point in this application.
Turning now to the procedure illustrated diagrammatically in FIG.
2, the anatomy of the lymph system is modified so that the thoracic
duct becomes the only lymph channel. This is optimally achieved by
a two-stage operative procedure before thoracic duct fistula and
before or after antigen administration or during the fistula
procedure by appropriated quantities of albumin or serum infusions,
or dextran or other osmotically active materials.
Referring specifically to FIG. 2, the central venous pressure of
the patient or subject (whether it be animal or human) is elevated
to between about 12 to 25 centimeters of water pressure by surgical
procedures or by the intravenous administration of albumin and/or
certain other replacement therapy chemicals, such as the
intravenous administration of salt solutions, serum or plasma or
dextran. Such administration is shown schematically in line 50a of
FIG. 3, line 81 in FIG. 4 and line 102 in FIG. 5. The amount of
administration of the replacement therapy medicaments and/or
albumin or serum or dextran is controlled by the desired venous
pressure. The venous pressure may be measured continuously by
electronic means. If it is below a critical and chosen value, the
subject receives a measured quantity of additional therapy
medicaments and/or albumin or serum or dextran during the
subsequent time period.
Additionally, the venous pressure may be increased by a controlled
increased resistance of the venous returned to the heart. If this
is found to be required the azygous veins are tied and remote
controlled cuffs are placed around the superior vena cava (SVC) and
inferior vena cava (IVC). After the subject has recovered from the
operation (approximately three days to one week) the constriction
will be applied to the IVC so as to increase the venous pressure to
approximately 15 to 25 centimeters of water pressure.
A fistula is then made in the thoracic duct 30a, as shown at zone
52 in FIG. 2, and an indwelling cannula (not shown) is inserted
into the fistula. The technique for the thoracic duct fistula has
been established in rats, rabbits, sheep, calves, man and other
animals and may require the use of a special double lumen catheter.
Heparin, or other blood anticoagulant, is infused up one lumen to
prevent clotting and to assure free flow of lymph from the fistula
52.
To this point, it will be seen that, because of the differential
venous lymphatic hydrostatic pressure the major lymph flow from
both major and minor lymphoid masses 26a and 32a, will occur, via
efferent lymph channels 28a, 34a, through the lymphatico-lymph
anastomic channels 40a, thence through the thoracic duct 30a and
finally flow through the thoracic duct fistula 52 rather than
through the multiplicity of alternate lymphaticovenous channels
36a. However, because antibody flow also occurs from the spleen 20a
directly to the venous circulation system 22a, by-passing the
thoracic duct 30a, for optimal results, and in order to prevent
antibodies from reaching the venous system and hindering the
augmentation of the antibody production system, the lymph flow from
the spleen 20a to the venous system 22a is preferably also
eliminated by a splenectomy, as shown diagrammatically at zone
62.
Splenectomy is the procedure for surgically removing the spleen.
This is achieved by an abdominal operation -- ligating and cutting
the splenic artery(ies) and vein(s) and then removing the spleen.
The splenectomy operation is not necessary for the increased
production of antibody, by virtue of the process of this invention,
but is preferred for optimal augmentation of antibody generation
and immune response.
The venous pressure can be elevated by the infusion of appropriate
amounts of albumin or serum or dextran or other osmotically active
materials. The venous pressure can also be elevated surgically by
methods which increase the resistance of the venous return to the
heart. If the venous pressure is elevated surgically it is
preferable that it be achieved by a two-stage procedure. The first
procedure is carried out prior to the fistula and consists of
ligation of the azygous veins and partial occlusion of the inferior
vena cava. The second procedure is the fistula and partial
occlusion of the superior vena cava.
The thoracic duct 30a becomes enlarged because of the increased
venous pressure and enables a larger cannula to be inserted in the
thoracic duct than would otherwise be possible. (The cannula is
inserted into the thoracic duct after the thoracic duct has
enlarged and reaches a plateau). The increased venous pressure also
prevents collateral lymphatico-venous channels from developing
during the fistula procedure and insures continuous lymph flow
through the thoracic duct. The increased venous pressure also
closes or reverses flow in the normal and already established
lymphatico-venous channels and enlarges established
lymphatico-lymph anastomic channels 40a.
The thoracic duct 30a is thus assured of being the primary lymph
channel and the fistula 52 performed will thereby allow the maximum
elimination of newly synthesized antibody through this primary
lymph channel.
Despite the foregoing procedures, it is possible that some newly
formed antibody will enter and circulate in the bloodstream,
especially when a splenectomy is not performed. Nonetheless, a very
significant proportion of such antibody will be lost through the
thoracic duct fistula 52.
In order that the process of this invention result in greatly
increased augmentation of antibody production and insure the health
of the subject, the cells contained in the lymph are required to be
returned intravenously in a viable and undamaged condition. This
may be achieved by a batch technique or by a continuously
centrifugal cell separator device, set forth in FIGS. 3, and 4, and
designated by numeral 70, and in FIG. 5 designated by numeral
108.
The lymph passes from the thoracic duct fistula 52 directly to the
centrifuge separator 70 (or 108) one type of which separator has
been described and patented in Australian Pat. No. 31,178/67 of
Kerby. The lymph fluid passing into the cell separator 70 (or 108)
shown in the above-identified Australian patent comes into contact
with surfaces within the cell separator, which do not denature or
alter the protein and other macromolecules within the lymph fluid.
Furthermore, frothing or foaming of the lymph fluid is avoided in
order to minimize conditions which might cause cell destruction and
denaturation of proteins. The temperature of the lymph fluid
passing through the cell separator is regulated between 25.degree.
C and 37.degree. C and care is taken to provide that the
temperature of the lymph does not rise appreciably as the cells
enter or leave the centrifuge.
In order to minimize any changes that might occur in the lymph and
cells when they are outside the body of the subject, the dead space
within the cell separator 70 (or 108) and ancillary equipment is
made as small as possible to minimize the time that the lymph cells
are outside the body. The lymph cells are retained outside the body
for 15 to 80 minutes at the most, and the rate of lymph flow, in
sheep and man, is preferably maintained at between 5 litres per day
and 12 litres per day.
The cell separator 70 (or 108) collects the lymph from the thoracic
duct fistula 52, separates the cells from the lymph fluid, washes
the cells and returns them to the subject, in an undamaged state,
intravenously, via line 71 in FIG. 3 or 4, and via line 114 in FIG.
5.
Complete sterility of the equipment is maintained and clotting
within the equipment is avoided by the injection of Heparin or
other anticoagulant. Build-up of fibrin particles and cellular
aggregates are avoided by automatic "flushing" of various critical
points of the apparatus. The centrifuge cell separator 70 (or 108)
continuously operates and may be monitored to record lymph flow,
temperature rise, and other major functions.
While the use of automatic continuous equipment has many advantages
over a batch technique, batch techniques may also be used to remove
the lymph fluid and return the lymph cells to animals. The
advantages of the continuously operating cell separator 70 (or 108)
include less manipulation of the cells, less chance of accidental
infection, and the shorter period for the lymphocytes to be out of
the body.
The cell-free lymph is pumped by any one of a number of pumps,
e.g., a tube compression pump from the centrifuge cell separator 70
(or 108), along line 79 to waste in FIG. 3 or along line 72, to an
immuno-adsorbent apparatus (IA), bearing numerical 74, in FIG. 4 or
along line 112 to immuno-adsorbent apparatus (IA) bearing numeral
110 on FIG. 5. In the latter two cases, the cell-free lymph fluid
comes into contact with, and agitated with, an immuno-adsorbent. An
insolubilized specific immunoadsorbent is chosen which adsorbs
gamma-globulin (FIG. 5) or other specific antibody (FIG. 4) from
the lymph fluid. The gamma-globulin, or other specific antibody,
adsorbed onto the immuno-adsorbent, is thus insolubilized and is
separated from the cell-free lymph fluid in a continuous flow
centrifuge which separates the insoluble material from the lymph
fluid automatically. The antibody free lymph is returned to the
subject via line 76 (FIG. 4), and line 120 (FIG. 5) after
preferably passing through a filter 78. The insolubilized
immunoadsorbent with attached antibody leaves IA apparatus 74 via
line 80 as in FIG. 4 or leaves IA 110 via line 115 in FIG. 5.
Examples of immuno-adsorbent materials are insolubilized horse (or
heterologous) anti Fc (sheep) which will remove sheep
gamma-globulin, and insolubilized rabbit albumin, which will remove
sheep anti-rabbit albumin.
Again, the removal of the gamma-globulin may be achieved by a batch
technique wherein hourly samples of cell-free lymph are adsorbed
into the appropriate immuno-adsorbent, and the remaining fluid is
returned to the subject.
The immuno-adsorbent after having been used in the IA apparatus may
be regenerated by a special piece of equipment which is not on line
with the main flow of body fluids. The regeneration can be achieved
by a batch technique either manually (repeated washing and
centrifugation and dispersion) or on a column.
The antigen to be given, of course, will vary depending upon the
specific type of antibody to be generated and produced in the
subject. This administration commenced, via line 75, is prior to
fistula. It is possible that continued production, in a highly
augmented fashion, of feedback antibody may not occur even under
the conditions set forth hereinabove, unless the subject is
repeatedly challenged by antigen. The continuation of antigen
administration after fistula has become operative, may be a
mandatory step in the process.
The immune status of the subject with respect to the normal
spectrum of antigenic stimuli which it may come in contact with, is
maintained by giving the subject appropriate amounts of pooled
plasma or serum or pooled gamma-globulin of that particular species
via line 50 in FIG. 3, line 50b in FIG. 4 or line 104 in FIG. 5.
While it is possible to specifically and selectively augment the
immune response against a specific antigen by removing from the
circulation the specific antibody, gamma-globulin (which contains
the antibody) as an entity may also be removed from the lymph fluid
of the subject.
The method depicted in FIG. 3, while not as complex as the process
described in FIG. 4 and FIG. 5 illustrates a basic concept of the
invention and is completely operable to augment antibody production
in the subject, which may itself, have therapeutic value. Referring
to FIG. 3 specifically, the thoracic duct fistula 52 and antigen
administration, via line 75, are followed as described earlier, and
lymph sent to separator 70 via line 52. The cells of lymph are
returned via line 71 to the subject. The total cell-free lymph
fluid is wasted, via line 79, and the essential replacement
ingredients, i.e., replacement therapy for the subject, entering
via line 50a, 50 are pooled plasma or serum of the same species as
the subject. Plasma or serum contains components necessary for life
and health of the subject as well as feed-back antibodies of all
kinds apart from the feed-back antibody related to the specific
antigen to which the subject has been deliberately immunized. The
plasma or serum will also increase the venous pressure if given in
appropriate amounts. This method does not require an
immuno-adsorbent apparatus (IA) or a centrifuge additional to the
centrifuge cell separator.
Another mode of treatment utilizing the concepts of this invention
is illustrated in FIG. 4.
Antigen is fed to the subject, via line 75, as described with
references to the FIG. 4 embodiment; lymph fluid is removed from
the thoracic duct fistula 52 and sent to the centrifuge cell
separator 70. The cells of the lymph are returned to the subject
via line 71, and the cell-free lymph sent on the IA apparatus 74,
via line 72. In this embodiment the immuno-adsorbent utilized is an
insolubilized specific antigen and a specific antibody (and only
the specific antibody) is thereby removed from the lymph fluid.
The immuno-adsorbent is fed to an immuno-adsorbent apparatus 74 via
line 86, and is intimately admixed with the cell-free lymph
entering IA apparatus 74 via line 72. The insolubilized
immuno-adsorbent with attached specific antibody is separated out
from the IA apparatus and leaves the IA along line 80, and the
cell-free, specific antibody-free lymph is returned to the
subject.
In FIG. 5, another variant of the basic invention is shown wherein
the subject is fed antigen and replacement therapy chemicals of a
specific type to be discussed, via lines 75, 102 and 104,
respectively. The thoracic duct fistula 106 is made as previously
described with reference to FIGS. 3 and 4. The lymph fluid is
separated from the lymph cells in a centrifuge cell separator 108,
the cell-free lymph proceeding to an IA apparatus 110, via line
112, and the cells returning to the subject via line 114. The
processing sequence and apparatus is the same as discussed with
reference to FIG. 4. The horse (or heterologous) anti Fc with its
attached gamma-globulin leaves the IA via line 115 and the
gamma-globulin free lymph is returned to the subject via line
120.
The immuno-adsorbent utilized for the process of FIG. 5 is horse
(or heterologous) anti-Fc, a specific insolubilized
immuno-adsorbent insolubilized by cross-linking with covalent
bonds. The horse (or heterologous) anti-Fc removes the
gamma-globulin (containing the specific antibody) from the lymph
and pooled gamma-globulin or pooled serum is utilized for the
replacement therapy (line 104) as well as the replacement
components, including albumin serum or dextran to raise the venous
pressure (line 102).
In the following Examples 1-32, the augmentation of antibody
production, and the collection thereof is illustrated with
reference to (a) antigen administration and (b) a thoracic duct
fistula performed but without splenectomy and without raising the
central venous pressure of the subject. Highly augmented production
of antibody results as compared with antibody production in the
control subjects. Examples 33 and 34 illustrate the toptimal mode
of procedure of this invention wherein splenectomy and increased
central venous pressures are employed.
Specific examples of the process of this invention follow:
EXAMPLES 11-32
In the following examples, 23 control rats and 9 experimental rats
were each administered antigen for a given period of time. A
thoracic duct fistula was performed on the experimental rats. No
thoracic duct fistula was performed on the control rats. No
splenectomy or increased central venous pressure procedures were
utilized with the experimental rats.
The total number of antibody cells in the spleen and in the
mesenteric lymph nodes in both central and experimental rats as
well as 19S plaque-forming cells and 7S antibody cells were
measured.
Details of the procedures are set forth below.
The animals used were Holtzman rats (250-350 gm. in weight).
Antigen administration comprised each rat receiving, intravenously,
10.sup.9 freshly washed Sheep Red Blood Cells (SRBC) in 1 ml. of
saline solution. The SRBC were given at the beginning of the
experiment and every 3 days thereafter.
Fistula procedures were performed on the experimental rats at
various times after the first antigen administration. The thoracic
duct fistula was performed by a standard technique except that the
catheter was made to have a double lumen. The larger lumen tube is
the tube through which the lymph flows, and has a polyethylene tip
which is attached to a silastic tube. The tip has a bevel for ease
of entry and a bump to ensure that the ties behind the bump keep
the catheter in position.
The silastic tube which forms the main part of the catheter provide
flexibility so that small errors of alignment are corrected without
tension on the duct. The smaller lumen is made from polyethylene
and is drawn out in a hot stream of nitrogen to a diameter of 100
microns. The nitrogen prevents oxidation of the surface of the
polyethylene during the drawing out procedure. The polyethylene
surface thus retains its excellent anti-clotting property.
The smaller tube of the catheter lies inside the main tube and
carries heparin in Ringers solution to the very tip of the
catheter. The Ringers solution, containing 10 units of heparin/ml.
was infused into the experimental rats at a rate of 1-2 ml./hr. up
the catheter. This prevented the lymph from clotting and prevented
the catheter from being blocked also by cellular aggregates.
Lymph collection is made by collecting it from the fistula in the
experimental animals in ice cold 50 ml. polycarbonate centrifuge
tubes, each containing 5 ml. of Ringers solution. (Each ml. of
Ringers solution contains 20 units of heparin, 0.1 mg. of
streptomycin and 100 units of penicillin). The lymph was collected
in 12-hourly batches, evenly suspended and a sample taken for a
white cell count (which was performed with a coulter counter). The
lymph was then centrifuged and the lymph fluid (mixed with Ringers
solution) was withdrawn, kept in separate containers and
deep-frozen for subsequent assays.
The lymph cells were suspended in 2 ml. of rat plasma and
immediately returned intravenously as follows:
A polyethylene catheter (polyethylene PE 50), was drawn down to 1/4
mm. was inserted into the femoral vein in such a way that the top
of the catheter was located in the iliac vein or inferior
vena-cava. It was found that, for success, the top of the venous
catheter should lie in a large vein so as to make possible the
returning of the cells, and the administration of medication
through the intravenous route over an extended period -- without
causing thrombosis of the vessel. Ringers solution, containing 2
units heparin/ml. was infused intravenously at the rate of 1-2
ml./hr. In addition approximately 10-25 ml. of rat serum was
infused each day.
Replacement therapy is provided as follows:
A silastic tube was inserted into the stomach of each rat and
sealed in position by a double row of purse string nylon atraumatic
sutures. Each rat received increasing amounts of high protein and
high calorie liquid food through the gastric tube. On the second
day of the fistula 10 ml. of this liquid food was given three times
per day. Between feedings, a continuous (1 ml./hr.) gastric drip of
Ringers solution was given.
The 4 tubes (thoracic duct cannula, heparin line to cannula,
intravenous catheter and gastric tube) pass from their sites of
insertion and run subcutaneously to emerge from beneath the skin of
the tail. In this way, when the animal is placed in a conventional
restraining cage, the experimentor has access to all catheters,
whereas the rat cannot interfere with these tubes.
The number of 19 -S haemolysin producing cells in the spleen and
mesenteric lymph nodes were estimated by localized haemolysis
(plaques) using agar as a thickening agent (following Jerne, N. K.,
and Nordin, A.A. -- Science 140: -405, 1963).
Antibody was assayed by the micro-titre agglutination technique.
7-S antibody was measured as the 2 ME resistant antibody. Blood was
collected by cutting tail veins. Serum was separated from the clot
after standing overnight at 2.degree.-5.degree. C and was
inactivated by heating at 56.degree. for 30 minutes. The titres
from a given experiment were determined simultaneously.
Lymph was collected in 12-hourly batches, evenly suspended, samples
withdrawn, the remainder centrifuged and the supernatant fluid
withdrawn. Each lymph sample was corrected for the dilution caused
by injection of Ringers (containing heparin) into the cannula and
by the volume (5 ml.) of Ringers, which was originally placed in
the centrifuge tubes. The agglutination titres in the successive
lymph samples of any rat were estimated simultaneously.
The rats were maintained in a fairly good health. Their degree of
hydration was normal and each rat only lost approximately 4-6
percent of his body weight.
TABLE 1 and GRAPH 1 (FIG. 7) show the number of 19S antibody
producing cells (Jerne plaque positive cells) in the spleens of the
control and experimental rats. In the 23 control rats, there is an
increasing number of plaque forming cells until day 8 (the sixth
day after antigen administration) followed by a sharp decline. The
thoracic duct fistula and cell-return procedure was performed on
the experimental rats as follows: 2nd day after antigen
administration (rats 4, 21, 22), on the 10th day (rat 41) and on
the 11th day, (rats 42, 43, 45), and finally on the 19th day (rats
33, 34), after the first antigen administration.
A 4-20 fold increase in the number of plaque cells in the spleens
of the experimental rats occurred (compared to the 23 control rats,
which were killed and analyzed after the same duration of repeated
antigen administration). The rate of increase of plaque forming
cells in spleens of rats operated 2 days after the first antigen
administration (rats 4, 21, 22) was faster than in the spleens of
rats operated at later dates.
TABLE I
__________________________________________________________________________
19-S PLAQUE FORMING CELLS IN SPLEEN OF CONTROLS AND EXPERIMENTAL
ANIMALS (PER 10.sup.6 SPLEEN CELLS)
__________________________________________________________________________
Days after repeated Controls antigen adminis- Experimental Rats
tration R 4 R 21 R 22 R 32 R 33 R 41 R 42 R 43 R 44 R
__________________________________________________________________________
45 0 0 1 38 65 2 84 90 * * * 3 98 do. do. do. 4 96 126 do. do. do.
5 346 450 do. **1800 **2240 6 712 **4530 7 576 8 847 1056 9 672 10
480 * 11 422 do. * * * * 12 336 do. do. do. do. do. 13 -- do. do.
do. do. do. 14 -- do. do. do. do. do. 15 -- do. do. do. do. do. 16
140 163 do. do. do. do. **452.sup.1 17 -- do. do. do. do. 18 --
**480.sup.2 do. do. do. 19 110 * * **1920 **1400 **2000 20 96 do.
do. 21 -- do. do. 22 83 **252 do. 23 -- do. 24 -- do. 25 63 **596
__________________________________________________________________________
*Indicates day of fistula. **Indicates day when rat was killed.
.sup.1 R 45 - Lymph stopped on 3rd day of fistula, flow
intermittent. .sup.2 R 41 - Lymph flow intermittent, stopped 2 days
prior to death.
TABLE II and FIG. 7 show that a generally similar result occurs in
the mesenteric lymph nodes as in the spleen.
Antibody Titres in Blood.
The total and 7S antibody (Agglutination Titre) in the blood of
control and experimental animals (Rat 42, 43, 44 -- 11 days after
first antigen administration) is shown in TABLE III and FIG. 8. In
the control rats there is a rapid rise in the blood titre until day
8, with a subsequent fall and plateau. (The rise in antibody titre
is measured as a ratio of amount on the day measured to day 1 of
the test). 19S antibody becomes undetectaable after day 10. In the
experimental rats there is a transient marked fall in the blood
titre followed by a rise and overshoot. In addition 19S antibody
reappears and transiently increases.
Daily Antibody Content and (Therefore Loss) in Lymph.
The volume of lymph lost was irregular and varied from 12-87
ml./day.
The average loss of total and 7S antibody in the lymph is shown in
TABLE IV (for rats 42, 43, 44) and TABLE V (for rats 33, 34). The
individual results of these animals are shown in TABLES VI, VII,
VIII, IX and X, as well as in FIG. 9. (total and 7-S antibody),
FIG. 10, (total antibody) and FIG. 11 (7-S antibody). In addition,
the ratio of total antibody lost on any day of the fistula compared
to day 1 is given in Table IV, column 6 (average for rat 42, 43,
44); TABLES V - VII, column 6 (individual values for rats 33, 34).
In all animals there is a short lag period followed by increasing
loss, reaching a value 19 times (average for rats 42, 43, 44) and
82 times (average for rats 33, 34) higher on the last as compared
to the first day of fistula.
Daily Antibody Lost in Lymph Compared to Total Antibody Content of
Blood:
The daily loss of antibody in the lymph increases, and is 10 - 30
times the total blood content on any one day (TABLE IV, average for
rats 42, 43, 44 -- compare column 4 and 8 for total antibody and
column 5 and 10 for 7-S antibody). Similar comparisons of
individual rats are given in Tables V, VI and VII. For rats 33 and
34 the antibody content of the blood was only estimated on the day
they were killed, that is after 7 days of lymph drainage. The
results are shown in TABLE VIII. The total antibody lost on the
last day of lymph drainage was 20 times the total blood
content.
TABLE II
__________________________________________________________________________
19-S PLAQUE FORMING CELLS IN MESENTERIC LYMPH NODES (PER 10.sup.7
__________________________________________________________________________
cells) DAYS AFTER RE- PEATED ANTIGEN CONTROLS EXPERIMENTAL RATS
ADMINISTRATION R4 R21 R22 R41 R42 R43 R45
__________________________________________________________________________
0 0 1 0 2 30 * * * 3 30 do. do. do. 4 30 do. do. do. 5 90 do.
**3260 **370 6 0 **560 7 60 8 35 9 57 10 13 * 11 5 do. * * * 12 --
do. do. do. do. 13 -- do. do. do. do. 14 -- do. do. do. do. 15 --
do. do. do. do. 16 20;18 do. do. do. **50.sup.1 17 -- do. do. do.
18 -- **50.sup.2 do. do. 19 30 **1200 **2270 20
__________________________________________________________________________
*Indicates day of fistula. **Indicates day when rat was killed.
.sup.1 R41 -- Lymph flow intermittent stopped 2 days prior to
death. .sup.2 R45 -- Lymph stopped on 3rd day of fistula, flow
intermittent. DAILY BLOOD ANTIBODY TITRES IN CONTROLS AND
EXPERIMENTAL ANIMALS -- AVERAGES
__________________________________________________________________________
DAYS AFTER Re- TOTAL ANTIBODY 7-S ANTIBODY TITRE PEATED ANTIGEN
CONTROL EXP. RATS CONTROL EXP. RATS ADMINISTRATION RATS (R
42,43,44) RATS (R 42,43,44)
__________________________________________________________________________
0* 0 0 1 0 0 2 1:2 0 3 1:8 1:1 4 1:32 1:2 5 1:64 1:8 6 -- -- 7
1:128 1:32 8 1:256 1:128 9 1:128 1:64 10 1:64 1:32 11 1:64 1:64**
1:32 1:32** 12 1:64 1:16 1:32 1:8 13 1:32 1:8 1:32 1:4 14 1:32 1:4
1:32 1:1 15 -- -- -- -- 16 1:32 1:16 1:32 1:4 17 1:32 1:32 1:32 1:8
18 1:32 1:32 1:32 1:16 19 1:32 1:64 1:32 1:64 20 1:32 1:32
__________________________________________________________________________
* Day 0 = Day before immunization ** Day of Fistula TABLE III
__________________________________________________________________________
AVERAGE OF DAILY VALUES: RATS 42; 43; 44.
__________________________________________________________________________
(1) (2) (3) (4) (5) (6) (7) (8) (9) (10) CONTENT OF ANTIBODY** IN
BLOOD ANTIBODY** LOST IN LYMPH DAYS POST LYMPH PER DAY TOTAL
ANTIBODY 7-S ANTIBODY LOST PER DAY TOTAL CON- EXPERI- CON- EXPERI-
IMMUNI- FIS- IN ML. TOTAL 7-S COMPARED TROL MENTAL TROL MENTAL
ZATION* TULA TO DAY 1 RATS RATS RATS RATS
__________________________________________________________________________
11 0 Nil Nil Nil 640 -- 320 -- 12 1 17 378 232 1 640 160 320 107 13
2 48 2,182 323 5.8 320 80 320 33 14 3 52 3,334 398 8.8 320 40 320
10 15 4 52 2,995 212 8.0 (a) (a) (a) (a) 16 5 58 2,430 308 6.3 320
160 320 47 17 6 60 4,325 673 11.5 320 320 320 160 19 8 67 7,148
7,148 19.0 320 533 320 533
__________________________________________________________________________
* Immunization: 10.sup.9 SRBC given i.v. 11 days prior to fistula
and every 3 days thereafter. ** Antibody measured in agglutinating
units; 1 agg. u. represents a positive hemagglutinin titre in
undiluted first tube, Example: Agg. u. = volume (of lymph/or blood)
.times. Serial dilution titre. Total agg. units of blood based on
estimated serum volume of 10 ml. per rat. (a) Not tested. TABLE IV
__________________________________________________________________________
AVERAGE OF DAILY VALUES: RATS 33 AND 34.
__________________________________________________________________________
(1) (2) (3) (4) (5) (6)
__________________________________________________________________________
DAYS POST ANTIBODY** LOST IN LYMPH PER DAY IMMUNI- LYMPH LOST TOTAL
7-S TOTAL COMPARED ZATION* FISTULA PER DAY IN ML. ANTIBODY ANTIBODY
TO DAY 1
__________________________________________________________________________
20 1 32 192 106 1 21 2 38 349 18 1.8 22 3 24 221 29 1.2 23 4 31 498
105 2.6 24 5 31 6,064 312 32.0 25 6 38 13,295 1,742 69.0 26 7 43
15,812 8,491 82.0
__________________________________________________________________________
TOTAL 7-S Antibody in blood at Control 480 480 Post-Mortem:
Experimental 800 480
__________________________________________________________________________
* Immunization: 10.sup.9 SRBC given i.v. 19 days prior to fistula
and every 3 days thereafter. ** Antibody measured in agglutinating
units: 1 agg. u. represents a positive hemogglutinin titre in
undiluted first tube (i.e.) agg. u. = volume (of lymph/or blood)
.times. serial dilution titre. Total agglutinating units of blood
based on estimated serum volume of 10 ml. per rat TABLE V
__________________________________________________________________________
DAILY RESULTS: RAT 42
__________________________________________________________________________
(1) (2) (3) (4) (5) (6) (7) (8) (7) (10)
__________________________________________________________________________
CONTENT OF DAYS POST LYMPH ANTIBODY**LOST IN LYMPH BLOOD TITRE
ANTIBODY** LOST PER DAY IN BLOOD PER DAY IMMUNI- FIS- IN ML. TOTAL
TOTAL 7-S TOTAL 7-S ZATION* TULA TOTAL 7-S COMPARED ANTI- ANTI-
ANTI- ANTI- TO DAY 1 BODY BODY BODY BODY
__________________________________________________________________________
12 1 25 360 360 1 1:16 1:8 160 80 13 2 42 1,500 261 4.2 1:8 1:4 80
40 14 3 31 2,592 111 7.2 1:4 1:1 40 10 15 4 46 1,334 83 3.7 (a) (a)
(a) (a) 16 5 45 1,350 85 3.8 1:16 1:8 160 80 17 6 63 3,975 598 11.0
1:32 1:16 320 160 18 7 40 6,040 2,195 16.8 1:32 1:16 320 160 19 8
64 7,259 7,259 20.1 1:32 1:32 320 320
__________________________________________________________________________
*Immunization: 10.sup.9 SRBC given i.v. 11 days prior to fistula
and every 3 days thereafter. **Antibody measured in agglutinating
units; 1 agg. u. represents a positive hemogglutinin titre in
undiluted first tube. Example: Agg. u. = volume (of lymph/or
blood), .times.Serial dilution titre. Total agg. units of blood
based on estimated serum volume of 10 ml. per rat (a) Not tested.
TABLE VI
__________________________________________________________________________
DAILY RESULTS: RAT 43
__________________________________________________________________________
(1) (2) (3) (4) (5) (6) (7) (8) (9) (10)
__________________________________________________________________________
CONTENT OF DAYS POST LYMPH ANTIBODY**LOST IN LYMPH BLOOD TITRE
ANTIBODY** LOST PER DAY IN BLOOD** PER DAY TOTAL TOTAL 7-S TOTAL
7-S IMMUNI- FIS- IN ML. TOTAL 7-S COMPARED ANTI- ANTI- ANTI- ANTI-
ZATION* TULA TO DAY 1 BODY BODY BODY BODY
__________________________________________________________________________
12 1 12 348 122 1 1:16 1:8 160 80 13 2 49 1,356 337 3.9 1:8 1:2 80
20 14 3 52 1,558 240 4.5 1:4 1:1 40 10 15 4 51 4,291 133 12.3 (a)
(a) (a) (a) 16 5 64 2,432 307 7.0 1:16 1:2 160 20 17 6 56 4,689 566
13.5 1:32 1:4 320 40 18 7 58 3,869 2,018 11.1 1:32 1:16 320 160 19
8 87 7,041 7,041 20.2 1:64 1:64 640 640
__________________________________________________________________________
*Immunization: 10.sup.9 SRBC given i.v. 11 days prior to fistula
and every 3 days thereafter ** Antibody measured in agglutinating
units; 1 agg. u. represents a positive hemogglutinin titre in
undiluted first tube. Example: Agg. u. = volume (of lymph/or blood,
.times. Serial dilution titre. Total agg. units of blood based on
estimated serum volume of 10 ml. per rat (a) Not tested. TABLE VII
__________________________________________________________________________
DAILY RESULTS: RAT 44. (1) (2) (3) (4) (5) (6) (7) (8) (9) (10)
ANTIBODY LOST IN LYMPH CONTENT OF DAYS POST LYMPH PER DAY BLOOD
TITRE ANTIBODY** LOST IN BLOOD PER DAY TOTAL TOTAL 7 - S TOTAL 7 -
S IMMUNI- FIS- IN ML. TOTAL 7 - S COMPARED ANTI- ANTI- ANTI- ANTI-
ZATION TULA TODAY 1 BODY BODY BODY BODY
__________________________________________________________________________
12 1 13 425 213 1 1:16 1:16 160 160 13 2 53 3,690 371 8.7 1:8 1:4
80 40 14 3 73 5,853 444 13.8 1:4 1:1 40 10 15 4 59 3,360 420 7.9
(a) (a) (a)(a) 16 5 64 3,450 532 8.1 1:16 1:4 160 40 17 6 56 4,312
855 10.1 1:32 1:8 320 80 18 7 56 7,220 1,939 16.9 1:64 1:16 640 160
19 8 51 7,143 7,143 16.8 1:64 1:64 640 640
__________________________________________________________________________
* Immunization: 10.sup.9 SRBC given i.v. 11 days prior to fistula
and every 3 days thereafter. ** Antibody measured in agglutinating
units; 1 agg. u. represents a positive hemagglutin titre in
undiluted first tube. Example: Agg. u. = volume (of lymph/or
blood)..times. Serial dilution titre. Total agg. units of blood
based on estimated serum volume of 10 ml. per rat. (a) Not tested
TABLE VIII
__________________________________________________________________________
DAILY VALUES: RAT 33. (1) (2) (3) (4) (5) (6) DAYS POST ANTIBODY**
LOST IN LYMPH PER DAY IMMUNI- LYMPH LOST TOTAL 7 - S TOTAL COMPARED
ZATION* FISTULA PER DAY IN ML. ANTIBODY ANTIBODY TO DAY 1
__________________________________________________________________________
20 1 16 260 130 1 21 2 37 395 0 1.5 22 3 26 266 0 1.02 23 4 30 435
103 1.7 24 5 39 9,179 448 35.3 25 6 39 10,482 544 40.3 26 7 48
20,844 10,260 79.0 TOTAL 7 - S Antibody in blood at Control 640 640
Post - Mortem: Experimental 1,280 640
__________________________________________________________________________
* Immunization: 10.sup.9 SRBC given i.v. 19 days prior to fistula
and every 3 days thereafter. Antibody measured in agglutinating
units: 1 agg. u. represents a positive hemagglutinin titer in
undiluted first tube (i.e.) agg. u. = volume (of lymph/or blood)
.times. serial dilution titer. Total agglutinating units of blood
based on estimated serum volume of 10 ml. per rat. TABLE IX
__________________________________________________________________________
DAILY VALUE: RAT 34. (1) (2) (3) (4) (5) (6) DAYS POST ANTIBODY**
LOST IN LYMPH PER DAY IMMUNI- LYMPH LOST TOTAL 7 - S TOTAL COMPARED
ZATION* FISTULA PER DAY IN ML ANTIBODY ANTIBODY TO DAY 1
__________________________________________________________________________
20 1 48 124 82 1 21 2 39 303 35 2.4 22 3 22 176 58 1.4 23 4 32 560
101 4.5 24 5 23 2,950 176 23.8 25 6 37 16,108 3,065 130.0 26 7 41
13,446 6,722 108.0 TOTAL 7 - S Antibody in blood at Control 320 320
Post - Mortem: Experimental 320 320
__________________________________________________________________________
* Immunization: 10.sup.9 SRBC given i.v. 19 days prior to fistula
and every 3 days thereafter. ** Antibody measured in agglutinating
units: 1 agg. u. represents a positive hemogglutinin titre in
undiluted first tube (i.e.) agg. u. = volume (of lymph/or blood)
.times.serial dilution titre. Total agglutinating units of blood
based on estimated serum volume of 10 ml. per rat. TABLE X
__________________________________________________________________________
Establishing a thoracic duct fistula, wasting the lymph fluid and
providing adequate replacement therapy therefor, and returning the
cells contained in the lymph, causes the following results in rats
who are given repeated injections of SRBC:
1. there is an increasing antibody loss in the lymph fluid with
duration of fistula.
2. The daily amount of antibody lost in the lymph fluid exceeds the
antibody content in the blood at any one time by a factor of
10-30.
3. The large loss of anitbody in the lymph causes a transient fall
in the antibody titre in the blood.
4. The fall in the blood titre can be interpreted best as acting in
the manner of reduced feed-back control on the immune system. This,
in conjunction with repeated antigen administration results in
a. an increase in the number of 19S plaque forming cells in spleen
and lymph nodes. (The number of 7S plaque forming cells was not
measured).
b. an increasing daily production of total and 7S antibody
(estimated by loss in lymph, not accountable for by decreasing
blood content).
c. the reappearance in blood and lymph of 19S antibody.
EXAMPLES 33 AND 34
The proportions of feedback anitbody produced can be greatly
increased over those set forth in Examples 1-32 by splenectomy
procedures as well as by the increase in systemic venous pressure,
as has been described previously. Examples incorporating these
additional procedures are set forth below.
Two Rats (R55 and R56) were splenectomized 3 weeks prior to the
first antigen administration. Systemic venous pressure was raised
by administration of rat serum intravenously. The pressure in the
venous system was raised to between 12 to 20 centimeters water
pressure above atmospheric. The venous pressure was measured three
times each day by a simple saline manometer attached to a central
venous catheter. The volume of a 60 percent serum solution
necessary to maintain the elevated venous pressure was estimated
from the previous lymph lost and the venous pressure. Each rat
received 50-80 ml. of 60 percent serum solution each day. The
venous pressure remained elevated to levels between 12 cms. and 20
cms of hydrostatic head.
Antigen administration and thoracic duct fistulas on R55 and R56
were performed as described with reference to Examples 1 through
32. Lymph production for R55 and R56 is shown in GRAPHS VII and
VIII (FIGS. 13 and 14, respectively), together with total anitbody
measured in agglutinating units. GRAPH IX (FIG. 14) is a comparison
of the net lymph production and the total agglutination units of
antibody in the lymph for R55 and R56 as compared with the best
production from R33 and R34. This, of course, provides a direct
comparison between procedures within venous pressures and
splenectomy procedures where only a thoracic duct fistula was made
without venous pressures and without splenectomy (R33 and R34).
The difference in antibody production between the two procedures is
evident and is of a higher order of magnitude when the venous
pressure is raised and splenectomy procedures are performed.
While various procedures are herein set forth, other modifications
and variations will occur to those skilled in the art. I intend,
therefore, to be bound only by the claims which follow:
* * * * *