Note: Descriptions are shown in the official language in which they were submitted.
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A PROCESS OF PROLONGING ORGAN ALLOGRA~T SURVIVAL
Description
Technical Field of the Invention
The field of the present invention is organ allograft survival.
More specifically, the field of the present invention is prolongation of organ
allograft survival. Organ allograft survival is prolonged by down-regulating
Thl activity in an organism receiving an organ allograft.
Back~round of the Invention
The development of novel immunosuppressive drugs has
caused a dramatic increase in the short term survival of organ transplants in
recent years. The use of such drugs, however, is associated with side effects
such as opportunistic infections, tumors and ultimately, chronic rejection.
The recent description of two subsets of T helper cells (Th),
with different cytokine secretion profiles and activities, may provide a new
paradigm for immunoregulation of organ allograft rejection (Mocm~nn et al.,
Immuno]. Todav 8:223, 1987~. The two Th cell subsets, de~ignated Thl and
Th2, can be characterized on the basis of their respective cytokine
production profiles. Th1 cells produce and secrete interferon-gamma (IFN-
y), lymphotoxin (LT) and interleukin-2 (IL-2). On the other hand, Th2 cells
produce and secrete interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6
(IL-6) and interleukin-10 (IL-10).
Acute organ allograft rejection may result from responses
associated with the pro-infl~mm~tory cytokines IL-2, IFN-y, and ~NF-~r,
derived from Thl ce]ls. Down-regulation of Thl activity, therefore, may be
a useful immunomodulatory therapy. It is also well known that inlEection by
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certain parasitic organisms can modulate the balance between Thl and Th2
activity. By way of example, Sher and coworkers have demonstrated that a
depression of Thl-type responses can be achieved by helminth infection
(Sher et al., J. Immunol., 147:2713, 1991). Helminth infection can also result
in up-regulation or stimulation of Th activity. Nematode induced up-
regulation can encc~mp~cs stimulation of IgE (and IgG1 in mice or IgG4 in
humans), mast cell hyperplasia and eosinophilia in all species, including
humans.
It is well known that the immune modulation associated with
helminth infection is unrelated to the worm antigensper se. For example,
greater than 80 percent of the IgE generated during Nippostrongylus
brasiliensis (N. brasiliensis) infection was found not to be directed to N.
brasiliensis (Jarrett el al., Clin. Exp. Immunol., 24:326, 1976).
Such induced up-regulation may be due to the cross-regulatory
actions of the Thl/Th2 system. The Thl and Th2 subsets have been found
to be cross-regulatory with respect to differentiation and activity. By way of
example, cytokines produced by Th2 cells (e.g., IL-4 and IL-10) inhibit Thl
cell growth and cytokine production and ablate the effects of Thl cytokines
(e.g., IFN-y) on their targets, while IFN-y inhibits Th2 function (Powrie et
al., Immunol. Today. 14:270, 1993).
The concept of resistance or susceptibility being related to
Thl/Th2 balance has also been suggested in a variety of other models,
including AIDS, and even successful pregnancy. The finding of pro-
infl~mm~tory cytokines IL-2, IFN-y, TNF-~r and IL-6 in rejecting kidney
allografts lends support to the hypothesis that Thl-type T cells orchestrate
the immune response, which culminates in rejection. It likely that a
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Thl/Th2 balance is established within the graft which changes over time,
~ differs among individuals, and is dependent on the treatment ~mini~tered
but that the cellular mech~ni~mc behind graft rejection, such as infi~tration
by macrophages and CI'L, are c~ e.~t with the activities of Thl cells.
s
Brief Summary of the Invention
In one aspect, the present invention provides a ~-ocess of
prolonging organ allograft survival in an organism COlll~li',illg down-
regulating Thl activity in the olg~i.~....
In a preferred embodiment, the down-regulation of Th1
activity is accomplished by infecting the organism with an effective Th2 up-
regulating amount of a nematode.
In another preferred embodiment, the down-regulation of Thl
activity is accomplished by ~-lmini~t~.ring to the org~ni~m an effective Th2
up-regulating amount of a soluble extract of a nematode.
Exemplary and preferred nematodes for use in a process of
the present invention are of the genus N'ippostrongvlus, Tric~uns, Ascaris or
Caenorhabditis. More preferred are Nippostrongylus brasiliensis, Trichuris
muris, Trichuris suis, Ascaffs lum~ffcoides, Ascaris suum or Caenorha~ditis
elegans. Most preferred is Nippostrongylus brasiliensis (N. brasiliensis). A
process of the present invention is particularly useful in prolonging hdney
allograft survival.
Brief Description of the Drawin~s
In the drawing, which forms a portion of the specification:
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FIG. 1 shows IgG1 levels following N. brasiliensZs infection or
treatment with N. br~siliens~s e~ctract.
.,
Detailed Descripbon of the Invention
I. The Invenbon
Infection with ~alasi~es such as nematodes induces strong IgE
responses not restricted to parasite antigens. In addition, certain he~minths
have profound immunomodulatory effects on infected ~nim~l~, inducing
strong Th2 responses while rliminiching Thl activity. The present invention
provides that infection of organisms with n.om~todes or the ~(1mini~tration of
soluble nematode extracts to organisms significantly prolonged organ
allograft survival in those org~ni~n~
II. Process of Prolonging Allograft Survival
In one aspect, the present invention provides a process of
prolonging organ al~ograft survival in an organism culllpli~ing down-
regulating Thl activity in the Ol~ iS~.
As used herein; the phrase "organ allograft" means a body
organ graft from a donor organism of the same species but a different
genotype than the recipient organism. As used herein, the term "organ"
means a vascularized internal organ such as heart, liver and kidney. An
exemplary and preferred organ allograft is a hdney allograft. As used
herein, an organ does not include skin. Skin differs from vascularized
organs contemplated by the present invention because a substantial
component of the graft vascular supply is derived from the donor rather
than the recipient. Thus, shn allografts are much less susceptible to certain
immune-mediated injuries (Bradley e~ aL, Immunol. Today. 13:434, 1992).
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As used herein, the terms "up-regulation" and "down-
~ regulation" and their gr~mm~t~c~l equivalents mean, respectively, shmulation
and inhibition. Thus, down-regulahon of Thl activity means a depression or
inhibition of the cascade of physio]ogical responses that acc~ pally a Thl
cytokine production profile and up-regulahon of Th2 activity means a
stimulahon or enhancement of the c~ de of physiological responses that
acco.l.~.al,y a Th2 cytokine production profile.
In a preferred embodiment, the down-regulation of Thl
activity is accomplished by infecting the o~ga~ - with an effective Th2 up-
regulating amount of a nematode. An effective amount is that amount
necessary to bring about the desired effect (e.g., down-regulation of Th1
activity). Means for determining an effective amount are well known in the
art. By way of example, an organism is infected with various amounts of a
particular nematode and the level of Thl activity monitored. An effective
amount will depend inter alia, as is well known in the art, on the nature of
the organism being injected and the nature of the particular nematode used.
Exemplary effective amounts using rodents and the nematode N. brasiliensis
are set forth hereinafter in t~e Examples.
In another plefelled embodiment, the down-regulation of Thl
activity is accomplished by ~mini~terjng to the organism an effective Th2
up-regulating amount of a soluble e~tract of a nem~tode. As set forth
above, means for determining an effective amount of a soluble extract are
well known and readily ascertainable by a skilled artisan. Means for making
a soluble extract are also well known in the art (See. e.g., Lee e~ aL,
Immunology, 55:721, 1985). By way of example, nematodes are
homogenized in an aqueous medium such as saline (0.9% NaCl). The
medium can further comprise buffers such as phosphate. Homogenization
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can be accomplished by mech~nir~l means or other disruptive means such as
sonication. The homogenate is ~pically cleared of particulate matter using
ultracentrifugation. The nematode is then filter sterilized to remove
bacteria.
s
A soluble nematode extract can be ~tlminictered to the
organism by parenteral routes of ~tlminictration as is well known in the art.
The e7~tract can be dispersed or suspended in a physiologically acceptable
diluent prior to ~f~minictratiom A preferred route of a-lminictration is
subcutaneous. An extract can be ~tlminictered prior to, simultaneously with
or after organ transplantation. In a ~lefelled embodiment, an extract is
minictered prior to, simultaneously with and after transplantation. One of
ordinary skill in the art can readily dete~nine the optimum ~minictratjon
schedule for a given transplant and treatment regimen.
Any nematode that down-regulates Thl activity can be used in
a process of the present invention. Exemplary and preferred nematodes for
use in a process of the present invention are of the genus Nippostrongylus,
Trichuris, Ascaris or Caenorhabditis. More preferred are Nippostrongylus
brasiliensis, Trichuris muris, Trichuris suis, Ascaris lumbricoides, Ascaris suum
or Caenorhabditis elegans. Most preferred is Nippostrongylus brasiliensis (~.
brasiliensis).
A process of the present invention prolongs allograft survival
when compared to allograft survival performed in the absence of nematode
treatment. In other words, a process of the present invention improves or 4
increases survival time of the allograft.
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A detailed description of the use of a process of the present
invention to prolong kidney allograft survival is set forth hereinafter in the
FY~mples. Briefly, rats were infected with N. brasilienszs (Nb) or treated
with a soluble extract of N. brasiliensis (Nb extract) and received a kidney
S allograft. The surviva] time of the kidney al]ograft was increased from 9.7 +
1.2 days (no treatment) to 32.0 + 10.0 days (Nb infection) and 21.5 + 4.6
days (Nb extract treatment). By day S post tr~n~pl~nt, untreated allografts
contained a substantial mononuclear cell infiltrate and kidney destruction
was advanced. In contrast, hdneys transplanted into Nb treated animals
had only a mild cellu]ar infiltrate and retained normal architecture. FACS
analysis revealed a significant decrease in the number of CD8+ cells
infiltrating Nb treated grafts. Prelimin~ry RT-PCR analysis of mRNA
expression by graft-infiltrating ce]ls indicated that the production of IL-4 wasup-regulated in Nb-treated kidneys as opposed to untreated allografts.
In additional studies, Nb treatrnent was associated with
substantial and significant increases in IgG1 levels (See Example 2).
The following Examples illustrate preferred embodiments of
the present invention and are not limiting of the claims or specification in
any way.
EX~MPLE 1: Kidnev Allo~raft Survival
Lewis (LEW,RT1l) and Brown Norway (BN, RT1n) rats were
chosen as recipients and donors, respectively. Male rats were purchased
from Harlan Sprague Dawley (Indianapolis, IN) and provided with water
and rat chow ad libitum. ~nim~lc receiving a ~.brasiliensis infection were
injected with 3~00 third stage infective larvae subcutaneously four days prior
to transplantation. ~nim~l~ receiving the nematode products were injected
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subcutaneously, at day -4, 0 and +4 (with day 0 being the day of transplant)
with 200 worm equivalents of adult worm homogenate. All procedures were
performed under sodium pentobarbital ~n~çst~esia (65 mglkg).
S The left kidney of the donor (BN) rat was perfused in situ withcold (4~C) saline, mobilized, and removed. It remained in cold saline during
the pl~a~ation of the recipient. Following left nephrectomy of the Lewis
recipient, the BN kidney was transplanted in a heterotopic position.
Ureteric reconstruction in these experiments was achieved by end-to-end
anastomosis. Four days after the transplants, the recipients were re-
anaesthetized and a right native nephrectomy was perforrned. ~nim~lc were
sacrificed when they showed signs of morbidity due to failure of the
transplanted kidney.
Graft survival data are shown below in Table 1.
TABLE 1
Kidney Allograft Survival Following Nematode Infection
Treatment Survival (days) Mean + SD
No treatment 10, 8, 11, 11
9,8,11,9,10 9.7+1.2
Nb infection 34, 27, 34, 23
25, 34, 25, 54 32.0 + 10.0
Nb extract 30, 21, 22, 20
16, 20 21.5 + 4.6
These results clearly show a significant prolongation of kidney
allograft survival in anirnals g*en either a N.brasiliensis infection or soluble
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worm extract. In untreated ~nim71~ kidneys survived a mean of only 9.7 +
1.2 (mean + SD, n=9) days, whereas kidneys in nematode infected rats
retained functional capacity much longer, resulting in a mean survival time
of 32.0 + 10.0 (mean + DS, n-8) days, with one animal retaining grafted
S kidney function up to 54 days post tr~nq-l~nt. In ~nim~l~ treated with the
worm extract the survival was also significantly longer (mean 21.5 + 4.6).
In addition to graft survival the histological appearance of the
transplanted kidneys was ~ccecse~ in the control versus ~. brasiliensis treated
recipients 5 days after transplant. Transplanted kidneys were flushed with
cold saline and cut into 2-3 mm thick slices before fixation in formalin.
Sections were taken and stained with hem~toxylin and eosin using standard
protocols.
The results demonstrate clear differences between the kidneys
taken from the N. brasiliensis infected and the control groups. The intensity
of the infiltration was, in a blinded evaluation, found to be much more
extensive in the control group when compared to the 1~. brasiiiensis infected
group. In the control group,'there was extensive infiltration of the interstitiaby mononuclear cells and evidence of damage to the tubular endothelium in
some tubules. In the kidneys isolated from N. brasiliensis infected recipients,
in contrast, there was only a small amount of mononuclear infiltrate around
major vessels and almost no interstitial infiltrate. Kidney architecture in the
N. brasiliensis infected recipients was virtually normal.
These data show that infection of rats by the nematode 1~.
brasiliensis or treatment with N. brasiliensis extracts significantly prolonged
kidney allograft survival across a known strong histocompatibility barrier.
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This prolongation likely results from the ability of N. ~rasiliensis to stronglyactivate Th2 responses with a resultant inhibition of Thl activity.
EX~MPLE 2: ~e,eulation Of I~G1 Response Bv Nematodes
Increases in the serum levels of total (non-specific) IgE and
IgG1 or IgG4 are associated with nematode infections in mice and humans
respectively. There are several points where this response can be regulated.
One of such, is during the process of Immunoglobulin class switch.
A group of five BALB/c mice was infected with 500 infective
larvae of the nematode Nippostrongylus brasiliensis and a second group of
five BALB/c mice (age matched) was injected subcutaneously with an extract
from N. brasiliensis (Adult Worm Homogenate, AWH) at a concentration of
200 worm equivalent emulsified in Freund's Incomplete Adjuvant. The
levels of total IgG1 in the serum obtained from the blood taken from these
mice, were measured weekly for ten weeks, by IgG1 caplure ELISA.
The data showed clearly that, the nematode extract, AWH
induces signiScant increases in the production of total serum IgG1. The
result agrees with the massive increase in total IgE aDd IgG1 levels
associated with infection with the larvae. As shown in FIG. 1, a ten fold
increase in the IgG1 levels was observed. Prior to the injection of AWH,
the total serum IgG1 level in the mice was 0.25mg/ml. This level increased
continuously from week 1 (0.30mg/ml) through week 4 (1.15 mglml).
Further increases in the serum IgG1 level upon re-injection with the
nematode extract at week 4, were also observed. It reached its maximum
(2.15 mg/ml) at week 7, before it started to decline. AT. ~rasiliensis extract
AWH, also induced significant increase iD total IgG1 levels in vivo.
