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Immunomodulatory agents from natural products |
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Principal Investigator : Shakti N Upadhyay Project Associates/Assistants Collaborators Immunomodulatory therapy, which relates to stimulation
or suppression of immune response of the host, is now being recognized as an
alternative to conventional chemotherapy for a variety of diseased conditions,
especially when host’s defense mechanisms have to be activated under the
conditions of impaired immune responsiveness or when a selective
immunosuppression has to be induced in situations like auto-immune disorders,
allergic reactions and organ transplantation. Since natural products,
especially the plants used in the Indian traditional medicine, are the
potential source of such immunomodulatory compounds, this project aims at
evaluating the compounds isolated from such plants for both immunopotentiating
and immunosuppressive activity with a view to developing leads for new
therapeutic products. In order to develop new therapeutic strategy, the work
is being carried out to identify plant derived molecules that i) inhibit the
expression of LPL by macrophages, ii) inhibit the LPL enzyme activity, iii)
inhibit expression of ICAM-1 on the endothelial surface, and iv) inhibit
pancreatic lipase activity. The project aims at i) identification and
isolation of immunopotentiating compounds from natural products based on their
stimulatory effect on the macrophage effector functions and ability to induce
Th 1/Th2 response, ii) identification and isolation of
anti-inflammatory/immunosuppressive/anti-atherosclerotic compounds from
natural products based on their inhibitory effect on the enzymes involved in
the signal transduction of immunological/inflammatory stimuli, and iii)
evaluation of the therapeutic potential of the immunomodulatory molecules
using appropriate animal models. NII-60 As mentioned last year, technical information and
commercial rights related to NII-60 were transferred to Gufic Biosciences
Limited, Mumbai, who have filed an application with the Drug Controller of
India to initiate clinical trials for prevention of septic shock in burn
patients. In the mean time, a collaborative work was initiated with the
Safdarjung Hospital, New Delhi to carry out evaluation of immunological status
of burn patients. Following ethical clearance obtained from appropriate
hospital authorities, blood samples were collected from burn patients
immediately upon arrival at the burn emergency unit and subsequent blood
samples were collected on alternate days, till discharge or survival of the
patients. Serum samples were analyzed for various immunological markers,
including cytokines and proliferative response of peripheral blood leukocytes
to PHA, LPS, tetanus toxoid and PPD. The result on some of the parameters
showed interesting correlation or distinction between survivor and
non-survivors. While IL-6, IL-10 levels were higher in survivors, TGF-b was
found to be higher in non-survivors. Similarly, proliferative response of
peripheral lymphocytes gradually increased in survivors on subsequent days, it
remained low in non-survivors. These parameters will be considered during the
clinical trials with NII-60. NII-60 was further
evaluated for LPS binding/neutralization property. NII-60 interacted with
different types of LPS obtained from E.coli, Shigella flexneri, Pseudomonas
aeruginosa and Salmonella typhimurium with same intensity. This data suggests
that NII-60 binds to a common interaction centre present in all types of LPS,
most likely the phosphorylated anionic lipid A moiety. The interaction of
NII-60 was quantitatively assayed by fluorescent method. The interaction of
NII-60 with LPS was established as ionic, as the relative binding intensity
decreased from 100% to 32.20 % in Polymyxin B displacement reaction at
equimolar concentration. Though this interaction was established at all pH,
the maximum binding was observed at pH 7.2 indicating its potential as LPS
antagonist at physiological pH. The neutralizing effect of NII-60 was further
demonstrated in Limulus gelation assay. To confirm the interaction of NII-60
with LPS in intact microorganisms, the lysozyme mediated outer membrane
permeabilization was evaluated in E.coli and compared with known cationic
membrane permeabilizer polymyxin B at same concentration. Interestingly NII-60
was also found to enhance the lysozyme mediated membrane permealization in
E.coli, like polymyxin-B. NII-18 A water-soluble salt of NII-18 has been prepared which
retained the biological activity. The inhibitory effect of this purified
NII-18 was also investigated against lipoprotein lipase and was found to be
reversible which follows the competitive inhibition kinetics pattern at IC50
1mM. Inhibition of NII-18 was observed at alkaline pH whereas there is no
effect or only slight inhibitory effect at acidic pH. Ligands such as Mg++,
Ca++, Cu++ reversed the inhibitory effect of NII-18, whereas Zn+ and glucose
maintained the inhibitory effect of NII-18 on lipoprotein lipase.
Interestingly high glucose concentration (10mM) sustains and increases the
inhibitory effect of NII-18 for longer time. Edible vaccine: expression of antigenic determinants
of Vibrio cholerae in tobacco or tomato and evaluation of their immunological
potential The aim of this study was to express cholera toxin B
subunit (ctxB) in tomato and tobacco and to determine its immunologic
potential. In this collaborative project, the construction of ctxB gene is
being carried out by CBT, Delhi and IMTECH, Chandigarh, and expression of the
antigens in plants is being carried out at the University of Delhi, South
Campus; immunological studies are being done at NII. Initial experiments were carried out to
check whether CT-4A is immunogenic or not by intra-dermal immunizations. CT-4A
immunized animals showed significantly higher cholera toxin specific IgG in
their serum as compared to those immunized with untransformed plant from the
2nd bleed onwards. Cholera toxin B was used as the positive control. However
it showed the presence of CT-specific IgG from the 1st bleed onwards only. T cell proliferation studies were carried out in these
same groups of animals. The group immunized with untransformed tobacco leaf
extract showed normal proliferation to Con-A, LPS and anti-CD3. However,
animals immunized with CT-4A and CTB showed selective suppression of T cells
in Con-A and anti-CD3 treated cells. The activity of LPS stimulated cells were
similar in all three groups of animals. Cytokine analysis was done on the supernatants
generated by the in vitro stimulation of the splenocytes isolated from these
different groups of animals. The cytokines that were studied are IL-10, IL-12
and TNF-a. Cholera toxin induced IL-10 production. However the amount produced
was significantly higher in CT-4A and CTB immunized groups. Addition of Con-A
and anti-CD3 reduced this amount. However LPS failed to show any suppressive
or synergistic action on the cytokine production levels. IL-12 and TNF-a
production was suppressed in CT-4A and CTB immunized groups and addition of in
vitro cholera toxin in the culture suppressed it even further. When the
experiment was repeated this trend was maintained. Mice were orally immunized with the same antigen. Then
these groups of mice were divided into 2 groups. In group (i) peyers patches
were taken out and stimulated in vitro with cholera toxin, and in group (ii)
these animals were given cholera holotoxin orally and were sacrificed after 24
hours. In these animals also studies were done in peyer’s patches.
Unstimulated cells were used as control. The studies included estimation of
CT-specific IgA and cytokine levels in the culture supernatants. The
experiments shows that only in CTB immunized group there was a significant
enhancement in the level of CT-specific IgA following oral challenge. However
the CT-4A immunized animals failed to induce significant CT-specific IgA
following either in vitro or oral challenge. The IL-10 also showed similar
profile, whereas IL-12 and TNF-a levels were very low. Publications Original peer-reviewed articles 1. Furmanova M, Gajdzis-Kuls D, Ruszowska J,
Czamocki Z, Obidoska G, Sadowska A, Rekha Rani and Upadhyay SN (2001) In vitro
propagation of Withania somnifera and isolation of withanolides with
immunosuppressive activity. Planta Medica (in press). 2. Yadav RP and Upadhyay SN (2001)
Environmental CO2 inversely regulates lipase-phospholipase production by
Aspergillous species. Ind J Microbiol (in press). 3. Agarwal Pujya, Yadav RP and Upadhyay SN
(2001) Growth medium for differentiation of pathogenic from non-pathogenic
aspergilli. J Med Microbiol (in press). Reviews/Proceedings 1.
Yadav RP, Agarwal Pujya and Upadhyay SN (2001) Microbial
lipases: Tool for drug discovery. J Sci Indus Res 59:977-987. Patents 1. Upadhyay SN, Wali N and Sarma DNK (2000) A
novel process for the preparation of mangiferin. and a composition used for
the prevention/treatment of parasitic diseases. Indian patent # 183,778
granted in Apr 2000. 2. Upadhyay SN, Sarma DNK, Saraswathi NK, Wali
N, Dhawan S and Yadav RP (2000) A novel process for isolation of berberine
from berberine containing plant material. Indian patent # 184,685 granted in
Sep 2000. 3. Upadhyay SN, Sarma DNK, Wali N, Saraswathi
NK and Dhawan S (2000) Method for suppressing allogenic immune response for
prevention/treatment of graft-vs-host disease or graft rejection. US patent
application # 08/789,464 granted in Oct 2000. 4. Upadhyay SN and Madan V (2001) Method for ex
vivo expansion of hematopoietic cells. US patent application # 09/339,759
granted in Mar 2001. |