National institute of immunology




НазваниеNational institute of immunology
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NATIONAL INSTITUTE OF IMMUNOLOGY

ANNUAL REPORT

2001 – 2002


CONTENTS

Terms of Reference 1

Preamble 3

Research Reports 9

Immunity and Infection 11

Gene Regulation 47

Molecular Design 63

Reproduction and Development 97

Ancillary Activities 117

Publications 121

Patents 129

Lectures Delivered on Invitation/Papers Presented 130

Distinctions/Honours 135

Membership of Extramural Bodies 137

Academic Courses and Training Programmes 141

Ph.D. Degrees Awarded to NII Scholars 146

Collaborations and Delegations 149

Distinguished Visitors 150

Seminars by Visiting Scientists & Guest Investigators 151

Supporting Units 153

Foundation Day 2001 159

Conferences/Symposia/Workshops Organized 160

Other Notable Activities/Events 161

Committees of the Institute 164

Staff of the Institute 169


TERMS OF REFERENCE

 To undertake, aid, promote, guide and co-ordinate research of high calibre in basic and applied immunology

 To carry out research for development of new vaccines and immunological reagents for communicable diseases

 To develop immunological approaches for regulation of male and female fertility

 To interact with industry for manufacture of vaccines and immunological reagents

 To organize postgraduate courses, workshops, seminars, symposia and training programmes of a specialized nature in the field of immunology, vaccine development and related areas

 To organize training programmes for technicians in immunological methods and related techniques

 To establish affiliation with recognized universities and institutions of higher learning for the purpose of enabling research scholars to register for postgraduate degrees

 To serve as a national reference centre for immunology and to provide consultancy services to medical and veterinary institutions, public health agencies and industries in the country

 To provide and promote effective linkages on a continuing basis between various scientific and research agencies/laboratories and other organizations working in the country in the field of immunology, vaccine development and related areas

 To collaborate with foreign research institutions, laboratories and other international organizations in fields relevant to the objectives mentioned above


PREAMBLE

The National Institute of Immunology (NII) strives to work steadily at furthering knowledge of the molecular mechanisms of interaction between biological systems and their environment, seeking clues for new intervention modalities of relevance to healthcare. Our strategy involves linking excellence in rigorous fundamental research with the pragmatic pursuit of emerging application possibilities in entrepreneurial partnerships. Thus, the range of areas being explored at NII span the cellular cross-talk, parasitic targets, and the precise modulation of bodily defense mechanisms at different levels of complexity which have, over the years, coalesced into four major themes: immunity and infection, gene regulation, molecular design, and reproduction and development. The salient features of our work during the reporting year 2001-02 are highlighted below.

Immunity and Infection

The use of a self-polymerizing coat protein from Johnson grass mosaic virus (JGMV) for presenting antigenic peptides from JEV is being explored. Fusions of four peptide sequences chosen from JEV E protein were made to JGMV CP using the recombinant DNA methods. The over-expressed fusion protein made in E. coli was purified and was shown to form virus-like-particles (VLPs). It was found that the VLPs induced vigorous anti-peptide and anti-JEV antibodies with high titers.

Multiple factors that include the host genes, the genetic make up of the virus and the relevant immunological responses influencing HIV-1 virulence, progression and pathogenesis are continued to be analysed. A unique feature about all the messenger RNAs in HIV-1 is the presence of a site for the HIV-1 regulatory protein TAT. Ribozymes targeted to cleave at this site have potential to down regulate TAT activity. The preliminary results suggest that the designed ribozymes indeed protect against, HIV-1 TAT-mediated apoptosis.

In order to examine the apoptotic pathways that determine the normal limit to the induction of T cell memory, various mechanisms that can possibly mediate the death signal in activated T cells are being investigated. It appears that autocrine free radical-mediated T cell activation-induced cell death may be crucial in limiting the degree of secondary responsiveness in antigen-responding primary T cell populations.

Long-lived peptide epitope mimics with high affinities for MHC are expected to ameliorate some of the difficulty with subunit vaccines consisting of peptidic epitopes. Indeed, the retro-inverso analogs of MHC class I specific T cell epitopes not only provided appropriate functional mimics of the corresponding native antigens, but also revealed intracellular mechanisms for peptide loading on MHCI.

The role of pharmacological agents in the development and activation of T lymphocyte responses is being investigated. It was found that the effects of pentoxifylline, a pan-phosphodiesterase inhibitor, on T cell priming were mediated through cAMP-dependent protein kinase A (PKA). It appears that the pentoxifylline-cAMP-PKA signalling may either extend an early event or modulate a late event in T cell priming to enhance secondary responsiveness.

While addressing the interface between mucosal and systemic immunity, an experimental model for tracking T cells primed in vivo following oral immunisation in the absence of adjuvant was established. The preliminary experiments with regard to the tracking of antigen-specific cells following i.p. immunisation of the chimeric mice with soluble antigen suggest that oral tolerance as a phenomenon may have less to do with the route of antigen uptake and more to do with the absence of adjuvant during initial T cell priming.

The investigations aimed at understanding the temporal relationships between anti-lymphocyte antibodies and autoantibodies of diverse specificities have resulted in two human monoclonal anti-lymphocyte antibodies. While one of these has cell surface reactivity towards a variety of cell lines, the other shows poor recognition of normal, healthy cells. The later appears to be an apoptosis-specific, rather than an activation-specific, antibody.

Engagement of Vi antigen in the interaction with the host cell surface receptors on intestinal epithelial cells as an early event during S. typhi infection has now been clearly established. However, there must be additional molecules which play a role in host-pathogen interaction during infection. Present data indicates that the signal to produce such molecules by the bacterium may come partly from contact with host cells and partly through interaction with one or more components of the serum.

The immuno-stimulatory potential of polymer particles was investigated in case of poor immunogens. It was shown that co-encapsulation of such antigens and carrier in polymer particles can be a substitute for chemical conjugation for improved immune response.

For the purpose of investigating novel ways of altering protective immunity against M. tuberculosis, an indigenous aerosol infection chamber has been designed. The device has been checked for sterility of air coming out of it. To ease the operation and to make it more reliable, the assembly is controlled by a microprocessor which controls the time of exposure by actuating relays and valves.

Gene Regulation

While addressing the comparative genomics of bubaline (Bubalus bubalis) it was shown that the satellite tagged transcribing sequences in the bubaline Bubalus bubalis genome undergo programmed modulation in the meiocytes. A 1378 bp Bam HI satellite DNA fraction (pDS5) from the bubaline Bubalus bubalis has been cloned, sequenced and its expression in different somatic tissues and germline was analysed. It is proposed that pDS5 satellite is involved in tandem multiplication of collagen gene, giving rise to about 2000 copies in the bubaline genome.

Genome-based approaches are being adopted to identify and exploit the Mycobacterium tuberculosis polyketide synthase gene clusters in the biosynthesis of various natural products using a two-pronged strategy. Four proteins from this cluster have been expressed in E. coli. Several other proteins from the gene cluster involved in the biosynthesis of phthiocerol dimycocerosate, a complex lipid exclusively found in pathogenic mycobacterial species, have also been made and characterized in order to address the cross-talk between FAS and PKS.

A fusion protein containing functionally significant or immunodominant regions of gp63 from L. donovani and LTB was cloned into secretory expression vector and the protein was purified in large amounts in active form as indicated from functions of LTB and gp63. It was shown that the anti-fusion protein antibodies block promastigote-macrophage binding.

Molecular Design

Towards understanding the pathogenesis of typhoid fever, the possibility of Salmonella proteins that might be involved in the recruitment of different factors from the host cell cytoplasm was investigated. It was shown that Salmonella specifically binds two proteins, Rab5 and actin. Rab5 specifically recognizes a type III secretory protein of Salmonella called SopE. SopE acts on a nucleotide exchange factor for Rab5 and also mediates the specific recruitment of Rab5 in GTP form on LSP, irrespective of prenylation, and thus promotes fusion of LSP with early endosomes and thereby inhibiting targeting of live Salmonella to the lysosomes and their eventual destruction.

It was demonstrated that the receptor-mediated delivery of biological response modifiers of macrophage metabolism regulated the levels of Rab7 and Rab5 at the transcriptional level. Although intracellular delivery of MDP may activate different signaling cascades, it appears that the downregulation of Rab5 concurrent with upregulation of Rab7 triggered by intracellular delivery of MDP is sufficient to target live Salmonella-containing phagosomes (LSP) to the lysosomes.

While exploring the heme acquisition mechanism in L. donovani, the 46 kDa hemoglobin-binding protein was further characterized. It has now been established that this is a hexokinase localized in the flagellar pocket of Leishmania.

Towards design and synthesis of the structural and functional mimics and inhibitors of GPI pathway, the efforts on devising a new convergent synthetic methodology towards GPI anchor and analogues have continued. The chiral protected myo-inositol, glycerolipid and glycan intermediates were brought together in correct linkages and stereochemistry to make GPI anchor, and its labeled analogues. This synthesis has enabled preparation of a hitherto inaccessible key GPI intermediate acylated at the C-2 position of inositol moiety.

A detailed computational analysis providing correlation of the sequences of various modular polyketide synthases with their substrate specificities has led to the development of a powerful web-enabled software for prediction of PKS domains in a given protein sequence.

While investigating the molecular mechanism of action of ribonucleolytic protein toxins, the structural and functional characterization of saporin mutants was continued towards understanding its mechanism of cytotoxicity. It is now established that the cytotoxic activity of saporin is a cumulative effect of its N-glycosidase and DNA fragmentation activity. The intracellular trafficking and processing of saporin was also investigated.

The sugar-peptide mimicry, established on the basis of polyclonal antibody cross-reactivity and structural similarity earlier, was further addressed with the help of a panel of monoclonal antibodies. All the IgM clones developed show cross-reactivity with the carbohydrate mimicking peptides as well as with the variety of other sugars. This is not the case with the secondary antibodies. Apparently, the specific sugar recognition of the antibodies is fine-tuned during maturation.

The model studies on reverse proteolytic condensation reactions were continued to be addressed. If the volume exclusion effect could facilitate the splicing of discontiguity sites in fragment complementing systems, was explored using RNase A and triosephosphate isomerase (TIM). It was found that whereas RNase S to RNase A conversion does not involve volume exclusion, considerable compaction is attained in TIM reformation consistant with the structural features in the two proteins.

Towards developing electrical property measurement based systems for monitoring biological processes, first prototype of a stand alone unit for the impediometric monitoring of Ag:Ab complexation has been developed. This unit is being evaluated as biosensor for detecting the spores of Bacillus anthraci. Also, a number of electrode designs have been developed for immunization using electroporation. The piezo ceramic-based device has been used to immunize mice using a recombinant hepatitis B surface antigen. It was found that mice do invoke an immune response when antigen was given by electroporation even without adjuvant.

Reproduction and Development

In the project addressing characterization of proteins important for fertility and cell death, the pathway used by estrogens to bring about germ cell apoptosis was identified for the first time. It was found that estrogens could induce increased germ cell apoptosis using both the Fas ligand and the cytochrome c to bring about cell death.

Studies concerning cell death in single-celled organisms e.g. L. donovani, showed that upon activation of death response by H2O2, a dose and time dependent loss of mitochondrial membrane potential (m) occurs. The importance of non-selective cation channels in altering oxidative stress induced changes in intracellular Ca2+ homeostasis that trigger downstream signaling cascades leading to apoptosis-like death in L. donovani promastigotes was also revealed.

Ongoing research efforts on studies concerning potentiation of the immune response in case of hCG vaccine by synthetic carriers and adjuvants were focussed on the new strategies such as use of Th-cell peptides as alternate carriers and inclusion of additional adjuvant with an aim to design a vaccine formulation that can elicit and maintain effective levels of antibody response in most recipients. Having earlier demonstrated consistently higher adjuvanticity of SBAS2 in inbred mice, potential of the compound was evaluated in an out-bred strain as well indicating that SBSA2 potentiate the antibody response in outbred population as well.

Cloning and expression of non-human primate and canine zona pellucida proteins in baculovirus expression system was achieved as a part of the studies on zona proteins in the context of fertility regulation. E. coli-expressed r-bmZP3, when subjected to structural analysis, showed a predominantly  sheet structure, though a reasonable  helical component was also observed.

The immune response to plasmid DNA encoding ZP glycoproteins was characterized. Mice immunized with VRbmZP1 plasmid DNA showed high titre anti-r-bmZP1 antibodies that were specific to zona. Also these antibodies inhibited the binding of human spermatozoa to the antibody treated human zona in a hemizona assay thus indicating their immunocontraceptive potential.

As part of the project concerning testis-specific proteins, the recombinant HSS protein was further characterized. The diverse assays revealed that HSS protein was present on the sperm surface on the acrosomal compartment. It was observed that the expression of hss transcript was observed only in round spermatids indicating post meiotic haploid gene expression.

Among the ancillary activities, technology support for the production of transgenic and knockout animals is being continued to be provided to a large number of groups within the Institute and outside, all over the country.

As in earlier years, both in terms of scientific strength as reflected in publications in highly competitive peer-reviewed international journals and technological competence as indicated by international patents, NII’s record shows steady growth this year as well. While maintaining consistency in the number of papers published in the journals included in Science Citation Index, quality of these publications was substantially enhanced this year. More than 30% of the published papers appear in the most prestigious journals. Three technological leads were granted international patents and three others were granted Indian patents during the year.

Practically all of NII’s research groups are now supported by substantive extramural grants from both national and international competitive sources. Five new principal investigators had their research programmes established this year. A large number of trainees including both young students and scientists with specific training needs have visited the Institute for various periods during this past year. The doctoral programme continues to draw about fifteen highly talented students in a nationwide entrance process. Intense research collaborations are continued to be established with various laboratories both, overseas and within the country.

The writing on the wall has been very clear for sometime now. In the emerging economic world order, our very survival as a free nation depends on acquiring the capability of gathering new mechanistic insights and translating those into globally competitive technologies, success in which depends on a strong base in creative research. We know we have a long way to go in this endeavor. However in this brief overview, I hope I have been able to convey the sense of urgency, purpose and drive that I and my colleagues in NII have about the work we do. We believe that the best service we can render to the nation that is supporting us is to keep that pace relentless.

15 October 2002 Sandip K Basu

Director

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