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Biomedical subjects

S K Datta

Publications and source records attributed to S K Datta.

At least 55 records · Page 3Linked to original sources
PubMed1996

Hyperexpression of CD40 ligand by B and T cells in human lupus and its role in pathogenic autoantibody production.

We investigated the role of the costimulatory molecules, CD40 and its ligand CD40L, in the pathogenesis of human SLE. In comparison to normal subjects or patients in remission, PBMC from active lupus patients had a 21-fold increase in the frequency of CD40L-expressing, CD4+T cells. However, the expression of CD40L induced in either lupus or normal T cells by mitogenic stimulation could be down-regulated equally well by CD40 molecules on autologous B cells. Active lupus patients also had a 22-fold increase in percentage of CD8+ T cells expressing CD40L, consistent with their unusual helper activity in SLE. Surprisingly, patients with active lupus had a 20.5-fold increase in B cells that spontaneously expressed high levels of CD40L, as strongly as their T cells. Although lupus patients in remission had low levels of CD40L+ cells in the range of normal subjects, mitogen-induced upregulation of CD40L expression in the T and B cells was markedly greater than normal, suggesting an intrinsic defect. A mAb to CD40L blocked significantly the ability of lymphocytes from lupus patients with active and established disease to produce the pathogenic variety of antinuclear autoantibodies in vitro, bolstering the possibility of anti-CD40L immunotherapy for lupus. Future studies on the hyperexpression of CD40L could elucidate a regulatory defect in the pathogenic T and B cells of lupus.

Adolescent↗
PubMed1996

Evidence for genomic changes in transgenic rice (Oryza sativa L.) recovered from protoplasts.

The occurrence of genomic modifications in transgenic rice plants recovered from protoplasts and their transmission to the self-pollination progeny has been verified with the random amplified polymorphic DNA (RAPD) approach. The plant was the Indica-type rice (Oryza sativa L.) cultivar Chinsurah Boro II. The analysed material was: (1) microspore-derived embryogenic rice cells grown in suspension culture, (2) transgenic plants recovered from protoplasts produced from the cultured cells and (3) the self-pollination progeny (two successive generations) of the transgenic plants. DNA purified from samples of these materials was PCR-amplified with different random oligonucleotide primers and the amplification products were analysed by agarose gel electrophoresis. Band polymorphism was scored and used in band-sharing analyses to produce a similarity matrix. Relationships among the analysed genomes were expressed in a dendrogram. The extensive DNA changes evidenced in cultured cells demonstrate the occurrence of somaclonal variation in the material used to produce protoplasts for gene transfer. Quantitatively reduced DNA changes were also found in the resulting transgenic plants and in their self-pollination progenies. While confirming the stability of the foreign gene in transgenic plants, this work gives molecular evidence for the occurrence of stable genomic changes in transgenic plants and points to in vitro cell culture as the causative agent. RAPDs are shown to be a convenient tool to detect and estimate the phenomenon at the molecular level. The methodology is also proposed as a fast tool to select those transgenic individuals that retain the most balanced genomic structure and to control the result of back-crosses planned to restore the original genome.

DNA, Plant↗
PubMed1996

An intron 1 regulatory region from the murine adenosine deaminase gene can activate heterologous promoters for ubiquitous expression in transgenic mice.

Ubiquitously expressed genes contain regulatory features which allow expression in virtually all cell types. In an effort to understand the molecular basis for this regulatory feature, the chromatin structure of the murine adenosine deaminase gene was examined by DNase I digestion in nuclei of several tissues. The promoter contained a strong hypersensitive site in all tissues examined, including those with very high and very low levels of ADA expression. Transgenic mouse studies revealed that a 3.3 kb EcoRI (3.3EE) fragment from intron I was required to generate a strong promoter DNase I hypersensitive site, and to produce ubiquitous expression. The 3.3EE fragment also contained a thymic enhancer activity which mapped to sequences conserved with the human ADA gene T-lymphocyte enhancer. Mutational analysis indicated that ubiquitous expression was not dependent on the presence of a functional thymic enhancer. Both the thymic enhancer and the ubiquitous activator within the 3.3EE fragment functioned with heterologous promoters in transgenic mice.

Adenosine Deaminase↗
PubMed1996

Role of natural effector cells in the prevention of radiation-induced leukemogenesis.

Four once-weekly exposures of 225 rads (R) of whole body X-irradiation causes a high incidence of lymphoma in C57BL/6 (C57) mice. The role of natural effector cells in radiation-induced leukemogenesis was investigated. Activity of natural killer (NK) and natural cytotoxic (NC) cells was depressed in irradiated mice over a prolonged period. Transplantation of bone marrow (BM) cells from normal C57 mice to irradiated mice restored their NK responses and prevented development of lymphoma. The effect of beige BM cell transfer to irradiated mice was not clear. Unlike short-term (4-hour) assay, results of long-term (20-hour) assay suggest partial restoration of NK and full restoration of NC activity in irradiated mice. Treatment of irradiated mice with polyinosinic: polycytidillic acid (poly I:C) increased NK activity and lymphoma resistance. Administration of small repeated doses of poly I:C to irradiated mice significantly prevented the development of lymphoma as well as significantly prolonging overall survival time in irradiated mice. These observations indicate that natural effector cells mediate resistance to radiation-induced leukemogenesis.

Animals↗
PubMed1995

Signal transduction by the B-cell antigen receptor.

The antigen receptor of B lymphocytes (BCR) plays important roles in recognition of foreign antigens and self-components to allow the immune system to make appropriate antibody responses. The BCR is a complex between membrane immunoglobulin and the Ig-alpha and Ig-beta heterodimer. Site-directed mutagenesis experiments have shown that the mu heavy chain transmembrane domain plays a key role in the association of mIgM with Ig-alpha/Ig-beta. In the absence of complex formation, mIgM is retained in the endoplasmic reticulum, and this function is also specified by the mu chain transmembrane domain. The ability of various mutant mIgM molecules to associate with Ig-alpha/Ig-beta correlates well with their ability to induce signal transduction reactions such as protein tyrosine phosphorylation and phosphoinositide breakdown. Thus, the signaling ability of the BCR appears to reside in the Ig-alpha/Ig-beta heterodimer. The cytoplasmic domains of Ig-alpha and Ig-beta each contain an ITAM sequence, which is defined by its limited homology with subunits of the T-cell antigen receptor and of Fc receptors. Moreover, chimeric proteins containing these ITAMs and surrounding sequences from the cytoplasmic domains of Ig-alpha or Ig-beta exhibit signaling function characteristics of the intact BCR. The Ig-alpha and Ig-beta chimeras are each capable of inducing all of the BCR signaling events tested and thus represent redundant functions. Cross-linking these chimeras leads to their phosphorylation and to binding of the intracellular tyrosine kinases Lyn and Syk. The BCR expressed in the nonlymphoid AtT20 cells, which express the Src-family tyrosine kinase Fyn but not Syk, was not able to trigger vigorous signaling reactions. Introduction of the active form of Syk into these cells restored some signaling events. These results are consistent with a model in which the ITAMs act to initiate the BCR signaling reactions by binding and activating tyrosine kinases.

Animals↗
PubMed1995

Interaction between CD40 and its ligand gp39 in the development of murine lupus nephritis.

We investigated the role of gp39-CD40 interaction in the development of glomerulonephritis in lupus mice. In contrast to normal mice, lupus mice had much higher percentages of intensely gp39+ T cells in their spleens even at the preautoimmune age of 1 mo, and the further increase in gp39 expression by anti-CD3 Ab stimulation was markedly greater in lupus T cells. The pathogenic autoantibody-inducing ability of Th clones and splenic Th cells from lupus mice could be blocked in vitro by anti-gp39 Ab. Acceleration of lupus nephritis by the transfer of pathogenic autoantibody-inducing Th clones in vivo could also be completely blocked by anti-gp39 Ab. Surprisingly, a brief treatment of lupus mice with anti-gp39 Ab had a sustained beneficial effect on their spontaneous disease long after the Ab had been cleared from their systems. Only three injections of anti-gp39 Ab given to prenephritic lupus mice at 3 mo of age markedly delayed and reduced the incidence of lupus nephritis up to 12 mo of age by which time almost all the control mice had developed severe glomerulonephritis. Remarkably, pathogenic Th cells were left intact in these anti-gp39-treated mice but their B cells could not produce pathogenic autoantibodies even 9 mo after the therapy. Our studies suggest that blocking the interaction between gp39 on pathogenic Th cells and CD40 on lupus B cells at a crucial window of time delays the expansion autoimmune memory B cells resulting in long-term therapeutic benefits.

Animals↗
PubMed1995

Lupus: key pathogenic mechanisms and contributing factors.

The past 30 years of research on murine and human systemic lupus erythematosus has served to identify an array of immunological aberrations--some shared, some unique, some primary, others secondary-- that may underlie this disease. In integrating these findings, it appears that at least four distinct pathogenic events characterize lupus: (1) Anti-DNA Abs and immune complexes induce renal damage; (2) B-cells produce pathogenic anti-DNA antibodies; (3) Th cells drive lupus B-cells; and (4) Increased concentrations and abnormal presentation of nucleosomes. The purpose of this review is to examine the roles of these four events in the pathogenesis of lupus and to identify the different factors that can precipitate these pathogenic events.

Animals↗
PubMed1995

Mechanisms of the pathogenic autoimmune response in lupus: prospects for specific immunotherapy.

A major step towards understanding the basic mechanism of systemic lupus erythematosus (SLE), the prototypic autoimmune disease that develops spontaneously, has been the identification of nucleosomes as a primary immunogen in this disease. The production of pathogenic autoantibodies in SLE results from an MHC class-II-restricted, cognate interaction between select populations of T helper cells and B cells that are specific for nucleosomal components. These observations pave the way for specific immunotherapy that blocks this pathogenic T and B cell interaction.

Animals↗
PubMed1995

Structure and specificity of T cell receptors expressed by potentially pathogenic anti-DNA autoantibody-inducing T cells in human lupus.

The production of potentially pathogenic anti-DNA autoantibodies in SLE is driven by special, autoimmune T helper (Th) cells. Herein, we sequenced the T cell receptor (TCR) alpha and beta chain genes expressed by 42 autoimmune Th lines from lupus patients that were mostly CD4+ and represented the strongest inducers of such autoantibodies. These autoimmune TCRs displayed a recurrent motif of highly charged residues in their CDR3 loops that were contributed by N-nucleotide additions and also positioned there by the recombination process. Furthermore, Th lines from four of the five patients showed a marked increase in the usage of the V alpha 8 gene family. Several independent Th lines expressed identical TCR alpha and/or beta chain sequences indicating again antigenic selection. 10 of these Th lines could be tested further for antigenic specificity. 4 of the 10 pathogenic anti-DNA autoantibody-inducing Th lines responded to the non-histone chromosomal protein HMG and two responded to nucleosomal histone proteins; all presented by HLA-DR molecules. Another Th line responded to purified DNA more than nucleosomes. Thus, these autoimmune Th cells of lupus patients respond to charged epitopes in various DNA-binding nucleoproteins that are probably processed and presented by the anti-DNA B cells they selectively help.

Amino Acid Sequence↗
PubMed1994

T cell receptor alpha-chain repertoire of pathogenic autoantibody-inducing T cells in lupus mice.

The production of pathogenic anti-DNA autoantibodies in mice with lupus nephritis is dependent on special autoimmune Th cells that can also transfer the disease into preautoimmune mice. In previous work, these pathogenic Th cells were cloned and their TCR beta-chains were sequenced to reveal a recurrent motif of anionic residues in their CDR3 loops. Accordingly, approximately half of the Th clones were found to be specific for nucleosomal Ag that contain cationic residues. Herein, we analyzed the TCR alpha-chain repertoire of 15 of these pathogenic Th clones and found them to be heterogeneous, even among the nucleosome-specific Th clones. Most of these autoimmune TCR alpha-chains contained anionic residues in their CDR3 in addition to cationic residues. Therefore, these pathogenic Th clones of lupus probably recognize epitopes with mixed charge runs that are derived from autoantigens, such as histone-DNA complexes. Interestingly, the V alpha gene segments used by 10 of these Th clones derived from the (SWR x NZB)F1 lupus mice differed from previously reported sequences indicating that they were new members or alleles of the respective V alpha gene family. One of the Th clones used a gene from an entirely new murine V alpha gene family, identified here as V alpha 23, which consisted of approximately two members that were conserved among strains with different V alpha haplotypes. Knowledge of the primary structure of the TCR expressed by these pathogenic Th clones of lupus would help in the analysis of their antigenic specificities and also would be essential for studying their regulation in transgenic mice carrying these autoimmune TCR genes.

Amino Acid Sequence↗
PubMed1994

Transgenic Indica rice (Oryza sativa L.) plants obtained by direct gene transfer to protoplasts.

We have established a reproducible procedure for transformation of protoplasts and regeneration of transgenic plants for an improved Indica rice cultivar IR43. Mature embryo-derived calli were placed in liquid culture medium containing maltose to establish meristematically active, embryogenic cell suspension lines. In order to obtain transgenic plants, a chimeric hygromycin phosphotransferase hph gene under the control of the cauliflower mosaic virus CaMV 35S promoter was introduced into protoplasts from these cell suspension lines using polyethylene glycol. Protoplasts were cultured in maltose-containing medium. Hygromycin B selection was applied to 14-day-old dividing cell colonies. Resistant calli were readily obtained after 3 weeks of selection. Seventy-three plantlets were regenerated from resistant calli from several independent experiments, and a few of the 29 plants grown in the greenhouse reached maturity. Stable integration of the transgene in the genome of these plants was confirmed by Southern blot analysis and the expression of the transgene in plants by hygromycin phosphotransferase assay. The procedure described yielded 5 to 18 resistant colonies and approximately four transgenic plantlets per million treated protoplasts.

Base Sequence↗
PubMed1994

Mechanism of B cell antigen receptor function: transmembrane signaling and triggering of apoptosis.

The antigen receptor of B lymphocytes (BCR) plays important roles in virtually every stage in the development, inactivation, or activation of B cells. The BCR is a complex of membrane immunoglobulin (mIg) and a heterodimer of two transmembrane polypeptides called Ig-alpha and Ig-beta. Site directed mutation of the mu immunoglobulin heavy chain has demonstrated that the mu transmembrane domain plays a key role in the assembly of mIgM with Ig-alpha/Ig-beta. In addition, there is a strong correlation between the ability of various mutant mIgM molecules to associate with Ig-alpha/Ig-beta and their ability to induce signal transduction reactions such as protein tyrosine phosphorylation and phosphoinositide breakdown. The cytoplasmic domains of Ig-alpha and Ig-beta share a region of limited homology with each other and with components of the T cell antigen receptor and of the Fc receptor. The presence of regions of the cytoplasmic domains of Ig-alpha or Ig-beta including this conserved amino acid sequence motif is sufficient to confer signaling function on chimeric transmembrane proteins. Both Ig-alpha and Ig-beta chimeras are capable of inducing all of the BCR signaling events tested. Based on these and related observations, we propose that the motifs act to initiate the BCR signaling reactions by binding and activating tyrosine kinases. Among the important events mediated by BCR signaling is induced expression of a series of genes referred to as early response genes. In B cells these include transcription factors and at least one component that regulates signaling events. One of these genes, c-myc, appears to play an important role in mediating apoptosis in B cells stimulated via the BCR complex.

Amino Acid Sequence↗
PubMed1994

Adenylosuccinate synthetase: a dominant amplifiable genetic marker in mammalian cells.

Adenylosuccinate synthetase (AdSS) functions at the branchpoint of purine nucleotide metabolism leading to the synthesis of AMP. The enzyme is inhibited by a metabolite of alanosine, an aspartic acid analog that is highly cytotoxic for most cells. We show here that it is possible to use alanosine selection to isolate from a population of transformants those cells having the highest levels of AdSS activity resulting from uptake and expression of AdSS minigenes. Transformants isolated in this way were selected for resistance to even higher concentrations of alanosine and resulted in the isolation of cells with highly amplified copies of the transfected AdSS minigenes. We demonstrated that nonselectable genes can be cotransferred and coamplified with AdSS minigenes. These findings indicate that AdSS minigenes can be used as dominant amplifiable genetic markers in mammalian cells.

Adenylosuccinate Synthase↗
PubMed1993

B-cell antigen receptor motifs have redundant signalling capabilities and bind the tyrosine kinases PTK72, Lyn and Fyn.

BACKGROUND: The 13 cell antigen receptor (BCR) is a multimeric protein complex consisting of an antigen recognition structure (membrane immunoglobulin) and two associated proteins, lg-alpha and Ig-beta It has been proposed that signalling through the BCR involves Ig-alpha and Ig-beta. Both of these proteins contain within their cytoplasmic domains an amino-acid motif that is present in a number of immune recognition receptors, including the BCR, T-cell antigen receptor and Fc receptor complexes. This motif, termed the antigen-receptor homology motif (ARH1), appears to have signal transduction ability. RESULTS: We now show that the presence of cytoplasmic regions containing the ARM motif from either Ig-alpha or Ig-beta is sufficient to confer signalling capability on an otherwise non-functional fusion protein. Both Ig-alpha- and Ig-beta-containing chimeras induced, in an apparently redundant fashion, signalling events seen upon membrane immunoglobulin crosslinking, including tyrosine phosphorylation of particular proteins, phosphoinositicle breakdown and calcium mobilization. Furthermore, crosslinking of the chimeras resulted in tyrosine phosphorylation of the Ig-alpha and Tg-beta tails and their association with the tyrosine kinases PTK72, p53/56(lyn) and p59(fyn). CONCLUSIONS: These observations indicate that Ig-alpha and Ig-beta are responsible for coupling membrane immunoglobulin to intracellular signalling components. Moreover, they demonstrate that a number of tyrosine kinases associate directly with the cytoplasmic domains of both Ig-alpha and Ig-beta. Stimulation of the chimeras, which results in tyrosine phosphorylation of the ig-alpha and Ig-beta tails, is a prerequisite for some of these associations. The implications of these findings for the mechanism by which the BCR initiates the signalling reactions are discussed.

Journal Article↗
PubMed1993

Nucleosome: a major immunogen for pathogenic autoantibody-inducing T cells of lupus.

Only a fraction (12%) of 268 "autoreactive" T cell clones derived from lupus-prone mice can selectively induce the production of pathogenic anti-DNA autoantibodies in vitro and accelerate the development of lupus nephritis when transferred in vivo. The CDR3 loops of T cell receptor beta chains expressed by these pathogenic T helper (Th) clones contain a recurrent motif of anionic residues suggesting that they are selected by autoantigens with cationic residues. Herein, we found that approximately 50% of these pathogenic Th clones were specific for nucleosomal antigens, but none of them responded to cationic idiopeptides shared by variable regions of pathogenic anti-DNA autoantibodies. Nucleosomes did not stimulate the T cells as a nonspecific mitogen or superantigen. Only the pathogenic Th cells of lupus responded to nucleosomal antigens that were processed and presented via the major histocompatibility class II pathway. Although the presentation of purified mononucleosomes to the Th clones could be blocked by inhibitors of endosomal proteases, neither of the two components of the nucleosomes--free DNA or histones by themselves--could stimulate the Th clones. Thus critical peptide epitopes for the Th cells were probably protected during uptake and processing of the nucleosome particle as a whole. The nucleosome-specific Th clones preferentially augmented the production of IgG autoantibodies to histone-DNA complex in vitro. In vivo, nucleosome-specific, CD4+ T cells were not detectable in normal mice, but they were found in the spleens of lupus-prone mice as early as 1 mo of age, long before other autoimmune manifestations. Immunization of young, preautoimmune lupus mice with nucleosomes augmented the production of autoantibodies and markedly accelerated the development of severe glomerulonephritis. Previously, crude preparations containing nucleosomes were shown by others to have polyclonal mitogenic activity for B cells from normal as well as lupus mice. Identification here of pure mononucleosome as a lupus-specific immunogen for the Th cells that selectively help the pathogenic anti-DNA autoantibody producing B cells of lupus could lead to the design of specific therapy against this pathogenic autoimmune response.

Amino Acid Sequence↗
PubMed1993

Genetic resistance to lymphoma-leukemia: role of natural killer cells in the rejection of lymphoma grafts.

Lethally irradiated genetically resistant (C57 x AKR) mice, unlike non-resistant (C3H x AKR) mice, which display genetic resistance to transplantation of parental C57 bone marrow grafts (GR to BMT), also reject transplanted parental AKR lymphoma grafts (GR to lymphoma). Depletion of natural killer (NK) cell activity of resistant (C57 x AKR) mice by anti-asialo GM-1 serum abrogated the resistance, whereas stimulation of NK cell activity of non-resistant (C3H x AKR) mice by polyinosinic: polycytidillic acid (poly I:C) induced the resistance to parental AKR lymphoma grafts. This shows that NK cells contribute to GR of (C57 x AKR) hybrid mice to parental AKR lymphoma grafts. Further, these results indicate that NK cells which confer genetic resistance to bone marrow transplantation (GR to BMT) in lethally irradiated (C57 x AKR) hybrid mice, also mediate GR to lymphoma transplantation in these mice, thereby linking GR to BMT with GR to lymphoma-leukemia.

Animals↗