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Biomedical subjects

A Kaushik

Publications and source records attributed to A Kaushik.

At least 37 records · Page 2Linked to original sources
PubMed1998

The primary antibody repertoire of kappa-deficient mice is characterized by non-stochastic Vlamda1 + V(H) gene family pairings and a higher degree of self-reactivity.

We have investigated the primary antibody repertoire of genetically manipulated 129/Sv kappa-deficient (JCkappaD) mice, in order to understand the contributions of the lambda-light chain, in the absence of an otherwise predominant kappa-light chain, to the development of humoral immunity. The expression of Vlambda1 gene (lambda1 and lambda3 subtypes) and the Vlambda1 + V(H) (J558, 36-60, V(H)11 and S107) gene family associations were studied in 7.43 x 10(3) mitogen-activated splenic B-lymphocyte clones of JCkappaD origin. Furthermore, the functional significance of the exclusive expression of the lambda-light chain, in the peripheral B-cell repertoire of JCkappaD mice, was analysed by determining natural autoantibody specificities in the circulating serum immunoglobulin and the frequency of autoreactive B-lymphocyte clones in the peripheral B-lymphocyte repertoire. These experiments revealed that: first, of the three available Vlambda genes at the lambda locus, the Vlambda1 gene is the one that is expressed most frequently (59.9%); second, non-random Vlambda1 + V(H) (J558, 36-60) gene family pairings occur in kappa-deficient mice; and third, a higher degree of self-reactivity is generated as a result of exclusive use of the lambda-light chain, as evidenced by higher levels of serum natural autoantibodies as well as a high frequency of autoreactive B-lymphocyte clones in kappa-deficient (129/Sv JCkappaD) mice. These observations suggest that the high murine kappa/lambda ratio in mice may, apart from high sequence diversity at the kappa-locus, be a result of endogenous selection against the lambda-light chain to restrict self-reactivity within the homeostatic threshold.

Animals↗
PubMed1997

CDRH3 length is the target of selection of disease-associated IgM autoantibodies.

The experiments outlined here provide evidence for positive selection of B-1 lymphocytes secreting IgM autoantibodies in the autoimmune mev mice as a result of the reduced threshold of responsiveness to autoantigens because of PTP1C deficiency, which is targeted at the CDR3 length of the variable region of the heavy chain. In addition, characteristic differences in the size, hydrophobicity pattern, and relative local charge of the CDR3 of the heavy chain of disease-associated IgM autoantibodies from moth-eaten mice help distinguish them from physiological natural autoantibodies present in normal mice.

Animals↗
PubMed1997

A single predominantly expressed polymorphic immunoglobulin VH gene family, related to mammalian group, I, clan, II, is identified in cattle.

In order to understand the generation of antibody diversity in cattle, seven cDNAs, from heterohybridomas secreting bovine IgM and IgG1 antibodies, were cloned and structurally analyzed for rearranged bovine VDJ genes. All of the seven bovine VH genes, together with four available bovine VH gene sequences, shared a high nucleotide sequence homology (84.2-93.5%). Based upon the criteria of nucleic acid homology > or =80%, all of the bovine VH gene sequences isolated from the expressed antibody repertoire constitute a single VH gene family, which we have designated as bovine VH1 (Bov VH1). An analysis of 44 bovine IgM-secreting mouse x cattle heterohybridomas, originating from polyclonally-activated PBLs from bovine leukemia virus-infected cattle, revealed that all of these expressed Bov VH1 (100%) based upon DNA sequencing and Northern dot blot. The bovine VH genes showed highest DNA sequence similarity, ranging between 81.5 and 87.6%, with a single sheep VH gene family (related to human VH4) and are, thus, closest to the VH genes from another ruminant species. The Bov VH1 gene family is most homologous to the murine VH Q-52 (71.8-78%) and human VH4 (67.4-69.8%) gene families, which belong to mammalian group, I, clan, II, VH genes. The CDR3 length of rearranged bovine VDJ genes is characteristically long (15-23 amino acids). The bovine JH gene segments were most homologous to human JH4 (82.1-87.2%) and JH5 (84.6-89.7%) genes, suggesting the existence of at least two JH gene segments. An analysis of CDRs provides evidence that somatic hypermutations contribute significantly to the generation of antibody diversity in cattle. Southern blot analysis of BamH I, EcoR I and Hind III digested genomic DNA from four cattle breeds (Holstein, Jersey, Hereford and Charolais) revealed three RFLP patterns; the genomic complexity of Bov VH1 ranged between 13 and 15 genes. These observations provide evidence for polymorphism at the bovine Ig-VH locus, similar to that seen in mice and humans.

Amino Acid Sequence↗
PubMed1997

Restricted CDR3 length of the heavy chain is characteristic of six randomly isolated disease-associated VH J558+ IgM autoantibodies in lupus prone motheaten mice.

To investigate the origin of disease-associated IgM autoantibodies (AAb), we compared the genetic and structural characteristics of IgM AAb from autoimmune prone motheaten (mev) mice with natural autoantibodies (NAAb) from normal background C57/BL6 strain. Six hybridoma-derived IgM molecules each were obtained both from mev mice, at the terminal stage of systemic autoimmune disease, and from mitogen-stimulated C57/BL6 mice. These were randomly selected for VH J558 gene expression (aberrantly expressed in mev mice). The variable regions of the IgM molecules, both from autoimmune and normal mice, were encoded by unmutated germline VH genes. Disease-associated AAb from mev mice were predominantly encoded by the J558 subfamily 186.2, whereas five J558 subfamilies were utilized in NAAb originating from normal mice. Junctional diversity as a result of N or P nucleotide insertions and D-D fusions was noted among IgMs originating from both mev (mostly B-1 lymphocytes) and C57BL/6 (mostly B-2 lymphocytes) mice. Interestingly, all six J558+ IgMs from mev mice showed a restricted CDR3 length of 10 amino acids, with similar hydrophobicity indices. Four unique V-D-J rearrangements were observed among these IgMs. None of the IgMs were polyreactive and three of the six were subsequently observed to express monospecific autoreactivity with synthetic peptides (residues 81-92 and 37-53) representing segments of the T cell CD4-accessory molecule. Three IgM antibodies had hydrophilic arginine residues in their CDR3 heavy chain region. By contrast, all six J558+ IgMs from C57/BL6 mice had variable CDR3 length, distinct VDJ rearrangements and a local negative charge in the CDR3 region. Four of these IgMs demonstrated polyreactivity with multiple conserved autoantigens and, hence, were classified as NAAb. These findings provide evidence for either positive or impaired negative selection of B-1 lymphocytes secreting disease-associated IgM AAb in mev mice. This likely results from a reduced threshold of responsiveness to autoantigens due to PTP1C deficiency, which is targeted at the CDR3 length of the variable region of the heavy chain. In addition, characteristic differences in the size and hydrophobicity pattern of the CDR3 of the heavy chain allow structural distinction between monospecific disease-associated IgM AAb and the polyreactive IgM NAAb.

Amino Acid Sequence↗
PubMed1997

Association of cerebral palsy with epilepsy.

Fifty patients of various types of cerebral palsy were studied to find out an association between cerebral palsy, EEG abnormalities and development quotient. Seventy-six per cent patients had spastic cerebral palsy. Hypotonic cerebral palsy was the next common type (14%). Athetosis and ataxic forms were found to be rare (2% each). Epilepsy was associated with 56% patients. Clinical types of seizures observed were: Generalised tonic-clonic (43%), myoclonic (17.9%), generalised tonic (10.7%), partial simple (10.7%) and partial complex (17.9%). The incidence of seizures was highest in hypotonic type in which 85.7% had epilepsy. Mean developmental quotient of cerebral palsy patients was 34.9% with maximum retardation in hypotonic cerebral palsy (25.14%). Sixty per cent of patients had abnormal EEG, out of these hypotonic patients had maximum (70%) chances of EEG abnormality followed by spastic patients (55%). Developmental retardation was more severe statistically in the patients with abnormal EEG than normal EEG.

Cerebral Palsy↗
PubMed1996

The primary antibody repertoire of normal, immunodeficient and autoimmune mice is characterized by differences in V gene expression.

During the last decade, the structure and organization of the immunoglobulin heavy and light chain locis have been defined in mice and humans. Studies on VH gene expression at different stages of development, in different organs and disease states have provided useful insight into the construction of a primary antibody repertoire in mice. Clearly, 3'VH genes 7183, Q52 and Vh11, which are conserved during evolution, are preferentially expressed during early development of the B-lymphocyte repertoire. A preferential use for the V kappa 4 gene family is evident during early B-cell development. The initial development of the primary antibody repertoire is therefore influenced by a restricted set of VH and V kappa gene elements. The restricted B-cell repertoire is subsequently normalized in the periphery, as revealed by stochastic VH gene expression, as a result of exposure to environmental antigens. Obviously, the peripheral B-cell pool characterized by stochastic VH gene expression is selectively replenished by newly generated B cells in bone marrow that preferentially expresses 3'VH genes. The V kappa genes are, however, expressed in a non-random manner in the neonatal and adult B-lymphocyte repertoire that is probably related to VH and V kappa association dynamics and/or positive or negative selection. Interestingly, these characteristics of neonatal and adult primary repertoire are noted in both B1 and B2 lymphocytes. No remarkable age-related differences are evident for VH and V kappa gene expression. In healthy mice, both the mitogen responsive (available) and unstimulated (expressed) B-cell repertoire show similar VH gene expression. Interestingly, VH gene expression varies in different organs which may reflect, or occur as a result of, the specialized function of each organ. For example, J558 gene expression is higher in the peripheral LN where B cells continuously encounter exogenous antigens. The skewed VH and V kappa gene expression noted in immunodeficient and autoimmune lupus-prone mice reflects the impairment of the primary antibody repertoire associated with immunodeficiency and autoimmune disorders.

Animals↗
PubMed1996

V lambda-light chain genes reconstitute immune responses to defined carbohydrate antigens or haptens by utilizing different VH genes.

The contribution of the lambda-light chain to the development of peripheral B cell repertoire and generation of specific antibodies to haptens and polysaccharide antigens was studied in genetically manipulated kappa-deficient and lambda 2-transgenic mice. The results clearly demonstrate a non-stoichiometric VH gene family expression in the absence of k-light chain and suggest a non-stochastic pairing between VH and V lambda genes, expressed in the peripheral B cell repertoire. A shift in VH gene utilization in the case of VI lambda + antibodies was evident in response to beta 2-6 fructosan and TNP hapten. These observations demonstrate the availability of compensatory mechanisms in the absence of VK genes and are consistent with the hypothesis that VH gene family expression is controlled by genetic factors from inside the VH locus. Furthermore, genetic factors from outside the VH locus, namely restricted available light chain diversity, may lead to a shift in VH gene utilization in the peripheral B cell repertoire.

Animals↗
PubMed1996

Homologues of murine Vh11 gene are conserved during evolution.

Since the murine Vh11 gene family shows 75% homology with group III VH gene families and is preferentially expressed in B-1 lymphocytes, we have analyzed if corresponding Vh11 genes existed across phylogenetically distant mammalian lineages. In a Southern blot, homologues of the murine Vh11 gene were detected in the genomic DNA from rats, pigs, sheep, cattle, horses, dogs and man. These observations suggest conservation of corresponding sequences of the Vh11 gene family during evolution, similar to group III VH genes, either because of strong selection pressures essential for species fitness and survival or molecular drive leading to these consequences.

Animals↗
PubMed1995

Diverse VH and V kappa genes encode antibodies to Pseudomonas aeruginosa LPS.

The molecular nature of the murine Ab response to Pseudomonas aeruginosa LPS was examined using a panel of 10 well-defined anti-LPS mAbs. Abs to P. aeruginosa LPS are encoded by diverse V-genes, with at least five VH and four V kappa gene families represented in these Abs. The Abs that bind to hydrophilic O-polysaccharide side chains of B-band LPS and A-band LPS are encoded by VH J558, SM7, and J606 gene families, while Abs to hydrophobic core and lipid A regions are encoded by X24, SM7, and Q52 gene families. All active JH and only two J kappa (J kappa 2 and J kappa 5) germ-line genes are utilized in the anti-LPS Abs examined. Four of six anti-P. aeruginosa mAbs used diversity genes of the DSP2 gene family. Interestingly, JH1 and JH2 use was observed in three mAbs that reacted with hydrophilic LPS epitopes (O-polysaccharide, A-band LPS), whereas JH3 and JH4 use was observed in three mAbs that bound to the more hydrophobic regions of LPS (core, lipid A). Point mutations were observed in framework and complementarity-determining regions (CDRs) of VH and VL genes, suggesting an Ag-driven maturation process in response to P. aeruginosa LPS. Mutations occurred in all heavy chain CDRs, as well as in CDR1 and CDR3 of the light chain, indicating an important role of these regions in binding to LPS. These data suggest that diverse VH and V kappa genes encode Abs to LPS from P. aeruginosa.

Amino Acid Sequence↗
PubMed1995

Contribution of the V lambda light chain to the development of the primary antibody repertoire.

The experiments outlined here provide evidence that most VH gene families are expressed in association with either of three V lambda genes available in kappa-deficient mice, though their expression is nonrandom. An exclusive use of V lambda L-chain genes, where predominantly expressed V lambda 1 and V lambda 2 differ by only seven amino acid substitutions in the germline, results in a higher degree of self-reactive antibodies to conserved natural antigens like albumin, thyroglobulin, and stress proteins. The higher levels of self-reactive antibodies, characteristic of preimmune B-cell repertoire, are likely to be a product of nonrandom VH gene family expression and their restricted pairing with three V lambda L-chain genes. These data suggest that a low lambda L-chain expression in normal mice is due, in part, to endogenous selection against lambda L chain in order to restrict self reactivity within the homeostatic threshold.

Animals↗
PubMed1995

The nude mutation results in impaired primary antibody repertoire.

We have studied the effect of the nude mutation and/or T lymphocytes on the development of V gene germ-line repertoire in neonatal athymic (nu/nu) and euthymic (+/nu) littermates. A total of 2.35 x 10(6) and 1.47 x 10(6) B lymphocyte clones from nu/nu and +/nu neonates, respectively, were examined for the expression of select VH (J558, J606, S107, 36-60, 7183 and Q52) and Vx (1, 2, 8 and 9) gene families as well as VH (J558, S107) + Vx (1, 9) associations. Data showed that the nude mutation, whether homozygous or heterozygous, significantly affects VH and Vx gene expression as well as VH and Vx pairings and, thus, provide evidence for a defective development of B cell repertoire in both athymic nude (nu/nu) and euthymic (+/nu) mice. In addition, an analysis of 3.34 x 10(6) B lymphocyte clones from adult C57BL/6 mice showed non-stochastic association between VHJ558 + Vx1 gene families that suggests restrictions on clonal population in order to maintain homeostasis in the immune system. Studies outlined here, therefore, describe an hitherto unknown defect in the development of B lymphocyte repertoire as a result of the nude mutation which is independent of thymic dysgenesis.

Animals↗
PubMed1995

An immunodominant epitope on 40 kilodalton outer membrane protein is conserved among different strains of Haemophilus (Histophilus) somnus.

Four murine monoclonal antibodies (mAb) were constructed against Haemophilus (Histophilus) somnus, an important bovine pathogen, and used to analyze immunologically significant antigenic determinants on these organisms. These mAbs specifically recognized immunodominant epitopes present on the 40 kilodalton (kD) fraction of the major outer membrane protein (OMP) of H. somnus. The 9D3 IgG1 kappa mAb recognized an immunodominant epitope on the 40 kD major outer membrane protein that is conserved or shared among all the three strains (septicemic, respiratory and uro-genital) of H. somnus. Two IgM kappa mAbs (4D6 and 10C2) recognized epitope(s) on the 40 kD OMP from septicemic and respiratory strains of H. somnus but none from the whole bacterial cell preparations. Another IgM kappa mAb 9D2 recognized an antigenic determinant on the 40 kD protein from the OMP as well as a whole bacterial cell preparation of a septicemic strain of H. somnus. These data demonstrate that, at least, three immunologically significant antigenic determinants on H. somnus are defined by mAbs against this bovine pathogen. Importantly, these studies suggest that the epitope present on the 40 kD major OMP, recognized by the 9D3 mAb, is immunodominant and conserved among septicemic, respiratory and urogenital strains, and is, therefore, suitable for further investigating its use in the development of an immunodiagnostic assay and also as a recombinant vaccine.

Animals↗
PubMed1995

Skewed VH and V kappa gene family expression and pairing occurs among B lymphocytes in autoimmune motheaten mice.

Motheaten mice homozygous for the recessive mev mutation develop a fatal immunodeficiency syndrome associated with hypergammaglobulinemia, thymic aplasia, production of autoantibodies and development of a severe lupus like systemic autoimmune disease. Most B lymphocytes in this mutant strain belong to B-1 subset. We have addressed the question if differences existed in the V-gene repertoire of autoimmune mev/mev mice as compared to phenotypically normal mev/+ and C57BL/6 background strain by examining the VH and V kappa gene family expression as well as the association of VH and V kappa gene families among B lymphocyte clones. The data outlined here demonstrate that both the expression of VH and V kappa gene families and their association is skewed in mev/mev mice, suffering from systemic autoimmune disease, and differs significantly from phenotypically normal mev/+ litter mates as well as the C57BL/6 background strain. In addition, VH+V kappa gene family pairs in phenotypically normal mev/+ differed from normal C57BL/6 mice suggesting that motheaten mutation, whether homozygous or heterozygous, alters the development of the B lymphocyte repertoire. These observations suggest positive selection of B-1 lymphocytes in autoimmune motheaten mice either as a result of selective processes, via receptor-ligand interactions, operating on the development of the primary antibody repertoire or defective B lymphocyte haematopoiesis due to the deficiency of haematopoietic cell phosphatase involved in determining the threshold by which B cells respond to self antigen(s).

Adjuvants, Immunologic↗
PubMed1993

Blood lead levels among populations differentially exposed to vehicular exhaust in Rohtak, India.

In the absence of local industrial sources of lead, leaded gasoline has been suggested as the major source of lead in the urban atmosphere of Rohtak town, situated near Delhi metropolitan city and with a high vehicular density. Blood lead levels were measured in 42 male volunteers from within the Rohtak area with varying degrees of exposure to vehicular exhaust. The occupationally exposed group with a daily exposure of 10-12 h in automobile workshops was found to have the highest levels of blood lead (mean value 21.26 microg dl(-1)) followed by roadside population (mean value 14.91 microg/dl(-1)). This group of people had a daily exposure of 8-10 h in their business establishments, situated at a distance of less than 5 to about 10 ft (< 1.5-c. 3.0 m) from the road with an average traffic density of 8000 vehicles day(-1). Urban residents were found to have higher blood lead levels (mean value 9.85 microg/dl(-1)) than the rural ones (mean value 3.34 microg/dl(-1)). The values were found to increase correspondingly with the increase in age and smoking habits within the particular categories of volunteers. The levels found in the present study are comparable to those reported from other major cities of India, but well within the tolerable limits as recommended by the European Economic Community.

Journal Article↗
PubMed1992

Malarial placental infection and low birth weight babies.

Two-hundred fifty-six mothers and their newborns were subjected to clinical and haematological tests for the evidence of malaria. Placentae of these were examined histopathologically for malarial parasites and malarial pigment. Forty six placentae showed scanty malarial pigment ingested by monocytes. These appearances were associated with focal syncytial necrosis and proliferation of cytotrophoblastic cells. Plasmodium falciparum was found in cord blood of six cases. The mean weight of newborns born to mothers having no evidence of malarial placental infection was 2.763 kg, while mean weight of newborns belonging to infected placentae was 2.143 kg. The difference was highly significant.

Birth Weight↗