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B S Prabhakar

Publications and source records attributed to B S Prabhakar.

At least 73 records · Page 4Linked to original sources
PubMed1991

Identification of a putative shared epitope between Coxsackie virus B4 and alpha cardiac myosin heavy chain.

Molecular mimicry is an important postulated mechanism for autoimmunity in viral myocarditis. The 356-1 monoclonal antibody neutralizes Coxsackie virus B4 by binding to the VP1 protein and cross-reacts with mouse alpha cardiac myosin heavy chain. We used this monoclonal antibody to screen a lambda gt11 expression library made from CD-1 mouse hearts. Of the 48 positive plaques/10(6) recombinant phages examined, 14 of the strongest-reacting clones were purified for additional studies. The inserts were amplified by polymerase chain reaction and the amplified products ranged from about 150 to 1400 bp in size. Northern hybridization using these inserts demonstrated that 11 out of 14 reacted with a message equivalent to that of cardiac myosin in size. Additional Southern hybridization studies suggested that these 11 inserts contained overlapping sequences in the light meromyosin fragment of cardiac myosin. Sequence analysis confirmed that these 11 independent, recombinant clones contained a common sequence representing amino acid residues 1299-1647. Within this fragment only one isoform-specific site matched the observed reactivity pattern of 356-1 among hearts from various species. Thus, we were able to identify a putative shared epitope represented by residues 1632-1647.

Amino Acid Sequence↗
PubMed1990

Characterization of the 70KDA component of the human Ku autoantigen expressed in insect cell nuclei using a recombinant baculovirus vector.

The Ku autoantigen is a human nuclear, DNA-binding heterodimer of 70kDa and 86kDa proteins. It is the target of autoantibodies in several autoimmune diseases. We now report the expression of a cDNA encoding the 70kDa Ku protein. Large amounts of protein were obtained using a recombinant baculovirus vector, in contrast with earlier unsuccessful attempts using other expression systems. We demonstrate that the 70kDa Ku protein is targeted to the nucleus and is associated with the nuclear matrix when expressed in the absence of the 86kDa Ku component. No post-translational modifications were observed. The 70kDa protein binds double and single-stranded DNA with very high affinity. Our results suggest that the baculovirus expression system may be of widespread use in the production and characterization of human autoantigens.

Animals↗
PubMed1990

Binding and functional effects of thyroid stimulating hormone on human immune cells.

The expression and functional relevance of thyroid stimulating hormone (TSH) receptors on human immune cells were studied. Flow cytometric analysis was used to study the binding of biotinylated TSH to human peripheral blood mononuclear cells (PBMC) and various purified lymphoid populations. Our results indicate that the hormone binds well to monocytes and natural killer (NK) cells and marginally to purified tonsillar T and B lymphocytes. There was a significant increase in the binding of TSH to purified B cells that were activated in vitro with Staphylococcus aureaus Cowan. In contrast, the binding of TSH to T cells was unaltered when they were stimulated with phytohemagglutinin (PHA). While TSH increases DNA synthesis and intracellular cAMP levels of FRTL-5 rat thyroid cells, it did not have such stimulatory effects on lymphocytes. However, there was a moderate increase in Ig production by activated B lymphocytes when they were cultured in the presence of the hormone. A possible function for TSH as a link between the immune system and the thyroid is discussed.

B-Lymphocytes↗
PubMed1990

A neutralizing monoclonal antibody against Coxsackievirus B4 cross-reacts with contractile muscle proteins.

A panel of Coxsackievirus B4 (CVB4) neutralizing monoclonal antibodies (mAbs) were tested against a panel of normal mouse tissues. One mAb, 356-1, reacted specifically with murine heart tissue. Immunohistochemical studies revealed an A band pattern of staining of the heart. Examination of sequential differential extracts of heart by Western immunoblotting showed that 356-1 predominantly reacted with the murine cardiac myosin heavy chain. A rather weak cross-reaction was found with actin. These observations were confirmed by the binding of 356-1 to purified cardiac myosin and actin. This antibody showed a higher affinity for murine cardiac muscle myosin than for skeletal muscle myosin. Examination of the reactivity of 356-1 with CVB4 polypeptides using Western immunoblotting revealed that 356-1 binds to the VP-1 capsid protein. These studies imply that molecular mimicry is one mechanism by which autoimmunity could develop during CVB4 induced myocarditis.

Animals↗
PubMed1990

Cell surface expression of the 70-kD component of Ku, a DNA-binding nuclear autoantigen.

The Ku complex, a heterodimer of 86- and 70-kD proteins, is a nuclear DNA-binding autoantigen. However, hydrophobicity analysis of the deduced amino acid sequence of the 70-kD protein had strongly suggested that this might also be a membrane protein. In the present study, using antibodies to synthetic peptides and a polyclonal antiserum to the purified protein, we show that the 70-kD protein of the Ku complex is present in isolated plasma membranes of human cells. By indirect immunofluorescence microscopy and fluorescein-activated cell sorting, we demonstrate that this autoantigen is exposed on the cell surface. In addition, we have identified several domains of the protein that are exposed. Our study provides one of the first demonstrations of a eukaryotic, nuclear DNA-binding protein that is also on the cell membrane. Moreover, our results might help explain how autoantibodies to the Ku autoantigen could target cells for an autoimmune attack.

Amino Acid Sequence↗
PubMed1989

Cloning and characterization of a cDNA that encodes a 70-kDa novel human thyroid autoantigen.

cDNA clones were isolated by screening a human thyroid carcinoma lambda gt11 library with immunoglobulins purified from serum of a patient with autoimmune Graves' disease. One clone (ML8) containing a 1.25-kilobase (kb) insert hybridized with a single 2.0-kb poly(A+) mRNA in human thyroid and lymphocytes but not in human brain, liver, kidney, or muscle. In addition, this probe also hybridized with a single 2.0-kb poly(A+) mRNA from a rat thyroid cell line (FRTL-5). An apparently full length 2,074-base pair (bp) human cDNA was obtained and sequenced. The nucleotide sequence of the 2,074-bp cDNA includes a 5'-noncoding sequence of 17 bp, a 1827-bp open reading frame, and a 222-bp 3'-noncoding sequence. The canonical polyadenylation signal AATAAA is present 18 bp upstream of the poly(A) tail. This cDNA encodes a 69,812-dalton protein with two potential N-linked glycosylation sites and at least one potential membrane spanning domain. Immunoprecipitation of the in vitro translated protein by sera from several patients with Graves' disease argues that the 69,812-dalton protein is an autoantigen.

Amino Acid Sequence↗
PubMed1989

Monoclonal antibodies against osteonectin show conservation of epitopes across species.

Several monoclonal antibodies were produced to bovine osteonectin, a major noncollagenous protein in the extracellular matrix of bone, and four were characterized. These antibodies showed different reactivities in Western immunoblots, immunoprecipitation, and indirect immunofluorescence, indicating that they recognize different epitopes on the protein. The data indicate that an epitope recognized by one of the antibodies is masked in interactions of osteonectin within cells and in the extracellular matrix. The high degree of cross-species immunoreactivity observed against bone osteonectin with these monoclonal antibodies indicates that these common epitopes have been conserved during evolution.

Animals↗
PubMed1989

Organ reactive autoantibodies from non-immunized adult BALB/c mice are polyreactive and express non-biased VH gene usage.

To examine the naturally activated autoreactive B cell repertoire, we analyzed a panel of hybridomas from unmanipulated adult BALB/c spleen cells for reactivity patterns and VH gene usage. We found a pattern of VH usage that was diverse and appeared to reflect the germline repertoire. Although all but one natural antibody hybridoma (NAb) were initially selected for organ rather than antigen binding, the majority of organ reactive IgM NAbs were polyreactive, expressing a broad range of reactivity patterns for both self and foreign antigens, that were unique for each NAb and were not indiscriminate. Our results are consistent with the hypothesis that many naturally activated adult B cells are highly polyreactive and that autoreactivity is a consequence of polyreactivity. We suggest that the population of NAbs exhibiting organ reactivity overlaps the populations of other IgM autoantibodies that have been described previously, and that these all derive from a pool of polyreactive IgM antibodies which are polyclonally activated in the early immune response. These polyreactive natural antibodies may represent a first line of defense and offer protection for the host against a variety of foreign agents.

Animals↗
PubMed1988

Human lymphocytes produce pro-opiomelanocortin gene-related transcripts. Effects of lymphotropic viruses.

Expression of the pro-opiomelanocortin (POMC) gene was examined in normal human lymphocytes and lymphocyte cell lines infected by lymphotropic viruses. POMC gene transcripts were detected in human lymphocytes using stringent RNA-RNA hybridizations. Low transcript levels were found in normal phytohemagglutinin-stimulated peripheral blood mononuclear cells and in tonsillar T and B cells. The highest levels were found in cells infected with Epstein-Barr virus (EBV). However, T cell lines infected with human T lymphotropic viruses did not have increased levels of transcripts. The transcript levels in an EBV-transformed B lymphocyte line were not affected by dexamethasone or corticotropin-releasing hormone, known regulators of anterior pituitary POMC gene expression. Therefore, it is possible that EBV infections could result in abnormal POMC expression.

Cell Line↗
PubMed1988

Probing the human B-cell repertoire: isolation of Epstein-Barr virus (EBV)-transformed human B lymphocytes making antibodies with a common idiotope that have different antigen-binding specificities.

Human peripheral B lymphocytes were transformed with Epstein-Barr virus and probed with an antiidiotypic antibody made against a human monoclonal autoantibody. Six cell lines were isolated that produced antibodies bearing a common idiotope. Despite the presence of this common idiotope, the antibodies showed antigen-binding specificities different from that of the parental antibody to which the antiidiotypic antibody was made. By probing Epstein-Barr virus-transformed cells with well-characterized antiidiotypic antibodies, it should now be possible to isolate and determine the frequency of B cells bearing specific idiotopes in the human repertoire and to study the antigen-binding properties of these antibodies.

Antibodies, Viral↗
PubMed1988

Spontaneously proliferating human B lymphocytes make autoantibodies.

Peripheral blood lymphocytes from 29 patients with autoimmune diseases and eight normal controls were cultured in vitro. Several weeks later, lymphocyte proliferation was observed in many of these cultures. Cell lines were obtained from about half of the donors, at a frequency of approximately 1 in 10(7) lymphocytes. Outgrowth occurred only in cultures from patients who had antibodies to the viral capsid antigen of Epstein-Barr virus (EBV). The proliferating cells also contained EBV nuclear antigen, an occurrence confirming that proliferation was a result of latent EBV infection of the donors. Supernatants from these cell lines were tested for autoantibodies by screening against normal tissues. About 30% of the cell lines made autoantibodies, many of which reacted with smooth muscle or epithelial cells. Because EBV is harbored in a latent form in the majority of the adult population, reactivation of latent EBV infection in vivo may explain the production of autoantibodies seen in a variety of immunoregulatory disorders.

Adolescent↗
PubMed1987

Human monoclonal multiple-organ-reactive autoantibodies distinguished by mouse monoclonal anti-idiotypic antibodies: expression of idiotopes in humans with and without autoimmune diseases.

Previously we reported on the production and characteristics of a number of human monoclonal autoantibodies. All of these autoantibodies were of the IgM class and reacted with antigens in multiple organs. In this study we generated IgG murine monoclonal anti-idiotypic antibodies against five human monoclonal autoantibodies, (i.e., MOR-h2, MOR-h3, MOR-h4, CG1, and CG2). These anti-idiotypic antibodies reacted strongly with the corresponding human monoclonal autoantibody, but minimally or not at all with other human monoclonal autoantibodies. By using these anti-idiotypic antibodies as probes, we screened sera obtained from normal individuals and patients with insulin-dependent diabetes mellitus, Hashimoto's thyroiditis, and systemic lupus erythematosus for the expression of idiotopes. Our study showed that the idiotopes recognized by three of the anti-idiotypic antibodies, i.e., anti-CG1, anti-CG2, and anti-MOR-h2, were not expressed, but the idiotopes recognized by two of the anti-idiotypic antibodies, i.e., anti-MOR-h3 and anti-MOR-h4, were expressed in normal individuals. In patients with autoimmune disorders, there was no increase in the expression of the CG1, CG2, and MOR-h2 idiotopes, but 45 and 23% of the patients with systemic lupus erythematosus showed a significant increase in the expression of the MOR-h3 and MOR-h4 idiotopes respectively. These findings show that there is widespread expression in the B cell repertoire of certain autoantibody-associated idiotopes.

Animals↗
PubMed1987

Selection of coxsackievirus B4 variants with monoclonal antibodies results in attenuation.

Inoculation of suckling mice with coxsackievirus B4 (CB4) results in the death of a majority of the animals. In this study we selected antigenic variants of CB4 in the presence of neutralizing monoclonal antibodies and tested them to see whether they were attenuated. Antigenic variants selected with a single antibody showed little or no attenuation by producing a high mortality (60 to 100%). A double variant selected with two antibodies showed considerable attenuation by causing only 25% mortality. A triple variant selected with three antibodies was almost completely attenuated (killed only 5% of the animals). Polypeptides from these variants were tested for their ability to interact with the monoclonal antibodies used for their selection. These studies showed that resistance of variant virus to neutralization in general was due to the inability of the antibody to bind to the virus. However, one of the antibodies could bind but not neutralize the virus, perhaps due to an alteration in the epitope. It is concluded that selection of CB4 variants using more than one neutralizing monoclonal antibody can lead to attenuation of the virus.

Animals↗
PubMed1986

Multiple organ-reactive IgG antibody induced by an antiidiotypic antibody to a human monoclonal IgM autoantibody.

MOR-h1 is a human multiple organ-reactive (MOR) monoclonal autoantibody (Ab1) that reacts with human growth hormone (hGH) and a 35 kD protein found in the anterior pituitary, thyroid, stomach, and pancreas. 4E6 is a mouse monoclonal anti-idiotypic antibody (Ab2) that reacts with the paratope of MOR-h1 and is ligand inhibitable. In the present study, we immunized a rabbit with 4E6 and purified an IgG fraction (anti-4E6) from the sera. Competitive inhibition experiments showed that anti-4E6 (Ab3) binds to the same epitope on 4E6 and to the same antigens (i.e., hGH and 35 kD protein) as does MOR-h1. By immunofluorescence, anti-4E6, an IgG antibody, shows the same multiple organ reactivity with tissues as does MOR-h1, an IgM antibody. From these and other studies, we conclude that the 4E6 paratope (Ab2) has a conformational resemblance to an epitope on hGH and the 35 kD protein. This raises the possibility that antibodies made in response to certain anti-idiotypic antibodies may be one of the mechanisms for triggering an autoimmune response.

Antibodies, Anti-Idiotypic↗
PubMed1986

Anti-idiotypic antibodies to monoclonal antibodies that neutralize coxsackievirus B4 do not recognize viral receptors.

We have made anti-idiotypic antibodies in rabbits against three mouse monoclonal neutralizing antibodies with specificities for independent epitopes on Coxsackievirus B4. Each of these anti-idiotypic antibodies was found to react specifically with the immunizing monoclonal antibody in radioimmunoassays and did not react with the other monoclonal antibodies. In addition, the anti-idiotypic antibodies specifically inhibited the function (i.e., virus neutralization) of the immunizing antibody. These anti-idiotypic antibodies were tested for their ability to recognize receptors for Coxsackievirus B4 as measured by their ability to inhibit the attachment of radiolabeled virus to cellular receptors. The anti-idiotypic antibodies did not block the binding of Coxsackievirus B4 to monkey kidney cells. Moreover, when tested for their ability to bind to other receptor positive cells, none of the anti-idiotypic antibodies bound above control levels. Anti-idiotypic antibodies did induce a small anti-anti-idiotypic antibody response in mice when tested by radioimmunoassay; however, little if any virus neutralizing activity was found in the sera of these mice. Our results contrast to those reported for anti-idiotypic antibodies in several other virus systems and suggest that not all anti-idiotypic antibodies made against neutralizing antibodies are capable of eliciting an antiviral immune response or binding to viral viral receptors on cells.

Animals↗