Patrick R. McClintock

Multiple organ-reactive monoclonal autoantibodies

Autoantibodies directed against a wide range of normal tissue antigens have been found in the sera of patients with autoimmune diseases1–8. It is generally thought that different and specific autoantibodies react with different tissues but the possibility exists that some autoantibodies may react with common antigens found in different tissues and organs. Recently, we showed that mice infected with reovirus developed a polyendocrine disease with autoantibodies to the pancreas, anterior pituitary, thymus and gastric mucosa9,10. Using hybridoma technology, we obtained a number of monoclonal autoantibodies11 which reacted with antigens in single organs. We now report the production and pattern of reactivity of seven multiple organ-reactive monoclonal autoantibodies. By using antibody-affinity columns, autoantigens also have been isloated and their molecular weights determined. The results suggest that monoclonal multiple organ-reactive autoantibodies react either with the same molecule present in several organs or with common antigenic determinants on different molecules in multiple organs. In either case, the existence of multiple organ-reactive antibodies may be a partial explanation for multiple organ autoimmunity.

Virus-induced Diabetes Mellitus: No Evidence for Immune Mechanisms in the Destruction of β-Cells by the D-Variant of Encephalomyocarditis Virus

A possible contribution of the immune system to the pathogenesis of virus-induced diabetes mellitus was investigated using the D-variant of encephalomyocarditis (EMC-D) virus. Studies on the F1 and backcross progeny of susceptible and resistant strains of mice gave no suggestion of a linkage between susceptibility and the major histocompatibility locus. Immunosuppression by antilymphocyte serum did not prevent the induction of EMC-D-induced diabetes. Athymic nude mice infected with EMC-D virus showed a nearly identical diabetogenic response as comparedwith heterozygous litter mates. Passive transfer of lymphocytes from mice made diabetic with EMC-D virus into normal mice failed to produce diabetes. From these and other studies, we conclude that the development of EMC-Dinduced diabetes is due to the direct destruction of β-cells by the virus and that the contribution of the immune response to the pathogenesis of this disease is, at the most, minor.

Expression and modulation of virus receptors on lymphoid and myeloid cells: Relationship to infectivity

Abstract Using a sensitive and specific assay for the binding of radiolabeled encephalomyocarditis (EMC) virus to cell surface receptors, we have measured the kinetics of virus attachment to murine lymphoid and myeloid cells, and have shown that receptors for this virus vary with the phase of cell growth and can be modulated or, in some cases, induced. The initial rate constant ( K R ) for resident peritoneal macrophages was 0.8 × 10 −9 cm 3 min −1 cell −1 , but after in vivo stimulation the rate of binding increased by a factor of 2. In contrast, unstimulated splenic T and B lymphocytes failed to bind EMC virus ( K R −9 cm 3 min −1 cell −1 ), but after exposure to appropriate mitogens, the initial rate constant increased to as high as 6.7 × 10 −9 cm 3 min −1 cell −1 . Studies with continuous cell lines showed that the binding of virus to receptors varied by up to 10-fold and was greatest during the exponential growth phase as compared to the stationary phase. Although the K R increased following exposure of lymphocytes to mitogens, immunofluorescence revealed that only a small fraction (less than 2%) of the cells actually contained viral antigens, suggesting that virus receptors either were induced in only a small subpopulation of lymphocytes or that postattachment restriction prevented viral replication in a larger fraction of the cells. A survey of six cloned BALL/c T and B cell lymphomas arrested at different stages of differentiation showed that two of these lymphomas possessed EMC virus receptors, arguing that receptors are present only at certain stages of cell differentiation. Evidence that receptors are functionally important comes from experiments which showed that only cells with a K R greater than 0.1 × 10 −9 cm 3 min −1 cell −1 are susceptible to infection. These studies suggest that factors which influence the expression and modulation of virus receptors may determine the outcome of certain viral infections.

Virus-induced diabetes mellitus. XXV. Difference in the RNA fingerprints of diabetogenic and non-diabetogenic variants of encephalomyocarditis virus.

Summary The genomes of diabetogenic and non-diabetogenic variants of encephalomyocarditis virus were analysed by nucleic acid hybridization and RNA fingerprinting. Hybridization and thermal elution profiles failed to show any difference between the RNAs of the two variants, whereas fingerprinting of the T1-digested RNAs revealed at least one oligonucleotide, 20 to 25 nucleotides long, missing in the non-diabetogenic variant.

Genomic and receptor attachment differences between Mengovirus and encephalomyocarditis virus

Abstract Antigenically, encephalomyocarditis (EMC) virus and Mengovirus cannot be distinguished by hyperimmune sera. Biologically, however, these two viruses are quite different. The D clone of EMC virus produces diabetes, but not encephalitis, whereas our 2T clone of Mengovirus produces a rapidly lethal encephalitis. Moreover, by thermal elution analysis of EMC virus and Mengovirus cDNA-RNA hybrids there was an estimated 20% difference in the nucleotide sequences of these two viruses. Receptor attachment studies revealed that the rate of binding of the neurovirulent Mengovirus was 5 to 10 times greater for neuronal cell lines than was the nonneurovirulent EMC virus. Receptor saturation experiments showed that unlabeled Mengo and EMC viruses effectively blocked the binding of labeled homologous, but not heterologous, virus. It is concluded from these and other studies that Mengo and EMC viruses are related, but distinct viruses that bind to different receptors on the cell surface.

Receptors for encephalomyocarditis virus on murine and human cells

Abstract The attachment kinetics of radiolabeled encephalomyocarditis virus were studied using established murine and human cell lines and murine and human erythrocytes. Unlabeled virus completely blocked the binding of labeled virus and virus subjected to pH 6.0 treatment bound at only one-tenth the rate of native virus. The initial rate constant at 0° calculated for human erythrocytes was 3.2 × 10 −10 cm 3 min −1 cell −1 . The rate constants for Friend leukemia cells and HeLa cells were, respectively, 200 and 2000 times faster. In contrast, no binding was observed to murine erythrocytes. The attachment of the virus to HeLa cells was found to be temperature independent over the range 0 to 40°. The attachment to murine cells (L-929 and Friend leukemia), however, was progressively reduced with increasing temperatures (0 to 40°) to about 0.01 of the rate at 0°. The decrease in binding at temperatures greater than 0° appears to be due to an increased rate of dissociation of the virus. It is proposed that both receptor number and viral affinity can influence pathogenicity.

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.

Human multiple organ-reactive monoclonal autoantibody recognizes growth hormone and a 35,000-molecular weight protein.

By fusing peripheral leukocytes from a patient with insulin-dependent diabetes with mouse myeloma cells, a heterohybridoma was isolated that, for over one year, has secreted a human monoclonal autoantibody, designated MOR-h1 (multiple organ-reactive human 1). This antibody reacts with antigens in several endocrine organs including the pituitary, thyroid, stomach, and pancreas. By double immunofluorescence, MOR-h1 was found to react specifically with growth hormone (GH)-containing cells in the anterior pituitary and, by enzyme-linked immunosorbent assay, MOR-h1 was shown to react with both natural and biosynthetic GH. Absorption experiments revealed that GH could remove the capacity of MOR-h1 to react not only with cells in the anterior pituitary, but also with cells in the thyroid, stomach, and pancreas. The demonstration with hyperimmune serum that these organs do not contain GH indicated that MOR-h1 was reacting with a different molecule(s) in these organs. By passing extracts of pituitary, thyroid, and stomach through an MOR-h1 affinity column and analyzing the eluted antigens by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a 35,000-mol wt polypeptide was isolated from each of these organs. In addition, a 21,500-mol wt polypeptide with an electrophoretic mobility identical to purified human GH was isolated from the pituitary, but not the other organs. It is concluded that MOR-h1 reacts with a 35,000-mol wt polypeptide present in the pituitary, thyroid, and stomach and that this antibody also recognizes a determinant on GH.

Anti-idiotypic antibodies against a human multiple organ-reactive autoantibody. Detection of idiotopes in normal individuals and patients with autoimmune diseases.

We have recently isolated and characterized a human monoclonal autoantibody, MOR-h1 (multiple organ-reactive human 1), that reacts with antigens in multiple organs and have shown that this antibody binds to human growth hormone and a 35,000-mol wt protein. In the present study we generated three monoclonal anti-idiotypic antibodies (4E6, 3E5, and 3F6) against MOR-h1. These anti-idiotypic antibodies specifically reacted with MOR-h1 and not with 26 other multiple organ-reactive monoclonal IgM autoantibodies nor with pooled human IgM (myeloma proteins). The binding of the anti-idiotypic antibodies to MOR-h1 was inhibited by both human growth hormone and the 35,000-mol wt protein, which strongly suggests that these antibodies react with epitopes at or near the paratope on MOR-h1. The results of competitive binding experiments revealed that the epitope recognized by 4E6 is distinct from that recognized by 3E5 and 3F6. Using these anti-idiotypic antibodies, lymphocytes and sera from normal individuals were tested for the presence of the 4E6 and 3E5/3F6 idiotopes. By indirect immunofluorescence, the 4E6 idiotope was detected on an average of 1.1% of normal circulating B lymphocytes, and by enzyme-linked immunosorbent assays, the 4E6 and to a lesser extent the 3E5/3F6 idiotopes were found on IgG molecules in sera of normal individuals. In spite of the expression of idiotopes known to be present on MOR-h1, no MOR-h1-like antibody activity was detected in normal sera. Examination of sera from patients with several autoimmune diseases failed to show an increased expression of the 4E6 idiotope as compared with normal controls. These data suggest that anti-idiotypic antibody 4E6 recognizes a public idiotope, the expression of which is not restricted to autoimmune disease.

Viral Receptors: Expression, Regulation and Relationship to Infectivity

The presence or absence of specific cell surface receptors can influence the host range and tissue tropism of viruses. For example, receptors for poliovirus are known to confer susceptibility on human and some primate cells, while cells of other species lack receptors and are resistant to infection [1]. Although postattachment restrictions are known, virtually all viruses appear to be restricted initially by appropriate receptor expression on the target cells. The most intensively studied viral receptors have been those for the picornaviruses, myxoviruses, and retroviruses. For these, detailed information on the mechanisms of attachment and penetration have been reported [2]. Even the chromosomal locations of the structural genes for some of these receptors are known and have been assigned to human chromosome 19 [3].