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.

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.

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.

Isolation of Escherichia coli mutants lacking methylcytosine-dependent restriction systems for cloning extensively methylated frog virus 3 DNA.

Many bacterial strains possess methylation-dependent restriction systems (MDRS) that demonstrate methylcytosine-dependent restriction endonuclease activity for the dinucleotide sequence, dCpdG. This makes these strains unsuitable for cloning methylated DNA. Some commercially available bacterial cells are recommended for cloning DNA fragments with methylated cytosines and adenines, e.g., Escherichia coli DH5-alpha MCR. Our attempts to clone frog virus 3 (FV3) DNA, which has the highest degree of cytosine methylation ever reported, using DH5-alpha MCR cells, were not successful. This and other observations suggested the existence of additional MDRS that have not yet been eliminated from DH5-alpha MCR cells. In order to isolate a mutant from this bacterial strain that is suitable to clone highly methylated FV3 DNA, we transformed these cells with a recombinant pUC19 plasmid containing a methylated 1.4-kb genomic DNA fragment from FV3, and selected for ampicillin (Ap) resistance. Three such attempts yielded only one colony that contained a fully methylated 1.4-kb FV3 genomic DNA fragment. Furthermore, plasmid-cured Ap-sensitive colonies originating from this clone were isolated and have been successfully employed to clone the highly methylated FV3 genomic DNA fragment.

Conditional expression of foreign genes by temperature-sensitive mutants of vaccinia virus.

To assess the utility of two temperature-sensitive (ts) mutant vaccinia viruses as vectors for the conditional in vitro expression of recombinant foreign genes, we have studied the kinetics of expression of foreign genes incorporated into these viruses. At nonpermissive temperature, 40 degrees C, these viruses were defective either in DNA synthesis or in virus assembly. Foreign gene expression was affected by the nature of the ts lesion and by the nature of the vaccinia promoter positioned upstream from the foreign gene. With both vector viruses, a foreign gene controlled by the p7.5 early-late promoter was expressed at both 33 degrees and 40 degrees C. With the DNA synthesis-defective vector virus, foreign gene expression controlled by the p11 DNA synthesis-dependent late promoter was inhibited at 40 degrees C, but could be turned on by shift to 33 degrees C. This ts expression system provides an alternative to use of drugs that inhibit DNA synthesis as a means for experimental manipulation of gene expression. Both vector viruses can be used with existing vaccinia virus expression technology.

Amphibian and piscine iridoviruses proposal for nomenclature and taxonomy based on molecular and biological properties.

We have compared a number of properties of the well-characterized iridovirus, frog virus 3, with two other iridoviruses from amphibia, bullfrog edema virus and Lucké triturus virus, and with a piscine iridovirus, goldfish virus (GFV), to provide information for developing taxonomic classification of these viruses and establishing their ecological niche. Purified virions had similar size and shape (icosahedral) for each virus, and the genomic DNAs of each virus were methylated by a virus-induced DNA methyltransferase. The three amphibian viruses replicated equally well in fish (FHM), hamster (BHK), and human (WI-38) cell monolayer with identical cytopathology, while GFV failed to replicate in these cell lines. However, GFV replicated albeit at a slow rate, in a goldfish cell line; there was no detectable replication by the amphibian viruses in these cells. The amphibian iridoviruses had virtually similar DNA sequences, while those of GFV were markedly different. Analyses of virus-induced polypeptides in infected cells corroborated the DNA analyses; the polypeptides of the amphibian viruses were similar and distinct from those of the fish virus. Nongenetic reactivation could only be accomplished between the three amphibian viruses but not with the piscine virus. Based on these data, we suggest taxonomic and nomenclature designations of amphibian and piscine iridoviruses.

Biogenesis of vaccinia: analysis by three-factor crosses reveals mutual influence on stability of drug resistance and temperature sensitivity when both markers occur in some recombinant virus isolates.

Recombination analysis with 5 temperature-sensitive (ts) mutants of vaccinia virus, relegated to the E category phenotype and mimicking closely the effects produced by the antibiotic rifampicin, was undertaken to determine whether the genetic loci determining this phenotype were clustered on the genetic map. Optimum conditions for analysis were established with respect to the MOI and duration of incubation. At an MOI of 5-10, consistent values of recombination frequency (RF) were obtained, but became significantly lower when the MOI was 1 or less. Constant RF values were recorded when the duration of the infection was 8-24 h. These values were increased 3-fold by extending the incubation to 36 h. Under the standard conditions for analysis adopted, each mutant partner was added at an MOI of 10 and incubation was for 18 h. The map of group E mutants drawn from RF values obtained by 2-factor crosses was confirmed by 3-factor cross analysis, in which recombinants carried both the ts mutations and the rifampicin-resistance (R) marker. Both 2- and 3-factor cross data revealed that the 5 group E mutations were not clustered on the vaccinia virus genome. Surprisingly, after recombining ts 7743 with rifampicin resistance (R), different isolates of ts 7743 R could be mapped at two loci to the right or left of another isolate, ts 9383. Characterization of five independent plaque isolates of the progeny arising from a ts 7743 R parent showed that one isolate possessed a cold-sensitive phenotype (33 degrees/40 degrees = 1:26), two isolates retained their original ts character, and two became phenotypically like wild type (wt) with respect to thermosensitivity.(ABSTRACT TRUNCATED AT 250 WORDS)

The replication cycle of tanapox virus in owl monkey kidney cells.

The growth kinetics of tanapox virus in owl monkey kidney cells was elucidated by single-step growth curves at multiplicities of 10, 1.0, and 0.1 plaque forming units (pfu) per cell at 37 and 33 degrees C. Virus replicated equally well at both temperatures and produced a cytopathic effect that was characterized by densely packed rounded cells with retrogressed monolayer and granular vacuolated cytoplasm. Single-step growth curves revealed that the eclipse period varied from 24 h postinfection (hpi) at a multiplicity of infection of 10 pfu/cell to 48 hpi at 0.1 pfu/cell. The length of the latent period also varied from 36 hpi at 10 pfu/cell to 48 hpi at 0.1 pfu/cell. The intracellular virus, extracellular virus, and total virus titers reached their maximums relatively early at 10 pfu/cell as compared with 0.1 pfu/cell. About 78% of the mature progeny virion is retained intracellularly at 10 pfu/cell at 96 hpi. We conclude that tanapox virus replication is similar to other poxviruses, but the replication cycle is longer when compared with vaccinia virus.

Mutation in a DNA-binding protein reveals an association between DNA-methyltransferase activity and a 26,000-Da polypeptide in frog virus 3-infected cells.

The DNA of frog virus 3 (FV3), an iridovirus, is highly methylated; more than 20% of the cytosine bases are methylated at the 5-carbon position by an FV3-induced DNA methyltransferase (DNA-mt). To determine the role of this enzyme in virus replication and regulation of gene expression, we have analyzed an FV3 mutant that lacks DNA-mt activity and is resistant to 5-azacytidine (an inhibitor of DNA-mt). Comparative polypeptide analysis, using cytoplasmic extracts from the wild-type FV3 and mutant-infected cells, revealed that a single protein of 26,000 (26K) molecular weight was altered in the mutant-infected cells. The altered polypeptide migrated faster in SDS-polyacrylamide gel as compared to the wild-type FV3 26K protein. Five spontaneous revertants derived from the mutant regained the migrational characteristic of the wild-type 26K protein, DNA-mt activity, and methylation of their DNA. We further show that the 26K polypeptide is a DNA-binding protein and that 80% of the enzyme activity can be eluted from an ssDNA affinity column. Taken together, these data support the conclusion that the 26K polypeptide is associated with DNA-mt activity.

Methotrexate-resistant Chinese hamster ovary cells contain a dihydrofolate reductase with an altered affinity for methotrexate.

Previous reports [Flintoff, W. F., Davidson, S. V., & Siminovitch, L. (1976) Somatic Cell Genet. 2, 245--261; Gupta, R. S., Flintoff, W. F., & Siminovitch, L. (1977) Can. J. Biochem. 55, 445--452] described a series of Chinese hamster ovary cells that were resistant to the cytotoxic action of methotrexate and contained a dihydrofolate reductase that was less sensitive to inhibition by the drug than wild-type enzyme. In this study, binding of labeled methotrexate to the reductase--NADPH complex and separation of free and bound drug by filtration through Sephadex G--25 have been used to demonstrate that clonal isolates of these resistant cells contain a dihydrofolate reductase varying between 2.5- and 6-fold lower in affinity for the drug than the wild-type enzyme. The apparent dissociation constant for the wild-type enzyme is 0.5 x 10(-9) M. Using two-dimensional polyacrylamide gel electrophoresis, 11 independently selected resistant isolates have been shown to contain a reductase with a similar overall net charge as the wild-type enzyme. Reductase purified from either wild-type or resistant cells contains two components after isoelectric focusing in polyacrylamide gels. The major component represents about 90% of the total protein and has a pI of about 8.0. The minor component representing about 10% of the reductase protein has a pI between 7.2 and 7.6.

Transactivation of methylated HIV-LTR by a frog virus 3 protein.

DNA methylation has been implicated in the suppression of transcription of a large spectrum of eukaryotic genes. Frog virus 3 (FV3) contains genomic DNA that is the most extensively methylated of all known animal viruses. However, FV3 gene expression is tightly regulated in a sequential fashion in infected cells. Therefore, FV3 must have evolved a mechanism(s) to overcome the inhibitory effects of DNA methylation. FV3 has been shown to induce expression of methylated foreign genes in transient transfections. This study was designed to establish if this FV3-induced expression of methylated genes could be demonstrated in stable cell lines which contain integrated foreign genes that are silenced by DNA methylation. Stably transfected simian Vero and human T-cells containing a single copy of the methylated and transcriptionally suppressed HIV-LTR CAT construct were either infected with FV3 or fused with FV3-infected fat head minnow cells. The results from these experiments lead us to conclude that FV3 infection does promote expression of a foreign, stably integrated gene (HIV-LTR), which was previously silenced by DNA methylation. We also observed that stably transformed human T-cells incubated at 30 degrees, unlike at 37 degrees, expressed minute but detectable HIV-LTR-directed CAT activity. Significance of this finding in HIV pathogenesis remains elusive.

Naturally occurring antibodies to liposomes. II. Specificity and electrophoretic pattern of rabbit antibodies reacting with sphingomyelin-containing liposomes.

Sera obtained from rabbits after immunization with a variety of unrelated antigens contain antibodies that induce complement- (C) mediated lysis of sphingomyelin-containing liposomes in the absence of the relevant antigen from the membrane. Absorption or inhibition with dimyristoyl-phosphatidyl choline-containing liposomes were less effective than with sphingomyelin-containing liposomes in decreasing or abolishing C-dependent lysis of target-liposomes. Phosphoryl choline chloride inhibited the C-dependent lysis mediated by these antibodies, but only when used in high molar excess and in the presence of low antibody concentrations. Purified anti-liposome antibodies displayed an isoelectric focusing pattern consistent with a polyclonal response. The findings confirm the antibody nature of the anti-liposome activity of rabbit sera and indicate that their predominant specificity is directed against conformations of the phospholipid molecule in which the polar (phosphoryl choline) group does not have a major contribution.

The genome sequence of Yaba-like disease virus, a yatapoxvirus.

The genome sequence of Yaba-like disease virus (YLDV), an unclassified member of the yatapoxvirus genus, has been determined. Excluding the terminal hairpin loops, the YLDV genome is 144,575 bp in length and contains inverted terminal repeats (ITRs) of 1883 bp. Within 20 nucleotides of the termini, there is a sequence that is conserved in other poxviruses and is required for the resolution of concatemeric replicative DNA intermediates. The nucleotide composition of the genome is 73% A+T, but the ITRs are only 63% A+T. The genome contains 151 tightly packed open reading frames (ORFs) that either are > or =180 nucleotides in length or are conserved in other poxviruses. ORFs within 23 kb of each end are transcribed toward the termini, whereas ORFs within the central region of the genome are encoded on either DNA strand. In the central region ORFs have a conserved position, orientation, and sequence compared with vaccinia virus ORFs and encode many enzymes, transcription factors, or structural proteins. In contrast, ORFs near the termini are more divergent and in seven cases are without counterparts in other poxviruses. The YLDV genome encodes several predicted immunomodulators; examples include two proteins with similarity to CC chemokine receptors and predicted secreted proteins with similarity to MHC class I antigen, OX-2, interleukin-10/mda-7, poxvirus growth factor, serpins, and a type I interferon-binding protein. Phylogenic analyses indicated that YLDV is very closely related to yaba monkey tumor virus, but outside the yatapoxvirus genus YLDV is more closely related to swinepox virus and leporipoxviruses than to other chordopoxvirus genera.

Multiple anti-cytokine activities secreted from tanapox virus-infected cells.

Tanapox virus (TPV) produces a mild disease in humans characterized by transient fever, one or more nodular skin lesions and regional lymphadenopathy. We demonstrate that TPV-infected cells, but not mock-infected cells, secrete an early 38 kDa glycopeptide that, unlike any other known protein, binds to human (h) interferon-gamma, hIL-2 and hIL-5. In concomitant experiments this polypeptide failed to bind to hIL-1 alpha, hIL-3, hIL-4, hIL-6, hIL-7, hIL-8 or hIL-10. Inhibition of hIL-2 and hIL-5 biological activities were demonstrated using a hIL-2-dependent mouse T cell line (HT-2) and a hIL-5-dependent erythroleukemia cell line (TF-1), respectively. The 38 kDa polypeptide also inhibited the bioactivity of interferon-gamma. Taken together, our results suggest that TPV has evolved multiple pathways to disarm both TH1 cell-mediated (IL-2 and interferon-gamma) and TH2-associated (IL-5) immune responses for its infectivity with remarkable genetic economy.

Possible mechanisms in the pathogenesis of virus-induced diabetes mellitus.

Insulin-dependent diabetes mellitus results from destruction of pancreatic beta cells. Viruses and autoimmunity have been implicated as possible causes of beta cell destruction in genetically predisposed individuals. The evidence for viruses comes largely from experiments in animals, but several studies in humans point to viruses as triggers in the pathogenesis of diabetes in some cases. In animal models, at least 4 different possible mechanisms for virus-induced diabetes have been proposed. The first mechanism is direct cytolytic infection of pancreatic beta cells. One group of viruses, including encephalomyocarditis virus, Mengovirus 2T, and Coxsackie B viruses, can directly infect and destroy pancreatic beta cells independent of autoimmune processes. The second mechanism is triggering of autoimmune responses. In contrast to the encephalomyocarditis virus-induced diabetes, reovirus type 1 and rubella virus seem to be somehow associated with autoimmunity in the genesis of a diabetes-like syndrome in a certain strain of suckling mice and hamsters, respectively. The third mechanism is cumulative environmental insults. The cumulative environmental insults with viruses and beta cell toxic chemicals can result in diabetes in genetically predisposed non-human primates and certain inbred strains of mice. The fourth mechanism is persistent infection. A certain virus, such as lymphocytic choriomeningitis virus, persistently infects murine pancreatic beta cells and produces hyperglycemia. The evidence that viruses cause diabetes in humans is more circumstantial.(ABSTRACT TRUNCATED AT 250 WORDS)

Transcription of methylated viral DNA by eukaryotic RNA polymerase II.

The genome of the large icosahedral DNA virus, frog virus 3 (FV3), is heavily methylated at the cytosine residues of dCdG dinucleotide pairs, with more than 22% of the total cytosine residues in the form of 5-methylcytosine (5mC). This methylation is carried out postreplicatively in the cytoplasm of infected cells by a virus-encoded DNA methyltransferase. DNA methyltransferase activity was shown to copurify with a 26 kD virus-induced, DNA-binding protein that had an altered mobility in extracts from cells infected with a DNA-methyl-transferase deficient mutant of FV3. Immediately after infection, the highly methylated parental DNA is transcribed in the nucleus by the host cell RNA polymerase II. As FV3 induces the synthesis of a protein that can override the inhibitory effect of methylation on the transcription of exogenous promoters methylation in vitro, we suggest that this protein is a factor evolved by this virus to allow transcription from methylated promoters by eukaryotic RNA polymerase II.

Selective inhibition of TNF-alpha induced cell adhesion molecule gene expression by tanapox virus.

Poxviruses encode virulence factors that have been identified as proteins that are secreted from infected host cells. Some of these secretory proteins impede host immune defences. We have previously demonstrated that tanapox virus (TPV) infected cells secrete an early 38 kDa glycopeptide that binds to human (h) interferon-gamma, hIL-2, and hIL-5. We now show an additional activity in the supernatant from TPV infected cells that down-regulates the expression of tumour necrosis factor-alpha (TNF-alpha) induced cell adhesion molecule gene expression. This activity was not detected in mock infected cells. Enzyme linked immunosorbent assays (ELISA) on primary human endothelial cells, show the induction of E-selectin, vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) following TNF-alpha or IL-1 beta treatment, as expected. Supernatant from TPV infected cells significantly decreased the TNF-alpha but not IL-1 beta-induced expression of these molecules. Mobility shift assays and Northern blot analyses further show that the supernatant from TPV infected cells inhibited TNF-alpha-induced activation of the nuclear transcription factor-kappa B (NF-kappa B) and transcriptional activation of the E-selectin, VCAM-1 and ICAM-1 genes. Based on TNF-alpha affinity chromatography, this activity appears to be associated with a 38 kDa glycopeptide.

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.

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 diseases. 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 mucosa. Using hybridoma technology, we obtained a number of monoclonal autoantibodies 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 isolated 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.