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J J Marchalonis

Publications and source records attributed to J J Marchalonis.

At least 91 records · Page 5Linked to original sources
PubMed1989

Flow cytometric analysis of human lymphocytes using affinity-purified antibody to T cell receptor beta synthetic J region peptide.

The purpose of this study was to use affinity-purified polyclonal antibodies produced against a synthetic peptide corresponding to the joining (J) region of a human T cell receptor beta chain to characterize antigen receptor expression on subpopulations of human lymphocytes. The synthetic peptide used was ANYGYTFGSGTRLTVV, corresponding to the J segment of the human beta-chain gene YT35. Biochemical characterization has previously demonstrated binding of anti-J beta peptide antibodies to the alpha/beta heterodimer and to certain immunoglobulin light chains. Flow cytometric analysis of normal human peripheral blood lymphocytes performed here, using affinity-purified antibodies to the J beta peptide, showed expression of the epitope on 50-60% of CD20 (B1)-positive B lymphocytes, and on 40-50% of CD8-positive T lymphocytes. Only background levels were observed on CD4-positive T cells.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1989

Immunoproteins in evolution.

All vertebrates respond to antigenic challenge by specific cellular reactions and by producing circulating antibodies, and they also contain recognition molecules that are evolutionary relics of primitive non-immune recognition. One such ancient recognition molecule is C-reactive protein (CRP), a member of the pentraxin family, that has homologs occurring in species as diverse as vertebrates, tunicates and the horseshoe crab (an ancient arachnoid). This molecule has lectin-like properties, can act as an opsonin and interact with complement and cells in a manner paralleling immunoglobulins (Igs). The horseshoe crab lectin and CRP, although unrelated to Igs, share functional idiotopes with classical antibodies. This finding reflects either an evolutionary convergence or a mechanism of "mini gene insertion" that allows molecules of distinct evolutionary histories to react to the same ligands. Serum antibodies of all vertebrates are polydisperse in charge and are composed of polypeptide chains comparable in mass to those of mammalian light and heavy chains. Molecular genetic studies of placoderm derived vertebrates are incomplete but are sufficient to allow the conclusion that Igs of these species are specified by variable (V), joining (J) and constant (C) gene segments and that rearrangement are an essential feature for the generation of antibody diversity. Here we present new evidence following from the use of antibodies directed against synthetic joining region peptides as probes in the study of rearranging Igs in evolution, the use of recombinant DNA technology to study T cell receptor V beta genes in a goldfish genomic library and the isolation and characterization of a gene fragment specifying sandbar shark light chain C region. We reached the following conclusions: (1) J region segments are the most conserved in evolution, and this most probably reflects the essential requirements for these gene segments in the formation of intact Ig genes by rearrangement; (2) the framework segments of V regions are highly conserved in vertebrate evolution for both T cell receptors and classical Igs; and (3) although C region segments of light chains of lower vertebrates are homologous to their mammalian counterparts, the degree of conservation of C region structure in phylogeny is apparently less than that for V regions. Essentially, phylogenetic trees can be built using C regions but not V regions. We have identified a molecule of approximate mass of 26 kDa in the hemolymph of the tunicate, Boltenia ovipera, that is serologically cross-reactive with shark heavy chain and with J region peptides.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1989

Solid-phase antigen binding by purified immunoproteins from antigen-specific monoclonal T cell hybridomas.

We have developed two distinct solid-phase immunoassays for the detection of antigen binding activity by products of antigen binding T cell hybridomas in the absence of MHC. Two suppressor T cell hybridomas studied (34s-18 and 34s-704) are specific for keyhole limpet hemocyanin, a protein antigen, and the other suppressor T cell hybridoma (51H7D) binds specifically to the arsonate hapten. We have adapted these hybridomas to growth in serum-free medium and have isolated molecules with antigen binding activity both from the cell membranes and from the culture fluid in which the cells had been grown. The antigen binding molecules (ABM) produced by the KLH-specific hybridomas bound best to native hemocyanin; binding was decreased when KLH was denatured by reduction and alkylation and no binding was found to an arthropod (Limulus) hemocyanin. The arsonate binding hybridoma, on the other hand, produced molecules specific for this hapten; they showed no capacity to bind KLH. The antigen binding molecules affinity-purified from all three T hybridomas have intact masses of either 145,000, 67,000 or 48,000 when run in SDS-PAGE under non-reducing conditions. Following reduction, ABM resolve in SDS-PAGE into a complex of polypeptide chains having apparent masses of 65,000, 56,000 and 49,000, with either a pair of bands at 26,000 and 22,000, or with a single band at 32,000, which is consistent with the size of translation products of mRNA previously isolated from these hybridomas. Two of the hybridomas, 34s-18 and 34s-704, used for isolation of antigen binding products in this study, were previously reported to lack detectable rearranged gamma or beta genes and therefore to lack expression of the alpha/beta or gamma/delta heterodimers. The antigen binding molecules react in solid-phase immunoassay with some antibodies specific for variable (first framework) region and joining (J) region peptide sequences predicted from T cell receptor gene sequence. Furthermore, the affinity-purified antigen binding molecules from mouse T cell hybridomas cross-react in ELISA with goat anti-rabbit IgG and not with protein G, thus allowing the use of these commercially available reagents in standard laboratory assays. Interestingly, ABM anchored in intact cell membranes, which could be shown to specifically bind antigen, did not cross-react with goat anti-rabbit IgG, indicating that the cross-reactive moiety is not detectable when the ABM are in this situation.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1989

A monoclonal antigen-binding T cell immunoprotein: antigenic relatedness to T cell receptor beta chain FR1 V and J peptide segments: physicochemical distinctiveness from classical immunoglobulins and T cell receptor heterodimers.

The monoclonal murine T cell hybridoma, 51H7D, was previously shown to bind the arsazobenzene hapten and to produce a soluble antigen-binding molecule. In this paper we characterize this antigen-binding immunoprotein for its relationship to known T cell receptors serologically, using antibodies specific for variable region framework, or joining region peptides predicted from gene sequence and by biochemical means. The 51H7D cell expresses a protein with subunit size of approximately 31,000, that reacts antigenically with affinity-purified antibodies directed against synthetic first framework and joining segment peptides, corresponding to the gene sequence of the T cell receptor beta chain, YT35. This molecule does not react with affinity-purified antibodies directed against murine immunoglobulin, framework 1 sequences of alpha and gamma T cell receptors, or with antibodies against synthetic heavy chain joining segments. The subunit of mol. wt. 31,000 can form higher aggregates, notably in the mol. wt range of 60,000-70,000, depending upon extraction conditions. The soluble form of the antigen-binding molecule bears the J beta cross-reactive determinant and occurs predominantly as a charge restricted molecular species of approximate mol. wt 60,000-70,000. The purified molecule has a blocked N-terminus, but quantitative statistical analysis of its amino acid composition indicates a closer relatedness to T cell receptor beta chains and other antigen-binding T cell products, than it has to alpha, gamma or delta TCR chains. No evidence for more than one type of polypeptide chain was found and the polymerization is not dependent upon the formation of disulfide bonds. These studies raise the possibility that antigen-binding soluble T cell molecules might belong to a new family of immunoproteins, that is related to, but distinct from, classical immunoglobulins and alpha beta or gamma delta heterodimers.

Amino Acids↗
PubMed1989

Characterization of interleukin-1 activity in tunicates.

1. Eight North American species of tunicates were examined for the presence of interleukin-1 (IL-1) like activity. 2. The tunicates studied produce molecules with readily detectable lymphocyte activation factor (LAF) activity. 3. G50 column chromatography separated molecular species that were directly mitogenic for thymocytes (mol. wt greater than 50,000) from those that were comitogenic in an IL-1 assay (mol. wt 20,000). 4. Tunicate fractions with LAF activity induced increased vascular permeability in rabbit skin. 5. Tunicate LAF activity was neutralized by polyclonal anti-human IL-1 antisera. 6. These data further support the conclusion that IL-1 is an ancient and functionally conserved molecule.

Animals↗
PubMed1989

Evolution of immunoglobulin light chains: cDNA clones specifying sandbar shark constant regions.

Sharks are living fossils that are indistinguishable morphologically from their Devonian ancestors of approximately equal to 400 million years ago. If parallel conservatism characterizes their biochemical evolution, characterization of their immunoglobulin chains could provide information regarding the primordial features of these essential defense molecules. Shark immunoglobulins are polydisperse like those of mammals, but these species lack homogeneous myeloma proteins. This heterogeneity has precluded direct determination of the sequence of elasmobranch light-chain proteins. We have sequenced four cDNA clones that contain the constant-region sequence as well as varying degrees of variable- or joining-region segments. The sandbar shark (Carcharhinus plumbeus) has at least four distinct light-chain constant regions, and these can be considered homologs of mammalian lambda chains. Approximately 40% identity was found in comparison from sharks to mammals. Certain stretches of sequence were remarkably conserved, whereas others varied in a manner consistent with accepted concepts of speciation. One hexapeptide (Ala-Thr-Leu-Val-Cys-Leu) occurred in lambda constant regions of all vertebrate species. There was a universal conservation of certain cysteines, phenylalanines, tryptophans, and glycines and strong identities in the block of residues from Ser-176 to Trp-186. Comparison of the shark sequence with that of the characterized human lambda myeloma protein Mcg indicates a strong conservation of three-dimensional structure in this light-chain domain representing species whose ancestors diverged early in vertebrate evolution. The shark light-chain sequence contains primordial features shared by mammalian kappa and lambda chains and by T-cell receptor beta chains.

Amino Acid Sequence↗
PubMed1989

Evolution of variable and constant domains and joining segments of rearranging immunoglobulins.

The rearranging immunoglobulins (Igs) are a family of recognition and defense proteins found in all vertebrate classes. These proteins consist of two types of polypeptide chains; each of these contains a variable (V) domain, a joining (J) segment, and a constant (C) region, which can itself consist of one to four domains. The distinction between light and heavy chains is an ancient one phylogenetically that is reflected in the structures of V, J, and C regions. Despite the early emergence of these genetic elements, conservatism is apparent in the peptide structures encoded by V, J, and C exons. C regions of heavy chains did not evolve as single units; rather the individual domains show their own clustering patterns, which apparently are independent of heavy-chain designation or species. C-region domains of light chains and the T cell receptor beta chain are similar to one another and to the most carboxyl-terminal domain of heavy chains. Comparison of the light chains of sharks, bullfrogs, chickens, and mammals indicated that a phylogenetic distinction can be made between kappa and lambda light chains. V and J segments of the rearranging T cell receptors alpha, gamma, and delta are homologous to the corresponding segments of Igs, but their C regions form a group that is markedly distinct from those of conventional Igs and Tcr beta.

Amino Acid Sequence↗
PubMed1989

Detection of human and marmoset immunoglobulin heavy chain by a polyclonal antiserum to a marmoset immunoglobulin-related T cell product.

We show that a polyclonal rabbit antiserum raised against a purified monoclonal T cell component from a lower primate (cotton-topped marmoset) reacts by immunoblot transfer (Western Blot analysis) with serum immunoglobulin of man and marmoset. The antigenic component had an approximate mass of 68 kilodaltons and was isolated by immune-affinity chromatography from culture fluid in which the marmoset T cell leukemia 70-N2 had been grown. The reaction with human serum immunoglobulin is with a subset of the IgG molecules and is localized to the gamma heavy chain. The reaction with marmoset serum immunoglobulin is predominantly directed against the heavy chain, but slight reactivity is also noted against the light chains. These results substantiate reports of serological cross-reactions between immunoglobulin-like T cell receptors and classical immunoglobulins and illustrate the similarity between immunoglobulins of man and those of a distantly related New World primate.

Animals↗
PubMed1988

Immunoglobulin G in multiple sclerosis brain.

We extracted free and bound IgG from plaques and normal-appearing white matter of multiple sclerosis (MS) brains. By isoelectric focusing (IEF), three patterns of IgG distribution were seen: (A) a restricted high-pI distribution with a specific band at low pH, (B) a restricted high-pI pattern, and (C) a broad pI pattern similar to that of the unbound IgG extracted at neutral pH. In one MS brain, we compared the IEF pattern of plaque material with that of normal-appearing white matter (NAWM); the low-pH extract of plaque material (PM) had a restricted pattern at high pI. In another MS brain, a specific band of bound IgG was found. These data suggest that MS lesions expose an antigen(s) unique to MS. B cells consequently might be stimulated by a disease-related antigen(s) in the MS lesion.

Brain↗
PubMed1988

Partial characterization of immunoglobulin light chains of carcharhine sharks: evidence for phylogenetic conservation of variable region and divergence of constant region structure.

Isolated light chains of IgM-type immunoglobulins of carcharhine sharks were analyzed by serological and biochemical means. When analyzed by isoelectric focusing analysis, light chains of the tiger shark (Galecerdo cuvieri), the galapagos shark (Carcharhinus galapagensis) and the sandbar shark (Carcharhinus plumbeus) showed a broad, but patterned, spectrum of bands ranging from pI 5.0 to 7.7 in which discrete families were observed. Serologically, light chains of the galapagos shark cross-reacted with rabbit antibodies against mouse immunoglobulin and a synthetic peptide corresponding to the J segment of T cell receptor beta chain. The latter cross-reaction is shared among light chains and T cell receptors. Although there was considerable heterogeneity in isoelectric focusing analysis, the light chains were homogeneous on the basis of apparent mass (23 kDa) and those of tiger shark and galapagos shark had relatively homogeneous dominant N-terminal sequences representing the first framework. The N-terminal sequences of these two shark light chains, were strongly homologous to one another and showed 75% identity to certain V kappa sequences of man and dog. Homology was also shown to V lambda sequences, but the degree of identity was approximately 50%. Following cleavage of the tiger shark light chain with o-iodosobenzoic acid which cleaves at tryptophanyl residues, a constant region peptide was isolated by gel filtration. It was possible to identify the homolog of this peptide within the constant regions of mammalian kappa and lambda chain, but the relationship to C kappa chain was stronger. The degree of identity among the corresponding C region peptides of mammalian, avian and elasmobranch species was much less than that observed for the framework 1 sequence of the light chain variable region. These data support the concept that variable and J region sequence have been conserved in the evolution of placoderm-derived vertebrates, but that constant regions show much greater phylogenetic variation.

Amino Acid Sequence↗
PubMed1988

Immunoglobulin epitopes defined by synthetic peptides corresponding to joining region sequence: conservation of determinants and dependence upon the presence of an arginyl or lysyl residue for cross-reaction between light chains and T-cell receptor chains.

Joining or J region sequences of rearranging immunoglobulins and T-cell receptors show considerable sequence homology, particularly in their C-terminal portion corresponding to the fourth framework region of immunoglobulin variable regions. In order to test the question of whether serological cross-reactions between immunoglobulin variable regions and T-cell receptors were due to antigenic similarities in their J regions, we synthesized synthetic peptides corresponding to immunoglobulin J regions and to J regions predicted from gene sequence of the T-cell receptor beta chain. We found that antibodies produced against a synthetic 16-mer J beta sequence reacted with T-cell receptor chains and also with immunoglobulin light chains. The cross-reactivity was dependent upon the J signature sequence FG()GT(R or K)L where the presence of a positively charged lysyl or arginyl residue was essential for cross-reactivity. We were able to classify J region determinants into two distinct antigenic sets; one corresponding to JH and the other corresponding to J kappa, J lambda, J beta and J alpha. Although considerable homology occurs between JH and JL (or J beta) sequences, little cross-reactivity was observed between these two J subsets. Antibodies raised against polyclonal murine IgG immunoglobulins contained antibody subpopulations specifically reactive with either JH or J beta peptides. The serological data derived here using antipeptide antibodies are consistent with computer modeling studies that indicate that the conformations of T-cell receptor variable regions resemble those of classical immunoglobulins. Our data comparing cross-reactivities restricted to the J region indicate that the expression of the J region by intact T-cell receptor beta chains is probably more similar to that of light chains than it is to the corresponding region of heavy chains.

Animals↗
PubMed1988

The occurrence of a peripheral T-cell lymphoma in a chronically immunosuppressed renal transplant patient.

A 32-year-old man received a cadavaric renal transplant in 1975 for end-stage renal disease and, thereafter, was treated with azathioprine and methylprednisolone for chronic immunosuppression. In 1985, he presented with fever and pancytopenia that persisted despite withdrawal of the immunosuppressive agents. Lymph node and liver biopsies demonstrated malignant lymphoma within the sinuses of the node and the sinusoids of the liver. A splenectomy was performed for persistent pancytopenia, and the spleen demonstrated malignant lymphoma of the diffuse mixed large and small cell type exclusively within the cords of the red pulp. The immunophenotype of the tumor cells was obtained by frozen section immunoperoxidase staining with monoclonal antibodies and flow cytometric analysis. The tumor cells were positive for the Pan T cell markers CD3 and CD2, but were negative for the subset markers CD4 and CD8. A DNA hybridization study conducted on the splenic tissue conclusively identified the clonal nature of the malignant T cells by demonstrating rearrangement of the T cell receptor beta gene. In spite of multiple chemotherapeutic regimens, the patient developed increasing peripheral blood involvement and died with disseminated lymphoma. This case appears to be unique in that it is the first report of a chronically immunosuppressed transplant recipient to develop a malignant lymphoma of the mature T cell type, and several of the pathologic features of the tumor have not been observed previously.

Adult↗
PubMed1987

Conservation among vertebrate immunoglobulin chains detected by antibodies to a synthetic joining segment peptide.

We used affinity purified antibodies produced against a synthetic peptide sequence corresponding to the entire J beta of a human T cell receptor gene to screen sera of man, mouse and other vertebrates to determine the presence of cross-reactive molecules. Little evidence for free alpha/beta heterodimers was found, but the antibody reacted with light chains of many vertebrate species, including characterized myeloma proteins of man and mouse. Some vertebrate orders, notably Aves, lacked polypeptide chains cross-reactive with J beta, but detectable determinants occurred in primitive vertebrates such as the galapagos shark (Carcharhinus galapagensis). In addition to the strong cross-reaction with purified light chains, human heavy chains reacted weakly with the antibody. The cross-reaction correlated with the sequence of the denatured immunoglobulins and was inhibitable with free peptide. These results establish the similarity of T cell receptor beta chains to immunoglobulin chains and support the conclusion that J region sequences were conserved, not only within mammalian immunoglobulins and T cell receptors, but in vertebrate evolution.

Amino Acid Sequence↗