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P Carmi

Publications and source records attributed to P Carmi.

At least 19 recordsLinked to original sources
PubMed2001

Autoantibodies to pancreatic hsp60 precede the development of glucose intolerance in patients with cystic fibrosis.

Persons expressing the genetic disease cystic fibrosis (CF) suffer from a high risk of developing impaired glucose tolerance and diabetes. The development of diabetes in CF has been attributed, in the past, to the destruction of pancreatic islets and their resident beta-cells secondary to the destruction of the surrounding tissue by mechanical clogging of the pancreatic exocrine ducts. However, the discovery that autoimmunity to the 60-kDa heat shock protein (hsp60) may cause type I diabetes in NOD mice raises the possibility that hsp60 autoimmunity may be involved in CF diabetes too; could the hyperimmunization to bacterial hsp60 characteristic of CF spread to self-hsp60 and hence to autoimmune diabetes? We now report that rising levels of IgG autoantibodies to hsp60 do indeed precede the appearance of glucose intolerance and diabetes in CF patients. We produced a recombinant human pancreatic hsp60 protein and investigated the IgG antibody response to hsp60 in prediabetic and non-diabetic patients with CF. To detect hsp60 autoantibodies in the presence of high levels of antibodies to bacterial hsp60, we absorbed test sera with the 60-kDa GroEL of Pseudomonas aeruginosa and used an immunostaining technique. Using this technique, 32 prediabetic CF patients were evaluated over a five-year period, three years, on the average, before the onset of glucose intolerance. We found that a significant increase in hsp60 autoantibody preceded impaired glucose tolerance (P=0.042, n=17), diabetes (P=0.011, n=15) and glucose intolerance (P=0.005, n=32). As has been observed in NOD mice and in type I diabetic patients, the hsp60 autoantibodies decline at the outbreak of glucose intolerance in the CF patients. The association of CF diabetes with the rise and fall of hsp60 autoimmunity suggests that the pathogenesis of the diabetes may not be merely mechanical, but arise in the wake of bacterial hyperimmunisation.

Adolescent↗
PubMed2000

Vaccination with empty plasmid DNA or CpG oligonucleotide inhibits diabetes in nonobese diabetic mice: modulation of spontaneous 60-kDa heat shock protein autoimmunity.

Nonobese diabetic (NOD) mice develop insulitis and diabetes through a process involving autoimmunity to the 60-kDa heat shock protein (HSP60). Treatment of NOD mice with HSP60 or with peptides derived from HSP60 inhibits this diabetogenic process. We now report that NOD diabetes can be inhibited by vaccination with a DNA construct encoding human HSP60, with the pcDNA3 empty vector, or with an oligonucleotide containing the CpG motif. Prevention of diabetes was associated with a decrease in the degree of insulitis and with down-regulation of spontaneous proliferative T cell responses to HSP60 and its peptide p277. Moreover, both the pcDNA3 vector and the CpG oligonucleotide induced specific Abs, primarily of the IgG2b isotype, to HSP60 and p277, and not to other islet Ags (glutamic acid decarboxylase or insulin) or to an unrelated recombinant Ag expressed in bacteria (GST). The IgG2b isotype of the specific Abs together with the decrease in T cell proliferative responses indicate a shift of the autoimmune process to a Th2 type in treated mice. These results suggest that immunostimulation by bacterial DNA motifs can modulate spontaneous HSP60 autoimmunity and inhibit NOD diabetes.

Adjuvants, Immunologic↗
PubMed1999

The 60-kDa heat shock protein modulates allograft rejection.

Allograft rejection is a process of immune reactivity triggered by foreign transplantation antigens. We now demonstrate that the 60-kDa heat shock protein (hsp60), a molecule that is identical in the donor and the recipient, can regulate allograft immunity. In wild-type mice, hsp60 expression was greatly enhanced in allografts being rejected. By using MHC class II (Ealpha) promoter hsp60 transgenic mice either as donors of skin with enhanced expression of hsp60, or as allograft recipients with decreased hsp60 autoimmunity, we found that augmented expression of mouse hsp60 in the allograft accelerated its rejection, whereas reduced autoimmunity to mouse hsp60 in graft recipients delayed the process. Moreover, in nontransgenic mice, therapeutic administration of hsp60 or hsp60 peptides, known to modulate naturally occurring hsp60 autoimmunity, led to delayed allograft rejection. Thus, we demonstrate that hsp60 expression and hsp60 autoimmunity can influence and modify the immune response to foreign antigens. Hence, autoimmunity to self-hsp60 epitopes is not necessarily an aberration, but may serve physiologically and therapeutically to modulate foreign immunity.

Animals↗
PubMed1998

Idiotypic immunization induces immunity to mutated p53 and tumor rejection.

The p53 molecule might serve as a common tumor-associated antigen, as the tumor suppressor gene p53 is mutated and the p53 protein is often over-expressed in tumor cells. We report that effective immunity to p53 can be induced through an idiotypic network by immunization of mice with a monoclonal antibody (PAb-240) specific for mutated p53, or with a peptide derived from the complementarity determining region (CDR) 3 of the variable domain of the light chain (VL) of this antibody. The immunized mice produced IgG antibodies to p53 and mounted a cytotoxic reaction to a tumor line bearing mutated p53. The idiotypically immunized mice were resistant to challenge with the tumor cells. Thus antibodies to p53 might serve as immunogens for activating resistance to some tumors. At the basic level, these findings indicate that a network of p53 immunity may be organized naturally within the immune system.

Animals↗
PubMed1997

SA-1, a nuclear protein encoded by one member of a novel gene family: molecular cloning and detection in hemopoietic organs.

We report the molecular cloning of a novel gene family. The first member of this family was cloned from a mouse lambda gt11 cDNA library using the B92 monoclonal antibody (mAb) raised against stromal cell extracts. This was followed by RACE-PCR using mRNA from the stromal cell line. A 4 kb cDNA was obtained encoding a unique protein sequence of 1258 aa, that we designate stromal antigen (SA)-1. The human SA-1 gene was cloned by homology from a thymus cDNA library and the sequence of the predicted protein was found to be highly homologous to the murine SA-1 (>98.9%). Another cDNA was cloned and the deduced protein (SA-2) was 71% homologous to SA-1. Northern blot and PCR analysis indicated that on the mRNA level the SA-1 gene is expressed in all tissues analyzed and probably encodes a single transcript. The identification of SA-1 protein in tissues and cells required combined immunoprecipitation and Western blotting using a polyclonal antiserum raised against a predicted peptide of SA-1 and the B92 mAb. Using this assay we identified a protein of about 120 kDa in hemopoietic organs. Subcellular fractionation indicated that SA-1 is a nuclear protein. Thus, despite the ubiquitous expression on the mRNA level, the protein was predominantly detected in hemopoietic organs and may therefore be controlled on a post-transcriptional level. The SA-1 gene described in this study is highly conserved between mouse and man. This implies a crucial function for this protein.

Amino Acid Sequence↗
PubMed1997

Hsp60 peptide therapy of NOD mouse diabetes induces a Th2 cytokine burst and downregulates autoimmunity to various beta-cell antigens.

A peptide of the human 60-kDa heat-shock protein (hsp60), designated p277, was found to be useful as a therapeutic agent to arrest the autoimmune process responsible for diabetes in nonobese diabetic (NOD) mice. The effectiveness of peptide treatment was associated with the induction of peptide-specific antibodies of the IgG1 but not of the IgG2a isotype, suggesting the possibility that a Th2-type response may have been induced. We now report that the effectiveness of p277 treatment is associated with the transient activation of anti-p277 splenic T-cells that produce the Th2 cytokines interleukin-4 (IL-4) and IL-10. The Th2 response to p277 was associated with reduced Th1-type autoimmunity to hsp60 and to two other target antigens associated with diabetes: GAD and insulin. The Th2 shift appeared to be relatively specific; spontaneous T-cell reactivity to a bacterial antigen peptide remained in the Th1 mode in the p277-treated mice. Moreover, treatment with the bacterial peptide did not induce a change in cytokine profile, and it did not affect progression of the disease. Thus, effective peptide treatment of the diabetogenic process associated with the induction of antibodies may be explained by selective and transient activation of Th2 autoimmune reactivity.

Animals↗
PubMed1995

The plasmacytoma growth inhibitor restrictin-P is an antagonist of interleukin 6 and interleukin 11. Identification as a stroma-derived activin A.

A stromal protein, designated restrictin-P, that specifically kills plasma-like cells was purified to homogeneity and shown to be identical with activin A. The specificity to plasma-like cells stemmed from the ability of restrictin-P/activin A to competitively antagonize the proliferation-inducing effects of interleukin (IL) 6 and IL-11. Restrictin-P further interfered with the IL-6-induced secretion of acute phase proteins by HepG2 human hepatoma cells and with the IL-6-mediated differentiation of M1 myeloblasts. A competition binding assay indicated that restrictin-P did not interfere with the binding of IL-6 to its receptor on plasma-like cells, suggesting that it may act by intervening in the signal transduction pathway of the growth factor. Indeed, concomitant addition of restrictin-P and IL-6 to cytokine-deprived B9 hybridoma cells was followed by sustained overexpression of junB gene until cell death occurred, while IL-6 alone caused a transient increase only. This altered response to IL-6 stimulation was accompanied by a moderate increase in STAT protein activation. Thus, in this study, we identified the plasmacytoma growth inhibitor, restrictin-P, as being activin A of stromal origin. It is shown that activin A is an antagonist of IL-6-induced functions and that it modifies the IL-6 signaling pattern.

Activins↗
PubMed1993

Structure-function relationship of a recombinant human galactoside-binding protein.

A galactoside-binding lectin (hL-31) containing a collagen-like sequence was identified in human tumor cells. It was found to be the homologue of the IgE-binding protein, the macrophage cell-surface Mac-2 antigen, and the murine CBP35, RL-29, and mL-34 lectins. Here we report on the expression in Escherichia coli and functional analysis of recombinant hL-31 (rhL-31). The rhL-31 was purified in one step through an asialofetuin affinity column. The rhL-31 was reactive to anti-lectin antibodies and retained its lactose-dependent hemagglutination of trypsin-treated glutaraldehyde-fixed rabbit erythrocytes. The rhL-31 elutes from an affinity column as a 31-kDa monomer and undergoes homodimerization at relatively high protein concentrations, comparable to those used to mediate hemagglutination. Electron microscopy showed that the rhL-31 appears as a Y-shaped structure. Lactoperoxidase-catalyzed iodination of murine tumor cell-surface proteins followed by collagenase treatment revealed that the lectin is probably a peripheral membrane protein whereby both the amino and the carboxy termini are exposed on the outer cell membrane. These results point to the membrane disposition and orientation of the lectin and suggest a mechanism for a structure-function relationship of lectin activity.

Amino Acid Sequence↗
PubMed1991

Purification of human tumor cell autocrine motility factor and molecular cloning of its receptor.

Tumor autocrine motility factor (AMF) has been detected in and purified from serum-free conditioned medium of human HT-1080 fibrosarcoma cells. Under nonreducing conditions, AMF migrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a single band of 55 kDa but under reducing conditions as a band of 64 kDa. Two-dimensional polyacrylamide gel electrophoresis of the purified AMF resolved two groups of polypeptides with isoelectric points of 6.1 and 6.2 (majors), 6.35 and 6.4 (minors). Purified AMF stimulated HT-1080 cell migration in a dose-dependent fashion. The motility stimulation of the fibrosarcoma cells with AMF is associated with the phosphorylation of the AMF receptor, a 78-kDa cell surface glycoprotein (gp78), suggesting protein kinase participation in migratory signal transduction. The gene encoding gp78 was cloned from an HT-1080 fibrosarcoma complementary DNA library. The deduced sequence encodes a polypeptide of 323 amino acids. The nucleotide and predicted amino acid sequence of the gp78 reveals significant homology with the human suppressor/oncogene p53 protein.

Amino Acid Sequence↗
PubMed1991

Molecular cloning and chromosomal mapping of a human galactoside-binding protein.

A human galactoside-binding protein with an Mr of 31,000 was cloned from the human HT-1080 fibrosarcoma complementary DNA library. A partial complementary DNA clone containing the complete coding region was characterized and the deduced sequence encodes a polypeptide of 242 amino acids with the characteristics of a carbohydrate-binding protein. The gene coding for the human galactoside-binding protein was mapped to the chromosomal band 1p13. The deduced amino acid sequence of the human galactoside-binding protein revealed 95 residues at the amino terminus, homologous to the predicted amino acid sequence of the second exon of the human L-myc gene.

Amino Acid Sequence↗
PubMed1990

Evidence for the role of 34-kDa galactoside-binding lectin in transformation and metastasis.

The endogenous Mr 34,000 galactoside-binding lectin (L-34) is found at elevated levels in a wide variety of neoplastic cells and correlative evidence suggests that it is involved in tumor metastasis in vivo and in transformation in vitro. We demonstrate here that introduction of recombinant L-34 into tumorigenic, weakly metastatic UV-2237-cl-15 fibrosarcoma cells results in an increased incidence of experimental lung metastases in syngeneic and nude mice. Transfection of normal BALB/c-A31 cloned fibroblasts with functional L-34 results in acquisition of anchorage-independent growth and in morphological transformation in vitro but not in tumorigenicity in vivo. These results provide direct evidence that the cellular expression of L-34 is associated with some aspects of transformation and with metastasis, but not with tumorigenicity per se.

Animals↗
PubMed1989

Identification of the metastasis-associated, galactoside-binding lectin as a chimeric gene product with homology to an IgE-binding protein.

We report the complete primary and secondary structures of a metastasis-associated Mr 34,000 galactoside-binding lectin. The polypeptide sequence (264 amino acids) was derived from the nucleotide sequence of three overlapping complementary DNA clones isolated from lambda gt11 and lambda gt10 phage libraries of UV-induced murine fibrosarcomas. Striking features of the polypeptide sequence are two distinct regions of beta-sheet and globular structures at the amino and carboxy terminals, respectively. Homology search suggests that the polypeptide is a chimeric gene product formed by fusion of the 5'-end of an Mr approximately 14,000 galactoside-binding lectin with an internal domain of the collagen alpha gene. Enzymatic treatment with collagenase confirmed the presence of a collagen-like structure in the polypeptide. Unexpectedly, the entire sequence is greater than 85% homologous to a rat low affinity IgE-binding protein.

Amino Acid Sequence↗
PubMed1988

Expression of two different endogenous galactoside-binding lectins sharing sequence homology.

The endogenous beta-D-galactoside-binding lectins of UV-2237-IP3 fibrosarcoma cells consist of two polypeptides with molecular weights of 14,500 (L-14.5) and 34,000 (L-34). Recently we constructed complementary DNA (cDNA) libraries from these cells in the expression vector lambda gt11 and isolated and characterized cDNA clones encoding the two lectin species (A. Raz et al., Exp. Cell Res., 173: 109, 1987). We report here on the similarity in structure and gene number in genomic DNA of the fibrosarcoma cells and syngeneic normal mouse lung DNA. The expression of mRNAs hybridizing to these cDNAs was evaluated in three pairs of normal versus their transformed cell variants. Messenger RNA corresponding to the Mr 34,000 lectin was present more abundantly in the transformed cells than in their normal counterparts, while no difference was detected between the cell pairs with respect to mRNA levels corresponding to the Mr 14,500 lectin. We have established the nucleotide sequences of the two cDNA clones. The deduced amino acid sequence of L-14.5 lectin cDNA clone (L-3) is significantly homologous to the sequence of six other L-14 vertebrate galactoside binding lectins. The sequence of the Mr 34,000 lectin cDNA clone (M5) revealed that it shares a stretch of 39 amino acids with all of the L-14 galactoside binding lectins, irrespective of origin of the species, suggesting both that they are all derived from a common ancestral gene, and that this domain is necessary for similar sugar binding properties.

Amino Acid Sequence↗
PubMed1987

Cloning and expression of cDNA for two endogenous UV-2237 fibrosarcoma lectin genes.

cDNA libraries from the murine UV-2237-IP3 fibrosarcoma cell line were prepared in the expression vector lambda gt11. Using specific antibodies, cDNA clones coding for the 14.5-kDa and the 34.0-kDa endogenous galactoside-specific lectins were isolated. In vitro translation and immunoprecipitation of mRNAs hybrid-selected by the cloned sequences from whole cell RNA provided confirmation for the clones. RNA and DNA blot analyses revealed that the two lectin proteins represent products of two different genes. We found differential levels of the specific mRNAs when low- and high-metastatic counterparts of the UV-2237 tumor system were compared, and these genes were not expressed in detectable amounts in normal mouse liver.

Animals↗
PubMed1985

Expression of ki-ras oncogene in tumor cell variants exhibiting different metastatic capabilities.

The expression of oncogenes in well-characterized B16 melanoma and UV-2237 fibrosarcoma cell variants that exhibit distinct metastatic properties was explored. The search for the expression of 11 different oncogenes revealed that the major oncogene in those two tumor systems is the Kirsten-ras (ki-ras). The results indicate that the amounts of specific ki-ras mRNA and the p21 protein are similar in both low- and high-metastatic counterparts. These results suggest that in these systems there is no apparent direct correlation between the amount and expression of the major cellular oncogene so far identified and the metastatic potential of these tumor cells.

Animals↗
PubMed1984

Monoclonal antibodies to endogenous galactose-specific tumor cell lectins.

A monoclonal antibody, 5D7, was obtained after immunization of syngeneic mice with B16 melanoma cell extracts enriched for endogenous lectin activity and screening for inhibition of lectin-mediated hemagglutination. Binding of this antibody to affinity-purified B16 melanoma galactoside-specific lectin was revealed by solid-phase radioimmunoassay and binding to the surface of viable B16 cells was demonstrated by indirect immunofluorescence. Inhibition of lectin activity and cell surface labeling by 5D7 antibody were also found with several types of cultured human and murine cells including melanoma, sarcoma and carcinoma. This monoclonal antibody should be useful for evaluating the role of tumor cell surface lectins in intercellular interactions and metastasis.

Animals↗