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Biomedical subjects

A L Notkins

Publications and source records attributed to A L Notkins.

At least 37 records · Page 2Linked to original sources
PubMed1998

cDNA encoding a single-chain antibody to HIV p17 with cytoplasmic or nuclear retention signals inhibits HIV-1 replication.

HIV-1 gag p17 protein is an attractive target for molecular intervention, because it is involved in the viral replication cycle at both the pre- and postintegration levels. In the present experiments, we targeted p17 by intracellularly expressing a cDNA encoding an Ab to p17. cDNA from a hybridoma-secreting Ab to p17 was cloned, sequenced, reconstructed as a single-chain Ab fragment (scFv), and expressed in the cytoplasm or nucleus with appropriate retention signals. The expressed scFvs had no effect on T cell growth or CD4 expression and bound specifically to HIV-1 p17. Human CD4+ Jurkat T cells that expressed scFvs and were infected with HIV-1 showed a marked reduction in virus replication compared with cells expressing vector alone. The inhibition of virus replication was more pronounced when scFvs were expressed in the cytoplasm rather than the nucleus. From these studies, we conclude that the intracellular expression of a single-chain Ab to p17 inhibits HIV replication; in addition, the degree of inhibition is related to the intracellular targeting site.

Amino Acid Sequence↗
PubMed1998

Cells transfected with a non-neutralizing antibody gene are resistant to HIV infection: targeting the endoplasmic reticulum and trans-Golgi network.

Plasmids containing single chain Fv (scFv) non-neutralizing human anti-HIV-1 gp41 Ab cDNA, with or without endoplasmic reticulum (ER) or trans-Golgi network (TGN) retention signals, were constructed. Stable transfectants expressing these scFvs then were generated from COS-7 cells and HIV-1-susceptible CD4+ human T cells (Jurkat). scFv without a retention signal was secreted from cells, whereas scFv with an ER or TGN retention signal remained primarily within targeted intracellular compartments. The expression of scFv, scFv-ER, and scFv-TGN did not adversely affect the appearance of uninfected cells, as measured by growth rate or CD4 expression. Pulse-chase experiments revealed that the t(1/2) of scFv-ER and scFv-TGN within cells was greater than 24 h and less than 9 h, respectively. The scFv-ER and scFv-TGN bound HIV gp160, and the scFv-ER-gp160 and the scFv-TGN-gp160 complexes were stable within HIV-infected transfectants. Further studies revealed that the maturation processing of gp160 into gp120 and gp41 was blocked in the scFv-ER transfectants, but not in the scFv-TGN transfectants. Moreover, HIV replication, as measured by p24, was inhibited by up to 99% in cells transfected with scFv-ER or scFv-TGN, but was not inhibited in cells transfected with the secretory form of scFv. It is concluded that the targeting of non-neutralizing anti-HIV-1 Abs to specific intracellular compartments blocks HIV replication and represents a potential therapeutic strategy for protecting uninfected lymphopoietic stem cells from HIV-1-infected patients.

Animals↗
PubMed1998

IA-2 and IA-2beta: the immune response in IDDM.

Pancreatic islet cell autoantigens associated with insulin-dependent diabetes mellitus (IDDM) include a recently identified family of protein tyrosine phosphatase-like molecules, notably IA-2 and IA-2beta. IA-2 is a 979 amino acid transmembrane protein located on human chromosome 2q35, whereas IA-2beta is 986 amino acids long located on human chromosome 7q36. Comparison of human IA-2 and IA-2beta showed 74% identity within the intercellular domains, but only 27% indentify within the extracellular domains. These IA-2 molecules are expressed predominantly in cells of neuroendocrine origin, particularly pancreatic islets and brain. Radioimmunoprecipitation with recombinant IA-2 and IA-2beta has been used to measure autoantibodies to these molecules and their intracellular fragments. Autoantibodies to IA-2 are detected in the majority (60% to 80%) of newly diagnosed IDDM patients and in less than 2% of controls. The major antigenic determinants of both IA-2 and IA-2beta reside within the C-terminus of their intracellular domains. In first-degree relatives of IDDM patients, the presence of autoantibodies to IA-2 is predictive of IDDM and in combination with autoantibodies to glutamic acid decarboxylase (GAD) the positive predictive value is in the 50% range. The role of IA-2 and IA-2beta in the pathogenesis of IDDM is still unclear. Identification of these antigens has extended our ability to predict the disease and may be valuable in the search for antigen-specific therapies to prevent IDDM.

Animals↗
PubMed1998

Polyreactive antigen-binding B cells are the predominant cell type in the newborn B cell repertoire.

Polyreactive antibodies bind to a variety of different self and non-self antigens. The B cells that make these antibodies express the polyreactive lg receptor on their surface. To determine the frequency of polyreactive antigen-binding B cells in peripheral blood, we incubated two different antigens, one (insulin) labeled with fluorescein isothiocyanate and the other (beta-galactosidase) with phycoerythrin, with peripheral B cells. The percentage of cells that bound these antigens was determined with the fluorescence-activated cells sorter. Approximately 21% of adult B cells bound insulin, 28% bound beta-galactosidase, and 11% bound both antigens. In contrast to B cells in the adult repertoire, 49% of B cells in cord blood bound insulin, 54% bound beta-galactosidase, and 33% bound both antigens. The properties of polyreactive antigen-binding B cells in adult and cord blood were similar, except for the fact that almost all the polyreactive antigen-binding B cells in cord blood were CD5 positive (93%), whereas only 40% of the polyreactive antigen-binding B cells in adult peripheral blood were CD5 positive, indicating that the CD5 marker is not directly linked to polyreactivity. The percentage of polyreactive antigen-binding B cells in patients with Sjögren's syndrome, systemic lupus erythematosus and rheumatoid arthritis was equal to or slightly below that found in the normal adult B cell repertoire. It is concluded that polyreactive antigen-binding B cells are a major constituent of the normal adult B cell repertoire and are the predominant cell type in the newborn B cell repertoire.

Adolescent↗
PubMed1998

Immunological heterogeneity in type I diabetes: presence of distinct autoantibody patterns in patients with acute onset and slowly progressive disease.

Type I diabetes mellitus may represent a heterogeneous disorder with a distinct pathogenesis in patients with young and adult onset of the disease. To investigate whether serological markers directed to different autoantigens have the potential to distinguish acute onset from slowly progressive Type I diabetes we analysed antibodies to tyrosine phosphatases IA-2/ICA512 (IA-2A) and IA-2beta/phogrin (IA2betaA), antibodies to GAD65 (GADA) and cytoplasmic islet cell antibodies (ICA) in a non-selected group of diabetic patients clinically classified as having Type I or Type II diabetes at diagnosis. Both IA-2A and IA-2betaBA were found to be positively associated with onset before the age of 20 years and the presentation of classical features of Type I diabetes. In Type I diabetes 56 % (112/200) of patients were positive for IA-2A and 38 % (76/200) for IA-2betaA. In contrast, only 1 of 785 (0.1 %) patients with Type II diabetes had IA-2A and all of them were negative for IA-2betaA (p < 0.001). Among the patients with Type II diabetes 7.6% (n = 60) were ICA positive and 2.8% (n = 22) had GADA suggesting the presence of slowly progressive Type I diabetes. GADA were found in 8 of 60 (13.3 %) ICA positive subjects which was lower than the percentage detected in patients with acute onset of diabetes (115/157 73.2%) (p < 0.001). Blocking of double antibody positive sera showed that only 3 of 8 (37.5 %) patients with slowly progressive diabetes had ICA restricted to GAD or IA-2 whereas ICA were completely inhibited in 12 of 20 (60.0 %) patients with Type I diabetes. Among 193 patients with Type II diabetes available for follow-up, 35 % of ICA positives, 58 % of GADA positives and 60 % of those positive for both markers required insulin by 3 years. However, using strict criteria for the switch to insulin treatment the corresponding sensitivity of each marker was only low (9%, 10% and 5%). We show that clinical subtypes of Type I diabetes are associated with distinct humoral autoimmunity. IA-2A and GADA were associated with classical features of Type I diabetes whereas GADA and an uncharacterized ICA subspecificity indicate slowly progressive disease.

Acute Disease↗
PubMed1998

Expression, characterization, processing and immunogenicity of an insulin-dependent diabetes mellitus autoantigen, IA-2, in Sf-9 cells.

Autoantibodies to a 64-kD protein and a 40-kD tryptic fragment from pancreatic islets have been detected at high frequency in the sera of patients with insulin-dependent diabetes mellitus (IDDM). IA-2, a newly isolated transmembrane protein tyrosine phosphatase, is a major islet cell autoantigen in IDDM and the precursor of a 40-kD tryptic fragment. To express large quantities of recombinant IA-2 protein and analyse post-translational modifications we expressed full-length human IA-2 in baculovirus-infected Sf-9 cells. IA-2 expression was analysed by Western blot and by immunoprecipitation of 35S-methionine-radiolabelled proteins with rabbit antisera or IDDM sera. A 120-kD IA-2 protein was detected during the early, but not the late, phase of the infection. Pulse-chase experiments showed that the 120-kD protein was processed into fragments of 64 kD and smaller fragments of approximately 50 kD, 38 kD and 32 kD. The 64-kD fragment appeared as a doublet. Tunicamycin and PNGase F treatment down-shifted the 120-kD protein and the 64-kD doublet into lower molecular weight bands, suggesting that both were glycosylated. Trypsin treatment converted the 120-kD protein and the 64-kD doublet into a 40-kD fragment. Baculovirus-expressed IA-2 was as sensitive or slightly more sensitive than in vitro translated IA-2 in detecting autoantibodies to IA-2: 66% of sera from newly diagnosed IDDM patients reacted with baculovirus-expressed IA-2 compared with 59% of the same sera which reacted with in vitro translated IA-2. It is concluded that baculovirus-expressed IA-2 is a good source of autoantigen and that a number of lower molecular weight fragments with which IDDM autoantibodies react are derived from the 120-kD full-length IA-2 molecule.

Animals↗
PubMed1998

Profile and differential expression of protein tyrosine phosphatases in mouse pancreatic islet tumor cell lines.

Protein tyrosine phosphatases (PTPs) play important roles in cell growth and differentiation of normal and tumor cells. In this study, we analyzed the PTP profile in two pancreatic islet tumor cell lines. Transcripts were isolated from alphaTC-1 (glucagon-secreting) and betaTC-1 (insulin-secreting) cell lines for templates. A pair of degenerative primers, based on the conserved regions of known PTPs, was used to amplify the transcripts by polymerase chain reaction. A total of 1,620 clones was examined by restriction enzyme analysis and cDNA sequencing. Twenty-one PTPs were identified, including nine cytosolic PTPs (TcPTP, P19PTP, PTP1B, PTPMEG, PTP1C, SYP, PTPH1, PTPL1, and PTPD1), nine transmembrane PTPs (PTPdelta, PTPgamma, PTPkappa, DEP-1, IA-2, LAR, PTPalpha, PTPNE3, and PTPepsilon), and three new PTPs--PTPmu-like PTPkappa-like, and IA-2beta. An RNase protection assay demonstrated that some of these PTPs were expressed predominantly in glucagonoma (i.e., PTPdelta and IA-2) and others in insulinoma (i.e., PTP1C, PTPkappa, and PTPNE3) cells. In this report, we present the first profile of PTPs in alpha and beta tumor cell lines.

Amino Acid Sequence↗
PubMed1998

Molecular cloning and characterization of a highly basic protein, IA-4, expressed in pancreatic islets and brain.

A substraction library was constructed from mouse insulinoma (betaTC-1) and glucagonoma (alphaTC-1) cell lines. Differential screening and sequencing revealed a novel cDNA clone, IA-4, which was expressed in the islets of Langerhans and the brain. IA-4 cDNA is 1,007 bp in length and predicts a protein of 187 amino acids with a molecular mass of 19,940 D. Examination of the amino acid sequence showed a high content of arginine (18.7%), proline (14.4%), alanine (16.0%), leucine (13.4%) and glycine (9.6%). The deduced pI value is 12.5 indicating a highly basic protein. Northern blot analysis revealed a 1-kb mRNA highly expressed in brain, trigeminal ganglia and cell lines of neuroendocrine origin. Rabbit polyclonal antiserum raised against a synthetic IA-4 peptide, designated Pep-1, not only reacted with IA-4 recombinant protein, but also immunostained the islets of Langerhans and large neurons of the hippocampus, cerebral cortex, spinal cord, dorsal ganglia and Purkinje cells of the cerebellum. The high expression of IA-4 protein in neuroendocrine cells and its unique amino acid sequence suggest that IA-4 may have an important, but still undetermined, function in these special cell types.

Amino Acid Sequence↗
PubMed1998

The relationship between humoral and cellular immunity to IA-2 in IDDM.

Autoantibodies to the neuroendocrine protein insulinoma-associated protein 2 (IA-2), a member of the tyrosine phosphatase family, have been observed in individuals with or at increased risk for IDDM. Because this disease is thought to result from a T-cell-mediated autoimmune destruction of the insulin-producing pancreatic beta-cells, we analyzed humoral and cellular immune reactivity to this autoantigen to further define its role in the pathogenesis of IDDM. Peripheral blood mononuclear cells (PBMC) from individuals with newly diagnosed IDDM or at varying levels of risk for the disease were stimulated in vitro with the entire 42-kDa internal domain of IA-2 (amino acids 603-979), a series of control antigens (glutathionine-S-transferase, tetanus toxoid, Candida albicans, mumps, bovine serum albumin), and a mitogen (phytohemagglutinin). The frequency and mean stimulation index of PBMC proliferation against IA-2 was significantly higher in newly diagnosed IDDM subjects (14 of 33 [42%]; 3.8+/-4.5 at 10 microg/ml) and autoantibody-positive relatives at increased risk for IDDM (6 of 9 [66%]; 3.9+/-3.2) compared with autoantibody-negative relatives (1 of 15 [7%]; 1.8+/-1.0) or healthy control subjects (1 of 12 [8%]; 1.5+/-1.0). The frequencies of cellular immune reactivities to all other antigens were remarkably similar between each subject group. Sera from 58% of the newly diagnosed IDDM patients tested were IA-2 autoantibody positive. Despite investigations suggesting an inverse association between humoral and cellular immune reactivities against islet-cell-associated autoantigens, no such relationship was observed (rs=0.18, P=0.39) with respect to IA-2. These studies support the autoantigenic nature of IA-2 in IDDM and suggest the inclusion of cellular immune responses as an adjunct marker for the disease.

Adolescent↗
PubMed1998

Determination of encephalomyocarditis viral diabetogenicity by a putative binding site of the viral capsid protein.

The molecular mechanism by which some, but not all, variants of encephalomyocarditis (EMC) virus selectively infect pancreatic beta-cells in mice and induce IDDM has been an enigma for more than a decade. We report here that the binding site of the EMC viral capsid protein VP1 determines viral diabetogenicity. Recombinant chimeric EMC viruses containing threonine, serine, proline, aspartic acid, or valine at position 152 of the major capsid protein VP1 bind poorly to beta-cells. In contrast, recombinant chimeric EMC viruses containing alanine or glycine at position 152 of the VP1 bind efficiently to and infect beta-cells, resulting in the development of diabetes. Three-dimensional molecular modeling reveals that the van der Waals interactions are greater and the residues surrounding position 152 of the VP1 are more closely packed in recombinant chimeric viruses containing threonine, serine, proline, aspartic acid, or valine at position 152 than in recombinant chimeric viruses containing alanine or glycine at the same position. Our studies reveal that the surface areas surrounding alanine or glycine at position 152 of the VP1 are more accessible, thus increasing the availability of the binding sites for attachment to beta-cell receptors and resulting in viral infection and the development of diabetes.

Animals↗
PubMed1997

Autoantibodies to IA-2 and IA-2 beta in insulin-dependent diabetes mellitus recognize conformational epitopes: location of the 37- and 40-kDa fragments determined.

IA-2 and IA-2 beta are major autoantigens in insulin-dependent diabetes mellitus (IDDM) and the precursors, respectively, of a 40-and 37-kDa tryptic fragment that reacts with IDDM sera. In the present study, by amino acid sequencing of recombinant IA-2 and IA-2 beta, we determined the tryptic cleavage sites involved in the generation of these fragments. Both cleavage sites are immediately after an arginine residue at position 653 for IA-2 and position 679 for IA-2 beta. The resulting tryptic fragments are 326 and 307 amino acids in length and retain their ability to react with IDDM sera. In contrast to IA-2 and IA-2 beta, other members of the protein tyrosine phosphatase (PTP) family (i.e., RPTP kappa, RPTPmu, NU-3, SHP, and 3CH134) are completely susceptible to digestion by trypsin. Sequence analysis revealed five conserved cysteine residues in IA-2 and IA-2 beta that are not present in other PTPs. Reduction and alkylation of IA-2 and IA-2 beta recombinant proteins resulted in loss of both resistance to digestion by trypsin and reactivity with autoantibodies in IDDM sera. It is concluded that disulfide bond formation plays a critical role in the maintenance of antigenic structure and that the autoantibodies to IA-2/IA-2 beta in IDDM sera recognize conformational epitopes.

Amino Acid Sequence↗
PubMed1997

Molecular characterization of the promoter region of a neuroendocrine tumor marker, IA-1.

IA-1 is an intronless gene, which encodes a 510 amino acid protein with a zinc-finger DNA-binding motif that is expressed in tumors of neuroendocrine origin. The 5'-upstream region of the IA-1 gene was recently sequenced. In this paper, the regulatory elements and the promoter region of the 5'-upstream region were analyzed by use of a series of deletion mutants (ranging from +26 bp to -2090 bp upstream of the IA-1 gene), which were tested in a pituitary tumor cell line, AtT-20, and Hela cells by transient transfection assays. These experiments showed that a 506 base pair upstream sequence was sufficient for maximal expression of a reporter gene. Multiple known regulatory elements were found within this region including three E boxes and a clustered Sp-1 site. In addition, Southwestern blot analysis, using a radiolabeled promoter sequence (extending from -108 bp to -66 bp) and nuclear extracts from both neuroendocrine and non-neuroendocrine cell lines, revealed four promoter binding proteins designated PBP1, PBP2, PBP3 and PBP4 with molecular weights of 55 kD, 32 kD, 29 kD, and 27/28 kD, respectively. These studies suggest that several different regulatory elements in the 5'-upstream region of the IA-1 gene and at least four different nuclear proteins may be involved in the cell-specific expression of IA-1.

Binding Sites↗
PubMed1997

Human chorionic gonadotropin hormone prevents wasting syndrome and death in HIV-1 transgenic mice.

At birth, transgenic mice, homozygous for the HIV-1 provirus pNL4-3, deleted in gag/pol, are normal in appearance and weight. Within several days after birth, the pups develop a syndrome characterized by dry, scaly, hyperkeratotic skin, growth failure, and death. The possibility that the homozygous embryos are being protected during gestation by a maternal factor led us to treat the newborn animals with various pregnancy-related hormones including human chorionic gonadotropin (hCG), estrogen, progesterone, and dexamethasone. Treatment with hCG prevented death, led to normal growth, and markedly reduced skin lesions. In contrast to the skin of the untreated homozygous pups, which expressed high levels of HIV mRNA and proteins (i.e., gp120 and Nef), the skin of the hCG-treated pups showed a marked reduction in both HIV mRNA and proteins. Discontinuation of hCG resulted in the reappearance of HIV transcripts and proteins, skin lesions, and growth failure resulting in death. In addition, HIV transcripts and proteins were reduced significantly in heterozygous mothers during pregnancy, but reappeared after parturition. Similarly, hCG treatment resulted in a decrease of HIV proteins in the skin of nonpregnant heterozygous transgenic mice. These findings suggest that the inhibiting effect of hCG on HIV expression may be clinically useful in the treatment of HIV infections, and may be responsible, during pregnancy, for the low transmission of HIV from infected mothers to their offspring.

Acquired Immunodeficiency Syndrome↗
PubMed1997

Immunization therapies in the prevention of diabetes.

Insulin-dependent diabetes (IDD), being an autoimmune disease, offers several opportunities for immunological interventions that may result either in the reduction of disease severity or in delaying diabetes onset. Among the various experimental preventative approaches, parenteral immunization with islet-specific autoantigens appears to be practically simpler and promising. We have previously shown that immunization with insulin, insulin B chain and B chain epitope (p9-23), but not insulin A chain, in incomplete Freund's adjuvant (IFA) and in alum (with B chain) delayed/prevented diabetes onset in NOD mice. Here we demonstrate the protective efficacy of affinity purified GAD65 in IFA. While both insulin B chain and GAD65 significantly delayed the onset of diabetes (P=0.001), a recently described tyrosine phosphatase (IA-2) antigen did not (P=0.38). Interestingly, B chain immunization reduced the incidence of cyclophosphamide (CY)-accelerated diabetes by about 50-55%. We also provide further evidence that B chain, upon increased adsorption to alum, could improve on its protective capacity in NOD mice.

Animals↗
PubMed1997

Comparison of IA-2 with IA-2beta and with six other members of the protein tyrosine phosphatase family: recognition of antigenic determinants by IDDM sera.

To study the expression of protein tyrosine phosphatases (PTPs) in pancreatic islets, a cDNA library from islet cells was constructed and analysed. Twenty-one different PTPs were found to be expressed in islet cells, including three previously unknown PTPs. One of these, IA-2beta, was cloned, sequenced, and found to be related to IA-2, a major autoantigen in insulin-dependent diabetes mellitus (IDDM). The intracellular and extracellular domains of IA-2beta were 74 and 27% identical, respectively, to the intracellular and extracellular domains of IA-2. Approximately 70 and 45% of sera from patients with IDDM had autoantibodies that immunoprecipitated recombinant IA-2 and IA-2beta, respectively. By use of deletion mutants, we were able to show that the autoantibodies reacted with the intracellular, and not the extracellular, domains of IA-2 and IA-2beta, and that the major antigenic determinants resided within the COOH-terminus of the intracellular domains. Further studies revealed that approximately 97% of the IDDM sera that reacted with IA-2beta also reacted with IA-2, whereas only 50% of IDDM sera that reacted with IA-2 also reacted with IA-2beta. In contrast to the reactivity of IDDM sera with the IA-2 and IA-2beta, IDDM sera did not react with six other members of the PTP family. It is concluded that many members of the PTP family are expressed in pancreatic islets, but thus far only IA-2 and IA-2beta appear to be recognized as autoantigens by IDDM sera.

Amino Acid Sequence↗
PubMed1997

EBV binds to lymphocytes of transgenic mice that express the human CR2 gene.

Epstein Barr virus (EBV) is unable to bind to or infect normal mouse lymphocytes. A construct containing the human complement receptor type 2 (CR2) gene, the receptor for EBV, was placed under the control of the IgH/c-fos enhancer/promoter and microinjected into single cell embryos. A total of five transgenic mouse lines were established and four expressed hCR2 mRNA. Flow cytometry and immunostaining revealed that approximately 15-30% of the lymphocytes from the thymus, spleen and lymph nodes expressed hCR2 protein on their surface and bound EBV. Despite this binding, less than 1% of the cells showed evidence that the virus was internalized or replicated. Transgenic mouse lymphocytes, expressing hCR2, could not be immortalized with EBV. It is concluded that the simple expression of hCR2 receptor on mouse lymphocytes is not sufficient for efficient infection.

Animals↗