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A L Notkins

Publications and source records attributed to A L Notkins.

At least 19 recordsLinked to original sources
PubMed1996

In vitro effects of anti-HIV immunotoxins directed against multiple epitopes on HIV type 1 envelope glycoprotein 160.

We have used a panel of anti-gp160 MAbs to construct anti-HIV immunotoxins by coupling antibodies to ricin A chain (RAC). The ability of the immunotoxins to kill HIV-1-infected cells and halt the spread of infection was tested in tissue culture on persistently and acutely infected cell lines and primary lymphocyte cultures stimulated with phytohemagglutinin (PHA blasts). Laboratory strains and clinical isolates of HIV both were tested. The constitution and antigen-binding capacity of the immunotoxins were confirmed by ELISA and indirect immunofluorescence. Immunotoxins that bind epitopes exposed on the cell surface effectively killed persistently infected cells, although killing was not directly proportional to binding of immunotoxin to cell. The activity of anti-gp41, but not anti-gp120, immunotoxins was markedly enhanced in the presence of soluble CD4 or peptides corresponding to the CDR3 region of CD4. CD4-mediated enhancement of anti-gp41 immunotoxin activity was observed for laboratory strains neutralized by sCD4 and for clinical isolates that were resistant to neutralization by sCD4. Immunotoxin action was potentiated by brefeldin A, bafilomycin A1, cortisone, and an amphipathic fusion peptide, but not by cytochalasin D, nocodazol, monodansyl cadaverine, or trans-retinoic acid. Anti-HIV immunotoxins are useful tool with which to study the functional expression of gp120/gp41 antigens on the surface of HIV-infected cells, as well as potential AIDS therapeutics. Because these studies relate to the accessibility of viral antigens to antibody-mediated attack, these studies also have relevance for vaccine development.

Anti-Bacterial Agents↗
PubMed1996

IA-2, a transmembrane protein of the protein tyrosine phosphatase family, is a major autoantigen in insulin-dependent diabetes mellitus.

IA-2 is a 105,847 Da transmembrane protein that belongs to the protein tyrosine phosphatase family. Immunoperoxidase staining with antibody raised against IA-2 showed that this protein is expressed in human pancreatic islet cells. In this study, we expressed the full-length cDNA clone of IA-2 in a rabbit reticulocyte transcription/translation system and used the recombinant radiolabeled IA-2 protein to detect autoantibodies by immunoprecipitation. Coded sera (100) were tested: 50 from patients with newly diagnosed insulin-dependent diabetes mellitus (IDDM) and 50 from age-matched normal controls. Sixty-six percent of the sera from patients, but none of the sera from controls, reacted with IA-2. The same diabetic sera tested for autoantibodies to islet cells (ICA) by indirect immunofluorescence and glutamic acid decarboxylase (GAD65Ab) by depletion ELISA showed 68% and 52% positivity, respectively. Up to 86% of the IDDM patients had autoantibodies to IA-2 and/or GAD65. Moreover, greater than 90% (14 of 15) of the ICA-positive but GAD65Ab-negative sera had autoantibodies to IA-2. Absorption experiments showed that the immunofluorescence reactivity of ICA-positive sera was greatly reduced by prior incubation with recombinant IA-2 or GAD65 when the respective antibody was present. A little over one-half (9 of 16) of the IDDM sera that were negative for ICA were found to be positive for autoantibodies to IA-2 and/or GAD65, arguing that the immunofluorescence test for ICA is less sensitive than the recombinant tests for autoantibodies to IA-2 and GAD65. It is concluded that IA-2 is a major islet cell autoantigen in IDDM, and, together with GAD65, is responsible for much of the reactivity of ICA with pancreatic islets. Tests for the detection of autoantibodies to recombinant IA-2 and GAD65 may eventually replace ICA immunofluorescence for IDDM population screening.

Animals↗
PubMed1996

IA-2, a transmembrane protein tyrosine phosphatase, is expressed in human lung cancer cell lines with neuroendocrine phenotype.

IA-2 is a transmembrane protein tyrosine phosphatase isolated recently from a human insulinoma subtraction library. Its expression in normal human tissues is restricted primarily to the pancreatic islets and brain. In this report, we describe the expression of IA-2 mRNA in a panel consisting of 20 lung tumor cell lines with neuroendocrine and non-neuroendocrine phenotype and 17 non-lung tumor cell lines. IA-2 mRNA was detected in 8 of 11 neuroendocrine small cell lung carcinomas, 4 of 4 non-small cell lung carcinomas with neuroendocrine phenotype, and 11 of 12 non-lung neuroendocrine tumor cell lines. In contrast, IA-2 mRNA was not detected in five non-neuroendocrine lung carcinomas, nor in a panel of other non-neuroendocrine tumor cell lines. The expression pattern of IA-2 mRNA suggests that IA-2 may represent a new marker for neuroendocrine differentiation In human lung cancer cells and perhaps other neuroendocrine tumors.

Autoantigens↗
PubMed1996

Identification of a second transmembrane protein tyrosine phosphatase, IA-2beta, as an autoantigen in insulin-dependent diabetes mellitus: precursor of the 37-kDa tryptic fragment.

A novel cDNA, IA-2beta, was isolated from a mouse neonatal brain library. The predicted protein sequence revealed an extracellular domain, a transmembrane region, and an intracellular domain. The intracellular domain is 376 amino acids long and 74% identical to the intracellular domain of IA-2, a major autoantigen in insulin-dependent diabetes mellitus (IDDM). A partial sequence of the extracellular domain of IA-2beta indicates that it differs substantially (only 26% identical) from that of IA-2. Both molecules are expressed in islets and brain tissue. Forty-six percent (23 of 50) of the IDDM sera but none of the sera from normal controls (0 of 50) immunoprecipitated the intracellular domain of IA-2beta. Competitive inhibition experiments showed that IDDM sera have autoantibodies that recognize both common and distinct determinants on IA-2 and IA-2beta. Many IDDM sera are known to immunoprecipitate 37-kDa and 40-kDa tryptic fragments from islet cells, but the identity of the precursor protein(s) has remained elusive. The current study shows that treatment of recombinant IA-2beta and IA-2 with trypsin yields a 37-kDa fragment and a 40-kDa fragment, respectively, and that these fragments can be immunoprecipitated with diabetic sera. Absorption of diabetic sera with unlabeled recombinant IA-2 or IA-2beta, prior to incubation with radiolabeled 37-kDa and 40-kDa tryptic fragments derived from insulinoma or glucagonoma cells, blocks the immunoprecipitation of both of these radiolabeled tryptic fragments. We conclude that IA-2beta and IA-2 are the precursors of the 37-kDa and 40-kDa islet cell autoantigens, respectively, and that both IA-2 and IA-2beta are major autoantigens in IDDM.

Amino Acid Sequence↗
PubMed1996

Transgenic models of human immunodeficiency virus type-1.

Transgenic models have provided significant insights into HIV-1 pathogenesis, particularly with regards to Kaposi's sarcoma. HIV-associated nephropathy, the tissue-restricted expression of CNS strains of HIV-1, and the function of Nef in vivo. Both multigenic and single gene constructs have contributed to our understanding of HIV-1-induced diseases. While failing to provide models suitable for vaccine development, these transgenic models have provided great insight into HIV pathogenesis and may yet provide a means for the development and testing of molecular based therapies for AIDS.

Animals↗
PubMed1996

Characterization and chromosomal localization of a new protein disulfide isomerase, PDIp, highly expressed in human pancreas.

Protein disulfide isomerase (PDI) catalyzes protein folding and thiol-disulfide interchange reactions. The enzyme is localized in the lumen of endoplasmic reticulum (ER) and is abundant in secretory cells of various tissues. In this study we describe the isolation and characterization from human pancreas of a new protein, PDIp, that is structurally and functionally related to PDIs. PDIp cDNA is 1,659 bp in length and predicts a protein with an open reading frame of 511 amino acids. PDIp amino acid sequence shows 46% identity and 66% similarity to that of human PDI. PDIp possesses two thioredoxin-like active sites (WCGHCQ and WCTHCK) and an endoplasmic reticulum retention signal sequence, KEEL, at the carboxyl terminus. Northern analysis of normal human tissues and various human tumor cell lines revealed PDIp mRNA (2.0 kb) expression only in the normal pancreas. Recombinant PDIp protein catalyzed reductive cleavage of insulin and renaturation of reduced RNaseA. Somatic cell genetics and fluorescence in situ hybridization localized the PDIp gene to the short arm of human chromosome 16. It is concluded that PDIp is a new member of the PDI family and is highly expressed in human pancreas.

Amino Acid Sequence↗
PubMed1995

Antigen-binding B cells and polyreactive antibodies.

The present experiments were initiated to see if cells capable of binding antigens could make polyreactive antibodies. Fluorescein isothiocyanate-labeled self and non-self antigens were incubated with B cells from normal individuals. Antigen-binding cells were separated from non-antigen-binding cells by flow cytometry, immortalized with Epstein-Barr virus and analyzed at the clonal level for their capacity to make polyreactive antibodies. Four to six times more cells making polyreactive antibodies were found in the B cell subset that bound antigens than in the B cell subset that did not bind antigens. The majority of the polyreactive antibodies were of the immunoglobulin (Ig)M isotype. Immunoflow cytometry revealed that cell lines making polyreactive antibodies bound a variety of antigens (e.g., insulin, IgGFc and beta-galactosidase), whereas cell lines making monoreactive antibodies bound only a single antigen. The binding of antigens to B cell lines that made polyreactive antibodies could be inhibited (range, 28%-57%) by both homogeneous and heterogeneous antigens. Both CD5+ and CD5- antigen-binding B cells made polyreactive antibodies, but the frequency was slightly higher in the CD5+ antigen-binding (85%) as compared to the CD5- antigen-binding (50%) population. Comparison of CD5+ B cells that bound antigens with CD5+ B cells that did not bind antigens showed that approximately 86% of the former, but only 15% of the latter, made polyreactive antibodies. It is concluded that cells capable of binding a variety of different antigens can make polyreactive antibodies and that antigen binding is a good marker for identifying polyreactive antibody-producing cells.

Antibody Formation↗
PubMed1995

Two-phase approach for the expression of high-affinity human anti-human immunodeficiency virus immunoglobulin Fab domains in Escherichia coli.

We describe here a two-phase approach for the development of high-affinity human anti-HIV immunoglobulin Fab domains in a bacterial expression system. The first phase of this technique involves the generation of human hybridoma cell lines producing high-affinity antibodies (MAbs). Anti-HIV-1 human MAbs from peripheral blood lymphocytes (PBLs) were prepared from an HIV-1-seropositive patient and from an HIV-1-seronegative volunteer immunized with HIV-1 rgp160. One MAb (T15G1), derived from the blood of the seropositive donor, was specific for HIV-1 gp41, recognized gp41 on the surface of HIV-1-infected cells and bound this antigen with an apparent dissociation constant of 4 x 10(-10) M. A second MAb (M7B5), developed from the immunized volunteer, was specific for HIV-1 gp120 with a dissociation constant on the order of 8 x 10(-10) M, but was unable to recognize cell surface antigen. In the second phase of this technique the Fab domains of these two MAbs were molecularly cloned into a bacterial expression vector. mRNA was isolated from the M7B5 and T15G1 hybridoma cell lines and used as a template for the production of cDNA. The cDNA was amplified using the polymerase chain reaction (PCR) technique, and then fused, in frame, into a bacterial expression vector. The recombinant Fabs (rFabM7B5 and rFabT15G1) were expressed as dicistronic messages in bacteria using the IPTG-inducible lactose promoter (LacZ). DNA sequencing was used to define the gamma chain isotypes and the VH and VL chain gene usage. The binding specificities of rFabM7B5 and rFabT15G1 were indistinguishable from their respective intact MAbs.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Transplantation of skin from human immunodeficiency virus type 1-transgenic mice to normal congenic mice results in graft rejection.

Skin from mice transgenic (Tg) for part of the human immunodeficiency virus type 1 (HIV-1) genome was transplanted onto normal mice of the same strain. All Tg grafts were rejected within 29 days. In contrast, skin from normal mice that was transplanted to HIV-1-Tg recipients remained viable for > 67 days. Histologic examination of Tg grafts on normal mice showed evidence of monocytic infiltrates. Monocytic infiltrates were not observed, however, when either normal or Tg skin was transplanted onto Tg mice. Immunohistologic staining verified the presence of gp120 protein expression in the Tg-transplanted skin but not in adjacent normal skin. It is concluded that the Tg mice are immunologically tolerant to the HIV-1 gene products they express.

Animals↗
PubMed1995

Both VH and VL chains of polyreactive IgM antibody are required for polyreactivity: expression of Fab in Escherichia coli.

Monoclonal polyreactive antibodies can bind to many structurally dissimilar self and non-self antigens. Neither the precise antigen-binding site on the polyreactive antibody molecule nor the molecular basis of polyreactivity has been elucidated. The present study was initiated to see whether antibody genes encoding the Fab fragment of a human monoclonal polyreactive IgM antibody (MoAb 67) could be efficiently expressed in Escherichia coli, and whether the bacterially expressed Fab fragments possessed biological activity. cDNA encoding the variable domains of the heavy and light chains of MoAb 67 were cloned, amplified by polymerase chain reaction (PCR) and expressed in E. coli. Neither the recombinant heavy nor light chain showed antigen-binding activity. In contrast, the recombinant Fab 67 fragment showed the same antigen-binding reactivity profile as the native IgM antibody. It is concluded that the antigen-binding activity of polyreactive antibodies resides in the Fab fragment, and that both the heavy and light chains are required for activity.

Antibodies, Anti-Idiotypic↗
PubMed1995

Maternal-fetal interactions affect growth of human immunodeficiency virus type 1 transgenic mice.

Infants vertically infected with human immunodeficiency virus type 1 (HIV-1) often manifest profoundly deficient growth with failure to thrive. The pathologic mechanisms that produce growth failure associated with pediatric HIV infection are not clear. Transgenic mice homozygous for a gag/pol deletion mutant of the infectious provirus pNL4-3 have been found to manifest a similar growth failure pattern. To explore the influence of HIV-1 on fetal growth and maternal-fetal interactions, we examined intrauterine growth of transgenic and nontransgenic mice and evaluated the consequence of embryo transfer into normal and heterozygous transgenic mothers. Mice homozygous for the HIV transgene had normal intrauterine and birth weights but uniformly displayed severe growth retardation postnatally. Transgene expression was prominent in transgenic fetuses and their placentas and in uteri of transgenic mothers, as determined by Northern analysis. Although embryo transfer did not affect intrauterine growth, the pregnancy rate in transgenic mothers was markedly lower than in nontransgenic controls. In both fetal and neonatal tissues, transgene expression was significantly greater in homozygous animals when compared with heterozygotes, but the difference was magnified postnatally. These results suggest that HIV gene expression affected both mother and neonate. In the mother, expression of the HIV-1 transgene reduced postfertilization pregnancy rate. Once the animal was pregnant, however, the effects of transgene expression on the homozygous fetus were overcome in utero, possibly by the contribution of maternal factors or by inhibition of HIV-1 gene expression by a fetal or maternal factor(s). In the neonate, HIV-1 transgene expression increased dramatically in homozygotes and was associated with profound growth failure.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Transgenic models of HIV-1.

Transgenic technology has been very successful at providing insights into possible processes involved in HIV-induced pathogenesis. The availability of these small animal models for the study of HIV-related syndromes including KS, epidermal proliferative lesions, HIV-associated nephropathy, AIDS-related growth failure and cachexia may well facilitate the development of novel therapies for these complications. Other phenotypes created in mice, such as cataracts and hepatic cancer [59], may not have human analogies but may still provide insight into pathogenesis. Thus, transgenic models have already provided resources to study many manifestations of AIDS and others are likely to be developed. The optimal strategy for designing future transgenic animals, however, is less clear. No transgenic mouse model has been generated to date that will provide an avenue for vaccine development. This advance awaits the further discovery of the host factors that facilitate the virus replicative cycle in humans and a better understanding of these pathways in the mouse. For the development of molecular-based therapy, however, the currently available models may well be adequate to test molecular inhibitors of transcription [7,60,61] and post-transcriptional processing of viral mRNA [62]. Whether single or multigenic constructs under the control of the LTR are better or worse for this purpose is a debatable issue. Transgenic technology may yet make an additional contribution to the development of molecular therapy for AIDS. The best method of demonstrating that a gene therapeutic strategy is safe to administer to patients has not been determined. By introducing potentially therapeutic constructs into mice as transgenes, their safety can be assessed in many different cell types in vivo, analogous to toxicological testing in rodents for systemically administered drugs. Thus, transgenic technology has already provided insights into the pathogenesis of HIV-1. While it has not yet proven its utility for vaccine development, transgenic technology holds the promise of being an active participant in the development of both safe and effective gene therapy approaches for the treatment of AIDS.

AIDS Dementia Complex↗
PubMed1994

Isolation, sequence and expression of a novel mouse brain cDNA, mIA-2, and its relatedness to members of the protein tyrosine phosphatase family.

This study describes the isolation of a putative transmembrane protein tyrosine phosphatase (PTP), mIA-2, from a mouse brain cDNA library. The cDNA encodes 979 amino acids containing a unique extracellular domain and a single intracellular catalytic domain. Expression of mIA-2 was found primarily in the central nervous system and in neuroendocrine cells. The sequence shares a high degree of homology with its human counterpart (92% identity), especially in the intracellular domain, which shows 99.3% identity between the two species. In both human and mouse IA-2, several substitutions were found in the highly conserved regions including an Ala to Asp substitution in the core sequence. Bacterial expression of a glutathione S-transferase fusion protein showed that mIA-2 had no enzyme activity with conventional substrates such as Raytide, myelin basic protein, angiotensin, RR-src and pNpp. When tested with the total tyrosine-phosphorylated cellular proteins isolated on an anti-phosphotyrosine antibody column, it also showed little, if any, enzyme activity. These findings suggest that mIA-2 is a new member of the transmembrane PTP family that either has very narrow substrate specificity perhaps requiring post-translational modification for enzyme activity or has a still unknown biological function.

Amino Acid Sequence↗
PubMed1994

Growth failure and AIDS-like cachexia syndrome in HIV-1 transgenic mice.

The mechanisms which predispose to growth failure in infants and children infected with immunodeficiency virus type-1 (HIV-1) are not fully understood. The contributions of viral replication and CD4+ T cell depletion to growth failure in an HIV-1 transgenic mouse model were investigated. Mice homozygous for the transgene, a gag-pol deletion mutant of the HIV-1 provirus pNL4-3, exhibited marked cachexia, growth retardation, lymphoproliferation with a reduction in the percentage of CD4+ T cells but an increase in the absolute number of splenic CD4+ and CD8+ T cells, thymic hypoplasia, and early death. Despite the absence of T cells, athymic nude mice, homozygous for the HIV transgene, displayed comparable growth failure. The results indicate that AIDS-like cachexia may be produced by expression of viral envelope or accessory genes, need not be accompanied by absolute depletion of CD4+ T cells, and may occur independent of T cell function.

Acquired Immunodeficiency Syndrome↗
PubMed1994

Genomic organization, 5'-upstream sequence, and chromosomal localization of an insulinoma-associated intronless gene, IA-1.

IA-1 is a novel cDNA originally isolated from a human insulinoma subtraction library (ISL-153). It encodes a protein containing both a zinc finger DNA-binding domain and a putative prohormone domain. IA-1 transcripts have been found thus far only in tumors of neuroendocrine origin. Clinical studies have shown that IA-1 is a sensitive marker for neuroendocrine differentiation of human lung tumors. In this study, we cloned and sequenced the entire IA-1 gene and its 5'-upstream region from a human liver genomic library. In situ hybridization localized the IA-1 gene to the short arm of human chromosome 20. Sequence analysis and restriction enzyme mapping showed that the IA-1 gene is uninterrupted and appears to be intronless. Evidence that IA-1 is an intronless gene that can translate into protein was obtained from in vitro translation studies that showed that both IA-1 cDNA and IA-1 genomic DNA yielded identical protein products of approximately 61,000 daltons. Examination of the 5'-upstream region (2090 base pairs) revealed several tissue-specific regulatory elements, including glucokinase upstream promoter elements and a Pit-1 factor binding site. The presence of several different upstream regulatory elements may account for IA-1 gene expression in different neuroendocrine tumors.

Base Sequence↗
PubMed1994

cDNA sequence and genomic organization of mouse secretin.

A murine insulinoma library was constructed by subtracting glucagonoma cDNAs from insulinoma cDNAs. Comparison of the nucleotide sequence of one of the clones with sequences from the GenBank database showed that it was a member of the secretin family. The clone, 490 bp long, encodes a protein of 133 amino acids consisting of a signal peptide, a N-terminal peptide, secretin, and a C-terminal peptide, which showed 88% and 80% homology with rat and porcine secretin precursor proteins, respectively. The translated secretin peptide showed a unique Met to Thr substitution at position 5 as compared to the secretins from other species. That the Met to Thr substitution was not tumor-related was demonstrated by the fact that an identical sequence was found in cDNA from normal mouse intestine. Studies on the mouse secretin gene revealed that it contains four exons separated by 81 bp, 110 bp, and 96 bp introns.

Amino Acid Sequence↗
PubMed1994

Modulation of the expression of CD5 antigen on the surface of human peripheral B lymphocytes.

In this study, we investigated the expression of CD5 molecules on the surface of mature human peripheral B lymphocytes. Human CD5+ B cells were isolated from tonsils and peripheral blood. Their terminal differentiation into plasma cells was induced with IL-2. In the presence of this lymphokine, a subset of CD5+ B cells ceased to express CD5 proteins on their surface. When CD5+ B cells and non-B cells were removed from the suspension. CD5 antigens were spontaneously reexpressed on the surface of CD5- B cells. This suggests that the CD5- phenotype of a subset of B cells in these suspensions resulted from pressure(s) exerted on them by the other cellular components of the suspensions. B cells which have reexpressed membrane CD5 in the absence of CD5+ B cells and non-B cells retained their ability to reprogress to CD5- phenotype and to undergo terminal differentiation into plasma cells when stimulated with IL-2. A short exposure (4 hr) of CD5- B cells to 5 nM IL-2 resulted in the loss of their capability to spontaneously reexpress surface CD5 in the absence of other lymphoid cells. Taken together, these data suggest that the expression of CD5 antigens on B cell membrane is governed by a network-type balance between all cellular compartments within the suspension. Fluctuations of this equilibrium results in the shuttling of B cells between CD5+ and CD5+ phenotypes until they become irreversibly engaged in the terminal differentiation pathway. The interconversion CD5+<==>CD5- on B cell membrane does not argue for CD5 molecule as the marker of a distinct B cell lineage.

Antigens, CD↗