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Biomedical subjects

A L Notkins

Publications and source records attributed to A L Notkins.

At least 109 records · Page 6Linked to original sources
PubMed1991

Transplantation of syngeneic transfected cells to probe the in vivo immune response to viral proteins.

BALB/3T3 cells were transfected with the glycoprotein D (gD) gene of herpes simplex virus (HSV) and a cell line expressing gD on the cell surface was isolated. In vitro, 51Cr release tests showed that the transfected cells were destroyed by anti-HSV antibody in the presence of complement. To investigate in vivo immune response, the gD-transfected cells were transplanted into the footpads of syngeneic HSV-immunized or unimmunized BALB/c mice. In unimmunized mice, transfected cells remained intact for 7 days or longer, and the site of injection showed only slight lymphocyte infiltration. In contrast, in immunized mice, transfected cells elicited massive lymphocyte infiltration and were mostly destroyed by day 4. Analysis of infiltrating cells revealed that they were mainly Thy1+ and CD8+ lymphocytes along with small numbers of CD5+, CD4+, and B lymphocytes. These studies show that transfected murine cells expressing gD can be used to study the in vivo immune response to single viral proteins and they argue that the immune response contributes to the pathogenesis of HSV infection.

Animals↗
PubMed1991

Polyreactivity and antigen specificity of human xenoreactive monoclonal and serum natural antibodies.

Naturally occurring antibodies that react with xenogeneic antigens are a clinically important subset of antibodies because they initiate hyperacute rejection of organs transplanted between disparate species. This currently precludes the use of nonprimate organs for human transplantation. Most antibodies that arise after immunization are monoreactive, i.e., bind only to the immunogen. Similarly, some "natural" antibodies, e.g., isohemagglutinins, bind in a monoreactive manner. In contrast, other natural antibodies, e.g., those that bind to actin, are polyreactive (i.e., bind to multiple ligands). Such polyreactive antibodies may be derived predominantly from CD5+ B cells. In this study, we demonstrate that the majority of xenoreactive natural antibodies in human serum are polyreactive, as indicated by the ability of ssDNA and thyroglobulin (ligands commonly used as targets of polyreactive antibodies) to block the binding of the antibodies to xenogeneic antigens, whereas these ligands could not block the binding of antitetanus antibodies to tetanus toxoid. Furthermore, we compared the ability of 8 polyreactive and 7 monoreactive human mAb to bind to porcine antigens. All of the polyreactive mAb reacted with porcine antigens at mAb concentrations less than 3 micrograms/ml, while none of the monoreactive mAb reacted at concentrations less than 3 micrograms/ml. Each polyreactive mAb reacted with partially overlapping, but distinct sets of porcine cell surface moieties. These results indicate that human polyreactive mAb can bind to multiple xenogeneic antigens in a selective manner and that xenoreactive natural antibodies in human serum are largely polyreactive.

Animals↗
PubMed1991

Transgenic mouse lines that are immunologically tolerant and nontolerant to herpes simplex virus glycoprotein D.

A construct containing the gene for glycoprotein D of herpes simplex virus (HSV-gD), under the control of the simian virus 40 early promoter, was microinjected into single-cell embryos, and four transgenic mouse lines were established. Three were homozygous (lines 75, 111, and 113) and one was hemizygous (line 108) for the HSV-gD gene. Examination of sera revealed that only one of the lines (line 75) spontaneously produced antibody to HSV-gD. Immunization of the other three lines with vaccinia virus-HSV-gD showed that one of them (line 113) responded by making antibody to HSV-gD, whereas the other two (lines 108 and 111) appeared to be immunologically tolerant. Evidence that tolerance was not absolute was obtained by immunization with infectious HSV, which resulted in an antibody response to HSV-gD in some of the animals from line 111. Examination of organs for HSV-gD mRNA revealed transcripts in the tolerant line (line 108) and in the partially tolerant line (line 111), but not in the nontolerant line (line 113), suggesting that the development of immunological tolerance requires active expression of the HSV-gD gene.

Animals↗
PubMed1990

Flow cytometry to identify cell types to which enzymes bind. Effect of lactic dehydrogenase virus on enzyme binding.

Flow cytometry was used to measure the binding of enzymes (i.e. lactate dehydrogenases 1 and 5, malate dehydrogenase, and asparaginase) to cells. Of the four enzymes studied, asparaginase showed the greatest binding. Single color analysis revealed that asparaginase bound best to preparations enriched in macrophages, and dual color analysis showed that the binding was to macrophages. Studies on continuous cell lines revealed that asparaginase bound to one mouse macrophage line, but not to another or to murine fibroblasts. Inoculation of mice with lactic dehydrogenase virus, a virus that infects macrophages, decreased the in vivo clearance of asparaginase from the circulation and the in vitro binding of asparaginase to peritoneal macrophages. It is concluded that flow cytometry can be used to study the binding of enzymes to cells, to identify the cell type to which the enzyme binds, and to measure changes in the capacity of cells to bind enzymes.

Animals↗
PubMed1990

Frequency of B cells committed to the production of antibodies to insulin in newly diagnosed patients with insulin-dependent diabetes mellitus and generation of high affinity human monoclonal IgG to insulin.

Circulating autoantibodies to insulin can be detected in patients with insulin-dependent (type I) diabetes mellitus (IDDM) at the onset of the clinical disease. To characterize the autoantibody response in IDDM patients, we determined the frequency of circulating B cells committed to the production of IgM, IgG, and IgA to insulin in 12 newly diagnosed IDDM patients and, for comparison, in 9 healthy subjects and 17 insulin-treated IDDM patients. We found that B cells committed to the production of anti-insulin IgG, but not IgM, autoantibodies are present at much higher frequency in the circulation of newly diagnosed IDDM patients before insulin treatment (0.209 +/- 0.142%, mean value +/- SD of total IgG-producing cell precursors) as compared with age-matched healthy controls (0.032 +/- 0.030% of total IgG-producing cell precursors). In IDDM patients who had been treated with insulin, cells producing IgG antibody to insulin were 0.177 +/- 0.139% of total IgG-producing cell precursors. Generation of IgG mAb from B cells of IDDM patients revealed that they were monoreactive, i.e., they bound to insulin, but to none of the other Ag tested, and displayed a high affinity for insulin (Kd approximately 10(-7) moles/liter). In contrast, the IgG mAb derived from healthy subjects were polyreactive, i.e., they bound to all Ag tested, and displayed a low to moderate affinity for insulin (Kd approximately 10(-5) to 10(-6) moles/liter). These findings show that lymphocytes committed to the production of high affinity IgG autoantibodies to insulin are common in the B cell repertoire at the onset of IDDM.

Antibodies, Monoclonal↗
PubMed1990

Characterization of the 70KDA component of the human Ku autoantigen expressed in insect cell nuclei using a recombinant baculovirus vector.

The Ku autoantigen is a human nuclear, DNA-binding heterodimer of 70kDa and 86kDa proteins. It is the target of autoantibodies in several autoimmune diseases. We now report the expression of a cDNA encoding the 70kDa Ku protein. Large amounts of protein were obtained using a recombinant baculovirus vector, in contrast with earlier unsuccessful attempts using other expression systems. We demonstrate that the 70kDa Ku protein is targeted to the nucleus and is associated with the nuclear matrix when expressed in the absence of the 86kDa Ku component. No post-translational modifications were observed. The 70kDa protein binds double and single-stranded DNA with very high affinity. Our results suggest that the baculovirus expression system may be of widespread use in the production and characterization of human autoantigens.

Animals↗
PubMed1990

Clonal analysis of a human antibody response. Quantitation of precursors of antibody-producing cells and generation and characterization of monoclonal IgM, IgG, and IgA to rabies virus.

We quantitated and characterized the changes in the human B cell repertoire, at the clonal level, before and after immunization with rabies virus. Moreover, we generated 10 monoclonal cell lines producing IgM, IgG, and IgA antibodies to the virus. We found that in healthy subjects, not previously exposed to the virus, nearly 2% of the circulating B lymphocytes were committed to the production of antibodies that bound the virus. These B cells expressed the surface CD5 molecule. The antibodies they produced were polyreactive IgM that displayed a relatively low affinity for the virus components (Kd, 1.0-2.4 x 10(-6) g/microliters). After immunization, different anti-virus (IgG and IgA) antibody-producing cells consistently appeared in the circulation and increased from less than 0.005% to greater than 10% of the total B cells committed to the production of IgG and IgA, respectively. Most of such B cells do not express CD5 and produce monoreactive antibodies of high affinity for rabies virus (Kd, 6.5 x 10(-9) to 1.2 x 10(-10) g/microliters). One of these IgG mAbs efficiently neutralized rabies virus in vitro and in vivo, as detailed elsewhere (Dietzschold, B., P. Casali, Y. Ueki, M. Gore, C. E. Rupprecht, A. L. Notkins, and H. Koprowski, manuscript submitted for publication). Hybridization experiments using probes specific for the different human V gene segment families revealed that cell precursors producing low affinity IgM binding to rabies virus utilized a restricted number of VH gene segments (i.e., only members of the VHIIIb subfamily), whereas cell precursors producing high affinity IgG and IgA to rabies virus utilized an assortment of different VH gene segments (i.e., members of the VHI, VHIII, VHIV, and VHVI families and VHIIIb subfamily). In conclusion, our studies show that EBV transformation in conjunction with limiting dilution technology and somatic cell hybridization techniques are useful methods for quantitating, at the B cell clonal level, the human antibody response to foreign Ags and for generating human mAbs of predetermined specificity and high affinity.

Antibodies, Monoclonal↗
PubMed1990

Binding and functional effects of thyroid stimulating hormone on human immune cells.

The expression and functional relevance of thyroid stimulating hormone (TSH) receptors on human immune cells were studied. Flow cytometric analysis was used to study the binding of biotinylated TSH to human peripheral blood mononuclear cells (PBMC) and various purified lymphoid populations. Our results indicate that the hormone binds well to monocytes and natural killer (NK) cells and marginally to purified tonsillar T and B lymphocytes. There was a significant increase in the binding of TSH to purified B cells that were activated in vitro with Staphylococcus aureaus Cowan. In contrast, the binding of TSH to T cells was unaltered when they were stimulated with phytohemagglutinin (PHA). While TSH increases DNA synthesis and intracellular cAMP levels of FRTL-5 rat thyroid cells, it did not have such stimulatory effects on lymphocytes. However, there was a moderate increase in Ig production by activated B lymphocytes when they were cultured in the presence of the hormone. A possible function for TSH as a link between the immune system and the thyroid is discussed.

B-Lymphocytes↗
PubMed1990

Biological characterization of human monoclonal antibodies to rabies virus.

Rabies virus antigen-specific human monoclonal antibodies (MAbs) that recognized either viral glycoprotein, ribonucleoprotein, or matrix proteins were generated. Only glycoprotein-specific MAb neutralized a variety of rabies viruses and protected laboratory rodents against lethal rabies virus infection. The determinant recognized by this MAb does not appear to reside in previously defined antigenic sites of the viral glycoprotein.

Animals↗
PubMed1990

Cell surface expression of the 70-kD component of Ku, a DNA-binding nuclear autoantigen.

The Ku complex, a heterodimer of 86- and 70-kD proteins, is a nuclear DNA-binding autoantigen. However, hydrophobicity analysis of the deduced amino acid sequence of the 70-kD protein had strongly suggested that this might also be a membrane protein. In the present study, using antibodies to synthetic peptides and a polyclonal antiserum to the purified protein, we show that the 70-kD protein of the Ku complex is present in isolated plasma membranes of human cells. By indirect immunofluorescence microscopy and fluorescein-activated cell sorting, we demonstrate that this autoantigen is exposed on the cell surface. In addition, we have identified several domains of the protein that are exposed. Our study provides one of the first demonstrations of a eukaryotic, nuclear DNA-binding protein that is also on the cell membrane. Moreover, our results might help explain how autoantibodies to the Ku autoantigen could target cells for an autoimmune attack.

Amino Acid Sequence↗
PubMed1989

High-affinity antibodies to ssDNA are produced by CD-B cells in systemic lupus erythematosus patients.

It was previously shown that human CD5+B cells are committed to the production of polyreactive "autoantibodies" that bind different self-Ag, including ssDNA, IgG Fc fragment, thyroglobulin, and insulin, as well as exogenous Ag, e.g., tetanus toxoid and other bacterial components. To define the contribution of the CD5+B cell subset to the production of antibodies to ssDNA in SLE, we quantitated, purified, and functionally characterized circulating CD5+ and CD5-B cells in patients and in healthy subjects. We found that in SLE patients circulating CD5+B cells were not increased in number, and both CD5+ and C5-B cells spontaneously secreted antibodies to ssDNA. Studies of the mAb we generated using CD5+ and CD5-B cells revealed that two types of antibody to ssDNA are produced in SLE. The first type, of the IgM class, is produced mainly by CD5+B cells and displays a moderate affinity for ssDNA (Kd, 10(-6) to 10(-7) mol/liter) and is polyreactive, not dissimilar to the autoantibodies inducible in the normal human B cell repertoire by polyclonal activation. The second type, mostly of the IgG and IgA class, is produced mainly by CD5-B cells and displays a higher affinity (Kd, 10(-8) mol/liter) for and reacts only with ssDNA. The functional features of the high affinity anti-ssDNA antibodies and the phenotype of their producing B cells mimic those characteristic of a "mature" Ag-driven response, such as that to tetanus toxoid in tetanus toxoid-vaccinated subjects.

Adult↗
PubMed1989

Nucleotide sequences of eight human natural autoantibody VH regions reveals apparent restricted use of VH families.

Eight full length cDNA were isolated from EBV transformed human PBL derived from different normal individuals. Five were derived from antibodies with the characteristics of natural polyreactive antibodies. Three were either monoreactive or bireactive. The most striking feature of the structure of these molecules was their utilization of VH families. Although three used the large VHIII family and one used the large VHI family, the other four used genes derived from two of the recently defined small human VH families VHIV and VHV. Three of the molecules represent VHIV expressed sequences and one is the first example of a VHV gene used in an antibody of defined specificity. The nucleotide sequences of some of the molecules were remarkably similar in their VH gene segments to previously described VH genes. The data suggest that natural autoantibodies may use a restricted portion of the VH repertoire, and, in addition, that some polyreactive antibodies may be germ line encoded. The implication of these findings for the origin and diversity of the human B cell repertoire is discussed.

Amino Acid Sequence↗
PubMed1989

Cloning and characterization of a cDNA that encodes a 70-kDa novel human thyroid autoantigen.

cDNA clones were isolated by screening a human thyroid carcinoma lambda gt11 library with immunoglobulins purified from serum of a patient with autoimmune Graves' disease. One clone (ML8) containing a 1.25-kilobase (kb) insert hybridized with a single 2.0-kb poly(A+) mRNA in human thyroid and lymphocytes but not in human brain, liver, kidney, or muscle. In addition, this probe also hybridized with a single 2.0-kb poly(A+) mRNA from a rat thyroid cell line (FRTL-5). An apparently full length 2,074-base pair (bp) human cDNA was obtained and sequenced. The nucleotide sequence of the 2,074-bp cDNA includes a 5'-noncoding sequence of 17 bp, a 1827-bp open reading frame, and a 222-bp 3'-noncoding sequence. The canonical polyadenylation signal AATAAA is present 18 bp upstream of the poly(A) tail. This cDNA encodes a 69,812-dalton protein with two potential N-linked glycosylation sites and at least one potential membrane spanning domain. Immunoprecipitation of the in vitro translated protein by sera from several patients with Graves' disease argues that the 69,812-dalton protein is an autoantigen.

Amino Acid Sequence↗
PubMed1989

Expression of a viral gene in insulin-producing cell lines renders them susceptible to immunological destruction.

The gene coding for the glycoprotein D of herpes simplex virus type 1 was cloned into plasmids under the transcriptional control of the SV40 promoter-enhancer or the rat insulin 1 promoter-enhancer sequences. These plasmids were transfected into rat insulinoma cells (RINm5F) and mouse NIH/3T3 cells and the expression of glycoprotein D was examined using cell surface immunofluoresence. The rat insulin 1 promoter-enhancer sequences directed efficient expression in RINm5F cells, but not in NIH/3T3 cells. In contrast, the SV40 promoter-enhancer sequences worked well in NIH/3T3 cells, but not in RINm5F cells. Expression of glycoprotein D did not interfere with insulin production by RINm5F cells. When stable cel lines expressing glycoprotein D were exposed to anti-herpes simplex virus type 1 antibodies and complement, they were destroyed. These studies provide additional evidence that specific promoter-enhancer elements are required for efficient gene expression in certain cell types and demonstrate that the expression of foreign antigens on the surface of insulin-producing cells can lead to their immunological destruction.

Animals↗
PubMed1989

Organ reactive autoantibodies from non-immunized adult BALB/c mice are polyreactive and express non-biased VH gene usage.

To examine the naturally activated autoreactive B cell repertoire, we analyzed a panel of hybridomas from unmanipulated adult BALB/c spleen cells for reactivity patterns and VH gene usage. We found a pattern of VH usage that was diverse and appeared to reflect the germline repertoire. Although all but one natural antibody hybridoma (NAb) were initially selected for organ rather than antigen binding, the majority of organ reactive IgM NAbs were polyreactive, expressing a broad range of reactivity patterns for both self and foreign antigens, that were unique for each NAb and were not indiscriminate. Our results are consistent with the hypothesis that many naturally activated adult B cells are highly polyreactive and that autoreactivity is a consequence of polyreactivity. We suggest that the population of NAbs exhibiting organ reactivity overlaps the populations of other IgM autoantibodies that have been described previously, and that these all derive from a pool of polyreactive IgM antibodies which are polyclonally activated in the early immune response. These polyreactive natural antibodies may represent a first line of defense and offer protection for the host against a variety of foreign agents.

Animals↗
PubMed1989

CD5+ B lymphocytes, polyreactive antibodies and the human B-cell repertoire.

The lock and key model of antigen-antibody reaction has traditionally been used to explain the specificity of antibodies and the need for antibody diversity. Recently it has become clear that certain antibodies are polyreactive and recognize a variety of self- and foreign antigens. It is now clear that these antibodies are made by a novel subset of B cells that bear the surface CD5 marker. Careful analysis has shown that about 20% of peripheral blood B lymphocytes in adults are CD5+ and, therefore, represent a major component of the normal human B-cell repertoire. The precise role of the antibodies produced by these cells is still not clear, but because of their polyreactivity they might function in clearing autoantigens from the circulation and/or as a rapid first line of defense against foreign invaders, such as bacteria and viruses. Sequence analysis showed that these antibodies use gene segments in germ-line configuration for their antigen-binding portion. In this article, Paolo Casali and Abner Notkins propose that polyreactive antibodies are what, for years, have been referred to as the 'natural antibodies' of serum and that under certain circumstances they may contribute to the pathogenesis of autoimmune diseases.

Antibody Specificity↗
PubMed1989

Scrapie-induced diabetes mellitus in hamsters.

Scrapie-infected hamsters had slightly elevated non-fasting plasma glucose levels, markedly abnormal glucose tolerance tests, and impaired release of insulin in response to a glucose load. Plasma cortisol levels were essentially the same in infected and uninfected animals. Histological examination of the pancreas revealed no morphological changes in infected animals with no alteration in distribution of cells secreting insulin, glucagon and somatostatin. In contrast, brains of scrapie-infected animals had the diffuse vacuolation typical of spongiform encephalopathy. These experiments suggest that scrapie-induced diabetes mellitus in hamsters may result from damage to the central nervous system.

Animals↗