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Biomedical subjects

D A Carson

Publications and source records attributed to D A Carson.

At least 217 records · Page 12Linked to original sources
PubMed1987

Synergistic inhibition of polyamine synthesis and growth by difluoromethylornithine plus methylthioadenosine in methylthioadenosine phosphorylase-deficient murine lymphoma cells.

The antiproliferative effects of the ornithine decarboxylase inhibitor alpha-difluoromethylornithine (DFMO) are limited by the inability of the compound to deplete completely cellular polyamine pools. 5'-Deoxy-5'-methylthioadenosine (MeSAdo), the purine end product of the polyamine biosynthetic pathway, is an inhibitor of spermine and spermidine synthesis. Furthermore, a substantial number of human tumors are deficient in MeSAdo phosphorylase, and cannot degrade MeSAdo. It therefore seemed possible that DFMO and MeSAdo could interact synergistically to inhibit polyamine synthesis in MeSAdo phosphorylase-deficient malignant cells. To test this hypothesis, we have analyzed the effects of DFMO, in combination with MeSAdo, on polyamine synthesis and growth in a MeSAdo phosphorylase-deficient murine lymphoma cell line (R1.1-H), and a MeSAdo resistant mutant (R1.1-H3). Cultivation of the R1.1-H3 cells in medium containing 250 microM DFMO and 500 microM MeSAdo caused profound depletion of putrescine, spermidine, and spermine, and the accumulation of both decarboxylated S-adenosylmethionine and its acetylated derivative to levels that exceeded by nearly 3-fold the total cellular content of S-adenosylmethionine. Similarly, DFMO sensitized the lymphoma cells to the growth inhibitory effects of MeSAdo. Supplementation of the medium with putrescine, spermidine, or spermine partially protected R1.1-H3 cells from the DFMO-MeSAdo drug combination. It is conceivable that MeSAdo, or related nucleosides, may potentiate the cytostatic effects of DFMO toward MeSAdo phosphorylase-deficient tumors.

Adenosine↗
PubMed1987

Pyridine nucleotide cycling and poly(ADP-ribose) synthesis in resting human lymphocytes.

NAD is a critical cofactor for the oxidation of fuel molecules. The exposure of human PBL to agents that cause DNA strand breaks to accumulate can deplete NAD pools by increasing NAD consumption for poly(ADP-ribose) formation. However, the pathways of NAD synthesis and degradation in viable PBL have not been carefully documented. The present experiments have used radioactive labeling techniques to trace the routes of NAD metabolism in resting PBL. The cells could generate NAD from either nicotinamide or nicotinic acid. PBL incubated with [14C]nicotinic acid excreted [14C]nicotinamide into the medium. Approximately 50% of a prelabeled [14C]NAD pool was metabolized during 6 to 8 hr in tissue culture. Basal NAD turnover was prolonged threefold to fourfold by 3-aminobenzamide (3-ABA), an inhibitor of poly(ADP-ribose) synthetase. Supplementation of the medium with 3-ABA also prevented the accelerated NAD degradation that ensued after exposure of PBL to deoxyadenosine plus deoxycoformycin at concentrations previously shown to cause DNA strand break accumulation. These results demonstrate that quiescent human PBL continually produce NAD and utilize the nucleotide for poly(ADP-ribose) synthesis.

Deoxyadenosines↗
PubMed1987

Substance P activation of rheumatoid synoviocytes: neural pathway in pathogenesis of arthritis.

Several clinical features are consistent with nervous system involvement in the pathogenesis of rheumatoid arthritis. The neuropeptide substance P is one possible mediator of this interaction, since it can be released into joint tissues from primary sensory nerve fibers. The potential effects of the peptide on rheumatoid synoviocytes were examined. The results show that substance P stimulates prostaglandin E2 and collagenase release from synoviocytes. Furthermore, synoviocyte proliferation was increased in the presence of the neuropeptide. Similar effects were observed with a truncated form of substance P. Synoviocytes were sensitive to very small doses of the neuropeptide (10(-9) M), and its effects were inhibited by a specific antagonist. Thus, the specific stimulation of synoviocytes by the neuropeptide substance P represents a pathway by which the nervous system might be directly involved in the pathogenesis of rheumatoid arthritis.

Arthritis, Rheumatoid↗
PubMed1987

Genetic analysis of human B cell hybridomas expressing a cross-reactive idiotype.

We have examined the genetic basis for the expression of a human cross-reactive idiotype (CRI) commonly found on monoclonal IgM rheumatoid factors. The CRI was identified with a monoclonal antibody (17.109) and has been localized previously to the kappa-variable region. By using the human lymphoblastoid cell line WI-L2-729-HF2, and mononuclear cells from several sources, a panel of hybridomas was generated that produced 17.109 CRI-positive Ig. A recently cloned human germ-line V kappa III gene, Humkv305, served as a probe to identify genes which were rearranged and expressed in 17.109 CRI-positive and -negative hybridomas. This probe, when hybridized to human genomic DNA under stringent conditions, identified only two to five germ-line bands. In 10 separate 17.109 CRI-positive hybridoma clones, an additional rearranged V kappa band was identified. The probe did not anneal to rearranged V kappa bands in hybridoma clones that produced kappa-chains lacking the CRI. RNA dot-blot studies provided evidence for expression of genes hybridizing to the Humkv305 probe. The results indicate that the 17.109 CRI is a serologic marker for a single V kappa gene, or a small family of closely related V kappa genes, which is identified by the Humkv305 probe.

B-Lymphocytes↗
PubMed1987

The glycine-alanine repeating region is the major epitope of the Epstein-Barr nuclear antigen-1 (EBNA-1).

The Epstein-Barr nuclear antigen-1 (EBNA-1) is a protein containing a large glycine-alanine repeat that has been shown to be antigenic. Antibodies to EBNA-1 can be detected by means of immunoblotting. Preincubation of antisera with purified EBNA-1 protein inhibits the binding of IgG antibodies in this system, indicating that those epitopes detected by immunoblots are also accessible on the native molecule. A number of synthetic peptides the sequences of which were derived from the glycine-alanine repeating region of EBNA-1 and from regions adjacent to it also inhibited antibody binding to EBNA-1. These showed, however, a 1000-fold variation in their inhibitory activities. Peptides containing only glycine and alanine were the most effective inhibitors. The anti-EBNA-1 antibodies did not react with several other peptides representing sequences from unrelated proteins. At saturating concentrations of peptide 85 to 100% of anti-EBNA-1, antibody binding was inhibited in all sera tested with one exception. Similar results are obtained when antibody binding is assayed by an enzyme immunosorbent assay by using partially purified EBNA-1 to coat the plates. Thus the glycine-alanine region, either through its primary structure or through conformations assumed by this region, forms the major epitope(s) of the EBNA-1 molecule.

Alanine↗
PubMed1987

Molecular basis for the cross-reactive idiotypes on human anti-IgG autoantibodies (rheumatoid factors).

High titres of anti-IgG autoantibodies (rheumatoid factors, RF) are characteristic of patients with rheumatoid arthritis, Sjögren's syndrome, and mixed cryoglobulinaemia, and may contribute to immune complex formation and tissue damage. The monoclonal RFs from cryoglobulinaemia patients frequently display cross-reactive idiotypes. The genetic basis for the cross-reactive idiotypes on RF autoantibodies has not been determined. To clarify structural and genetic relationships among RFs from unrelated subjects, a series of anti-peptide antibodies have been generated that define primary sequence-dependent idiotypes on RF heavy and light chains. Multiple monoclonal and polyclonal RFs from unrelated individuals have been probed by Western blotting with the anti-idiotypic reagents. The results show that sequences in the kappa light chain variable region represent a common structural element among RF autoantibodies. This hypothesis is confirmed by the cloning and sequencing of the conserved germline variable region gene which encodes human RF kappa chains.

Antibody Specificity↗
PubMed1987

Expression of a germline human kappa chain-associated cross-reactive idiotype after in vitro and in vivo infection with Epstein-Barr virus.

The mouse monoclonal antibody 17.109 recognizes a cross-reactive idiotype (CRI) associated with kappa IIIb light chains of human IgM-rheumatoid factor (RF) paraproteins. The 17.109 idiotypic determinant is encoded by one or a group of closely related V kappa genes. The association of the idiotype with IgM- and IgA-rheumatoid factors in certain autoimmune diseases necessitates an understanding of how human B lymphocytes can be induced to express the idiotype. To investigate the cellular expression of the 17.109 CRI, peripheral blood lymphocytes from normal donors were stimulated in vitro with Epstein-Barr virus (EBV) and pokeweed mitogen (PWM). EBV induced greater expression of IgM-associated 17.109 CRI than did PWM. The 17.109 CRI was preferentially associated with IgM rather than with IgG. In vivo EBV infection was studied in college students with infectious mononucleosis and displayed similar elevation of IgM-associated 17.109 CRI in sera obtained at presentation of clinical illness. Later, IgM levels declined while IgG-associated 17.109 CRI rose. The 17.109 idiotype was unrelated to antibodies against the Epstein-Barr virus nuclear antigen and the viral capsid antigen and was probably due to generalized activation of early B cells. These observations support the hypothesis that the 17.109 CRI is expressed by in vitro and in vivo EBV-infected cells. The 17.109 idiotype identifies a highly conserved V kappa gene product, which is expressed preferentially after EBV infection, but not exclusively with RF autoantibodies.

Animals↗
PubMed1987

High-frequency expression of a conserved kappa light-chain variable-region gene in chronic lymphocytic leukemia.

Malignant B lymphocytes from several patients with chronic lymphocytic leukemia (CLL) were examined for reactivity with murine monoclonal antibody 17.109. This antibody, prepared against the rheumatoid factor (RF) paraprotein Sie, recognizes a crossreactive idiotype on 48% of human IgM RF paraproteins, but does not react with IgM paraproteins without RF activity or substantially with normal pooled immunoglobulin. The 17.109-reactive idiotype is a marker for a kappa III variable-region gene, designated V kappa RF, that is conserved in outbred human populations. In a limited study of 31 CLL patients, the leukemic cells from 5 of 20 patients with kappa light chain-expressing CLL were recognized by the 17.109 monoclonal antibody. Despite having malignant cells specifically reactive with this antibody, patients with 17.109-positive CLL did not have elevated serum levels of circulating antibody bearing 17.109-reactive determinants. Total RNAs isolated from the CLL B lymphocytes, or from hybridomas produced by fusing the CLL cells with the WI-L2-729-HF2 cell line, were fractionated electrophoretically and examined by blot hybridization. Under stringent hybridization conditions capable of discerning a single base-pair mismatch, RNA from the 17.109-idiotype-positive CLL cells hybridized to synthetic oligonucleotide probes corresponding to framework and complementary-determining regions in the V kappa RF gene. The high frequency of the 17.109-associated idiotype and the V kappa RF gene in CLL suggests that the disease may arise from B lymphocytes that express a restricted set of inherited immunoglobulin variable-region genes with little or no somatic mutation.

B-Lymphocytes↗
PubMed1987

Rheumatoid factor and immune networks.

Rheumatoid factors represent a normal component of the immune network. The autoantibodies promote complement fixation and clearance of immune complexes. They amplify the avidity of polyclonally induced IgG. Genes related to the primary structure of rheumatoid-factor light chains are widely distributed in the human population and have been conserved during the evolution and dispersion of the species. Products of these genes may be detected with anti-idiotypic antibodies against synthetic peptides corresponding to individual hypervariable regions on rheumatoid-factor light chains. Such anti-peptide antibodies provide unique reagents for analyzing the genetics of immunoglobulins in outbred populations. Precursors of rheumatoid factor are abundant among immature B lymphocytes. Some of these cells may tend to localize to mucosal surfaces, where they are stimulated directly by pathogenic microorganisms with polyclonal B cell-activating properties. Synthesis of rheumatoid factor regularly accompanies all secondary immune responses but is usually transient. Production of the autoantibody is T-cell dependent. The T cells may recognize antigen in an IgG-antigen immune complex that is processed and presented by B-cell precursors of rheumatoid factor. Rheumatoid factor-associated light-chain idiotypes are rare in serum IgG and on IgG myeloma proteins. They are common among monoclonal IgM proteins and on the surface of the malignant B cells from patients with chronic lymphatic leukemia. The rheumatoid factors that are produced by patients with mixed cryoglobulinemia, or primary Sjogren's syndrome can share idiotypic antigens with monoclonal rheumatoid factors. Rheumatoid factor synthesis in the diseases may reflect an abnormal proliferation of B-cells that is not antigen-driven and that can degenerate into malignancy. The rheumatoid factors in patients with rheumatoid arthritis are diverse and almost certainly represent the outcome of antigen-induced, T cell-dependent mechanisms. The antigens that drive the T cells have not been identified but could represent exogenous microorganisms, self components, or idiotypic antigens that fortuitously interact with rheumatoid factors.

Autoantibodies↗
PubMed1987

Human autoantibodies to poly(adenosine diphosphate-ribose) polymerase.

The chromatin-bound enzyme poly(ADP-ribose) polymerase (ADPRP) is strongly stimulated by DNA with single- or double-stranded breaks, and transfers the ADP-ribose moiety of NAD to nuclear proteins. The activation of ADPRP is important for DNA repair and replication, and also has been postulated to play a role in the pathogenesis of lymphocyte dysfunction associated with chronic inflammatory diseases, and inborn errors of nucleoside metabolism. We have detected high titers of IgG autoantibodies to the ADPRP protein in six patients with rheumatic complaints. No other autoantibodies were detected in any of the six sera. The specificity of the anti-enzyme antibodies was established by (a) immunoprecipitation of ADPRP activity, (b) immunoprecipitation and immunoblotting of both the native 116-kD enzyme and its proteolytic digestion products. ADPRP was purified from human thymus and calf thymus. The autoantibodies reacted equivalently with both enzymes. The anti-ADPRP antibodies had a distinctive immunofluorescent pattern with HEp-2 cells, reacting intensely with nucleoli and metaphase chromosomes, and diffusely with the nucleus. Autoantibodies to ADPRP have not been described previously. The presence of a specific immune response against an enzyme that has been associated with various immunodeficiency syndromes raises intriguing possibilities concerning the relationship between DNA damage, immunodeficiency, and autoimmunity.

Autoantibodies↗
PubMed1987

The use of defined peptides in characterizing idiotypes.

The structural correlates of idiotypes have been sought in several antibody systems. The cumulative results suggest that the hypervariable regions (or complementarity-determining regions) of the heavy and light chains are the structural basis of idiotypes. However, in most cases, it is exceedingly difficult to associate a particular idiotypic determinant with a specific amino acid sequence. Recently, synthetic peptides were used to induce antibodies specific for predefined determinants in intact proteins. These findings led us and others to use synthetic peptides corresponding to the hypervariable regions/complementarity-determining regions to induce anti-idiotypes. These novel anti-idiotypic antibodies are easy to prepare, and are ideal reagents for structural and genetic studies of antibody responses.

Amino Acid Sequence↗
PubMed1987

Effects of recombinant human interferons on rheumatoid arthritis B lymphocytes activated by Epstein-Barr virus.

We evaluated the effects of all 3 classes of recombinant human interferon (IFN) on Epstein-Barr virus (EBV) infection of purified B lymphocytes from patients with rheumatoid arthritis (RA). After EBV infection, RA B cells secreted more IgM and significantly more IgM rheumatoid factor (RF) than normals. Spontaneous (no EBV) proliferation, IgM, and IgM RF were also higher in RA. All 3 types of IFN inhibited dose dependently EBV induced B cell activation. In RA, however, higher doses of each class of IFN were necessary to obtain 50% inhibition. IFN gamma was most potent in normals and RA. Four IgM RF production IFN gamma was significantly more potent than IFN alpha and IFN beta in reducing the spontaneous activation of RA B cells, and a similar trend was seen in B cell proliferation. These findings are discussed in the context of ongoing clinical trials with IFN gamma in RA.

Adult↗
PubMed1987

Rheumatoid factor.

Many human RF-associated crossreactive idiotypes (CRIs) have been defined. Since these CRIs probably are the phenotypic markers of Ig variable region genes, the data suggest that human RFs employ several Ig variable region genes which are common among outbred human populations. There are at least four types of stimuli which can trigger RF production. They are aggregated IgG in the form of antigen-antibody complexes, exogenous antigens bearing cross-reactive determinants to human IgG, autologous antibodies which mimic the Fc region of human IgG, and polyclonal B cell activators. Transient RF secretion is regularly induced by antigen-antibody complexes during secondary immune responses. In normal human subjects, RFs may be produced primarily by the B cells which bind mouse red cells, or express Leu l markers on their surface. Leu l is the human homolog of Ly l in mice. Upon polyclonal stimulation, these Ly l B cells secrete IgM autoantibodies. Combined protein and DNA sequencing studies reveal that human RFs utilize a small number of light chain variable region genes, together with several heavy chain variable regions genes. In contrast, by mRNA sequencing, murine RFs were found to employ many heavy and light chain variable region genes which belong to various gene families. Two human RF-related light chain variable region genes have been cloned and characterized. One germ-line Vk gene (designated Humkv325) is identical to four RF light chain sequences from unrelated individuals, and differs from the other eight RF light chains by one to seven amino acid residues. These results demonstrate that this RF-related Vk gene is both widely distributed and conserved among outbred human populations.

Amino Acid Sequence↗
PubMed1986

Human kappa light chain subgroup analysis with synthetic peptide-induced antisera.

All human kappa light chains belong to one of four subgroups, classified according to their amino acid sequences or by reactivity with adsorbed heteroantisera. The structural basis for the subgroup distinction by antisera is unknown. Therefore, to create anti-kappa subgroup antibodies with predefined specificity, we immunized rabbits with synthetic peptides which correspond to sequences within the first framework region of prototype kappa I, II, III, and IV light chains. The peptide-induced antisera recognized primary sequence-dependent kappa subgroup determinants. They correctly predicted the amino acid sequence in the first framework region of two kappa light chains. By Western immunoblotting and enzyme-linked immunoassay the antisera also identified previously typed, monoclonal light chains of different subgroups with complete specificity. These reagents, define a site of kappa subgroup distinction and represent a potent tool for the characterization of light chain heterogeneity.

Amino Acid Sequence↗
PubMed1986

A conserved human germline V kappa gene directly encodes rheumatoid factor light chains.

The full-length gene that encodes the light chain variable regions of an idiotypically related group of human IgM kappa rheumatoid factors (RFs) has been cloned and sequenced. The deduced amino acid sequence is identical to four separate RF proteins. These results prove that genes capable of encoding human anti-IgG autoantibody light chains without any somatic mutation are present in the kappa gene repertoire of normal people.

Amino Acid Sequence↗
PubMed1986

Modulation of diphthamide synthesis by 5'-deoxy-5'-methylthioadenosine in murine lymphoma cells.

The histidine derivative diphthamide occurs uniquely in eukaryotic elongation factor 2 (EF-2), and is the specific target for the diphtheria toxin mono(ADP-ribosyl)transferase. The first step in diphthamide biosynthesis may involve the transfer of aminocarboxypropyl moiety from S-adenosylmethionine to the imidazole ring of histidine in EF-2, to yield 2-(3-carboxy-3-aminopropyl)histidine and 5'-deoxy-5'-methylthioadenosine (MeSAdo). As the possible nucleoside product of the initial reaction in the diphthamide biosynthetic pathway, MeSAdo could be an inhibitor of diphthamide formation. In the present experiments, we have analyzed the effects of MeSAdo on diphthamide synthesis in a MeSAdo phosphorylase-deficient mutant murine lymphoma cell line (R1.1, clone H3). As measured by susceptibility to diphtheria toxin-induced ADP-ribosylation, MeSAdo inhibited the formation of diphthamide in EF-2. The inhibition was not due to a nonspecific effect on protein synthesis. Indeed, exogenous MeSAdo substantially protected the lymphoma cells from the lethal effects of diphtheria toxin. These results suggest that MeSAdo can specifically modulate the biosynthesis of diphthamide in EF-2 in murine malignant lymphoma cells.

Adenosine↗