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Biomedical subjects

D A Carson

Publications and source records attributed to D A Carson.

At least 91 records · Page 5Linked to original sources
PubMed1994

Defining the genetic origins of three rheumatoid synovium-derived IgG rheumatoid factors.

A major diagnostic marker in most rheumatoid arthritis (RA) patients is the rheumatoid factor (RF), an autoantibody that binds to the Fc region of IgG. To delineate the Ig genes and the underlying mechanism for RF production in RA patients, we applied a systematic approach to define the genetic origins of three IgG RFs derived from the synovial fluid of two RA patients. The results show that two of three IgG RF have substantial numbers of somatic mutations in their variable (V) regions, ranging from 13 to 23 mutations over a stretch of 291-313 nucleotides, resulting in a frequency of 4.4-7.8%. However, one IgG RF has only one mutation in each V region. This result indicates that an IgG RF may arise from a germline gene by very few mutations. The mutations occur mainly in the complementarity-determining regions (CDRs), and the mutations in the CDRs often lead to amino acid substitutions. Five of the six corresponding germline V genes have been found to encode either natural autoantibodies or autoantibodies in other autoimmune disorders; and three of the six V genes have been found in fetal liver. Taken together with other results, the data show that (a) several potentially pathogenic RFs in RA patients arise from natural autoantibodies, and (b) only a few mutations are required to convert the natural autoantibodies to IgG RFs.

Amino Acid Sequence↗
PubMed1994

Stimulation of HIV replication in mononuclear phagocytes by leukemia inhibitory factor.

This study examined the effects of leukemia inhibitory factor (LIF) on human immunodeficiency virus (HIV) replication in mononuclear phagocytes (MNP). LIF induced a dose-dependent increase in p24 antigen production in the chronically infected promonocytic cell line U1. The magnitude and time kinetics of the LIF effects were similar to interleukin 1 (IL-1), IL-6, and tumor necrosis factor (TNF), other cytokines known to induce HIV replication in this cell line. To characterize mechanisms responsible for these LIF effects, levels of HIV mRNA, activation of the DNA binding protein nuclear factor (NF)-kB, signal transduction pathways, and potential interactions with other cytokines were analyzed. LIF increased steady-state levels of HIV mRNA at 2.0, 4.3, and 9.2 kB. This was detectable by 24 h and persisted until 72 h. The DNA binding protein NF-kB is a central mediator in cytokine activation of HIV transcription. NF-kB levels were higher in unstimulated U1 cells as compared to the parent cell line U937. In both cell lines LIF increased NF-kB activity. Induction of NF-kB and HIV replication by cytokines are at least in part dependent on reactive oxygen intermediates. The oxygen radical scavenger N-acetyl-L-cysteine, but not an inhibitor of nitric oxide synthase, inhibited LIF-induced HIV replication. LIF induces the production of other cytokines in monocytes but its effects on HIV replication were not inhibited by antibodies to IL-1, TNF, or IL-6. These results identify LIF as a stimulus of HIV replication.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcysteine↗
PubMed1993

Autoantigen inhibits apoptosis of a human B cell leukemia that produces pathogenic rheumatoid factor.

We studied a variant CD5- B cell chronic lymphocytic leukemia (CLL) cell population that produces pathologic IgM kappa rheumatoid factor autoantibodies. In contrast to common CD5+ B cell CLL, this variant leukemia cell population displays intraclonal diversity in its expressed Ig V genes, similar to that noted for follicular B cell non-Hodgkin's lymphomas. Also, in contrast to common B cell CLL, these leukemia cells rapidly undergo cell death hours after being placed in tissue culture. We find that addition of Ag (aggregated human IgG) enhances significantly the survival of these cells in vitro. Leukemia cell survival also could be enhanced by exogenous IFN-gamma or anti-CD40 presented on Fc gamma RII (CDw32)-expressing L cells, but not by exogenous IL-4, IL-6, or monomeric human IgG. We find that Ag acts directly on the leukemia B cells to inhibit apoptosis. This effect could be mimicked by cross-linking the leukemia cells' surface IgM receptors with immobilized murine mAb specific for human Ig mu-chains, but not by immobilized mAb of irrelevant specificity. In contrast to most follicular NHL, this leukemia B cell population does not have evidence of bcl-2 gene rearrangement. Also, in contrast to non-Hodgkin's lymphomas and most B cell CLL, these cells do not express detectable amounts of bcl-2. Finally, although capable of inhibiting apoptosis, surface Ig receptor cross-linking does not induce expression of bcl-2 in these variant leukemia cells. We hypothesize that the lack of bcl-2 expression may render these leukemia cells particularly dependent upon the survival signal(s) derived from surface Ig receptor cross-linking. This state may represent an early stage in leukemia/lymphomagenesis, possibly accounting for the intraclonal diversity observed in the Ig V genes expressed by certain CD5- B cell leukemias and lymphomas.

Antigens, CD↗
PubMed1993

New adenosine kinase inhibitors with oral antiinflammatory activity: synthesis and biological evaluation.

Several 5-iodotubercidin analogues in the pyrazolo[3,4-d]pyrimidine ring system were synthesized as potential inhibitors of adenosine kinase by a direct Lewis acid-catalyzed glycosylation procedure using both the preformed carbohydrate and the heterocyclic base as starting materials. The 5'-hydroxyl, -chloro, -azido, -deoxy, -amino, and -fluoro derivatives were prepared and evaluated in three systems for biological activity relative to adenosine, the true substrate, and 5-iodotubercidin, a known inhibitor. First, each compound was studied kinetically for inhibition of purified human placental adenosine kinase activity. The order of potency was: iodotubercidin > hydroxyl > amino > or = deoxy > fluoro > chloro >> azido. The Ki values for the 5'-hydroxyl and 5'-amino compounds, the two most potent inhibitors, were 80 and 150 nM, respectively. The inhibition appeared to be essentially competitive in nature, although a noncompetitive component of significance for the more potent inhibitors cannot be ruled out. Second, a bioassay was conducted in which the toxicity of 6-methylmercaptopurine riboside toward human CEM lymphoblasts was reversed by varying concentrations of the compounds. The order of effectiveness of the compounds in this system, representing a functional inhibition of adenosine kinase in cultured cells, was about the same as that with the purified enzyme, except that the 5'-chloro and 5'-fluoro compounds were ineffective. Third, the 5'-hydroxyl derivative was evaluated in vivo in a rat pleurisy inflammation model and displayed biological activity at a dose of 30 mg/kg given orally. Finally, the in vitro toxicity of each compound was assessed in CEM lymphoblasts. Results indicated that the two most potent inhibitors in the pyrazolo[3,4-d]pyrimidine ring system, the 5'-hydroxyl (7) and the 5'-amino (20), were 15-fold and 75-fold, respectively, less growth inhibitory than 5-iodotubercidin.

Adenosine Kinase↗
PubMed1993

Ca2+/Mg(2+)-dependent endonuclease from human spleen: purification, properties, and role in apoptosis.

A major event in apoptosis is the digestion of chromatin into oligonucleosomal fragments. However, the enzymes responsible for the DNA degradation have not been well characterized. Here we report the purification of an endonuclease from human spleen cell nuclei that is likely to be responsible for DNA digestion in apoptosis. Enzyme activity was measured by a sensitive fluorometric assay, which assesses the conversion of plasmid DNA from a supercoiled to an open form. The endonuclease was extracted from isolated nuclei with NaCl between 100 and 350 mM and was further purified by chromatography on columns of phosphocellulose, Superdex 75, and chelating Sepharose (Zn2+ form). By gel filtration, the apparent molecular mass was 22-26 kDa; on SDS-polyacrylamide gel electrophoresis, the purified enzyme showed a single 27-kDa band. The enzyme required both Mg2+ (optimum, 5 mM) and Ca2+ (optimum, 2 mM) for activity. It was inhibited by Zn2+ (100% inhibition at 50 microM) and by high (> 10 mM) concentrations of Ca2+. Aurintricarboxylic acid, spermine, p-(hydroxymercuri)benzoate, and N-ethylmaleimide were also endonuclease inhibitors. No inhibition was observed with iodoacetamide, G-actin, or nucleoside 3',5'-bisphosphates. An optimum pH of 8.0 was found. When added to human CCRF-CEM lymphoblast nuclei, that do not contain the endonuclease, the purified splenic enzyme digested the chromatin into the mono- and oligonucleosomal fragments that are characteristic of apoptosis. On the basis of this result, and the observation that the activators and inhibitors of the purified endonuclease closely parallel those that affect apoptosis, it seems likely that this enzyme is involved in the apoptotic degradation of DNA in human lymphocytes.

Apoptosis↗
PubMed1993

Induction of antibodies to a kappa V region by gene immunization.

Direct gene transfer into muscle can lead to sustained gene expression at the injected sites. Here we tested the ability of in vivo gene transfection to immunize mice against an isolated human IgV region. The Humkv325 germline kappa L chain V gene encodes the kappa L chain in V regions of several human IgM autoantibodies and is used frequently in chronic lymphocytic leukemia. This gene was inserted into a mammalian expression vector, pREP7, to produce pREVk3. Mice injected i.m. with pREVk3 produced antibodies against the V region of Glo, a human monoclonal IgM paraprotein whose kappa L chain is encoded by Humkv325. Co-injection of an expression vector encoding IL-2 enhanced anti-Glo antibody production fivefold and induced a localized delayed hypersensitivity reaction. Antibody production also was induced when vectors encoding Humkv325 and IL-2 were injected s.c. These experiments demonstrate that gene immunization vectors can stimulate immune responses to antibody V region determinants.

Animals↗
PubMed1993

Microtiter format gene quantification by covalent capture of competitive PCR products: application to HIV-1 detection.

We have developed a simple gene quantification system using the competitive polymerase chain reaction (CPCR) followed by microtiter format analysis. CPCR is carried out using a mutant competitor with the same size as the target DNA product, and a minimal base exchange to insure the same amplification kinetics. One primer is aminated at the 5' end to produce PCR products that are captured onto carboxylated wells of microtiter plates through peptide bond formation. The non-aminated DNA strands are stripped off from the wells by alkali washing, and the remaining aminated strands are hybridized with either a digoxigenin-labeled wild type-specific oligonucleotide probe or a competitor-specific probe. To standardize the hybridization conditions of the probes, a DNA construct containing wild type and mutant competitor sequences in tandem is captured at different concentrations, hybridized with the probes, and used to generate a standard curve. Bound probes are detected by anti-digoxigenin antibody conjugated with peroxidase and chromogen. Optical densities are recorded with a conventional microtiter plate reader and converted to concentrations according to the standard curves. The ratios of wild type DNA to mutant competitor are used to determine the initial amounts of wild type DNA in the samples. This method was used successfully to quantify human immunodeficiency virus type 1 (HIV-1) env gene in human lymphocytes. It only requires a thermal cycler and a conventional microtiter plate reader, and can be readily done on a large scale. Potential applications include detection of other pathogens, diagnosis of genetic disorders and studies of gene expression.

Base Sequence↗
PubMed1993

Apoptosis and disease.

The intercellular connections that characterise advanced forms of life would not be possible without a mechanism to remove individual cells that are no longer needed, or that function abnormally. Such physiological cell death, in the absence of inflammation, is achieved by apoptosis, a structurally distinct programmed cell death pathway. Defective regulation of programmed cell death may play a part in the aetiology of cancer, AIDS, autoimmune diseases, and degenerative diseases of the central nervous system. The pharmacological manipulation of apoptosis offers new possibilities for the prevention and treatment of these illnesses.

Acquired Immunodeficiency Syndrome↗
PubMed1993

Systemic immunological effects of cytokine genes injected into skeletal muscle.

Somatic gene therapy is an interesting approach for the delivery of cytokines for prolonged periods. The present experiments show that direct injections into mouse skeletal muscle of cDNA expression vectors encoding interleukin 2 (IL-2), IL-4, or type beta 1 transforming growth factor (TGF-beta 1) induce biological effects characteristic of these cytokines in vivo. Mice injected intramuscularly with a vector encoding IL-2 had enhanced humoral and cellular immune responses to an exogenous antigen, transferrin, that was delivered at a separate site. These IL-2 effects were abolished by coadministration of a vector directing synthesis of TGF-beta 1. The TGF-beta 1 vector by itself depressed the anti-transferrin antibody response and caused an 8-fold increase in plasma TGF-beta 1 activity. The TGF-beta 1 plasmid injection did not cause muscle infiltration with monocytes or neutrophils and there was no evidence for fibrotic changes. Muscle injection with a cDNA encoding IL-4 selectively increased IgG1 levels but did not alter the cellular immune response to transferrin. In lupus-prone mice (MRL/lpr/lpr), injection with IL-2 expression vectors increased and TGF-beta 1 vectors decreased auto-antibodies to chromatin. These results demonstrate that intramuscular injection of cytokine genes, in the absence of infectious viral vectors, can regulate humoral and cellular immune responses in vivo.

Animals↗
PubMed1993

Methylthioadenosine phosphorylase deficiency in human non-small cell lung cancers.

Methylthioadenosine (MeSAdo) phosphorylase, a purine metabolic enzyme, is present in all normal mammalian tissues. A deficiency of this enzyme has been reported in some human leukemias and lymphomas and in a few solid tumors. In the present study, a specific immunoassay was used to assess the enzyme levels in human non-small cell lung cancer cell lines and primary tumors. We also tested the effects of MeSAdo phosphorylase-selective chemotherapy on the in vitro growth of enzyme-positive and enzyme-negative lung cancer cell lines. Of 29 non-small cell lung cancers, 9 (6 cell lines and 3 primary tumors, 31%) lacked detectable immunoreactive enzyme protein. Both 5,10-dideazatetrahydrofolate, an inhibitor of de novo purine synthesis, and methionine depletion, combined with MeSAdo, prevented the growth of the enzyme-negative non-small cell lung cancer cells under conditions in which enzyme-positive cells utilized MeSAdo to endogenously synthesize purine nucleotides and methionine. Our data suggest that MeSAdo phosphorylase deficiency is frequently found in non-small cell lung cancers and can be exploited in designing enzyme-selective chemotherapy.

Adult↗
PubMed1993

Relationship of deoxycytidine kinase and cytoplasmic 5'-nucleotidase to the chemotherapeutic efficacy of 2-chlorodeoxyadenosine.

The agent 2-chlorodeoxyadenosine (2-CdA) has chemotherapeutic activity in hairy cell leukemia (HCL) and in refractory chronic lymphocytic leukemia (CLL). The cytotoxic activity of 2-CdA requires the intracellular accumulation of 2-CdA nucleotides. Deoxycytidine kinase (dCK) and cytoplasmic 5'-nucleotidase (5'-NT) are the principal enzymes that phosphorylate 2-CdA and dephosphorylate 2-CdA 5'-monophosphate, respectively. The net accumulation of 2-CdA nucleotides may therefore depend on both dCK and 5'-NT. The purpose of the present experiments was to determine if there is a relationship between pretreatment levels of dCK and 5'-NT in HCL and in CLL cells, and the clinical outcome of 2-CdA treatment. As measured by a direct immunoassay for dCK in 25 CLL patients, and by a 5'-NT activity assay in 23 patients, mean dCK levels were significantly higher in 2-CdA responders than in nonresponders (P < .01), whereas mean 5'-NT levels were significantly lower in 2-CdA responders than in nonresponders (P < .05). Mean dCK levels were higher in six HCL 2-CdA responders than in one nonresponder, whereas mean 5'-NT levels were lower in the 2-CdA responders than in the nonresponder. These results suggest that both dCK and 5'-NT are determinants of 2-CdA responsiveness.

5'-Nucleotidase↗
PubMed1993

Function of B cells expressing a human immunoglobulin M rheumatoid factor autoantibody in transgenic mice.

We have generated transgenic mice that express the immunoglobulin (Ig)M heavy chain and kappa light chain genes coding for a human IgM rheumatoid factor (RF), Les. Transgenic B cells expressing human IgM RF show striking similarities to their counterparts in normal humans. They comprise a significant proportion of the adult B cell population, but secrete only low levels of RF into the serum. The RF transgene-expressing B cells localize to primary B cell follicles and the mantle zone regions of secondary follicles in the spleen. Using these mice we have been able to show that one of the central functions of normal RF-expressing B cells may be to act as highly efficient antigen-presenting cells for low concentrations of immune-complexed antigen. High levels of secretion of IgM RF can not be induced under normal circumstances, although RF-expressing B cells proliferate well in vitro to both aggregated human IgG and anti-human IgM antibodies. However, these mice are not intrinsically secretion deficient. By crossing the RF transgenic mice with the autoimmune MRL/lpr background, we find a dramatic increase, > 200-fold, in levels of serum RF. The results strongly suggest that a major function of normal resting RF B cells is unrelated to antibody secretion. Rather, the RF B cells in the follicles may play a role in antigen presentation and regulation of immune responses to antibody-bound nonself-, and possibly self-antigens. This physiologic role of RF B cells may be disrupted in RF-associated autoimmune disease.

Animals↗
PubMed1993

Divergent T cell receptor gamma repertoires in rheumatoid arthritis monozygotic twins.

OBJECTIVE: To determine if the expressed T cell receptor (TCR) gamma repertoire is altered in rheumatoid arthritis (RA). METHODS: Peripheral blood lymphocytes were collected from monozygotic twins who were either concordant or discordant for RA, or from a normal twin pair. TCR gamma-specific complementary DNA libraries were constructed using the anchored polymerase chain reaction. Gene usage was analyzed by plaque hybridization and sequencing. RESULTS: The expressed TCR V gamma repertoires both in RA patients and normal subjects were extremely diverse. Monozygotic twins who were concordant for RA expressed very different frequencies of TCR V gamma genes. CONCLUSION: RA does not lead to a specific clonal expansion or deletion of TCR V gamma genes in peripheral blood.

Adult↗
PubMed1993

Generation and molecular analyses of two rheumatoid synovial fluid-derived IgG rheumatoid factors.

OBJECTIVE: To study the Ig genes that encode IgG rheumatoid factor (IgG-RF) from rheumatoid synovial fluid. METHODS: We used rheumatoid synovial fluid B cells to generate IgG-RF-secreting hybridomas. We then characterized their binding properties and determined their nucleotide sequences. RESULTS: Two monospecific IgG-RFs were obtained. Sequence analysis of the RFs revealed a new V lambda gene family (designated V lambda 9) and extensive somatic diversification, including a duplication-insertion of 18 nucleotides (6 amino acid residues) into a hypervariable region. CONCLUSION: The data provide further support for an antigen-driven response in the sustained production of potentially pathogenic IgG-RFs in rheumatoid synovium.

Amino Acid Sequence↗
PubMed1993

The expressed T cell receptor V gene repertoire of rheumatoid arthritis monozygotic twins: rapid analysis by anchored polymerase chain reaction and enzyme-linked immunosorbent assay.

Because of heterogeneity in the outbred human population, it has been difficult to determine the genetic factors that influence the expressed T cell receptor (TcR) repertoire in autoimmune diseases. To overcome this problem, we have developed a combination of anchored polymerase chain reaction (APCR) and enzyme-linked immunosorbent assay (ELISA) that can accurately assess TcR V gene frequencies in numerous clinical samples. The results are independent of amplification efficiency, and V gene usage can be readily analyzed with an ELISA plate reader and associated software. Using this method, the TcR V beta gene repertoires in peripheral lymphocytes from nine sets of identical twins, normal, concordant or discordant for rheumatoid arthritis (RA), were studied. The TcR V beta results were compared with TcR V gamma frequencies in the same specimens as determined by APCR-ELISA and cDNA sequence analysis. The results showed a marked similarity in the TcR V beta gene repertoires between identical twins, compared to unrelated subjects (p < 0.05) whether or not they were concordant or discordant for RA. In contrast, the TcR V gamma gene repertoires in the monozygotic twins differed as much as in controls. The data imply that (a) the human TcR V beta gene repertoire in peripheral blood is genetically controlled, whereas (b) the TcR V gamma gene repertoire is primarily influenced by environmental stimuli, and (c) RA causes no consistent change in TcR V beta repertoire of peripheral blood. The APCR-ELISA method, in the context of large-scale family and population studies, should facilitate a more precise delineation of the genetic factors that regulate human TcR V beta expression.

Adult↗