Biomedical subjects

D A Carson

Publications and source records attributed to D A Carson.

At least 109 records · Page 6Linked to original sources

PubMed1993

Transforming growth factor-beta (TGF beta) inhibition of Epstein-Barr virus (EBV)- and interleukin-4 (IL-4)-induced immunoglobulin production in human B lymphocytes.

This study reports on the effects of TGF beta on the secretion of Ig isotypes by highly purified (> 99% CD20-positive) human peripheral blood B cells. Stimulation of these B cell preparations with EBV resulted in the secretion of IgM, IgG, and IgA and the addition of IL-4 induced readily detectable levels (> 100 ng/ml) of IgE between 10 and 25 days of culture. TGF beta 1 and TGF beta 2 showed similar dose-dependent suppression of IgM, IgG, and IgA, and the relative proportion of IgG and IgA remained unchanged in the presence of TGF beta. IgE production induced by EBV and IL-4 was significantly inhibited by TGF beta. TGF beta effects on Ig secretion were not related to inhibition of B cell proliferation by this cytokine. In contrast to these TGF beta effects on EBV activation of primary B cells, the constitutive Ig secretion by EBV-transformed B cells was resistant to TGF beta, while the increase in Ig secretion induced by IL-6 was inhibited by TGF beta. Thus, TGF beta inhibits the EBV-induced secretion of the major Ig isotypes in peripheral blood B cells and has differential effects on Ig secretion by transformed B cells.

B-Lymphocytes↗

PubMed1993

Characterization of two highly homologous autoantibody-related VH1 genes in humans.

Previously, we showed that the Humha 1lr rearranged gene was almost identical to the consensus amino acid sequence of several G6 idiotype-positive rheumatoid factor (RF) heavy chains, and to the VH gene-encoded region of the fetally expressed 51P1 cDNA. The finding led us to suggest that the ha 1lr-corresponding germline gene encodes the heavy chains of many human IgM RFs. We now report the isolation of the proposed germline gene, designated Humhv1051; it is identical to the consensus sequence of the G6 heavy chain V regions. During this experiment, we also isolated unexpectedly an additional VH1 gene, termed Humhv1051K; it differs from hv1051 by one amino acid residue. Importantly, hv1051K is identical to Humha113, the rearranged VH1 gene of a natural anti-cardiolipin antibody.

Amino Acid Sequence↗

PubMed1993

Regulation of the mature human T cell receptor gamma repertoire by biased V-J gene rearrangement.

To delineate how gene rearrangement influences the expressed human gamma delta T cell repertoire, we generated T cell receptor gamma (TCR gamma) V domain-specific cDNA libraries from the peripheral lymphocytes of eight donors and sequenced a total of 232 TCR gamma gene transcripts. The libraries consisted of both in-frame and out-of-frame rearranged TCR gamma genes. The in-frame TCR gamma gene transcripts were used to determine the diversity of functional T cells, whereas the out-of-frame transcripts, primarily derived from alpha beta T cells, were used to assess the frequencies of TCR V gamma-J gamma rearrangements in progenitor T lymphocytes. The results showed that both sets of transcripts exhibited strikingly restricted V gamma-J gamma combinations. Only 11 of 40 potential V gamma-J gamma rearrangements were common ( > or = 3% of total). The pattern of gene usage in the functional and nonfunctional transcripts was similar and did not differ markedly among donors. The only exception was the predominance of V gamma 9-JP in potentially functional transcripts from seven of eight individuals. These results show that V gamma-J gamma rearrangement is nonrandom and suggest that the diversity of TCR gamma genes in the functional gamma delta T cell repertoire partly depends upon preferentially rearranged V gamma-J gamma gene combinations. However, the expansion of V gamma 9/V gamma 2 T cells in adult peripheral blood can only be explained by antigenic selection of relatively rare V gamma 9-JP recombinants.

Adult↗

PubMed1993

2-Chlorodeoxyadenosine dose escalation in nonhematologic malignancies.

PURPOSE: We performed a dose-escalation study of 2-chlorodeoxyadenosine (2-CdA) in solid tumors to determine the maximum-tolerated dose (MTD) and define its toxicity profile at higher doses. PATIENTS AND METHODS: Twenty-one patients, seven with malignant astrocytoma, twelve with metastatic melanoma, and two with metastatic hypernephroma, were enrolled onto the study. Patients were entered onto cohorts that received 0.10, 0.15, or 0.20 mg/kg/d of 2-CdA by continuous intravenous infusion for 7 days every 28 days. 2-CdA levels were determined by radioimmunoassay. In tumor tissue samples, deoxycytidine kinase (dCK) levels were measured by both enzyme activity and immunoreactive protein analysis. RESULTS: Of seven patients treated with 2-CdA at 0.1 mg/kg/d, one experienced grade 3 or 4 myelotoxicity. Of 11 patients treated at 0.15 mg/kg/d, four experienced myelotoxicity, two after a single course of 2-CdA. All three patients who received 2-CdA at 0.2 mg/kg/d experienced myelosuppression. Neurologic events occurred in two patients, both with malignant melanoma. Two of seven patients (28.6%) with astrocytomas obtained partial responses with a median duration of 8 months. 2-CdA penetrated the blood-brain barrier. An association was found between dCK levels as measured by enzymatic activity and immunoreactive proteins, but this did not correlate with 2-CdA tumor responsiveness. CONCLUSION: The MTD for 2-CdA delivered as a 7-day intravenous infusion in patients with nonhematologic malignancies was determined to be 0.1 mg/kg/d, the same as the MTD for patients with hematologic malignancies. There was no clinical correlation with dCK expression and response to 2-CdA. The responses noted in patients with malignant astrocytoma warrant further phase II study.

Adult↗

PubMed1993

2-Chlorodeoxyadenosine: hairy cell leukemia takes a surprising turn.

2-Chlorodeoxyadenosine (2CdA; Cladribine; Leustatin) was developed as a result of the discovery that the death of lymphocytes in hereditary adenosine deaminase deficiency was the result of the accumulation of deoxynucleotides, specifically in lymphocytes, because of their unique enzymatic profile. This deoxyadenosine analog was selected from 25 candidate compounds because it was resistant to deamination by ADA and because it had a favorable therapeutic index. The lymphoid specific action of this drug is a function of the high deoxycytidine kinase and low 5' nucleotidase activity of lymphocytes. The drug appears to trigger apoptosis, very likely by preventing the repair of nicks in double-stranded DNA. Accordingly, 2-CdA has been found effective against a variety of lymphoid neoplasms. Hairy cell leukemia has proved to be most sensitive to its action, with approximately 80% of patients achieving a complete, long-lasting remission after a single five- or seven-day infusion. 2-CdA also shows promise in the treatment of autoimmune disorders.

2-Chloroadenosine↗

PubMed1992

Quantitative immunoassay of human deoxycytidine kinase in malignant cells.

Deoxycytidine kinase (dCK) is necessary for the activity of several nucleosides used for the chemotherapy of cancer and AIDS. However, the measurement of dCK catalytic activity in crude cell extracts may be imprecise, due to the presence of phosphatases and nucleotidases that degrade the enzyme products. We describe a simple immunoassay for dCK that can measure accurately as little as 5 ng enzyme protein in crude tissue extracts. The assay enabled us to show (i) that mutant cells deficient in dCK activity lack immunoreactive dCK protein, (ii) that dCK catalytic activity and immunoreactivity correlate closely in human tumors, and (iii) that immunoreactive dCK is particularly high in lymphocytes and lymphoid malignancies, although certain solid tumors may also contain the enzyme. The immunoassay of dCK could prove useful in the selection and monitoring of patients who are being treated with nucleosides that are activated by this enzyme.

Animals↗

PubMed1992

A polymerase chain reaction-based method for isolation of gene-specific sequences from the interferon-alpha gene cluster.

The interferon-alpha gene is a gene family of over 20 distinct genes having 80-95% homology with one another at a nucleotide level. Because of the high homology in the gene cluster, the available interferon-alpha gene probes can hybridize to multiple bands of different size on Southern blot analysis of restricted human genomic DNA. We used the polymerase chain reaction with the primers synthesized from Alu repetitive sequence and the conserved sequences of the interferon-alpha gene cluster to generate specific probes for individual interferon-alpha genes. The amplification products were subcloned into a plasmid vector and analyzed by DNA sequencing and Southern blotting of the restricted human placental DNA. One clone, which derived from interferon-alpha 14 gene, produced a single 5.2-kb band in Southern blots of the HindIII-restricted human placental DNA. This stands in contrast to the 10 bands of different size that were detected with a cDNA for the interferon-alpha I' gene. Our results indicate that a polymerase chain reaction-based method can be used to isolate gene-specific sequences from the interferon-alpha gene cluster. Since a variety of human cancers has been found to have the complete or partial deletion of the interferon-alpha gene cluster, the gene-specific probe generated by this method may aid in determining the breakpoints in the vicinity of the gene cluster.

Animals↗

PubMed1992

2-Chlorodeoxyadenosine: an active agent in the treatment of cutaneous T-cell lymphoma.

Cutaneous T-cell lymphomas are disfiguring malignant lymphoproliferative disorders for which standard therapy has been principally palliative. 2-Chlorodeoxyadenosine (2-CdA), a new purine analogue resistant to degradation by adenosine deaminase that has substantial activity against lymphoid neoplasms, was administered to 16 patients with cutaneous involvement by T-cell lymphoma. All patients had failed topical treatment modalities and/or systemic therapies. Fifteen patients were evaluable; one patient was not evaluable due to incomplete therapy and follow-up. The overall response rate was 47%. Three of 15 patients (20%) achieved complete responses and four of 15 patients (27%) achieved partial responses. The median duration of response was 5 months. One patient remains in unmaintained complete remission at 52+ months. Therapy was well tolerated. Myelosuppression was the principal toxicity encountered, occurring in 8 of 15 (53%) patients. 2-CdA is an effective new agent for the treatment of cutaneous T-cell lymphoma and warrants further study both as a single agent and in combination regimens.

2-Chloroadenosine↗

PubMed1992

Immunoglobulin V gene expression in CD5 B-cell malignancies.

Chronic lymphocytic leukemia (CLL) and small lymphocytic lymphomas (SLL) generally are malignancies of CD5 B cells. Immunophenotypic and clinicopathologic data, however, are required to distinguish subtypes that apparently have a different cytogenesis than that of conventional CLL or SLL. In addition to expressing CD5, neoplastic cells of the latter are also distinctive in that they frequently coexpress surface immunoglobulin (Ig), bearing one or more cross-reactive idiotypes (CRIs) (e.g. 17.109, G6,) that commonly are found on monoclonal IgM autoantibodies. The frequent occurrence of such CRIs reflects both the biased rearrangement and subsequent selected expression of Ig V genes with little or no somatic mutation. IgM/L CLL, for example, frequently (8/33) harbor abortive Ig rearrangements involving Humkv325, the VK gene encoding the 17.109-CRI. Also, the VH1 gene(s) encoding the G6 CRI accounts for over 10% of all VH genes and over 60% of all the VH1 genes used in randomly selected common CLL/SLL. Furthermore, comparison with the Ig expressed by nonmalignant G6 CRI+ B cells reveals an apparent restriction in the CDR3 of IgH expressed by G6 CRI+ CLL. Coupled with the observed potential bias in antibody light chain and heavy chain pairing in B-CLL, these data suggest that the autoantibodies expressed in this disease are selected based on antigen-binding activity. Collectively, our studies indicate that nonstochastic Ig V gene rearrangement and subsequent selection may influence the Ig repertoire expressed in this common B-cell malignancy.

Animals↗

PubMed1992

Oral antilymphocyte activity and induction of apoptosis by 2-chloro-2'-arabino-fluoro-2'-deoxyadenosine.

2-Chlorodeoxyadenosine (CdA) is active in chronic lymphocytic leukemia, hairy-cell leukemia, and low-grade lymphomas. In part, this spectrum of activity may be attributable to the selective toxicity of CdA to nondividing lymphocytes and monocytes. However, CdA is unstable at acidic pH and is degraded by bacterial nucleoside phosphorylases. The present experiments demonstrate that the 2'-arabino-fluoro derivative of CdA, designated CAFdA, is also directly toxic to quiescent lymphocytes and macrophages. Unlike CdA, CAFdA was stable at pH 2 and resisted degradation by Escherichia coli nucleoside phosphorylase. Cell killing was preceded by the formation of DNA strand breaks and could be prevented by supplementation of the medium with deoxycytidine. The initial DNA damage initiated the pattern of oligonucleosomal DNA fragmentation characteristic of apoptosis. Mutant lymphoblasts, deficient in deoxycytidine kinase, with elevated cytoplasmic 5'-nucleotidase, or with expanded deoxynucleotide pools secondary to increased ribonucleotide reductase activity, were cross-resistant to both CAFdA and CdA toxicity. One-week oral treatment with CAFdA (1 mg/ml in drinking water) achieved an average plasma concentration of 0.56 microM and eliminated 90% of chronic lymphocytic leukemia cells transplanted into severe combined immunodeficiency (scid) mice. Under the same conditions, CdA was much less active. Collectively, these results suggest that CAFdA could be effective as an oral agent in indolent lymphoproliferative diseases and in autoimmune diseases where lymphocyte and monocyte depletion is desirable.

2-Chloroadenosine↗

PubMed1992

Evidence for somatic selection of natural autoantibodies.

Natural autoantibodies are primarily immunoglobulin M (IgM) antibodies that bind to a variety of self-antigens, including self-IgG. Accounting for a large proportion of the early B cell repertoire, such polyspecific autoantibodies are speculated to contribute to the homeostasis and/or competence of the primary humoral immune system. Recent studies indicate that the leukemia cells from most patients with chronic lymphocytic leukemia (CLL) also express such IgM autoantibodies. Similarly, the leukemia cells from many CLL patients react with murine monoclonal antibodies (mAbs) specific for crossreactive idiotypes (CRIs) associated with human IgM autoantibodies. In particular, leukemic cells frequently react with G6, a mAb specific for an Ig heavy chain (H chain)-associated CRI, and/or with 17.109, a mAb that defines a kappa light chain (L chain)-associated CRI. Generated against IgM rheumatoid factor (RF) paraproteins, G6 and 17.109 each recognize a major CRI that is present in many IgM RF paraproteins. Furthermore, over 90% of the IgM paraproteins found to bear both H and L chain-associated CRIs also are found to have RF activity. Molecular characterization of these CRIs demonstrates that each is a serologic marker for expression of a highly conserved Ig V gene. As such, the frequent production of IgM polyspecific autoantibodies in CLL simply may reflect the frequent use of such highly conserved autoantibody-encoding Ig V genes with little or no somatic mutation. To test this hypothesis, we generated murine transfectomas to pair the 17.109-reactive kappa L chain of SMI, a 17.109/G6-reactive CLL population, with the Ig H chain of SMI or other G6-reactive leukemia cells or tonsillar lymphocytes. Cotransfection of vectors encoding the Ig H and L chains of SMI generated transfectomas that produce IgM kappa RF autoantibodies reactive with human IgG1 and IgG4. In contrast to G6/17.109-reactive IgM kappa RF Waldenstrom's paraproteins, the SMI IgM kappa also reacts with several other self-antigens, including myoglobin, actin, and ssDNA. However, cotransfection of the SMI L chain with a vector encoding any one of 10 different G6-reactive Ig H chains generated transfectomas that produce IgM kappa antibodies without detectable polyspecific autoantibody activity. These results indicate that polyspecific antiself-reactivity of G6/17.019-reactive Ig is dependent on the somatically generated Ig third complementarity determining region. Collectively, these studies imply that selection may be responsible for the frequent expression of polyspecific autoantibodies in CLL and early B cell ontogeny.

Amino Acid Sequence↗

PubMed1992

Genetic analysis of self-associating immunoglobulin G rheumatoid factors from two rheumatoid synovia implicates an antigen-driven response.

Although much has been learned about the molecular basis of immunoglobulin M (IgM) rheumatoid factors (RFs) in healthy individuals and in patients with mixed cryoglobulinemia and rheumatoid arthritis, little is known about the genetic origins of the potentially pathogenic IgG RFs in the inflamed rheumatoid synovia of patients. Recently, we generated from unmanipulated synovium B cells several hybridomas that secreted self-associating IgG RFs. To delineate the genetic origins of such potentially pathogenic RFs, we adapted the anchored polymerase chain reaction to rapidly clone and characterize the expressed Ig V genes for the L1 and the D1 IgG RFs. Then, we identified the germline counterparts of the expressed L1 IgG RF V genes. The results showed that the L1 heavy chain was encoded by a Vh gene that is expressed preferentially during early ontogenic development, and that is probably located within 240 kb upstream of the Jh locus. The overlap between this RF Vh gene and the restricted fetal antibody repertoire is reminiscent of the natural antibody-associated Vh genes, and suggests that at least part of the "potential pathogenic" IgG RFs in rheumatoid synovium may derive from the "physiological" natural antibody repertoire in a normal immune system. Indeed, the corresponding germline Vh gene for L1 encodes the heavy chain of an IgM RF found in a 19-wk-old fetal spleen. Furthermore, the comparisons of the expressed RF V genes and their germline counterparts reveal that the L1 heavy and light chain variable regions had, respectively, 16 and 7 somatic mutations, which resulted in eight and four amino acid changes. Strikingly, all eight mutations in the complementarity determining regions of the V gene-encoded regions were replacement changes, while only 6 of 11 mutations in the framework regions caused amino acid changes. Combined with L1's high binding affinity toward the Fc fragment, these results suggest strongly that the L1 IgG RF must have been driven by the Fc antigen.

Amino Acid Sequence↗

PubMed1992

Generation and characterization of two monoclonal self-associating IgG rheumatoid factors from a rheumatoid synovium.

OBJECTIVE: To study IgG rheumatoid factor (RF) from rheumatoid synovium. METHODS: We fused the K6H6/B5 human-mouse heterohybridoma with unstimulated rheumatoid synovial B cells to generate IgG-RF-secreting hybridomas. RESULTS: The RFs from 2 such hybridomas bound specifically to the Fc fragment of human IgG and self-associated to form immune complexes. Such immune complexes are a major characteristic of the pathogenic IgG-RFs in rheumatoid synovium. CONCLUSION: IgG-RF-secreting hybridomas have been obtained. Analyses may reveal the underlying mechanisms of the induction of IgG-RF.

Animals↗

PubMed1992

A systematic approach to defining the germline gene counterparts of a mutated autoantibody from a patient with rheumatoid arthritis.

OBJECTIVE: To define the germline counterparts of potentially highly mutated autoantibodies in disease states. METHODS: We developed a systematic approach by first characterizing a rearranged Ig gene and its upstream flank, and then designing suitable primers to amplify specifically the putative germline counterpart. RESULTS: We identified and characterized the germline counterpart of a rheumatoid factor heavy chain variable region. CONCLUSION: We showed unequivocally that the heavy chain of a rheumatoid factor, derived from synovial tissue, has 4 replacement mutations from the corresponding germline gene. The technique allows quick assessment of the degree of somatic mutation in many autoantibodies, and thus can help to elucidate the underlying mechanisms for the induction and sustained production of such antibodies in patients.

Amino Acid Sequence↗

PubMed1992

Transfer of human chronic lymphocytic leukemia to mice with severe combined immune deficiency.

B chronic lymphocytic leukemia (CLL) cells were transferred into mice with severe combined immunodeficiency (SCID). Leukemia cells injected into the peritoneal cavity of these animals may survive for at least 10 weeks in vivo. In contrast, leukemia cells do not survive for long periods when injected intravenously. Despite the longevity of CLL cells injected i.p., these cells apparently do not migrate to other lymphoid tissues. Eight to sixteen weeks after receiving CLL cells, SCID mice develop human IgG autoantibodies to human red blood cells and/or high serum levels of human Ig. Soon thereafter, these animals develop lethal human B-cell tumors. In contrast to the original CLL cells, these human B-cell tumors are CD5-negative, have genomic DNA of Epstein-Barr virus (EBV), express antigens associated with latent EBV infection and have distinctive Ig gene rearrangements by Southern. We conclude that bystander B cells may generate tumors in CLL-reconstituted SCID mice that emulate the EBV-associated lymphoproliferations noted in SCID mice reconstituted with normal human PBL.

Animals↗

PubMed1992

Genetic factors in the etiology and pathogenesis of autoimmunity.

Family and population studies indicate that several different genes can increase susceptibility to autoimmune diseases. Established genetic risk factors include genes encoding histocompatibility molecules, complement proteins, immunoglobulins, peptide transporter proteins, and genes controlling the production of sex hormones. Each factor may independently enhance the immunogenicity of autoantigens, either by increasing their processing and presentation by B lymphocytes and macrophages or by increasing the chance for recognition by autoreactive T and B lymphocytes. Genetic factors may also influence immune responses to infectious agents that can trigger autoimmunity. Because of the somatic generation of immune diversity, genetically identical individuals have different immune systems. The ability of genetic diagnosis to predict autoimmune disease in outbred populations cannot easily exceed the disease concordance rates in monozygotic twins, which usually are less than 50%. However, genetic diagnosis can target populations that should be monitored for serologic evidence of autoimmunity, which may precede clinical signs and symptoms. In the future, it may be possible to match different forms of immunotherapy with specific genetic defects.

Amino Acid Sequence↗

PubMed1992

Inheritance of monoclonal rheumatoid factor cross-reactive idiotypes in primary Sjögren's syndrome (pSS). Comparative studies of a rabbit polyclonal anti-idiotype and 17109 monoclonal anti-idiotype.

A comparison of the presence of two idiotypes, one identified by a rabbit polyclonal antiidiotypic antibody, first found on a cryoprecipitable IgM chi rheumatoid factor (RF) from an SS patient (3rd SS) and the 17109 idiotype, identified by a monoclonal antibody was performed in 106 sera and eight minor salivary gland biopsies of Sjögren's syndrome (SS) patients and 125 sera from age-sex matched normal controls. Of 106 of SS patients' sera 36 had immunoglobulins positive for the 3rd SS idiotype. 17109 activity was more prevalent in SS patients positive for the polyclonal anti-idiotype 3rd SS, than those with negative idiotype (9/36 VS 2/70 chi 2 = 12.53 P less than 0.005). Cross inhibition studies, however, revealed that the polyclonal anti-idiotype binding was not inhibited by the 17109 moAb. 3rd SS and 17109 anti-idiotypes were reacted with immunoglobulins in the serum of 3.5% and 1.7% of normal human sera respectively. Immunohistologic studies demonstrated that 4/8 and 2/6 minor salivary gland biopsies had infiltrating plasma cells containing immunoglobulins bearing the 3rd SS and the 17109 idiotypes, respectively. The inheritance of both idiotypes was investigated in sera of 4 SS kindreds. In two kindreds with 3rd SS positive probands, the idiotype was detected in 3 first degree relatives of the same generation. 17109 activity was detected in the serum of a sister of the positive proband who had a high RF titer. These results suggest that the 17109 moAb recognizes a different epitope of that of the 3rd SS. The idiotypes of monoclonal RFs are not inherited and probably are produced by plasma cells infiltrating the labial minor salivary glands of SS patients.

Animals↗

PubMed1992

Greatly enhanced inhibition of human immunodeficiency virus type 1 replication in CEM and HT4-6C cells by 3'-deoxythymidine diphosphate dimyristoylglycerol, a lipid prodrug of 3'-deoxythymidine.

3'-Deoxythymidine (3dT) is a weakly active dideoxynucleoside in human immunodeficiency virus (HIV)-infected cells because of its slow phosphorylation by cellular thymidine kinase. 3dT diphosphate dimyristoylglycerol (3dTDP-DMG), a phospholipid prodrug, was synthesized and found in vitro to be 18- to 50-fold more effective than 3dT in CEM and HT4-6C cells. In CEM cells, the selectivity index of 3dTDP-DMG was 270 versus 48 for 3dT, an increase of 5.6-fold. In thymidine kinase-deficient mutant CEM cells infected with HIV, 3dT and zidovudine (AZT) were virtually inactive but 3dTDP-DMG retained substantial activity, suggesting that its greatly increased antiviral activity is due in part to bypass of thymidine kinase. 3dTDP-DMG was 14- to 37-fold more active than 3dT in AZT-sensitive and AZT-resistant clinical isolates of HIV; no cross-resistance with AZT was noted. The results suggest that lipid prodrugs may be utilized in some cases to confer unique metabolic advantages over the corresponding free nucleoside; in the case of 3dTDP-DMG, an 18- to 50-fold increase in antiretroviral activity was observed in LAV-infected cells. The strategy would seem to be especially useful for antiviral nucleosides which are poorly phosphorylated.

Antiviral Agents↗