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Biomedical subjects

D A Carson

Publications and source records attributed to D A Carson.

At least 73 records · Page 4Linked to original sources
PubMed1996

Identification of human tumour suppressor genes by monochromosome transfer: rapid growth-arrest response mapped to 9p21 is mediated solely by the cyclin-D-dependent kinase inhibitor gene, CDKN2A (p16INK4A).

Microcell transfer of intact normal human chromosomes into immortal mouse and hamster fibroblast cell lines has revealed growth suppressive activity associated with a small sub-set of the human complement. Here, we describe the results of a detailed study aimed at identifying the gene or genes responsible for the rapid growth-arrest response obtained with human chromosome-9. Initially, STS-PCR deletion mapping of segregants arising in monochromosome transfer experiments was used successfully to localize the active sub-chromosomal region to 9p21. Subsequent fine-structure deletion mapping of previously uniformative hybrid segregants, employing additional markers between D9S162 and D9S171, provided strong evidence that the cyclin-dependent kinase (cdk) inhibitor gene CDKN2A (p16INK4A) was solely responsible for the chromosome-9 effect; 9p21 microdeletions in a significant proportion of segregant clones were restricted to a single CDKN2A exon. Transfection experiments with CDKN2A and CDKN2B cDNA expression vectors, using mouse A9 cells and three human malignant melanoma cell lines as recipients, provided further evidence in support of this hypothesis. Collectively, our results indicate that expression of human CDKN2A (controlled either by its natural regulatory elements, or by a cytomegalovirus promoter) is incompatible with in vitro proliferation in immortalized rodent cells and in human melanoma cell lines. The rapidity of the growth inhibitory effects of CDKN2A was inconsistent with a mode of action involving induction of replicative cell senescence via telomerase repression, but was consistent with a mechanism based on cell cycle arrest through cdk inhibition. The study described here has generated a panel of microdeleted monochromosome-9 donor hybrids which may prove valuable in functional investigations aimed at identifying other important tumour suppressor genes located on human chromosome-9.

Animals↗
PubMed1996

Effect of immune globulin on the prevention of experimental hepatitis C virus infection.

The efficacy of postexposure prophylaxis for the prevention of hepatitis C virus (HCV) infection was studied in experimentally infected chimpanzees. Three chimpanzees were inoculated with HCV: Two were treated 1 h later with anti-HCV--negative intravenous immune globulin (IGIV) or hepatitis C immune globulin (HCIG), and a third animal was not treated. HCV infection was detected in all 3 animals within a few days of inoculation. Once passively transferred anti-HCV declined in the HCIG-treated animal, there was an increase of HCV antigen (Ag)--positive hepatocytes followed by reappearance of anti-HCV; HCV Ag disappeared concordant with the development of acute hepatitis. Acute hepatitis C developed in both the IGIV-treated and untreated chimpanzees, with peak liver enzyme activity on day 59, but was delayed in the HCIG-treated animal until day 146. Postexposure HCIG treatment markedly prolonged the incubation period of acute hepatitis C but did not prevent or delay HCV infection. IGIV had no effect on the course of HCV infection.

Animals↗
PubMed1996

Influence of the rheumatoid arthritis-associated shared epitope on T-cell receptor repertoire formation.

Rheumatoid arthritis is associated with several human leukocyte antigen DRB1 types that express a common five-amino acid sequence called the shared epitope. Here we show that the human leukocyte antigen DRB1 shared epitope expands naive T lymphocytes that express the same T-cell-receptor variable region-joining region combinations that are prevalent in the synovia of rheumatoid arthritis patients. Thus, the shared epitope could affect rheumatoid arthritis disease susceptibility by selecting in the premorbid state specific T-cell subsets that contribute to synovial inflammation.

Arthritis, Rheumatoid↗
PubMed1995

Cancer progression and p53.

In a complex organism, somatic cells are under intermittent selection pressure for the emergence of mutants that can survive environmental insults and that can grow autonomously despite adverse conditions. Repeated rounds of mutation, selection, and proliferation may lead to cancer. The organism prevents malignant transformation by assuring accurate DNA repair before cell division, by forcing the death of cells with excessive DNA damage, and by placing limits on the replicative lifespans of most somatic cells. The p53 gene is a "guardian of the genome"--it regulates multiple components of the DNA damage control response and promotes cellular senescence. Disabling mutations and deletions of p53 occur in 50% of human tumours. p53-deficient cancers are often unstable, aggressive, and resistant to therapy.

Animals↗
PubMed1995

Human immunoglobulin (IgG) induced deletion of IgM rheumatoid factor B cells in transgenic mice.

The singular ability of immunoglobulin genes to hypermutate their variable regions, while permitting the generation of high-affinity antibodies against foreign antigens, poses a problem in terms of maintenance of immunological self-tolerance. Immunoglobulin gene hypermutation driven by a foreign antigen has the potential to generate antibodies that cross-react with self-components. Consequently, there must exist a mechanism in the periphery for inactivation of mature autoreactive B cell clones. The classical experimental system used to address this problem is the induction of tolerance to soluble, deaggregated human IgG. We have analyzed the mechanism of induction of tolerance to human IgG using transgenic mice that express a human IgM rheumatoid factor (IgM RF) on a large proportion of their B cells. Injection of deaggregated human IgG caused a specific deletion of those B cells that express an intact IgM RF on their cell surface. The degree of RF B cell deletion was proportional to the reduction in the proliferative response of splenocytes to antigen (aggregated human IgG), or to F(ab')2 fragments of anti-human IgM antibodies. Control experiments showed that IgG administration had little effect on the numbers of mouse Ig-bearing cells or their ability to proliferate to a nonspecific mitogen. Thus, the effects of IgG on the human IgM RF B cell are antigen specific and are not due to nonspecific toxic effects of the human IgG preparation. These experiments demonstrate that peripheral exposure to IgG induces deletion of reactive B cells, without any evidence for anergy, and differ from data obtained by other investigators studying tolerance to soluble protein antigens. The results imply that human Igs have distinct properties as soluble antigens, and that peripheral nonresponsiveness to IgG may be due to lymphocyte deletion.

Animals↗
PubMed1995

Oral 2-chlorodeoxyadenosine in psoriatic arthritis. A preliminary report.

OBJECTIVE: To determine if weekly oral 2-chlorodeoxyadenosine (2-CdA) can induce selective lymphocytopenia, and reduce inflammation, in patients with refractory psoriatic arthritis. METHODS: Seven patients with psoriatic arthritis were treated with oral 2-CdA at weekly dosages of 0.3 mg/kg to 0.45 mg/kg for 12 weeks, followed by monthly maintenance therapy. The patients were evaluated after 6 months. RESULTS: The drug treatment produced selective lymphocytopenia, and reduced lymphocyte infiltration into involved skin. One patient did not complete 12 weeks of therapy because of perceived lack of efficacy. Four of the 6 remaining patients had improved joint disease, and 5 of 6 had improved psoriasis. CONCLUSION: Weekly oral 2-CdA appears to be a well-tolerated regimen for the inducement of peripheral lymphocytopenia in patients with psoriatic arthritis. Larger-scale, controlled trials may be warranted.

Administration, Oral↗
PubMed1995

Positive selection in autoimmunity: abnormal immune responses to a bacterial dnaJ antigenic determinant in patients with early rheumatoid arthritis.

A novel 'multistep molecular mimicry' mechanism for induction of rheumatoid arthritis (RA) by bacterial antigens that activate T lymphocytes previously 'educated' by peptides derived from a class of human histocompatibility antigens is reported here. These antigens have the amino acid sequence QKRAA, which is also present on the Escherichia coli heat-shock protein dnaJ. Synovial fluid cells of early RA patients have strong immune responses to the bacterial antigen, but cells from normal subjects or controls with other autoimmune diseases do not. The activated T cells may cross-react with autologous dnaJ heat-shock proteins that are expressed at synovial sites of inflammation. Our findings may have direct relevance to new strategies for the immune therapy of RA.

Amino Acid Sequence↗
PubMed1995

Bacterial lipopolysaccharide copurifies with plasmid DNA: implications for animal models and human gene therapy.

During the course of gene therapy experiments in rodents, using intramuscular injections of plasmid DNA derived from Escherichia coli, we noted dose-related toxicity. This observation prompted a search for possible contaminants of DNA samples. We used the highly specific and sensitive limulus amoebocyte lysate assay (LAL), to monitor endotoxin bioactivity in DNA samples, and found plasmid DNA derived from standard E. coli bacterial strains, using traditional DNA isolation protocols, to be heavily contaminated with endotoxin, or lipopolysaccharide (LPA). Standard DNA isolation procedures resulted in the copurification of up to 500 micrograms/ml of LPS. LPS is a potent inducer of cytokines and other inflammatory mediators, and may complicate the use of naked DNA in gene therapy. The copurification of endotoxin with plasmid DNA also has important implications for in vitro transfection studies and microinjection of DNA into embryos. A simple and efficient protocol to reduce LPS contamination of plasmid DNA was developed. The conversion of intact bacteria to spheroplasts prior to the isolation of plasmid DNA, incubation with lysozyme, treatment with the detergent n-octyl-beta-D-thioglucopyranoside (OSPG) and polymyxin-B (PMB) chromatography, allowed the isolation of plasmid DNA containing less than 50 ng/ml LPS. This represents a 10,000-fold reduction in LPS contamination, compared to conventional methods of plasmid DNA purification, avoids potentially toxic reagents such as ethidium bromide, and produces a higher yield of plasmid DNA.

Animals↗
PubMed1995

Population and family studies of three disease-related polymorphic genes in systemic lupus erythematosus.

The contribution to systemic lupus erythematosus (SLE) of three lupus-associated polymorphisms (involving the C4A2 complement component, Humhv3005 and the T cell antigen receptor alpha chain gene) are investigated in 81 individuals from 14 multiplex SLE families, 41 unrelated lupus patients, and 88 unrelated healthy controls. The results show a strong association between C4A deletion and SLE in these families. While the current study confirms the previously reported association between hv3005 deletion and sporadic SLE, the study fails to support this association in familial SLE patients. Moreover, no correlation is detected between the occurrence of hv3005 deletion and C4A null alleles in lupus patients, suggesting that the effects of these genetic polymorphisms on predisposition to lupus are independent. The previously reported lupus-associated T cell receptor (TCR) alpha chain polymorphism is not detected in any of the individuals studied here. The combined data suggest that C4A null alleles predispose strongly to development of lupus, whereas the influence of hv3005 deletion is relatively weak. The results also suggest that contributions of weak susceptibility genes such as hv3005 to disease predisposition may be obscured by the effects of stronger genetic factors and thus need to be examined in patients lacking these factors.

Base Sequence↗
PubMed1995

Modulation of disease activity in murine systemic lupus erythematosus by cytokine gene delivery.

Somatic gene therapy is a novel approach with the potential to achieve prolonged increases in circulating levels of peptide hormones and cytokines. The present study evaluates the effects of monthly, intramuscular injections of cDNA expression vectors encoding for transforming growth factor beta (TGF beta) or interleukin 2 (IL-2) on disease activity in the MRL/lpr/lpr murine model of systemic lupus erythematosus (SLE). Monthly injections of plasmids cDNA between 6 and 26 weeks significantly elevated the serum levels of TGF beta (P < 0.005) and IL-2 (P < 0.05) compared with a control plasmid without insert. TGF beta encoding plasmid had beneficial effects in murine SLE with a prolonged survival of 70% at 26 weeks compared with 40% in the control group, decreased anti-chromatin and rheumatoid factor antibodies and a 50% decrease in total IgG production. Renal function was improved with reduced BUN levels and kidney inflammation as estimated by an histologic score. Those beneficial effects occurred in the apparent absence of local or systemic side-effects. In contrast, IL-2 cDNA injections appeared harmful with a decreased survival to 20% at 26 weeks, enhanced total IgG synthesis and autoantibodies production with a 4.5-fold increase in antichromatin antibodies. These results indicate that somatic gene therapy may provide a simple, inexpensive and effective mechanism for the long-term control of autoimmune diseases.

Animals↗
PubMed1994

Higher frequency of alterations in the p16/CDKN2 gene in squamous cell carcinoma cell lines than in primary tumors of the head and neck.

Sixty-eight primary head and neck squamous cell carcinomas and nine head and neck squamous cell carcinoma cell lines were examined for mutations and homozygous deletions of the p16/CDKN2 gene. Homozygous deletions of the p16/CDKN2 gene were found in three lines, and a mutation was detected in another cell line. In contrast, none of the primary tumors showed homozygous deletions and 11 of 68 tumors had missense or nonsense base changes. Seven tumors contained somatic mutations. Five tumors, including one that also had a somatic mutation, had a probable polymorphism at codon 140 leading to an amino acid change from Ala to Thr. Three of these also contained an apparent polymorphism at codon 98, which did not lead to an amino acid change. The frequency of mutations and deletions detected differs markedly between cell lines (44%) and primary tumors (10%) suggesting that while p16/CDKN2 may play a role in tumorigenesis in some head and neck squamous cell carcinomas, inactivation of p16/CDKN2 probably occurs more frequently in cell lines as a result of adaptation to cell culture.

Base Sequence↗
PubMed1994

Intradermal gene immunization: the possible role of DNA uptake in the induction of cellular immunity to viruses.

The skin and mucous membranes are the anatomical sites were most viruses are first encountered by the immune system. Previous experiments have suggested that striated muscle cells are unique among mammalian cell types in their capacity to take up and express free DNA in the absence of a viral vector or physical carrier. However, we have found that mice injected into the superficial skin with free (naked) plasmid DNA encoding the influenza nucleoprotein gene had discrete foci of epidermal and dermal cells, including cells with dendritic morphology, that contained immunoreactive nucleoprotein antigen. A single intradermal administration of 0.3-15 micrograms of free plasmid DNA induced anti-nucleoprotein-specific antibody and cytotoxic T lymphocytes that persisted for at least 68-70 weeks after vaccination. Intradermal gene administration induced higher antibody titers than did direct gene injection into skeletal muscle and did not cause local inflammation or necrosis. Compared with control animals, the gene-injected mice were resistant to challenge with a heterologous strain of influenza virus. These results indicate that the cells of the skin can take up and express free foreign DNA and induce cellular and humoral immune responses against the encoded protein. We suggest that DNA uptake by the skin-associated lymphoid tissues may play a role in the induction of cytotoxic T cells against viruses and other intracellular pathogens.

Amino Acid Sequence↗
PubMed1994

Deletions of the cyclin-dependent kinase-4 inhibitor gene in multiple human cancers.

Cytogenetic abnormalities of chromosome 9p21 are characteristic of malignant melanomas, gliomas, lung cancers and leukaemias. From a panel of 46 human malignant cell lines, we localized by positional cloning the most frequently deleted region on 9p21. Sequence analysis of the isolated fragment reveals two open reading frames identical to the recently described complementary DNA for the inhibitor of cyclin-dependent kinase 4 (CDK4). Polymerase chain reaction and Southern blot analysis confirmed the frequent deletion or rearrangement of the CDK4-inhibitor gene in melanomas, gliomas, lung cancers and leukaemias, and the absence of detectable gene transcripts. One carcinoma had a deletion entirely within the CDK4-inhibitor gene. The CDK4-inhibitor gene from a patient with dysplastic nevus syndrome had a germ-line nonsense mutation. The CDK4 inhibitor is thought to be a physiological suppressor of proliferation. Cells unable to produce the inhibitor may be prone to neoplastic transformation.

Base Sequence↗
PubMed1994

Competitive RT-PCR ELISA: a rapid, sensitive and non-radioactive method to quantitate cytokine mRNA.

We have developed a non-radioactive method to quantitate precisely levels of gene expression. This method is based on RT-PCR (reverse transcriptase-polymerase chain reaction) with an RNA competitor, followed by the covalent capture of the amplified DNA onto the wells of microtiter plates, and the quantitation of the PCR product by oligonucleotide hybridization and ELISA (enzyme-linked immunosorbent assay). The assay can reproducibly detect 1 zeptomole mRNA. The assay was successfully used to quantitate mRNA levels of the T cell derived cytokines interleukin-2, interleukin-4 and interferon-gamma in resting and stimulated human lymphocytes. Because it is performed in a microtiter ELISA format, this rapid, sensitive and non-radioactive method should facilitate measurements of gene expression, particularly in large clinical studies.

Base Sequence↗
PubMed1994

Solid-phase polymerase chain reaction.

The polymerase chain reaction (PCR) has facilitated the diagnosis of infectious diseases and genetic disorders, because of its ability to amplify minute amounts of nucleic acids. To distinguish genes of interest from nonspecifically amplified DNA, PCR products commonly are fractionated by electrophoresis, transferred to membranes, and then probed with a labeled internal sequence-specific oligonucleotide. Alternatively, the PCR products have been labeled directly, and hybridized to immobilized oligonucleotide probes. These methods require the tedious physical transfer of the PCR products. In order to amplify and immobilize genes simultaneously, we have developed a simple solid-phase PCR method. The technique enabled us to detect the HIV envelope gene readily without any transfer of amplified DNA.

Base Sequence↗
PubMed1994

Immune responses to the Escherichia coli dnaJ heat shock protein in juvenile rheumatoid arthritis and their correlation with disease activity.

Patients with juvenile rheumatoid arthritis frequently have abnormal immune responses to the hsp65 class of bacterial heat shock proteins. However, lymphocytes from children with other inflammatory diseases may also recognize hsp65, and the role of these antigens in juvenile rheumatoid arthritis remains controversial. We have studied humoral and cellular immune responses to a distinct, recently described bacterial heat shock protein, designated dnaJ. The Escherichia coli dnaJ gene was cloned and expressed, and the purified recombinant protein was used as an antigen. Neither normal children nor children with various chronic inflammatory diseases had lymphocyte proliferative responses to recombinant dnaJ. However, lymphocytes from patients with polyarticular, pauciarticular, and systemic manifestations of juvenile rheumatoid arthritis responded strongly to the antigen. Cellular immune responses to dnaJ were higher in synovial fluid than in blood and higher in children with active disease than in children in remission. These data show that increased immune reactivity to dnaJ is characteristic of juvenile rheumatoid arthritis and that the magnitude of the immune response is linked to disease activity. The results suggest that an abnormal immune response to antigens on commensal gut bacteria may contribute to the generation of chronic inflammation in juvenile rheumatoid arthritis.

Adolescent↗