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D Kelleher

Publications and source records attributed to D Kelleher.

At least 109 records · Page 6Linked to original sources

Conventional protein kinase C isoforms are not essential for cellular proliferation of a T cell lymphoma line.

The role of conventional protein kinase C (PKC) isoforms in the proliferation of a T cell lymphoma line was investigated, PKC isozyme-deficient cell lines were generated from HuT 78 which expresses PKC isoforms alpha, beta, epsilon and zeta. The first of these, K-4, expresses PKC alpha, epsilon and zeta but not beta. L-2, a mutant of K-4, expresses PKC alpha and zeta but expresses neither PKC alpha, beta, gamma, epsilon nor zeta when cultured continuously in PMA (L-2/PMA). All four cell lines continued to proliferate although at reduced rates in the presence of the PKC inhibitors staurosporine and H-7. K-4 and HuT 78 undergo growth arrest when activated with PMA (rate of proliferation decreases by 76 and 95%, respectively). While PMA-induced growth arrest occurs in L-2 cells which possess PKC alpha and zeta, PMA-induced growth arrest does not occur in L-2/PMA which is deficient in these isoforms. As these cell lines continue to proliferate in the presence of PKC inhibitors and since proliferation occurs in the absence of PKC isoforms (L-2/PMA), the results suggest that neither conventional PKC nor the epsilon and zeta isoforms are essential for proliferation of these cells. These data further suggest that PKC alpha and/or zeta may be involved in PMA-induced growth arrest.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

Flow cytometric analysis of surface major histocompatibility complex class II expression on human epithelial cells prepared from small intestinal biopsies.

A technique for preparing viable, single cell suspensions of the epithelial layer of small intestinal tissue obtained endoscopically is described. Constant agitation of four biopsies for 60 min in the presence of chelating and reducing agents gave yields of 1.2-6.7 x 10(6) cells, of which 11-30% were intraepithelial lymphocytes (IEL). Passage through a nylon wool column removed dead cells. This preparation was suitable for flow cytometric analysis. Using this technique, surface MHC class II molecule expression was studied in 14 patients with normal small intestinal mucosa. Fluorescence labelling of these cells showed strong HLA-DR expression by epithelial cells (EC), DP was expressed less strongly, while little DQ expression could be detected. This technique demonstrates that small intestinal biopsies taken during routine endoscopy can yield adequate numbers of viable epithelial cells to perform flow cytometric analysis.

Adolescent↗

Interferon-gamma and tumour necrosis factor production in patients with Helicobacter pylori infection.

The production of the cytokines, interferon-gamma and tumour necrosis factor by human antral mucosa cells and stimulated peripheral blood mononuclear cells were determined by enzyme linked immunosorbent assay and L929 bioassay respectively. Tumour necrosis factor production by peripheral blood mononuclear cells in response to Helicobacter pylori stimulation was depressed in Helicobacter pylori positive individuals, compared to Helicobacter pylori negative individuals (P < 0.05). There was no difference in tumour necrosis factor production by peripheral blood mononuclear cells in response to purified protein derivative. However, tumour necrosis factor production by cells isolated from gastric mucosa during short term culture was significantly higher in Helicobacter pylori positive patients (P < 0.05) than negative patients, indicating a probable macrophage response. Levels of interferon-gamma did not differ significantly in the gastric explant culture from the two groups. The results show that Helicobacter pylori negative patients have a stronger peripheral cellular immune response to Helicobacter pylori infection. The higher levels of tumour necrosis factor production by antral mucosa cells in Helicobacter pylori positive patients may reflect the infiltration of T lymphocytes and macrophages within the local mucosa.

Adult↗

Regulation of CD3 expression in a protein kinase C isozyme-deficient T-cell line.

Using the T-cell lymphoma line HuT 78, and a clone derived from HuT 78, designated K-4, the role of protein kinase C (PKC) isozymes in the expression of a variety of T-cell surface antigens was investigated. HuT 78 expresses PKC isozymes alpha and beta while K-4 expresses only PKC alpha. Flow cytometric analysis revealed that incubation of HuT 78 cells with phorbol 12-myristate 13-acetate (PMA) results in significant down-regulation of surface expression of CD3. While K-4 cells expressed reduced amounts of CD3, a similar reduction in CD3 expression was not observed when these cells were stimulated with PMA. The regulation of expression of CD11a (LFA-1), CD44, CD45RA and CD45RO and of the class II molecules DR and DP in response to PMA, was similar in both cell lines.

Antigens, Surface↗

Neurofilament expression in human T lymphocytes.

The expression of intermediate filaments in normal cells is mainly determined by their embryonal developmental origin. Flow cytometry using monoclonal antibody RT97 demonstrated that neurofilament was detectable in the human HuT 78 T-cell line and on resting T lymphocytes. Expression was greatly increased on lymphocytes activated for 3 days with phorbol ester. Western blotting confirmed the presence of the 200,000 MW form of neurofilament in T lymphocytes. Stimulation of peripheral blood T cells with phorbol myristate acetate (PMA) or with anti-CD3 monoclonal antibodies resulted in a marked increase in detection of phosphorylated neurofilament on Western blotting. Stimulation of HuT 78 cells with anti-LFA-1 resulted in redistribution of neurofilament from a perinuclear spheroid core into dendritic processes. These data indicate that T cells activated through the T-cell receptor associated complex express an intermediate filament usually associated with neurally derived cells. The finding that neurofilament expression and organization are regulated by T-cell surface molecules suggests a role for this intermediate filament in T-cell function.

Blotting, Western↗

Development and characterization of a protein kinase C beta-isozyme-deficient T-cell line.

In the human T-cell lymphoma line, HuT 78, proliferation and phorbol ester-induced growth arrest and differentiation were inhibited by the protein kinase C (PKC) inhibitor, staurosporine. By contrast, an alternative PKC inhibitor, H-7, inhibited proliferation but not phorbol ester-induced growth arrest. The cell line was found to contain both alpha and beta isoforms of PKC by Western blot techniques. A cell line, K-4, was cloned from HuT 78 in the presence of H-7 and this clone was found to be positive for PKC-alpha only. PKC-beta did not return on cultivation in the absence of H-7. Proliferation of K-4 was insensitive to inhibition with both H-7 and staurosporine. However, phorbol ester-induced growth arrest remained staurosporine sensitive. Phorbol-stimulated IL-2 secretion was minimal in the PKC-beta-deficient cell line. These data suggest that PKC-beta may be a regulatory enzyme for proliferation and stimulated interleukin-2 secretion in HuT 78 cells. Heterogeneity of responses to PKC activation may reflect the use of different isozymes in different intracellular pathways. The K-4 cell line should provide a useful tool in the dissection of involvement of PKC isozymes in cellular function.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

Leukocyte function-associated antigen 1 (LFA-1) is a signaling molecule for cytoskeletal changes in a human T cell line.

The HuT 78/2 clone is a human T cell clone which has an activated phenotype. When this line was stimulated with immobilized antibody to LFA-1, the majority of cells developed dendritic processes. These processes were demonstrated to contain cytoskeletal elements including microtubules and intermediate filaments. Phorbol myristate acetate synergized with and prolonged this effect. Thus, the LFA-1 molecule may function as a signaling molecule for cytoskeletal reorganization in a human T cell clone.

Antibodies, Monoclonal↗

Tolerance of the liver to the effects of Yttrium-90 radiation.

There are no reliable data documenting the tolerance of the human liver to ionizing radiation from a continuous Yttrium-90 source. As Yttrium-90 incorporated into microspheres is being used to treat patients with liver cancer, it is imperative that the tolerance of the human liver to this form of radiation damage be determined. Four patients with metastatic liver cancer were treated with Yttrium-90 to deliver radiation doses above that considered tolerable when given by conventional external sources. Patients were monitored with serial estimations of liver function tests and between 7 and 9 months after treatment liver biopsies were performed. Histological examination of the liver biopsies confirmed only minimal changes in the normal liver parenchyma. These data indicate that the human liver may tolerate relatively large radiation doses when delivered by Yttrium-90 microspheres embedded in the liver parenchyma as a number of discrete point sources.

Adenocarcinoma↗

Chemiluminescence by polymorphonuclear leucocyte subpopulations in chronic inflammatory bowel disease. Influence of the cell separation procedure.

Chemiluminescence (CL) is a simple quantitative assay of polymorphonuclear leucocyte (PMNL) oxidative metabolism. PMNL CL was found to be significantly higher in patients with chronic inflammatory bowel disease than in normal controls (167 +/- 60 vs. 139 +/- 50 mV/10(5) cells, p less than 0.05). There were no significant differences between patients with ulcerative colitis and Crohn's disease. Disease controls with rheumatoid arthritis and with bronchiectasis also demonstrated elevated CL. These results were obtained using a two-step gelatin/Ficoll-Hypaque procedure for PMNL separation. However when PMNLs were prepared using a one-step Ficoll-Hypaque procedure PMNL CL was found to be depressed in chronic inflammatory bowel disease (CIBD). It was demonstrated that this disparity was caused by the elimination of low-density neutrophils with high CL production by the one-step procedure. These data indicate that reports of abnormal in vitro neutrophil function in CIBD should be interpreted with caution since separation techniques which are satisfactory in normal individuals may significantly influence results in patients with inflammatory diseases. Furthermore these data indicate the presence of a subpopulation of activated low-density PMNL in patients with CIBD.

Cell Separation↗

Enhanced secretion of prostaglandin E2 by tissue-fixed macrophages in colonic carcinoma.

Prostaglandin E2 (PGE2) secretion by peripheral blood and tissue-fixed macrophages from patients with colorectal carcinoma was assessed. There was no significant difference between PGE2 production by peripheral blood mononuclear cells between patients with colorectal carcinoma and normal controls. However, secretion of PGE2 by tissue-fixed macrophages from within the colorectal carcinomata in response to opsonised zymosan was significantly higher than in the uninvolved colonic tissue. PGE2 production by tissue-fixed macrophages from within colonic polyps was found to be normal. These results could not be explained on the basis of increased availability of substrate arachidonic acid since addition of excess arachidonic acid resulted in similar findings. The enhanced production of PGE2 correlated with Dukes staging but not the level of differentiation. The production of PGE2 from epithelial cells in response to ionophore A23187 was not significantly enhanced. Leukotriene B4 secretion by intestinal macrophages in response to opsonised zymosan was not significantly elevated in the colonic tumour tissue. Modulation of levels of prostaglandin production within colonic tumours may play a role in the rate of growth and vascularity of these tumours and in the regulation of the local immune response to malignancy.

Calcimycin↗

Development and characterisation of T lymphocyte lines from human small intestinal biopsies.

T lymphocyte lines were developed from human small intestinal biopsies obtained at the time of gastroduodenoscopy. Lines were established as outgrowths from biopsy specimens in microculture using a combination of T cell mitogens, indomethacin, interleukin (IL-2) and autologous irradiated feeder cells. The predominant phenotype of T cells after six to 12 weeks in culture was CD2, CD3, and CD4 positive. Functionally, these T cell lines secreted IL-2 in response to stimulation with phytohemagglutinin (PHA) and phorbol 12-myristate 13-acetate (PMA) in combination, but not to PHA or PMA alone. Two to 7% of cells in each line expressed natural killer (NK) cell-associated markers--that is, Leu 7, CD16, and Leu 19. Moreover, such cells were cytotoxic for NK sensitive targets, but not for NK resistant targets or allogeneic or autologous Epstein Barr Virus (EBV) transformed B cells. Sufficient numbers of small intestinal lymphoid cells for study were previously available only from patients undergoing surgical resections of the small bowel. The ability to isolate and culture human small intestinal T lymphocytes from gastroscopic intestinal biopsies provides an important new tool for studies of human small intestinal lymphocytes.

Cell Line↗

Defective suppression in the autologous mixed lymphocyte reaction in patients with Crohn's disease.

T helper and suppressor cell control of autologous immunoglobulin production was measured in 14 patients with Crohn's disease (CD) using autologous B cells or monocytes to stimulate regulatory T-cell activity. A pronounced defect in suppressor cell function was observed in the patient group but not in matched controls irrespective of whether B cells or monocytes were used as the stimulus. This defect was observed for IgG, A and M. This defect was seen both in patients with active disease and with inactive CD suggesting the possibility that a primary regulatory defect might exist in this disease. The patient group displayed normal helper cell function.

Adolescent↗

An 80 to 85 kilodalton human phosphoglycoprotein associated with cell activation.

This report describes the identification and biochemical characterization of a new proliferation- and activation-associated membrane Ag. The M21C5 Ag (Mr 80 to 85 kDa) initially was immunoprecipitated from 125I-cell surface-labeled HT29 human tissue culture colon cancer cells by using a monoclonal antibody (M21C5) prepared from HT-29 immunized BALB/c mice. The M21C5 Ag is a glycoprotein as shown by metabolic labeling with 3H-leucine and 2-[3H]-mannose. It has a broad distribution on most proliferating tissue culture cell lines tested, but is absent from several normal human tissues that were examined. Although not detected on unstimulated PBL, the expression of the M21C5 Ag could be induced by stimulation of PBL with the T cell mitogens PHA or Con A. Two-color fluorescence analysis showed that M21C5 is expressed on both CD4 and CD8 activated T cells. After mitogen stimulation, the expression of the M21C5 Ag was delayed relative to the expression of IL-2 and transferrin receptors. M21C5 glycoprotein was shown to be an integral membrane protein that is phosphorylated primarily on serine residues. Based on its biochemical and tissue distribution properties, M21C5 phosphoglycoprotein appears distinct from other known proliferation and activation-associated molecules.

Animals↗

An extended HLA-D region haplotype associated with celiac disease.

Celiac disease has one of the strongest associations with HLA (human leukocyte antigen) class II markers of the known HLA-linked diseases. This association is primarily with the class II serologic specificities HLA-DR3 and -DQw2. We previously described a restriction fragment length polymorphism (RFLP) characterized by the presence of a 4.0-kilobase Rsa I fragment derived from an HLA class II beta-chain gene, which distinguishes the class II HLA haplotype of celiac disease patients from those of many serologically matched controls. We now report the isolation of this beta-chain gene from a bacteriophage genomic library constructed from the DNA of a celiac disease patient. Based on restriction mapping and differential hybridization with class II cDNA and oligonucleotide probes, this gene was identified as one encoding an HLA-DP beta chain. This celiac disease-associated HLA-DP beta-chain gene was flanked by HLA-DP alpha-chain genes and, therefore, was probably in its normal chromosomal location. The HLA-DP alpha-chain genes of celiac disease patients also were studied by RFLP analysis; 84% of HLA-DR3, -DQw2 patients had a 16-kb Xba I fragment that was present in only 36% of HLA-DR3, -DQw2 controls. Moreover, 79% of these patients had both alpha- and beta-chain polymorphisms in contrast to 27% of controls. Thus, celiac disease is associated with a subset of HLA-DR3, -DQw2 haplotypes characterized by HLA-DP alpha- and beta-chain gene RFLPs. Within the celiac-disease patient population, the joint segregation of these HLA-DP genes with those encoding the serologic specificities HLA-DR3 and -DQw2 indicates: (i) that the class II HLA haplotype associated with celiac disease is extended throughout the entire HLA-D region, and (ii) that celiac-disease susceptibility genes may reside as far centromeric on this haplotype as the HLA-DP subregion.

B-Lymphocytes↗

Phorbol myristate acetate induces IL-2 secretion by HUT 78 cells by a mechanism independent of protein kinase C translocation.

The protein kinase C (PKC) inhibitor H-7 was used to study the role of PKC in IL-2 secretion. In Jurkat E6.1, a phenotypically non-activated human T-cell line, IL-2 secretion in response to a combination of PMA and the calcium ionophore, ionomycin, or to the mitogen phytohaemagglutinin (PHA) was completely inhibited by H-7. In contrast, HUT-78, a phenotypically activated T-cell line, secreted IL-2 in response to PMA alone, and IL-2 secretion was not inhibited by doses of H-7 that completely blocked PKC activity. We conclude that PMA can stimulate IL-2 secretion independent of the translocation of PKC activity, and suggest that there is an alternative mechanism of action for phorbol esters in activated T cells.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

The effect of calcium channel blockade with nifedipine on splanchnic and systemic haemodynamics in cirrhosis.

The splanchnic and systemic haemodynamic effects of a single sublingual dose of nifedipine (slow calcium channel blocker) in nine patients with cirrhosis of the liver and portal hypertension were studied. Nifedipine produced a significant reduction in the mean arterial blood pressure (98 +/- 5.3 vs. 86 +/- 5 mmHg, P less than 0.05) but did not alter the mean heart rate, portal venous pressure or total liver blood flow. The systemic antihypertensive effect of nifedipine can be achieved without altering liver blood-flow in patients with chronic liver disease and portal hypertension.

Hemodynamics↗

An HLA-D region restriction fragment length polymorphism associated with celiac disease.

This study is the first to describe a molecular marker that distinguishes the celiac disease HLA-D region haplotype from a serologically identical haplotype in unaffected controls. Using a DQ beta chain cDNA probe and the restriction endonuclease Rsa I, we have detected a polymorphic 4.0 kb fragment which, in DQw2 individuals, is associated with a 40-fold increased relative risk of developing celiac disease. This finding should permit the identification of the celiac disease susceptibility gene(s) in the HLA-D region and facilitate a more precise dissection of the molecular and immunogenetic mechanisms involved in the pathogenesis of that disease.

Celiac Disease↗