Curla S. Walters

Aberrations in T lymphocytes and natural killer cells in vitiligo: A flow cytometric study

Twenty-five patients with vitiligo and twenty-five healthy control subjects were evaluated with the use of flow cytometry to compare percentages of peripheral T lymphocytes and natural killer cells. The percentages of total T lymphocytes, helper T cells, suppressor T cells, and natural killer cells were evaluated with the use of OKT3, OKT4, OKT8, and Leu-7 monoclonal antibodies, respectively. Mean total T lymphocytes and helper T cells were markedly depressed; mean natural killer cells were markedly elevated and mean suppressor T cells were moderately elevated in patients with vitiligo in comparison with control subjects. These results indicate that cell-mediated immunity is subject to some defect in regulation in patients with vitiligo. It remains to be determined whether these abnormalities are a direct cause or a result of vitiligo. Antibody-dependent cytotoxicity, utilizing killer cells with recently reported antimelanocyte antibodies found in patients, may be responsible for pigment cell destruction in vitiligo. Helper T cells may be reduced because of low levels or faulty production of T lymphocyte-stimulating factors in patients or because of a serum factor in patients that is toxic to helper T cells. The presence or absence of autoimmune and/or endocrine disease in patients with vitiligo had no effect on lymphocyte populations. There seemed to be a trend toward lower levels of helper T cells in patients having vitiligo for the shortest amount of time. In summary, the data indicate immunologic abnormalities in patients with vitiligo.

Helicobacter pylori inhibits gastric cell cycle progression

Helicobacter pylori infection of the gastric mucosa is associated with changes in gastric epithelial cell proliferation. In vitro studies have shown that exposure to H. pylori inhibits proliferation of gastric cells. This study sought to investigate the cell cycle progression of gastric epithelial cell lines in the presence and absence of H. pylori. Unsynchronized and synchronized gastric epithelial cell lines AGS and KatoIII were exposed to H. pylori over a 24-h period. Cell cycle progression was determined by flow cytometry using propidium iodide (PI), and by analysis of cyclin E, p21, and p53 protein expression using Western blots. In the absence of H. pylori 40, 45, and 15% of unsynchronized AGS cells were in G(0)-G(1), S, and G(2)-M phases, respectively, by flow cytometry analysis. When AGS cells were cultured in the presence of H. pylori, the S phase decreased 10% and the G(0)-G(1) phase increased 17% after 24 h compared with the controls. KatoIII cells, which have a deleted p53 gene, showed little or no response to H. pylori. When G1/S synchronized AGS cells were incubated with media containing H. pylori, the G(1) phase increased significantly (25%, P < 0.05) compared with controls after 24 h. In contrast, the control cells were able to pass through S phase. The inhibitory effects of H. pylori on the cell cycle of AGS cells were associated with a significant increase in p53 and p21 expression after 24 h. The expression of cyclin E was downregulated in AGS cells following exposure of AGS cells to H. pylori for 24 h. This study shows that H. pylori-induced growth inhibition in vitro is predominantly at the G(0)-G(1) checkpoint. Our results suggest that p53 may be important in H. pylori-induced cell cycle arrest. These results support a role for cyclin-dependent kinase inhibitors in the G(1) cell cycle arrest exerted by H. pylori and its involvement in changing the regulatory proteins, p53, p21, and cyclin E in the cell cycle.

Natural killer cell and lymphokine-activated killer cell activity against melanocytes in vitiligo

BACKGROUND Vitiligo is a common disease of unknown cause. Previous studies have shown abnormalities in natural killer (NK) cell cytotoxicity in patients when NK-sensitive erythroleukemic cell lines were used as target cells. OBJECTIVE The purpose of this study was to use melanocytes directly as target cells to determine NK and lymphokine-activated killer (LAK) cell cytotoxicity in patients with vitiligo and to determine whether NK or LAK cells can be implicated in any destructive mechanism for melanocyte cytotoxicity in vitro in this disease. METHODS Twenty-one patients with vitiligo were compared with a control group by studying NK cell activity (NKCA) and LAK cell activity (LAKCA) on several target cells. These included K562 cells, neonatal melanocytes, and malignant melanoma cells for NKCA and neonatal melanocytes and malignant melanoma cells for LAKCA. Cytotoxicity was measured with the standard chromium 51-release assay. RESULTS No significant differences were found between vitiligo patients and control subjects in NKCA against K562 cells or in NKCA and LAKCA against melanocytes. CONCLUSION NK cells and LAK cells are probably not responsible for melanocyte destruction in vitiligo.

p53 and p14 Increase Sensitivity of Gastric Cells to H. Pylori-Induced Apoptosis

H. Pylori infection of the gastric mucosa is associated with increased epithelial cell apoptosis. In vitro, interferon-γ and TNF-α have been shown to increase the sensitivity of cells to apoptosis induced by H. Pylori. The p53 tumor suppressor gene is frequently mutated in many cancers, including gastric cancer. Since p53 protein can induce apoptosis, we sought to determine whether or not p53 increases the ability of gastric epithelial cells to undergo apoptosis in response to H. Pylori-induced cell injury. Human gastric epithelial cell lines, AGS (p53 wild-type) cells and AGS cells infected with HPV E6 gene (AGS-E6) to inactivate p53 were exposed to H. Pylori. The p53, p21, and p14ARF proteins were measured in gastric epithelial cells by immunoelectrophoresis. Gastric epithelial cell apoptosis was measured by DNA end-labeling assay (TUNEL) and subG0 cell fractions using flow cytometry, and by agarose gel electrophoresis of DNA. Exposure to H. Pylori increased the levels of p53, p21, and p14ARF proteins two fold in AGS cells. Gastric AGS cells with fragmented DNA increased from 1.1% to 68% in after exposure to H. Pylori for 24 hr. However, AGS-E6 cells were relatively resistant to apoptosis induced by H. Pylori (only 15% of cells underwent apoptosis). In additional experiments, mouse embryonic fibroblasts (MEFs) were used to further investigate the role of ARF in stabilizing p53 after exposure to H. Pylori. Wild-type and p19ARF−/− MEFs were exposed to H. Pylori and evaluated for activation of p53, p19ARF, and apoptosis. As with AGS cells, H. Pylori stimulated a 2-fold increase in p53 and p19ARF in wild-type MEFs; however, there was no increase in p53 in ARF-null MEFs. H. Pylori easily stimulated apoptosis in wild-type MEFs, although, the absence of p19ARF significantly reduced the ability of H. Pylori to induce apoptosis in these cells. Activation of ARF by H. Pylori is important in stabilizing p53 resulting in increased apoptosis. Thus, inactivation of either ARF or p53 in gastric cells may reduce their ability to undergo apoptosis in response to injury induced by H. Pylori.

Relationship between peripheral blood lymphocytes and their functional capacity in sarcoidosis

Abstract Sarcoidosis is a disease of unknown etiology characterized by granuloma formation in tissues and anergy to delayed hypersensitivity. In 34 sarcoidosis patients and 16 normal persons, T cells from peripheral blood were quantitated and their functional capacity assessed by sheep red blood cell (SRBC) rosette assay and mitogen response, respectively. Effect of Kveim-Siltzbach suspension (KSS) on rosette formation and its ability to induce blast formation were also examined. Normal spleen cell suspension (NSS) was used as control. The results show that 84% of patients had suppressed T-cell numbers, while 79% had suppression of both active (rosettes formed immediately on incubation with SRBC) and total (rosettes formed after a 1-hr incubation on ice) T cells. Mean active was 30% in sarcoidosis patients vs 44% in controls, while mean total was 48% in sarcoidosis patients vs 64% in controls, while mean total was 48% in sarcoidosis patients vs 64% in control subjects. Suppressed mitogen response to phytohemagglutinin was seen in only 34% of patients and of this number, 90% had decreased T-cell numbers (active and total), suggesting that T-cell suppression does not necessarily reflect functional abnormality in these cells; however, the reverse is true. KSS caused no blastogenesis or change in rosette formation when incubated with lymphocytes from sarcoid patients (KSS active 36%; total 48%: NSS active 34%; total 48%). Either the cells are insensitive to KSS stimulation or sensitive cells have migrated to sites of granuloma formation.