Bystander T cells participate in specific response to cockroach antigen (CR) in vitro.

Allergic reactions due to whole body, body parts and fecal products of cockroach (CR) are characterized by inflammatory reaction that may lead to symptoms of rhinitis or asthma in atopic individuals. Although the majority of T cells at the site of CR hypersensitivity are not antigen specific, the cellular subset and cytokine receptors that participate and control the outcome of the reaction have not been fully studied. In this study, we have used fluorescent activated cell sorter (FACS) analysis to characterize the activation marker and cytokine profile of antigen specific and bystander T cells after in vitro stimulation of peripheral blood lymphocytes with whole body extract of CR antigen. There was significant enhancement of CD69 on blast and bystander T cells in all atopic subjects compared to non-atopics. Both antigen specific and bystander T cells showed increased expression of HLA-DR, CD25 and CD71 in 9 of 11 atopic patients compared to control. There was also an increase in CD45RA+ and a decrease in CD45RO+ cells following antigen stimulation. These results correlated with the increase in the early apoptotic cells observed in patients as measured by Annexin V stain. Our data revealed that there was no difference in the expression of CD95 in both stimulated and bystander T cells. However, there was enhancement of FasL by CR antigen, suggesting that the increased apoptosis that was observed was probably due to the Fas/FasL interaction. Positive intracellular IL2, IL-4 and IFN-gamma in T cells were observed in only the antigen specific blast cells in 83% of patients studied. These results suggest interplay of memory T cell response, apoptosis, and activated bystander T cells activities in maintaining cellular homeostasis during allergic reaction in cockroach sensitive atopic subjects.

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.

C5b-7 and C5b-8 precursors of the membrane attack complex (C5b-9) are effective killers of E. coli J5 during serum incubation.

The finding that C9-deficient sera (C9D) can kill serum sensitive strains of Gram-negative bacteria by us and other investigators, questions the role of C9 in the membrane attack complex as necessary for cell death. In these studies we have demonstrated that C5b-8 complexes generated on E. coli J5 during incubation in C9-depleted and C9-neutralized sera are effective in killing Gram-negative bacteria. In the same study, we extended our investigations to show that the deposition of C5b-7 complexes (from C8-deficient [C8D], C8 depleted and C8-neutralized sera) is also effective in killing Gram-negative bacteria. In all cases, these studies demonstrated that when E. coli J5 was incubated with C8D, C9D and pooled normal human serum [PNHS], deposited C5b-9 complexes from PNHS produced more killing than C5b-7 or C5b-8 complexes alone. These experiments clearly demonstrated that C5b-7 and C5b-8 complexes are bactericidal and that multimeric C9 within C5b-9 is not an absolute requirement for inner membrane damage and cell death of Gram-negative bacteria.

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.

Banding resolution of amniotic cell chromosome preparations for prenatal diagnosis.

Fifty consecutive routine female karyotypes of amniotic fluid cells were analyzed to determine: (1) the banding resolution of each metaphase in routine preparations, (2) whether all haploids in the same metaphase had the same level of banding resolution, and (3) which chromosomes showed the least/most variation in regard to banding resolution. For each karyotype, the number of dark bands for each right-hand-side chromosome were counted and compared with an ideogram (ISCN 1985). Banding resolution was separated into three groups: 400-band, 550-band, and 850-band. It was found that all 50 metaphases showed at least a 400-band resolution, and 1 metaphase showed a banding resolution between a 550-band and 850-band level. Although the chromosome preparations were sufficient for routine purposes, additional effort and banding techniques are required to detect small chromosomal abnormalities that require a higher level of resolution.

Prednisone when used as treatment for rejection correlates with poor outcome.

Since January 1974, 195 of 202 (95%) renal transplants have been performed on blacks at the Howard University Hospital Transplant Center. Hypertension is the most common cause of end-stage renal disease (ESRD) at this center (57%). The immunosuppressive regimens utilized were divided into four eras. The first era (1974-1980) consisted of the prophylactic administration of prednisone, Imuran (AZA), and Minnesota antilymphocyte globulin (MAG) with high prednisone dosage used to treat rejection. One-year, two-year, and five-year patient survival rates were 59% 54%, and 41%, respectively. Graft survival rates for the same period were 53%, 47%, and 36%. In the second era (1980-1983), the same immunoprophylaxis was used but only MAG was used to reverse rejection. One-year and two-year patient survival rates were 90% and 84%. Graft survival rates for the same period were 72% and 64%. When era 1 is compared with era 2, statistically significant improvement in patient survival is evident (P less than 0.005). Graft survival rates are statistically significant for one-year graft survival (P less than 0.05). In the third era (1983-1986), cyclosporine was the principal immunosuppressive agent used along with prednisone. Rejection in this era was treated by adjusting the cyclosporine dose to keep the level between 100 ng to 150 ng per mL and in addition to high prednisone. One-year patient survival and graft survival rates were 83% and 55%, respectively. The fourth era began April 1986 and was initiated because of previous bad experiences with high doses of prednisone to treat rejection in era 1.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

The influence of ethionine-supplemented soy protein diet on cell-mediated and humoral immunity.

These studies were designed to investigate the influence of ethionine, a suspected carcinogen, on cell-mediated (CMI) and humoral immunity. It is believed that ethionine, an analog of methionine which is produced by intestinal bacteria, could have significant relevance to health. To study the effect of ethionine on immune responsiveness, three groups of mice were allowed to feed ad libitum for 5 weeks on one of the following regimens: diet 1, a basal diet of 16% soy protein; diet 2, soy protein supplemented with 0.6% dl-methionine; and diet 3, soy protein supplemented with 0.1% dl-ethionine. The immunological parameters measured were responsiveness to mitogens, [phytohemagglutinin (PHA), concanavalin A (Con A), pokeweed mitogen (PWM), and lipopolysaccharide (LPS)], delayed-type hypersensitivity (DTH) to dinitrofluorobenzene (DNFB), and antibody formation to sheep red blood cells (SRBC). There were no significant differences in mitogen and antigen responses in mice maintained on diets 1 and 2 as measured by thymidine uptake in proliferating lymphocytes. However, there was a significant suppression in mitogen responsiveness in mice that received diet 3. DTH was also suppressed in mice on diet 3. Antibody levels were similar in all groups. Thus, there was clear evidence of suppression of CMI by ethionine in these studies.

Reactivity of subpopulations of mouse spleen cells to concanavalin A in vitro.

Subpopulations of spleen cells of mice bearing virus-induced mammary tumors were assessed for responsiveness to Concanavalin A (Con A) under in vitro xenogeneic and syngeneic culture conditions. When cultured with human A+ plasma (xenogeneic), spleen cells of normal, nontumor-bearing BALB/c mice exhibited a high degree of synthesis as compared to unstimulated cells in response to Con A. Spleen cells of mice inoculated with tumor exhibited response exceeding that of their normal counterparts by 59%. When normal BALB/c sera (syngeneic) was used as the supplement, no response of normal spleen cells was observed upon stimulation by Con A; however, spleen cells from tumour bearing individuals demonstrated heightened activity. Tumor-bearer spleen cell response to Con A of mice inoculated with 1 X 10(6) viable cells was biphasic. The peak of the first phase appeared as early as 10 days postinoculation and declined within 3 days after its appearance. The peak of the second phase appeared within 6 days following decline of the first and declined drastically within 3 days after its appearance. Coincidental with the onset of the second phase, the tumor became ulcerated, purulent, and necrotic. Both phases were predominantly T-cell mediated.

Neonatal IgE values in the black population.

Ninety-three cord blood samples from black neonates were evaluted for the total IgE level using the PRIST method. The results reveal values comparable to the IgE levels in other racial groups, suggesting that the influence of racial differences was not obvious at this stage.

Failure of ovalbumin associated with allogeneic spleen ceels to stimulate proliferation of lymph node cells of immune mice.

Ovalbumin-pulsed spleen cells were found to stimulate thymidine uptake of lymph node cells of syngeneic mice immunized with ovalbumin in complete Freund's adjuvant after treatment of spleen cells with Mitomycin C but not after heating the spleen cells at 56degrees for 30 min. Ovalbumin-pulsed spleen cells of allogeneic mice failed to stimulate the immune lymph node cells more than unpulsed cells, although a net increase in the thymidine uptake above the allogeneic stimulation was observed when free ovalbumin was added to the mixed culture. To eliminate the high background of the mixed lymphocyte reaction, F1 mice were made chimeric with bone marrow of one of the parental strains. Using lymph node cells of the immunized chimeras, the stimulation by pulsed spleen cells was much greater when antigen was presented on cells of the parental strain used for bone marrow injection than when presented on cells of the other parental strain.

Age-related changes in cell-mediated immunity in BALB/C mice.

The effect of increasing age on various tests of cell-mediated immunity was investigated in BALB/c mice both in vitro and in vivo with four different assay systems. The following results were obtained. 1) In contact sensitivity to DNFB, old mice (age 60 to 80 weeks) showed no differences in sensitization when compared to young adult mice (age 8 to 12 weeks). (In contrast, old NZB/W mice showed impaired contact sensitization when compared with young NZB/W MICE.)2) Unlike the reaction in contact sensitivity, cells from old BALB/c mice were defective in eliciting a graft-vs-host reaction. This was true also when a partially purified population of T cells was transferred. 3) In the mixed lymphocyte reaction, cells from old mice were as efficient or better than cells from young adult BALB/c mice in responding to or stimulating allogeneic cells. 4) Responses to PHA and Con A (Both T cell mitogens) were greatly reduced when old cells were cultured as compared with cells from young adult mice. Thus, we have found that within the same batch of mice, increasing age was associated with increased capabilities in some measures of cell-mediated immunologic function and decreased capabilities in other measures of the same.

Immunologic reactions to haptens on autologous carriers. I. Participation of both thymus-derived and bone marrow-derived cells in the secondary in vitro response.

Both thymus-derived (T) and bone marrow-derived (B) lymphocytes participate in the response to a hapten 4-hydroxy-3-iodo-5-nitrophenylacetic acid (NIP), coupled to a nonimmunogenic isologous carrier, mouse gamma globulin (MGG). Spleen cells from mice immunized with NIP-MGG show increased DNA synthesis in vitro when cultured with NIP-MGG. The participation of and requirement for T cells in the response was demonstrated by treating the spleen cells with anti-theta serum. This treatment resulted in a 77% inhibition of the antigen response. Furthermore, adoptively transferred normal thymus cells could be specifically "activated" by NIP-MGG in vivo and they responded secondarily to the antigen in vitro. The active participation of B cells in the secondary response was demonstrated by passing the immune spleen cells through a column coated with polyvalent anti-MGG serum. Column filtration reduced the number of NIP-specific plaque-forming cells and NIP-specific rosette-forming cells (both functions of B cells) and produced a 47% inhibition of the NIP-MGG response. The ability of the cells to respond to phytohemagglutinin (PHA) was not affected by column filtration showing that T cells were not being selectively removed. The participation of B cells in the in vitro NIP-MGG response was also shown by treatment of the spleen cells with antiserum specific for MGG and MGG determinants. B cells were removed by treatment with anti-IgM or polyvalent anti-MGG serum plus complement, resulting in a respective 46 and 49% inhibition of the response to NIP-MGG. (Treatment with anti-IgM serum had no effect on T cells.) The contribution of the hapten NIP to stimulation of T cells was investigated using NIP-MGG-activated thymus cells. These activated T cells responded in vitro very well to the NIP-MGG complex but not to the MGG carrier alone demonstrating the requirement of the hapten for T cell stimulation. The response was also partially inhibited (41%) by incubating the activated cells with NIP coupled to a single amino acid (epsilon-aminocaproic acid) before addition of NIP-MGG. These results demonstrated that T cells recognize the hapten NIP when it is coupled to the isologous carrier MGG.

Cells producing low-molecular-weight antibody to Escherichia coli lipopolysaccharide.

CD-1 mice immunized with sheep red blood cells (SRBC) or Escherichia coli lipopolysaccharide (LPS), developed splenic plaque-forming cells (PFC) producing low-molecular-weight antibody; these cells were detected by means of purified rabbit antisera to mouse gamma(1), gamma(2a), and gamma(2b) immunoglobulins. In contrast to SRBC, the primary gamma(1) response to LPS was absent and gamma(2a) and gamma(2b) PFC were detected irregularly. Both immunogens elicited a secondary cellular response in all three subclasses without a corresponding increase in "direct" or gamma(M) PFC. An increase in serum bactericidal antibody following a second injection of LPS was not parallelled by an increase in splenic gamma(M) PFC; it might therefore involve the synthesis of gamma(2) complement-fixing antibody.