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Epigallocatechin gallate modulates cytokine production by bone marrow-derived dendritic cells stimulated with lipopolysaccharide or muramyldipeptide, or infected with Legionella pneumophila.

The primary polyphenol in green tea extract is the catechin epigallocatechin gallate (EGCG). Various studies have shown significant suppressive effects of catechin on mammalian cells, either tumor or normal cells, including lymphoid cells. Previous studies from this laboratory reported that EGCG has marked suppressive activity on murine macrophages infected with the intracellular bacterium Legionella pneumophila (Lp), an effect mediated by enhanced production of both tumor necrosis factor-α (TNF-α) and γ-interferon (IFN-γ). In the present study, primary murine bone marrow (BM)-derived dendritic cells (DCs), a phagocytic monocytic cell essential for innate immunity to intracellular microorganisms, such as Lp, were stimulated in vitro with the microbial stimulant lipopolysaccharide (LPS) from gram-negative bacteria, the cell wall component from gram-positive bacteria muramyldipeptide (MDP) or infected with Lp. Production of the T helper cell (Th1)-activating cytokine, interleukin-12 (IL-12) and the proinflammatory cytokine, tumor necrosis factor-α (TNF-α), produced mainly by phagocytic cells and important for antimicrobial immunity, was determined in cell culture supernatants by enzyme-linked immunosorbent assay (ELISA). Treatment of the cells with EGCG inhibited, in a dose-dependent manner, production of IL-12. In contrast, enhanced production of TNF-α occurred in a dose-dependent manner in the DC cultures stimulated with either soluble bacterial product or infected with Lp. Thus, the results of this study show that the EGCG catechin has a marked effect in modulating production of these immunoregulatory cytokines in stimulated DCs, which are important for antimicrobial immunity, especially innate immunity. Further studies are necessary to characterize the physiologic function of the effect of EGCG on TNF-α and IL-12 during Lp infection, and the mechanisms involved.

Legionella pneumophila infection up-regulates dendritic cell Toll-like receptor 2 (TLR2)/TLR4 expression and key maturation markers.

ABSTRACT Dendritic cells (DCs) have a critical role in linking innate to adaptive immunity, and this transition is regulated by the up-regulation of costimulatory and major histocompatibility complex (MHC) molecules as well as Toll-like receptors. These changes in DCs have been observed to occur following microbial infection, and in the present study, we examined the effect of Legionella pneumophila infection on the expression of these DC markers. We showed that bone marrow-derived DC cultures from BALB/c mice infected with live L. pneumophila resulted in the up-regulation of Toll-like receptors 2 and 4 and the activation of CD40, CD86, and MHC class I/II molecules.

A quantitative immunofluorescence test for the detection of anti-Candida antibodies☆

A quantitative immunofluorescence assay for anti-Candida antibodies has been developed using a recently introduced system that includes an automatic fluorometer and a special immunoadsorbent for antigen coating. A commercially available cytoplasmic antigen preparation was adsorbed into the substrate, and after incubation with sera from patients with systemic candidiasis or from normal controls, the antibodies bound to the antigen-coated immunoadsorbent were revealed by the use of fluorescein-labeled antisera to human immunoglobulins. Using doubling dilutions of a high titer serum, a positive relation was found between antibody concentration and the logarithm of the intensity of fluorescence. Quantitative assays of unknown samples were performed using a calibration curve constructed from dilutions of that strongly positive sample; the results of antibody determinations were expressed as percentages of the control. Seven of 9 sera from patients with systemic candidiasis, and only 2 of 42 from asymptomatic individuals, had antibody levels considered significant in this assay. Precipitating antibodies were detected by counterimmunoelectrophoresis in all patients and in 18 of the asymptomatic controls; measurable antibody levels were also found in 14 controls showing no precipitating antibodies. This assay is simple, sensitive and inexpensive, and its quantitative nature makes it useful in the investigation of the immune response to C. albicans.

Bovine dialyzable lymph node extracts have antigen-dependent and antigen-independent effects on human cell-mediated immunity in vitro.

Abstract We have studied the effects of dialyzable lymph node extracts (DLNE), from bovines immune or nonresponsive to tuberculin, or human cell-mediated immunity to purified protein derivative of tuberculin (PPD) in vitro . The effects of bovine DLNE (B-DLNE) on the immune responsiveness of human cells were evaluated using both the direct and indirect agarose leukocyte migration inhibition (LMI) assays to measure the migration of human leukocytes, and by determining the effects of B-DLNE on the rate of regeneration of receptors for sheep red blood cells (SRBC) on human thymus-derived (T) lymphocytes after trypsinization. The effects of human dialyzable leukocyte extracts (H-DLE) were also evaluated in both systems, as controls. The results indicated that both B-DLNE and H-DLE contain a “transfer factor-like” activity which can be detected in vitro with the agarose LMI assay. Both B-DLNE and H-DLE contained components which promoted measurable specific antigen responsiveness in previously nonimmune lymphocytes; specificity was shown by analyses of the migration of human leukocytes in the presence of PPD, coccidioidin, or candida antigen in the presence and absence of H-DLE or B-DLNE from donors specifically responsive or nonresponsive to the various antigens. The components promoting specific antigen responsiveness were dialyzable and were derived only from lymphoid tissues. Dialysates from brain, liver, or skin failed to promote similar effects. When the amount of B-DLNE tested was less than that found to be optimal for promotion of antigen-specific responsiveness, pronounced enhancement of migration of human polymorphonuclear neutrophils (PMN) was seen, similar to the results obtained previously with H-DLE. Both B-DLNE and H-DLE significantly increased the rate of SRBC receptor generation on human T cells (up to a threefold increase at optimum concentrations). This activity, termed “mononuclear leukocyte-derived maturation factor” (LDMF), was found preferentially in lysates of lymphoid or reticuloendothelial organs and cells.

Carcinoembryonic antigen and cystic fibrosis protein in blood from cystic fibrosis homozygotes and heterozygote carriers.

Carcinoembryonic antigen (CEA) activity was measured by radioimmunoassay in blood from cystic fibrosis (CF) homozygotes, heterozygote carriers of CF, normal healthy controls, and other patient controls with carcinomas involving gastrointestinal organs. All samples were also screened by electrofocusing for cystic fibrosis protein (CFP), a metabolic marker previously shown to be associated with the CF gene. Significantly increased levels of CEA activity were found in all CFP‐positive groups; however, with one exception all patient controls with marked increases in CEA activity were CFP‐negative. Immunodiffusion of perchloric acid extracts of CEA‐like material from heterozygote carrier blood indicated that the CEA‐like material, which was elevated in homozygotes and heterozygotes for CF, showed only partial identity with two separate CEA preparations obtained from colon carcinomas and was not identical to either A, B, or O (H) blood group substances. This glycoprotein material did, however, react with three different anti‐CRA antisera. Our finding of an abnormally increased glycoprotein in cystic fibrosis, taken together with previous reports demonstrating abnormalities in the carbohydrate portion of glycoproteins found in various exocrine secretions in CF, further suggests that the primary defect in this disease is manifested partly as a defect in glycoprotein metabolism. This defect may result from an abnormality in one or more of the glycosyltransferases, possibly caused by a more primary defect in polyamine metabolism.

Alpha-1-antitrypsin (Pi) types in Down's syndrome.

Alpha‐1‐antitrypsin (Pi) phenotypes have been determined in 40 patients suffering from Downs syndrome. Thirty‐six of the patients were found to have a normal M phenotype, whereas two deficient phenotypes of the MS variety were observed. In addition, two M variants were noted. The significance of an M variant phenotype in some patients with Downs syndrome is not completely understood and is currently under investigation. Since the majority of the patients had a normal alpha‐1‐antitrypsin phenotype, the results of this study indicate that a deficiency in alpha‐1‐antitrypsin plays no role in the respiratory fragility of individuals with Downs syndrome.

White cell subpopulations in autoimmune infertile men and their wives with cytotoxic sperm antibodies.

Sixty six infertile men with cytotoxic sperm antibodies had fewer and lower percentages of OKT4-positive helper/inducer T lymphocytes p = 0.004 and 0.003, respectively), than the fertile (n = 14) and infertile (n = 12) antibody-negative controls. Ratio of OKT4/OKT8 was decreased (p less than 0.01), while numbers of OKDR and BD-Leu12 positive predominantly B lymphocytes were increased (p less than 0.0001) in infertile men with sperm antibodies. T lymphocytes positive for OKT3, OKT11 and OKT4 and the OKT4/OKT8 ratio were decreased in infertile women with sperm antibodies, while B lymphocytes positive for OKDR and BD-Leu12 were increased in infertile women with or without sperm antibodies (p less than 0.0001, versus fertile controls). Lymphocyte responses to phytohemagglutinin (PHA), concanavalin A (ConA) and pokeweed mitogen (PWM), however, were similar in 20 fertile and 136 infertile couples. Lymphocytes from infertile men with sperm antibodies had an enhanced stimulatory response to autologous sperm and seminal plasma. It is concluded that sperm antigens from autoimmune infertile men stimulate heightened immune responses to sperm antigens and altered distribution of white cell populations in both partners.

Enzyme-linked immunosorbent assay for detection of immunoglobulin G and M antibodies to teichoic acid in intravascular staphylococcal disease

An enzyme-linked immunosorbent assay (ELISA) for detection of IgG and IgM antibodies to cell-wall teichoic acids ofStaphylococcus aureus and three defined coagulase-negative staphylococci was tested using serum samples from 11 cases of intravascular coagulasenegative staphylococcal infections, 13 cases ofStaphylococcus aureus endocarditis, and 24 patients with no evidence of infection. IgG antibody titers to all four teichoic acids in the 13 patients withStaphylococcus aureus endocarditis were significantly different from those in noninfected control patients (p<0.0001). In contrast, IgG antibody titers in serum from 11 cases of intravascular coagulase-negative Staphylococcal infection were not significantly different from those in control sera. There were no differences in IgM antibody titers of the three groups. Although the ELISA was sensitive in detectingStaphylococcus aureus endocarditis, it was not reliable in the detection of intravascular coagulasenegative Staphylococcal infections, even when tested with specific teichoic acid.

Lymphocyte Subsets in Lymph Node Hyperplasias and B Cell Neoplasms as Determined by Fluoresceinated Antibodies and Flow Cytometry

In an attempt to see how monoclonal antibody staining and cell flow cytometry could be applied to lymphocyte suspensions made from surgically obtained tissues, 21 hyperplastic lymph nodes and 19 B cell neoplasms were analyzed. Information was obtained concerning percentages of lymphocyte subsets in different patterns of B cell hyperplasia and the correlation of helper/suppressor ratios in lymph nodes as compared to peripheral blood. In B cell neoplasms the immunotype of the neoplasm was identified and the composition of residual nonneoplastic/-reactive lymphocytes was determined. The expression of differentiation and activation antigens, e.g. transfemn receptor and OKTlO, in certain cases was also determined. In some cases of B cell neoplasm, peripheral blood was also analyzed and assessment of blood involvement was made as well as correlation of the distribution of lymphocyte subsets between the tissue compartment and blood compartment.

Immunoglobulin levels and liver function tests in normal and toxemic pregnancies

Abstract Serum immunoglobulin G and M levels were measured and liver function studies were carried out in 12 normal pregnant women, 36 pre-eclamptic women, and 5 women with pre-eclampsia superimposed upon chronic hypertension. Although IgG levels were slightly lower and IgM levels were slightly higher in the pre-eclamptic subjects, there was no significant difference in immunoglobulin levels between the normal and pre-eclamptic groups. Age and parity were not found to affect immunoglobulin levels in pre-eclamptic patients. There was no appreciable difference in liver function tests between the normal and pre-eclamptic groups except alkaline phosphatase levels in the toxemic primigravida group which were slightly higher than normal pregnancy levels.