Identification of Outer Membrane Vesicles Derived from Orientia tsutsugamushi

Orientia tsutsugamushi, a causative pathogen of Scrub typhus, is a gram-negative intracellular bacterium. Outer membrane vesicles (OMVs) are produced from the membrane of bacteria and play many roles related to the survival of the pathogen. However, there have been no reports confirming whether O. tsutsugamushi indeed produce OMVs. O. tsutsugamushi boryong was cultured in ECV-304 cells for the purification of OMVs. Western blot analysis and immunoenrichment using anti-O. tsutsugamushi monoclonal antibody and electron microscopy were employed for identification and characterization of OMVs. We confirm the presence of OMVs derived from O. tsutsugamushi, and also found that those OMVs contain a major surface antigen of 56-kDa protein and variant immunogenic antigens.

Effect of Cysticercus cellulosae fractions on the respiratory burst of pig neutrophils

Neutrófilos, eosinófilos e macrófagos são células que interagem com os parasitas no corpo do hospedeiro desenvolvendo atividade antiparasitária. A reação inicial destes leucócitos é a geração de espécies reativas de oxigênio (ERO) a fim de expulsar os parasitas. No presente trabalho estudou-se o efeito da fração total, de escolex e de membrana de Cysticercus cellulosae sobre a explosão respiratória de neutrófilos de suínos. A produção de peróxido de hidrogênio (H2O2) pelos neutrófilos incubados com as frações de C. cellulosae apresentou acréscimo de 190% (extrato total), 120% (escolex) e 44% (membrana). Alta atividade de catalase (33%, 28% e 28% para extrato total, escolex e membrana respectivamente) foi observada nos neutrófilos incubados com as frações de metacestodeo, podendo representar a própria proteção celular do neutrófilo. Frações de escolex e de membrana aumentaram a capacidade fagocitária dos neutrófilos (44% e 28%, respectivamente). Por outro lado, a fração total do cisticerco não alterou a capacidade fagocitária dos neutrófilos, o que pode estar relacionada com modificações na função da membrana celular causadas pela alta produção de ERO na presença da fração total. O extrato total de C. cellulosae é tóxico para os neutrófilos, indicada pela diminuição da capacidade fagocitária, provavelmente pela indução de alto nível de ERO. A diferença de toxicidade do extrato total, de escolex e de membrana para os neutrófilos pode ocorrer pelo efeito antigênico presente no fluido vesicular no extrato total de C. cellulosae.

Immunohistopathological reactions for liver-specific membrane lipo-protein in experimental autoimmune hepatitis.

Antibody to the hepatocyte membrane protein, was induced in inbred strain C57BL/6 and C3H mice by immunisation with 100,000 g supernatant of syngeneic liver homogenate in CFA. Three weekly intraperitoneal injection of 200 ul of liver homogenate with CFA for continuous 4 weeks gave the best possible result. Histopathological changes were characterised mainly by perivascular inflammatory infiltrates and hepatocyte necrosis which mimicked human autoimmune hepatitis. In one of the immunological parameters, antibody to hepatocyte membrane protein (LSP) has been demonstrate by ouchterlony method in the test serum of those animals, who had received weekly doses of liver antigen. Thus in experimental autoimmune liver disease, semi-purified syngeneic liver fluid (S-100) leads to hepatic destruction and to an inflammatory process with several features in common with human chronic aggressive hepatitis. The presence of antibody against syngeneic liver antigen (S-100) in the test sera emphasizes that hepatocyte membrane protein does have an important role in liver tissue pathogenesis and disease process in experimental model. In this study we tried to prove that hepatocyte membrane protein may act as a target antigen in developing experimental autoimmune hepatitis.

An aminopeptidase regulates LPS stimulated interleukin-8 receptor on the surface of human neutrophils.

A large number of inflammatory diseases are mediated by interleukin-8, an inflammatory neutrophil chemotactic agent. Since the cytokine acts through a cell surface receptor, detailed knowledge about the regulation of receptor expression is very important. We found that LPS in serum became activated and triggered the expression of IL-8 receptor by more than two folds within 30 min. After that period, the receptor attained normal level within 2 hr of SA-LPS stimulation. EDTA and bestatin could block this downregulation of IL-8 receptor. Intracellular Ca2+ level was increased till 45 min of SA-LPS stimulation and then the level was reduced. Addition of CaCl2 accelerated and depletion of Ca2+ inhibited the downregulation of the IL-8 receptor. The ligand could fully protect the loss of receptor from downregulation. It suggests that during SA-LPS stimulation, increase in intracellular Ca2+ level activates an aminopeptidase which presumably cleaves the N-terminal region of the receptor, critically essential for the function of IL-8. Thus the activated aminopeptidase regulates the functions of IL-8. The study is important for understanding the regulation of IL-8 receptor expression by LPS during bacterial infection.

Amphiphilic and hydrophilic domains of human sperm membrane antigens recognized by immunoinfertile sera.

Selected sera from married couples with immunological infertility were used to identify antigens on hydrophilic and amphiphilic domains of human sperm membrane. Out of eight sera, six recognized proteins from the hydrophilic as well as amphiphilic regions of the sperm membrane. Sera were either reactive to acrosome or to equator and tail of human sperms in indirect immunofluorescence assay.

Preservaçäo de interferon de membrana amniótica humana / Preservation of the human amniotic membrane interferon

Foram realizadas investigaçöessobre a presença do interferon de membrana amniótica humana (IFN-AM) a fim de permitir pesquisas posteriores a respeito de sua purificaçäo, propriedades químicas e biológicas e ensaios clínicos.Testes de inativaçäo rápida foram realizados para ensejar resultados imediatos.IFN-AM foi preparado pela infecçäo de âmnios pelo vírus da doença de Newcastle.A atividade anti-vírica do IFN-AM foi determinada em um sistema de células Vero-vírus da encefalomiocardite de camundongo ou vírus Sindbis.Para os testes de inativaçäo, preparaçöes de IFN-AM contendo 0;1 e 2 por cento de soro de carneiro foram aquecidas em temperaturas de 35ºC a 100ºC em tempos variáveis e foi medida a atividade antivírica residual.Destes dados, foi calculada a constante de Arrhenius de queda de atividade pelo calor, que foi representada graficamente contra a temperatura absoluta.Os dados de temperaturas altas(45ºC e acima) foram extrapolados para temperaturas baixas(37ºC e abaixo).Os resultados mostraram que o IFN-AM preservou-se melhor em pH 2 em presença de soro.Os dados obtidos nas temperaturas de 45º, 55º e 65ºC permitiram o cálculo de constante de Arrhenius de queda para baixas temperaturas por extrapolaçäo.A -4, 2ºC näo deverá ocorrer perda de ativadade do IFN-AM.

Solubilizacion de la membrana del espermatozoide humano con n-dodecil sarcosinato de sodio: I.accion del detergente / Solubilization of the membrane human spermatozoa: I. action of the detergent

Espermatozoides humanos fueron lavados con PBS y tratados com n-dodecil sarcosinato de sodio (Sarkosyl). El insoluble remanente estaba compuesto por cabezas, cuyas colas fueron cortadas a la altura del cuello o del segmento intermedio y cuyas membranas no sufrieron mayores danos, estando el acrosoma intacto y por pequenos corpusculos que podrian ser acrosomas aislados. Los resultados indican que la accion del detergente es de dos tipos claramente diferenciados: a)diseccion del espermatozoide con separacion de la cola y b)disolucion del mismo dejando el acrosoma intacto. El residuo conserva actividad inmunologica e inmunobiologica

Detection of liver membrane antibodies in patients with non-insulin-dependent diabetes mellitus.

Circulating liver membrane antibodies (LMAb) were examined in 71 patients with non-insulin-dependent diabetes mellitus (NIDDM) without liver dysfunction and 16 cases of KK mouse as a model of obese diabetic animals. LMAb were detected in 10 NIDDM patients (14.1%). Fasting blood glucose, glycosylated serum albumin (G-Alb), and glycosylated hemoglobin A1 (HbA1) levels were compared with LMAb-positive and negative groups. The G-Alb levels and HbA1 levels of the LMAb-positive group were significantly higher (p less than 0.05 and p less than 0.01, respectively) than those of the LMAb-negative group. In addition, there were no differences in histological findings in KK mouse liver between the LMAb-positive and negative cases. These results revealed that the state of continued high blood glucose directly or indirectly influence the autoimmunity and clinical features of NIDDM patients.

Immunopharmacological studies on picrorhiza kurroa Royle-Ex-Benth. Part V: Anti-inflammatory action: relation with cell types involved in inflammation.

Relative importance of various cells involved in inflammation and in anti-inflammatory action of P. kurroa extract (PK) was investigated in albino rats. Effects of chemical depletion of macrophages and polymorphs and a functional deprivement of mast cells and platelets were examined on carrageenin induced pedal inflammation as well as on anti-inflammatory effect of PK treatment in this test. Such depletions/functional deprivements altered the inflammatory response in conformity with the known role of these cells. The anti-inflammatory effect of PK treatment was counteracted at 1 hr, 3 hr and 5 hr post-insult intervals by mast cell, neutrophil and macrophage depletion respectively. Manipulation of platelets was without effect.

Differentiation antigen on murine B lymphocytes defined by monoclonal antibodies.

Spleen cells from an AKR/J X DBA/2J F1 mouse immunized with NZB/BIN spleen cells were fused with SP2/0-Ag14. Two hybrid cell lines, B220-1 and B220-2, were established that secreted antibody to the B-lineage specific B220 antigen. B220-1 and B220-2 are present on 45-55% of splenic and bone marrow lymphocytes and absent from thymus. By flow cytometry, all immunoglobulin-bearing cells were stained by these monoclonal antibodies. Although these monoclonals do not stain thymocytes, they do react weakly with Lyt-2+ peripheral T cells. Dual parameter analysis of B lymphocytes using RA3-3A1 or 14.8 show that these monoclonals recognized the same population. Prior incubation with RA3-3A1 or 14.8 was unable to completely block the binding of B220-1 or B220-2, implying that the epitopes recognized are different from the previously described monoclonal antibodies. Immunoprecipitation of the splenic lymphocyte reveals a molecule which migrates on SDS-PAGE as a single band with MW of 220,000 daltons. Expression of the distinct antigens recognized by B220-1 and B220-2 varied among mouse strains, indicating previously unappreciated polymorphism of the B220 molecule. These monoclonals are useful for cytotoxic elimination of B cells and for three-color flow cytometry.

Surface Properties of Cell Membrane Tested by Lectin Induced Cytoagglutination (I)

This report describes surface properties of several cell membranes tested by lectininduced agglutination reactions which were quantitated using the microquantitative particle counter agglutination assay of Davis et al. (1976). The quantitative assays of concanavaalin A (con A) induced agglutination were performed for rabbit erythrocyte, rat erythrocyte, human erythrocyte, and sarcoma 180 mouse ascites cells. The percent agglutination versus the con A concentration revealed a sigmoid curve in all cases, but the steepness of the sigmoid curve is variable depending on the cell types. It varies even with the same cell but in different species. Optimum cell concentration was (0.92-0.95) x 10(7) cells/ml final concentration in the hanging drop, for rabbit erythrocytes, (0.77-1.64) x 10(7) cells/ml for rat erythrocytes, (1.59-2.7) x 10(7) cells/ml for human erythrocytes and (0.23-0.39) x 10(7) cells/ml for sarcoma 180 mouse ascites cells. When minimal and maximal agglutination percentages were defined as the concentration of con A/ml/1 x 10(6) cells corresponding to 10% and 95% agglutination, minimal and maximal agglutination occured at 0.56 ug, 19.98 ug for human erythrocytes at 0.56 ug, 224 ug for rat erythrocytes at 0.08 ug, 1.43ug for rabbit erythrocytes at 0.12 ug, 14.8 ug for sarcoma 180-mouse ascites cells respectively. The order of inhibitory activity of alpha-methyl-D-mannopyranoside (alphaMM) for each corresponding cells from the highest inhibition was human erythrocytes, rat erythrocytes, sarcoma 180 mouse ascites cells and rabbit erythrocytes. The concentrations of alphaMM required for 50% inhibition per ml of the final concentration in the hanging drop per 1 x 10(7) cells were 0.565 umoles for rabbit erythrocytes, 0.072 umoles for rat erythrocytes, 0.018 umoles for human erythrocytes and 3.677 umoles for sarcoma 180 mouse ascites cells, respectively. From our experimental results we conclude that the cytoagglutination activity was increased with con A, the inhibitory activity with alphaMM in the presence of con A was decreased, however the sarcoma 180 mouse ascites cells revealed a contradictory result, and might be due to the topological distribution of agglutination site changes to a distribution more favorable for agglutination.

Initial characterization of Trypanosoma lewisi surface membrane antigen.

The purified T. lewisi were subjected to hypotonic lysis plus freezing and thawing in acetone dry ice bath. The trypanosome ghosts were obtained after repeated washing and centrifugation. The homogenized ghost suspension was assayed for enzyme Na++K+ ATPase activity to ratify the presence of the trypanosome surface membrane. Membrane solubilized in sodium dodecyl sulfate were fractionated by gel filtration on Sephadex columns equilibrated with the detergent and electrophoresis in polyacrylamide gel. Crude trypanosome surface membrane antigens were tested for their immunogenicity, administered to rats in Fraund's complete adjuvant. The results of these experiments indicated that the protective immunogen is tightly bound to the membrane since the use of strong anionic detergent is necessary in its extraction.