Prokaryotic expression and immunogenicity analysis of latent infection antigen Rv2628c-Rv1737c fusion protein of Mycobacterium tuberculosis / 中国人兽共患病学报

According to the codon characteristics of the prokaryotic system,we synthesized the optimized Rv2628c-Rv1737c nucleic acid sequence and constructed the expression plasmid pET24a-Rv2628c-Rv1737c.After sequencing,expression in E.coli was induced.The fusion protein was found in inclusion bodies.After renaturation and affinity chromatography purifi-cation,the Rv2628c-Rv1737c recombinant protein,with a molecular weight of 57 kDa and a purity exceeding 90％,was ob-tained.The recombinant protein was used to stimulate PBMC cells from different patients,and the differences in IFN-γ mRNA expression were analyzed with q-PCR.The Rv2628c-Rv1737c fusion protein stimulated patients with TB,particularly latent TB infection(LTBI).The level of IFN-γ mRNA in PBMC cells was higher than that in healthy controls(P＜0.05).Mice immu-nized with BCG+Rv2628c-Rv1737c/DMT showed significant induction of high levels of IgG antibodies.Rv2628c-Rv1737c re-combinant protein,as a latent infection antigen,is recognized by PBMCs cells infected with TB,with strong immunogenicity,and thus may serve as a potential TB subunit vaccine target antigen.This protein may be used to prevent TB infection,particu-larly latent infection,and to perform laboratory diagnosis.

EFFECT OF TLR7 LIGAND ON SIGNAL TRANSDUCTION OF INTERFERON GAMMA / Шинэ санаа Шинэ нээлт

@#BACKGROUND: Toll like receptors (TLRs) are a class of proteins that key role in the innate immune system. TLR7 is expressed on monocytes, macrophages and dendritic cells, T cell, B cell and eosinophiles. TLR7, originally identified as recognizing imidaquinoline, loxibrine, broprimine and ssRNA, ssRNA viruses such as vesicular stomatitis virus, influenza A virus and human immunodefiency virus. It is known that virus ssRNA affects signaling molecule of IFN-y. Objective: To determine gene and protein activation of IFN-y signal transduction by TLR7 ligand in the endothelial cells. MATERIAL: In study we used mouse aortic linear endothelial cell which is cultured (END-D) in 5% heat- inactivated fetal calf serum (FCS), medium (DMEM) containing antibiotic mix(penicillin G, streptomycin, amphotericin B) at 37°C (5% CO2). Endothelial cells treated with synthetic IFN-γ and imiquimodligands, then the NO (nitric oxide) concentration in the supernatant is determined by Griess reagent. Endothelial cells are cultured in 6 well cell culture plate and in each well 2*104cells are expected to be grown for 24 hours of culture. Then, the cells are treated with synthetic IFN-γ and имиквимод ligand for 6 hours and the NO signaling gene activation iNOS mRNA expression which is induced by IFN-γ is determined by RT-qPCR. Endothelial cells are cultured in 12 well cell culture plate and in each well 2*104 cells are expected to be grown for 18 hours of culture. Then, the cells are treated with synthetic IFN-γ and imiquimodligands for 24 hours and the NO signaling protein iNOS expression which is induced by IFN-γ is determined by western blotting. The experiment was conducted as representation mean of at least three test results. The difference between statistical probabilities is determined by the “Students” t test. The p<0.01 value is assumed to be statistically different. RESULTS: TLR7 ligand imiquimodaugmented interferon gamma induced nitric oxide production TLR7 ligand imiquimodincreased interferon gamma induced iNOS mRNA gene expression. TLR7 ilgand imiquimodup-regulated interferon gamma induced iNOS protein expression. CONCLUSIONS: TLR7 ligand imiquimod augments IFN-γ signaling in the endothelial cells. This synergistic effect has revealed in the levels of gene and protein expression.

Valoración de las pruebas diagnósticas para tuberculosis bovina en un rebaño bovino ubicado en zona de alta incidencia del estado Zulia Venezuela / Valuation of diagnostic tests for bovine tuberculosis in cattle herd located in a high incidence area of Zulia state Venezuela

Las pruebas de tuberculina son las de uso generalizado para el diagnóstico y el control de la tuberculosis (TBC) en el hombre y en los animales. Se caracteriza por una compleja mezcla de antígenos de mycobacterias capaces de inducir reacciones de hipersensibilidad en animales infectados, incluso con mycobacterias diferentes al Mycobacterium bovis, por efectos de reactividad cruzada. La preparación del derivado protéico purificado (PPD), es similar a la de la tuberculina, a diferencia de la concentración de proteínas, las cuales se separan por precipitación con agentes químicos y no por calor, aumentando su especificidad. Los primeros resultados obtenidos con las pruebas serológicas para el diagnóstico de tuberculosis bovina muestran que existe una gran reactividad antigénica cruzada entre las especies de mycobacterias, por lo que se requiere de antígenos más específicos. Se implementó un ELISA-TBC para la detección de anticuerpos anti M. bovis. El ensayo inmunoenzimático para el IFN-y bovino cuando se utilizó conjuntamente con el sistema de cultivo de sangre completa resultó en un ensayo in vitro rápido y sensible para detectar la reactividad de la inmunidad mediada por células al M. bovis en el ganado infectado. A partir de estas pruebas se compararon los resultados obtenidos para establecer la sensibilidad y especificidad utilizando como prueba oro, los datos obtenidos en el cultivo bacteriológico y la reacción en cadena de la polimerasa (PCR). Los animales reaccionantes a la tuberculina incluyeron animales positivos a PPD-B y PPD-A, así como animales negativos a cultivo bacteriológico y PCR. Los PPD-B positivos, no son en su totalidad, los mismos reaccionantes al IFN-y o al ELISA-TBC. Aún cuando su sensibilidad es baja, muestra mayor especificidad y concordancia que el resto de las pruebas utilizadas.

The tuberculin tests are widely used for diagnosis and control of tuberculosis (TB) in humans and animals. It is characterized by a complex mixture of mycobacteria antigens able to induce hypersensitivity reactions even in animals infected with mycobacteria other than M. bovis, for purposes of cross-reactivity. The preparation of purified protein derivative (PPD) is similar to the tuberculin, unlike the concentration of proteins which are separated by precipitation with chemical agents and not by increasing its specific heat. The first results obtained with the serological tests for diagnosis of bovine tuberculosis show that there is a great antigenic cross-reactivity between mycobacterias species so it requires more specific antigens. It implemented a cattle IFN-g test and ELISA-TBC to detect anti M. bovis activity. The immunoassay test for IFN-g used in conjunction with the cropping system of whole blood resulted in an essay in vitro rapid and sensitive to detect the reactivity of the cell-mediated immunity to M. bovis in livestock infected. Comparative test of the tuberculina, test of Gamma Interferon (INF-y) and a test ELISA-TBC, soon was taken to slaughter house to take linfoides weave samples and nodules, to which the test of chain reaction of Polimerasa was applied to them, to bacteriological culture and (PCR), for the identification from the pathogen. From these tests the patterns of immune response settled down and the obtained results of the different tests were compared to establish sensitivity and specificity using with t gold standard, the data collected in culture and PCR. The results were analyzed using the statistical method of analysis of variance for nonparametric tests. The PPD B-positive, are not the same reacting to IFN-y or at ELISA-TBC. Although its sensitivity is low, it shows greater specificity and consistency as the rest of the tests used.

Citosinas sericas (IL-4, IL-6 e IFN-y) en embarazadas de la ciudad de Valencia Venezuela / Serum cytokines (IL-4, IL-6 e IFN-y) in pregnant women from Valencia city

A fin de evaluar el comportamiento de algunas citosinas o interleukinas (IL-4, IL-6, IFN-y) durante el embarazo, seestudiaron 25 mujeres embarazadas durante los tres trimestres, 12 adolescentes y 13 adultas. Las determinaciones de las interleukinas se realizaron por el método de ELISA, tipo sándwich, observándose incremento significativo (p<0,05) para la IL-4 entre el 1er y 2do trimestre (8,18±2,43 vs 53,42±17,25 pg/ml) y entre el 1er y el 3er (8,18±2,43 vs 42,47 ± 6,27 pg/ml) pero no entre el 2do y el 3er (53,42 ± 17,25 vs 42,47 ± 6,27 pg/ml) para todas las embarazadas, con un patrón similar en las adolescentes y adultas, pero la comparación entre ellas por edad sólo mostró diferencia significativa para la evaluación del 2do trimestre, correspondiendo a las adultas niveles más altos (60,53± 17,47 vs 45,71±13,85 pg/ml). Con relación a la IL-6, se observó que los valores se incrementan a medida que avanza el embarazo con diferencia significativa (p<0,05) entre los trimestres (2,01±3,46; 9,25±7,22; 11,54±9,40 pg/ml) pero similar para ambos grupos etarios sin diferencia estadística significativa entre ellos en ninguno de los periodos. Para el IFN-y se observó un ligero aumento para todas las embarazadas que no alcanzó diferencia significativa entre los trimestres ni para los grupos etarios considerados. Se concluye que el embarazo induce aumentos significativos de IL-4, de IL-6 y en menor grado de IFN-y y que estos cambios no están asociados a la edad de la embarazada.

In order to assess interleukin levels (IL-4, IL-6, IFN-y) during pregnancy, 25 pregnant women (12 adolescents and13 adults) along the three trimesters were evaluated. An ELISA test was performed to determine serum interleukin. A significant increase (p<0.05) for IL-4 between first and second trimester (8.18±2.43 vs. 53.42±17.25 pg/ml) and between first and third (8.18±2.43 vs. 42.47 ± 6.27 pg/ml) but not between second and third (53.42 ± 17.25 vs. 42.47 ± 6.27 pg/ml) was observed. Similar pattern of increase was found for adolescents and adults, but comparison by age showed significantly higher levels in adults at second trimester (60.53± 17.47 vs. 45.71±13.85 pg/ml). Regarding IL-6, increase during pregnancy was also observed, reaching significant differences between trimesters (2.01±3.46; 9.25±7.22; 11.54±9.40 pg/ml): A similar pattern was found for adolescents and adults, but no differences by age were found at any moment. For IFN-y, a slight increase was observed but it did not reach significant differences either between trimesters or by age groups. It is concluded that pregnancy induced significant increases of IL-4 and IL-6, and in a lesser degree for IFN-y but age of the women was not related to the changes.

Canges of Peripheral IFN-r-Producing CD4(+) and CD4(-) Cell Frequencies in Kawasaki Disease

PURPOSE: This study was performed to analyze the frequencies of peripheral IFN- gamma -producing cells at the single cell level, and to determine concentrations of circulating interferon- gamma (IFN- gamma) in the acute and subacute phases of Kawasaki disease(KD). METHODS: Ten patients with KD were studied and seven healthy children were selected as control subjects. Using immunofluorescent detection of intracellular IFN-gamma in CD4(+) and CD4(-) the cells, the frequencies of IFN-gamma -producing cells in peripheral blood mononuclear cells(PBMC) were studied. Circulating IFN-gamma levels were measured by enzyme-linked immunosorbent assay. RESULTS: The frequencies of peripheral blood CD4(+) and CD4(-) IFN- gamma -producing cells in acute phase KD patients were significantly lower than in subacute phase KD patients and control children(P<0.05). CD4(-) cells, thought to be mainly composed of CD8(+) cells, appeared to be more responsible for the reduced frequencies of total IFN- gamma -producing cells than CD4(+) cells. There were, however, no differences in frequencies of IFN- gamma -producing cells between KD patients in the subacute phase and control group children. In contrast, serum IFN- gamma levels were higher in KD patients in the acute phase than in the subacute phase(P<0.05). Conclusion The above results show an increased level of circulating interferon-gamma and a decreased emergence of peripheral IFN-gamma-producing cells in acute KD patients, suggesting transient infiltration of activated IFN-gamma-producing cells into the inflammatory sites during acute KD. These findings also support the hypothesis that IFN-gamma plays an irnportant role in the pathogenesis of KD-related vasculitis.

Change of expression of IL-4, IL-5, and IFN-r mRNA in Der p I-specific T-cell clones / 천식및알레르기

BACKGROUND: It is known that immunotherapy promotes the development of allergen-specific Thl-like lymphocytes whose products are effective in inhibiting clinical response of sensitized atopic patients to allergen exposure. At the single cell level in short term culture, however, IL-4 and IL-5 are co-expressed, while IL-4 and IFN-y are exclusively expressed. OBJECTIVE: IL-4, IL-5, and IFN-y mRNA were measured in Der pI-specific T-cell clones (TCCs) to evaluate whether expression of cytokine in allergen-specific TCC is fixed regardless of stimuli. METHOD: Seven Der pI-specific TCCs were made from two asthmatics sensitive to D. pteronyssinus. IL-4, IL-5, and IFN-y mRNA were measured by RT-PCR in these TCCs after antigen-specific (Der pI) and nonspecific (PHA + TPA) stimuli. RESULTS: IL-4 and IL-5 mRNA were expressed in four and six of seven TCCs, but IFN-y mRNA was not expressed in any TCCs after Der pI-specific stimuli. Meanwhile, after the stimulus of TPA plus PHA, IFN-y mRNA as well as IL-4 and IL-5 mRNA were expressed in four of seven TCCs, and in one TCC, only IFN-y mRNA was expressed without expression of IL-4 mRNA. CONCLUSION: The expression of cytokine may be variable in allergen-specific TCC according to the type and amount of stimuli.

A Study of Serum Cytokines in Nephrotic Syndrome

PURPOSE: It has been reported that patients with nephrotic syndrome have high serum IgE value and IL-4 involve in IgE synthesis, IL-6 is an autocrine growth factor for the proliferation of mesangial cells. We studied association between serum cytokines and of nephrotic syndrome. METHODS: We measured serum IL-6, IL-4, IFN-Y, CD23 in 14 children with nephrotic syndrome and 3 healthy children by ELISA. And we also measured serum IgG, IgA, IgM and IgE in 14 children with nephrotic syndrome. RESULTS: 1) The serum IL-4 and CD23 levels of nephrotic syndrome were significantly higher than that of control (P<0.01, P<0.05 respectively). 2) There were no differences in serum IL-6 and IFN-Y levels between control and nephrotic syndrome. 3) The serum value of IgG in nephrotic syndrome were more decreased than that in control group. 4) The serum value of IgE in nephrotic syndrome were more increased than that in control group. 5) There was no differences in the serum values of IgA and IgM between nephrotic syndrome and control group. CONCLUSIONS: The children with nephrotic syndrome had higher serum IL-4, CD23, IgE level than healthy children. In conclusion, nephrotic syndrome is highly associated with the cytokines. I expect that the further study of cytokines in nephrotic syndrome is useful for treatment and prediction of prognosis of nephrotic syndrome.

A Study of Serum Cytokines in Nephrotic Syndrome

PURPOSE: It has been reported that patients with nephrotic syndrome have high serum IgE value and IL-4 involve in IgE synthesis, IL-6 is an autocrine growth factor for the proliferation of mesangial cells. We studied association between serum cytokines and of nephrotic syndrome. METHODS: We measured serum IL-6, IL-4, IFN-Y, CD23 in 14 children with nephrotic syndrome and 3 healthy children by ELISA. And we also measured serum IgG, IgA, IgM and IgE in 14 children with nephrotic syndrome. RESULTS: 1) The serum IL-4 and CD23 levels of nephrotic syndrome were significantly higher than that of control (P<0.01, P<0.05 respectively). 2) There were no differences in serum IL-6 and IFN-Y levels between control and nephrotic syndrome. 3) The serum value of IgG in nephrotic syndrome were more decreased than that in control group. 4) The serum value of IgE in nephrotic syndrome were more increased than that in control group. 5) There was no differences in the serum values of IgA and IgM between nephrotic syndrome and control group. CONCLUSIONS: The children with nephrotic syndrome had higher serum IL-4, CD23, IgE level than healthy children. In conclusion, nephrotic syndrome is highly associated with the cytokines. I expect that the further study of cytokines in nephrotic syndrome is useful for treatment and prediction of prognosis of nephrotic syndrome.

Effects of Mycobacterium leprae , M . leprae Phenolic Glycolipid - 1 and Cytokines on the Nitric Oxide Generation of RAW 264 . 7 Macrophages / 대한피부과학회지

Background: Nitric oxide(NO) has been reproted to play an important role in macrophage-mediated microbicidal capacity for a variety of intracellular pathogens. NO generation is used as an indicator of microbicidal function of macrophages. OBJECTIVE: Our purpose is to investigate the production of NO rom macrophages phagocytized with Mycobacterium leprae or M. leprae phenolic glycolipid-1(PGL-1) for the purpose of elucidating the pathogenesis of leprosy. METHODS: We used a murine macrophage cell line, RAW 264.7. Macrophages were incubated with dead M. leprae or PGL-1, respectively and then treated with interfer n-gamma(IFN-r) and/or tumor necrosis factor-alpha(TNF-a). The release of NO was determined spectrophotometrically by measuring nitrite. RESULTS: M. Leprae and PGL-1 failed to stimulnte NO secretion execept at high bacteria-to-cell rations(50:1)and at the higheat concentrat,ion(100pg/ml) of PGL-1. IFN-r or IFN-r plus TNF-a markedly stimulated macrophages phagocyt,ized with M. leprae or PGL-1 to release NO . CONCLUSION: Defective IFN-r-dependent NO production of macrophages may be an important factor in the pathogenesis of leprosy.

The Antitumor Effects of Mice Peritoneal Macrophages Transferred IL-2 and IFN-Y Gene in vivo / 中国肿瘤生物治疗杂志

The eukaryotic expression vector pREP-8-IL-2 or and pREP-8-IFN-? was injected i.p. into mice by calcium phosphate coprecipitation method and IL-2 gene or IFN-? gene was successfully transfected into peritoneal M?s whose expression products could enhance the cytotoxicity of M?s, which secrete some TNF, IL-1 and NO, activate the nonspecific immunity and inhibit the growth of tumor effectively. In particular, IL-2 gene in combination with IFN-? gene transfection were successfully transfected into peritoneal M?s whose high expression products not only significantly enhance the M?s cytotoxicity and made it secrete high level TNF, IL-1 and NO, but also activated the CTLs of spleen and initiated specific immunity and nonspecific immunity . This would produce synergic antitumor effects. The results showed that IL-2 and IFN-? gene transfection produced more antitumor effects than IL-2 or IFN-? gene transfection alone.

Antiproliforative effect and HLA-DR induction of recombinant gamma interferon on cultured human keratinocytes / 대한피부과학회지

The authors investigated the antiproliferative effect and expression of HLA-DR an- tigen by recombinant gamma-interferon (r-IFN-y) on cultured human keratinocytes (KC). The results were as follows, 1. From 10l.J/ml of r-1FN-p exposure, the proliferation of KC decreased in a concentration dependent fashion. But there was little difference of antiproliferative effect above 30U/ml of r-IFN-y exposure. 2. The expression of HLA-DR antigen on KC increased in a concentration and time dependent fashion of r-IFN-p exposure. E3ut t,here was little difference of HLA-DR antigen expression on KC above 30tJ/ml and most of HLA-DR antigen were expressed within 48hr. 3. The opt,imal condition for HLA-DR antigen induction on KC by r-IFN-p was likely t,hat HLA-DR KC was observed at 48hr under the our exposure of 30U/ml of r-IFN p. 4. After 4hr exposure of 30U/ml of r-IFN-p, KC expresed HLA-BR. antigen, reaching a maximum intensity at 3 days. At, 7 days, the loss of HI A-DR KC showed over 90% of maximum intensity.