Detection and characterisation of circulating cysticercal antigens in human cerebrospinal fluid

With biotin labelled and unlabelled immunoglobulin fraction of anticysticercal antibodies raised in rabbits, tandem-enzyme linked immunosorbent assay (T-ELISA), capture-dot immunobinding assay (C-DIA) and reverse passive haemagglutination (RPHA) tests were developed for the detection of cysticercal antigens. The sensitivity levels were respectively, 9 ng ml−1, 2 ng ml−1 and 45 ng ml−1. All three methods were of equal specificity as none of the antigens of Mycobacterium tuberculosis, Japanese encephalitis virus and Echinococcus granulosus reacted with anticysticercal IgG. Cysticercal antigens were detected in the cerebrospinal fluid (CSF) of confirmed neurocysticercosis at sensitivity levels of 91·6% by T-ELISA, 83·33% by C-DIA and 75% by RPHA and specificity levels of >93%. Western analysis of these antigens in CSF showed mainly antigens of 64–68 kDa and 24–28 kDA. By crossed immunoelectrophoresis (CIE) with an intermediate gel technique, five circulating antigens were found to be released from scolex and fluid.

Demonstration of IgE Antibodies to Nucleic Acid Antigens in Patients with Sle

Using a combination of avidin-biotin microELISA and solid phase radioimmunoassay, we examined sera from 23 patients with systemic lupus erythematosus (SLE), two patients with established sensitivity to ingested shrimp, and 15 healthy normal subjects. In addition to IgG antibodies, varying amounts of IgE antibodies specific for native DNA (nDNA), denatured or single-stranded DNA (dnDNA), RNA, and tRNA were demonstrable in the sera of SLE patients, but not in the sera of normal subjects. A comparison of the specificity of nucleic acid-specific IgE antibodies present in the sera of shrimp-sensitive patients with those present in the sera of seven SLE patients revealed that the IgE antibodies in the sera of shrimp-sensitive patients specifically recognized shrimp tRNA but not yeast tRNA, calf thymus RNA, or calf thymus DNA, while those present in the sera of patients with SLE recognized all these nucleic acid antigens. The IgE antibodies directed against nDNA, dnDNA, RNA, and tRNA may mediate mast cell and basophil degranulation and thus contribute both to immediate-type hypersensitivity phenomena including hives seen in patients with SLE and to the localization of IgE-nucleic acid complexes in target

Development and characterization of monoclonal antibodies against WbSXP-1 for the detection of circulating filarial antigens

The importance of developing effective assays to diagnose, monitor and evaluate human lymphatic filariasis has been emphasized by the World Health Organization. Presently, few immunodiagnostics are available for filarial monitoring programmes. The Wuchereria bancrofti (Wb) SXP-1 parasite protein, with 84% homology to Brugia malayi (Bm) SXP-1, was found to be highly immunogenic. WbSXP-1 is one among the diagnostic candidate molecules that were used for developing a rapid-antibody-flow-through diagnostic kit for filariasis. Studies were initiated with the aim of developing monoclonal antibodies against recombinant WbSXP-1 and prospective applications for the detection of both circulating Wb and Bm antigens in serum samples from infected individuals. The monoclones 1A6C2 of subclass IgG1k, and 2A12F8 of class IgM, specifically detected Wb and Bm microfilaria isolated from patients and did not show cross-reactivity with other filarial recombinant antigens. We anticipate that this work will address the problems faced in the rapid diagnosis of human lymphatic filariasis in endemic areas in developing countries.

HLA-B*57 and Gender Influence the Occurrence of Tuberculosis in HIV Infected People of South India

Background. Substantial evidence exists for HLA and other host genetic factors being determinants of susceptibility or resistance to infectious diseases. However, very little information is available on the role of host genetic factors in HIV-TB coinfection. Hence, a longitudinal study was undertaken to investigate HLA associations in a cohort of HIV seropositive individuals with and without TB in Bangalore, South India. Methods. A cohort of 238 HIV seropositive subjects were typed for HLA-A, B, and DR by PCR-SSP and followed up for 5 years or till manifestation of Tuberculosis. HLA data of 682 HIV Negative healthy renal donors was used as control. Results. The ratio of males and females in HIV cohort was comparable (50.4% and 49.6%). But the incidence of TB was markedly lower in females (12.6%,) than males (25.6%). Further, HLA-B* 57 frequency in HIV cohort was significantly higher among females without TB (21.6%, 19/88) than males (1.7%, 1/59); P = 0.0046; OR = 38. CD4 counts also were higher among females in this cohort. Conclusion. This study suggests that HIV positive women with HLA-B* 57 have less occurrence of TB as compared to males.

Pattern recognition and cellular immune responses to novel Mycobacterium tuberculosis-antigens in individuals from Belarus

Background: Tuberculosis (TB) is an enduring health problem worldwide and the emerging threat of multidrug resistant (MDR) TB and extensively drug resistant (XDR) TB is of particular concern. A better understanding of biomarkers associated with TB will aid to guide the development of better targets for TB diagnosis and for the development of improved TB vaccines. Methods: Recombinant proteins (n = 7) and peptide pools (n = 14) from M. tuberculosis (M.tb) antigens associated with M.tb pathogenicity, modification of cell lipids or cellular metabolism, were used to compare T cell immune responses defined by IFN-gamma production using a whole blood assay (WBA) from i) patients with TB, ii) individuals recovered from TB and iii) individuals exposed to TB without evidence of clinical TB infection from Minsk, Belarus. Results: We identified differences in M.tb target peptide recognition between the test groups, i.e. a frequent recognition of antigens associated with lipid metabolism, e.g. cyclopropane fatty acyl phospholipid synthase. The pattern of peptide recognition was broader in blood from healthy individuals and those recovered from TB as compared to individuals suffering from pulmonary TB. Detection of biologically relevant M.tb targets was confirmed by staining for intracellular cytokines (IL-2, TNF-alpha and IFN-gamma) in T cells from non-human primates (NHPs) after BCG vaccination. Conclusions: PBMCs from healthy individuals and those recovered from TB recognized a broader spectrum of M.tb antigens as compared to patients with TB. The nature of the pattern recognition of a broad panel of M.tb antigens will devise better strategies to identify improved diagnostics gauging previous exposure to M.tb; it may also guide the development of improved TB-vaccines.

Identification of immuno-dominant antigens of Trypanosoma evansi for detection of chronic trypanosomosis using experimentally infected equines

Trypanosoma evansi is the most extensively distributed trypanosome responsible for disease called surra in livestock in many countries including frequent outbreaks in India. The prevalence of this disease is most commonly reported by standard parasitological detection methods (SPDM); however, antibody ELISA is being in practice by locally produced whole cell lysate (WCL) antigens in many countries. In the present investigation, we attempted to identify and purify immuno dominant, infection specific trypanosome antigens from T. evansi proteome using experimentally infected equine serum by immuno blot. Three immuno dominant clusters of proteins i.e. 62-66 kDa, 52-55 kDa and 41-43 kDa were identified based on their consistent reactivity with donkey sequential serum experimentally infected T. evansi up to 280 days post infection (dpi). The protein cluster of 62-66 kDa was purified in bulk in native form and comparatively evaluated with whole cell lysate antigen (WCL). ELISA and immuno blot showed that polypeptide of this cluster is 100% sensitive in detection of early and chronic infection. Further, this protein cluster was also found immuno reactive against hyper immune serum raised against predominantly 66 kDa exo antigen, revealed that this is a common immunodominant moieties in proteome and secretome of T. evansi.

Infection of human amniotic and endothelial cells by Japanese encephalitis virus: Increased expression of HLA-F

Productive infection of human amniotic and endothelial cell lines with Japanese encephalitis virus (JEV) was established leading to the induction of NF kappa B and HLA-F, a non-classical MHC molecule. Induction of the HLA-F gene and protein in JEV-infected cells was shown to be NF kappa B dependent since it was blocked by inhibitors of NF kappa B activation. ShRNA targeting lentivirus-mediated stable knockdown of the p65 subunit of NF kappa B inhibited JEV-mediated induction of HLA-F both in the amniotic cell line, AV-3 as well as the human brain microendothelial cell line, HBMEC. The induction of HLA-F by treatment of AV-3 with TNF-alpha was also inhibited by ShRNA mediated knockdown of NF kappa B. TNF-alpha treatment of HEK293T cells that were transfected with reporter plasmids under the control of HLA-F enhancer A elements resulted in significant transactivation of the luciferase reporter gene. NF kappa B-mediated induction of HLA-F following JEV infection and TNF-alpha exposure is being suggested for the first time. (C) 2014 Elsevier Inc. All rights reserved.

Inhibition of ERK and proliferation in NK cell lines by soluble HLA-E released from Japanese encephalitis virus infected cells

Productive infection of human endothelial cells with Japanese encephalitis virus (JEV), a single stranded RNA virus induces shedding of sHLA-E. We show here that sHLA-E that is released upon infection with this flavivirus can inhibit IL-2 and PMA mediated ERK 1/2 phosphorylation in two NK cell lines, Nishi and NKL. Virus infected or IFN-gamma treated cell culture supernatants containing sHLA-E were found to partially inhibit IL-2 mediated induction of CD25 molecules on NKL cells. It was also found that sHLA-E could inhibit IL-2 induced H-3]-thymidine incorporation suggesting that, similar to cell surface expressed HLA-E, sHLA-E could also inhibit NK cell responses. Hence JEV-induced shedding of sHLA-E needs further investigation to better understand immune responses in JEV infections since it may have a role in viral evasion of NK cell responses. (C) 2014 Elsevier B.V. All rights reserved.

An evaluation of antigen capture assays for detecting active filarial antigens

Lymphatic filariasis is a parasitic disease of tropical countries. This is a disfiguring and painful disease contracted in childhood, but the symptoms become apparent only in later years. Diagnosis of filarial infection is very crucial for the management of the disease. The main objective of this study was to develop a filarial antigen-based immunological assay for the diagnosis and surveillance of the disease. Monoclonal and polyclonal antibodies were raised to the recombinant protein Brugia malayi vespid allergen homologue (VAH). Capture enzyme-linked immunosorbent assay (ELISA) was standardized utilizing various combinations of antibodies and evaluated with serum samples of endemic normal (EN, n = 110), microfilaraemic (MF, n = 65), chronic pathology (CP, n = 45) and non-endemic normal (NEN, n = 10) individuals. Of the 230 samples tested, VAHcapture assay detected circulating antigen in 97.91% of bancroftian and 100% of brugian microfilaraemic individuals, and 5% of endemic normal individuals, comparable to the earlier reported SXP-1 antigen detection assay. However, the combination of VAH and SXP-1 (VS) capture ELISA was found to be more robust, detecting 100% of microfilaraemic individuals and with higher binding values. Thus an antigen capture immunoassay has been developed, which can differentiate active infection from chronic infection by detecting circulating filarial antigens in clinical groups of endemic areas.

Haloarchaeal gas vesicle nanoparticles displaying Salmonella antigens as a novel approach to vaccine development

A safe, effective, and inexpensive vaccine against typhoid and other Salmonella diseases is urgently needed. In order to address this need, we are developing a novel vaccine platform employing buoyant, self-adjuvanting gas vesicle nanoparticles (GVNPs) from the halophilic archaeon Halobacterium sp. NRC-1, bioengineered to display highly conserved Salmonella enterica antigens. As the initial antigen for testing, we selected SopB, a secreted inosine phosphate effector protein injected by pathogenic S. enterica bacteria during infection into the host cells. Two highly conserved sopB gene segments near the 3'- region, named sopB4 and sopB5, were each fused to the grIpC gene, and resulting SopB-GVNPs were purified by centrifugally accelerated flotation. Display of SopB4 and SopB5 antigenic epitopes on GVNPs was established by Western blotting analysis using antisera raised against short synthetic peptides of SopB. Immunostimulatory activities of the SopB4 and B5 nanoparticles were tested by intraperitoneal administration of SopB-GVNPs to BALB/c mice which had been immunized with S. enterica serovar Typhimurium 14028 ApmrG-H111-D (DV-STM-07), a live attenuated vaccine strain. Proinflammatory cytokines IFN-y, IL-2, and IL-9 were significantly induced in mice boosted with SopB5-GVNPs, consistent with a robust Thl response. After challenge with virulent S. enterica serovar Typhimurium 14028, bacterial burden was found to be diminished in spleen of mice boosted with SopB4-GVNPs and absent or significantly diminished in liver, mesenteric lymph node, and spleen of mice boosted with SopB5GVNPs, indicating that the C-terminal portions of SopB displayed on GVNPs elicit a protective response to Salmonella infection in mice. SopB antigen-GVNPs were also found to be stable at elevated temperatures for extended periods without refrigeration. The results show that bioengineered GVNPs are likely to represent a valuable platform for antigen delivery and development of improved vaccines against Salmonella and other diseases.

基于PROFIBUS-DP的激光加工系统数字化控制

提出采用PROFIBUS-DP现场总线技术实现对激光柔性加工系统的数字化控制，给出了控制实现过程中的DL动态链接库方案，详述了控制中的同步问题，并将其应用于汽车覆盖件模具激光表面硬化，编程采用VC++面向对象工具加以实现。

Evaluation of live-attenuated Salmonella vaccines expressing Campylobacter antigens for control of C. jejuni in poultry

Campylobacter jejuni is a zoonotic bacterial pathogen of worldwide importance. It is estimated that 460,000 human infections occur in the United Kingdom per annum and these involve acute enteritis and may be complicated by severe systemic sequelae. Such infections are frequently associated with the consumption of contaminated poultry meat and strategies to control C. jejuni in poultry are expected to limit pathogen entry into the food chain and the incidence of human disease. Toward this aim, a total of 840 Light Sussex chickens were used to evaluate a Salmonella enterica serovar Typhimurium ΔaroA vaccine expressing the C. jejuni amino acid binding protein CjaA as a plasmid-borne fusion to the C-terminus of fragment C of tetanus toxin. Chickens were given the vaccine at 1-day-old and two weeks later by oral gavage, then challenged after a further two weeks with C. jejuni. Across six biological replicates, statistically significant reductions in caecal C. jejuni of c. 1.4 log10 colony-forming units/g were observed at three and four weeks post-challenge relative to age-matched unvaccinated birds. Protection was associated with the induction of CjaA-specific serum IgY and biliary IgA. Protection was not observed using a vaccine strain containing the empty plasmid. Vaccination with recombinant CjaA subcutaneously at the same intervals significantly reduced the caecal load of C. jejuni at three and four weeks post-challenge. Taken together these data imply that responses directed against CjaA, rather than competitive or cross-protective effects mediated by the carrier, confer protection. The impact of varying parameters on the efficacy of the S. Typhimurium ΔaroA vaccine expressing TetC-CjaA was also tested. Delaying the age at primary vaccination had little impact on protection or humoral responses to CjaA. The use of the parent strain as carrier or changing the attenuating mutation of the carrier to ΔspaS or ΔssaU enhanced the protective effect, consistent with increased invasion and persistence of the vaccine strains relative to the ΔaroA mutant. Expression in the ΔaroA strain of a TetC fusion to Peb1A, but not TetC fusions to GlnH or ChuA, elicited protection against intestinal colonisation by C. jejuni that was comparable to that observed with the TetC-CjaA fusion. Our data are rendered highly relevant by use of the target host in large numbers and support the potential of CjaA- and Peb1A-based vaccines for control of C. jejuni in poultry. © 2009 Elsevier Ltd. All rights reserved.

扩散抛物化(DP)NS方程组的意义及其在计算流体力学中的应用

本文首先讨论扩散抛物化(DP)NS方程组的早期研究工作:它的提出、数学性质、意义和在CFD中的应用,然后讨论扩散抛物化理论的一些新发展。这些新发展是对NS方程组数值计算进行物理分析的基础上得到的,其中包括NS方程组差分计算时,粘性剪切流对网格间距和格式精度的要求;粘性项只保留剪切粘性项的广义扩散抛物化(GDP)NS方程组,它的性质和应用。由于高Re数流动在NS方程组的差分计算中,网格Re数彼此相差悬殊的特点,产生了计算离散单元守恒方程组的新的算法思路,即离散流体力学(DFD)算法。在DFD算法中需要同时计算三种不同的守恒方程组(Euler,DPNS和NS方程组)。本文讨论了DFD格式的构造、它的优点和应用。并以超声速绕前后台阶流动为算例,来说明GDPNS方程组的用处和DFD算法的优点。DPNS方程组、GDPNS方程组、DFD算法是高智提出的,对这些问题他和合作者从

Avaliação do PRA e CD30s no transplante renal intervivos. Acompanhamento no 1 ano e após 6 anos em pacientes do Hospital Federal de Bonsucesso (Rio de Janeiro, Brasil)

O CD30 solúvel (CD30s) é uma glicoproteína transmembrana da família do fator de necrose tumoral expressa na superfície das células T. Quando este marcador é clivado ele torna-se solúvel, sendo detectado na circulação. Atualmente, o valor de CD30s pré-transplante vem sido demonstrado como um bom preditor de rejeição aguda (RA) e perda do enxerto. Poucos estudos foram realizados para sua avaliação no pós-transplante e sua correlação com sobrevida e TFG. Avaliar a eficácia da determinação dos marcadores laboratoriais CD30 solúvel (CD30s) e anticorpos reativos contra painel HLA (PRA) em seis meses, um ano e seis anos pós-transplante renal em receptores de doadores vivos, correlacionando estes marcadores com episódios de rejeição aguda, eventos infecciosos no pós-transplante, perda do enxerto e óbito do paciente transplantado. E, avaliar a correlação destes marcadores com a sobrevida do enxerto renal nestes períodos. Os pacientes estudados foram transplantados renais com doadores vivos no Hospital Federal de Bonsucesso (HFB) do Rio de Janeiro no ano de 2006 e do período de agosto de 2010 a maio de 2011, sendo uma extensão de um trabalho realizado previamente. CD30s e PRA foram analisados nas amostras coletadas no pré-transplante e com 7, 14, 21 dias, 1, 3, 6, 12 meses após o transplante e nos pacientes transplantados em 2006 amostras após 6 anos de transplante. A taxa de filtração glomerular (TFG) foi estimada utilizando MDRD e CKD-epi e 6 meses, 1 ano e 6 anos após o transplante. Os pacientes foram agrupados em 5 grupos: sem eventos, com perda do enxerto, óbito, rejeição aguda e pacientes com quadros infecciosos. Estes grupos foram avaliados com relação ao CD30s, PRA I e II e comparados dois a dois. O teste qui quadrado foi utilizado. Quando necessário aplicou-se a correção de Yates, o teste de Fisher, o teste de Kruskal-wallis. Foi considerado estatisticamente significante p<0,05. As análises foram feitas pelo programa EPI-Info (versão 3.5.3). Setenta e seis pacientes com doadores vivos foram incluídos no estudo 47 pacientes não tiveram nenhum evento (grupo 1), 7 pacientes perderam o enxerto (grupo 2), 3 pacientes faleceram (grupo 3), 11 pacientes ficaram no grupo de rejeição aguda (grupo 4) e oito pacientes tiveram infecção por CMV e herpes (grupo 5). Os pacientes do grupo de RA tiveram correlação positiva com os valores tanto de CD30s Pré-transplante (p=0,01), quanto do CD30s pós-transplante (p=0,002) e PRA I e II (p<0,001), respectivamente, quando comparados com pacientes sem eventos. A TFG tanto com MDRD e CKD-Epi não mostrou correlação com CD30s pré e pós-transplante e nem PRA I e II. A TFG com as duas fórmulas foi menor no grupo com RA comparado com o grupo sem evento após 6 anos de transplante (p=0,006). CD30s é um bom preditor de RA, assim como PRAI e II. E, também mais uma ferramenta que pode ser utilizada no acompanhamento pós-transplante Renal. A RA é um preditor isolado para diminuição de TFG no transplante.