Immune responses to Leptospira infection: roles as biomarkers for disease severity

Various leptospiral components have been identified and shown to be involved in tissue destruction. In addition, immune responses to leptospires have been implicated in target organ damages in severe leptospirosis cases. Several inflammatory mediators were shown to be higher in susceptible animals than in resistant hosts. Moreover, cytokines/chemokines and serum proteins induced following Leptospira infection were suggested to be biomarkers for disease severity in human leptospirosis. This review focuses on the role of immune responses in the severity of leptospirosis. Studies in both animal models and humans are discussed.

Comparison of a slide agglutination test, LeptoTek Dri-Dot, and IgM-ELISA with microscopic agglutination test for Leptospira antibody detection.

A slide agglutination test (SAT), LeptoTek Dri-Dot and IgM-ELISA were compared with a microscopic agglutination test (MAT) for the detection of Leptospira antibodies. Paired sera from 10 patients whose leptospirosis was clinically suspected and diagnosed by MAT, were evaluated in this study. Our data, especially from acute samples, demonstrate the SAT and Dri-Dot were more sensitive as initial screening tests than MAT. IgM-ELISA has an advantage over MAT, SAT, and Dri-Dot since the results can be interpreted from a single serum testing if the results of the test are positive. Eight of the ten cases could be diagnosed by IgM-ELISA. Our data suggest that IgM-ELISA may be used for the diagnosis of leptospirosis. However, the agglutination test is useful for screening and for secondary infection cases for which IgM antibodies may be undetectable. MAT can be performed as a reference test and when information regarding the causative serovar is required.

Myristica fragrans Houtt. methanolic extract induces apoptosis in a human leukemia cell line through SIRT1 mRNA downregulation.

BACKGROUND: Myristica fragrans Houtt. (nutmeg) contains antibacterial, antiviral and anti-cancer activities. However the mechanisms underlying those activities have not been clearly explained. OBJECTIVE: To study the effect of Myristica fragrans Houtt. methanolic extract on Jurkat human leukemia T cell line. MATERIAL AND METHOD: Methanol extract of Myristica fragrans Houtt. (Myristicaceae) was used to study the effect on Jurkat cell metabolic activity using an MTT assay and on apoptosis using annexin V staining. Expression of SIRT1 gene was determined by RT-PCR. RESULTS: At the concentrations 50 and 100 ig/mL, the methanol extract of Myristica fragrans Houtt significantly inhibited Jurkat cell proliferation and induced apoptosis as detected by annexin V staining. Downregulation of SIRT1 mRNA expression in Jurkat cells was observed even when the amount of methanol extract was 10 microg/mL. CONCLUSION: Methanol extract of Myristica fragrans Houtt induced apoptosis of Jurkat leukemia T cell line in a mechanisms involving SIRTI mRNA downregulation.

Binding of Leptospira to extracellular matrix proteins.

BACKGROUND: Leptospirosis is a zoonotic disease of global importance. Pathogenesis caused by this infectious disease remains unclear. Attachment of pathogenic leptospires to host tissues is a crucial initial step to establish the infection. OBJECTIVE: Study the binding of the spirochete to three types of extracellular matrix (ECM), collagen type IV, fibronectin, and laminin, which are major components of target organs. MATERIAL AND METHOD: ELISA-based experiments were performed to determine binding of pathogenic (serovar icterohaemorrhagie) and non-pathogenic (serovar Patoc) serovars, to purified ECM. RESULTS: Both pathogenic and non-pathogenic serovars bound to all three types of ECM in the dose-dependent manner and the binding to fibronectin is higher than to collagen and laminin (p < 0.005). CONCLUSION: Pathogenic leptospires can bind to various types of ECM and the binding of leptospires to fibronectin was higher than to collagen and laminin. However, this capability may not be the only mechanism that makes leptospires virulent since non-pathogenic leptospire can bind the ECM as well.

Analysis of connective tissue growth factor promoter polymorphism in Thai children with biliary atresia.

OBJECTIVE: Connective tissue growth factor (CTGF) has been proposed to play a key role in the pathogenesis of hepatic fibrosis in biliary atresia (BA). The aim of the present study was to determine the single nucleotide polymorphism (SNP) in the promoter region of CTGF gene in a Thai population, and to investigate the possible role of CTGF promoter polymorphism in the susceptibility of BA. MATERIAL AND METHOD: Genomic DNA was obtained from 84 patients with BA and 142 healthy controls. The -447 G/C and -132 C/G in CTGF promoter were amplified and examined by amplification-refractory mutation system (ARMs) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis, respectively. The test of Hardy-Weinberg equilibrium (HWE) was performed using HWE program of SNPAnalyzer. Statistical analysis was carried out with SPSS and Epi Info. RESULT: According to the previous experiment, there were two SNPs, which were at position -447 and -132 on the promoter. However, there was only one SNP at the position -447 in the Thai population. No significant differences in genotype and allele frequency were observed between BA and controls or with BA subgroups. CONCLUSION: The present study demonstrated that CTGF polymorphism at -447 G/C was not associated with BA and the jaundice status of the postoperative BA patients.

LipL32, an outer membrane protein of Leptospira, as an antigen in a dipstick assay for diagnosis of leptospirosis.

Microscopic agglutination test (MAT), as well as other serological assays that aimed at detecting antibodies to Leptospira, supplements the leptospirosis diagnosis based on the clinical features. Nevertheless, false positive results have been occasionally reported when the crude antigen was used in those antibody-based tests due either to the presence of antibodies stimulated by other antigenically related pathogens in the patient's serum, or the antibodies in the serum may be stimulated by a previously unrecognized Leptospira infection, especially in the disease endemic areas. Thus, the more refined antigen should improve the serodiagnostic accuracy. Among Leptospira spp., LipL32, which is a pathogenic Leptospira outer membrane protein (OMP), expressed by the bacteria grown both in vitro and in vivo. In this study, recombinant LipL32 protein was tested by a dipstick method for its potential in serodiagnosis of leptospirosis. Preliminary results suggest that the recombinant LipL32 is a good diagnostic detection reagent for specific Leptospira IgG. Diagnostic sensitivity and specificity of the Lip32 dipstick assay, when compared to those of MAT, were 100% and 98.33%, respectively.

Induction of apoptosis by herpes simplex virus in Jurkat cells is partly through caspase-3, -8 and -9 activation.

Herpes simplex virus (HSV), a large DNA containing virus, is endemic in all human populations investigated. After infection of mucocutaneuos surfaces, HSV establishes a latent infection in nerve cells. Various immune evasion mechanisms have been shown to be utilized by HSV including apoptosis induction in Tlymphocytes. However, the mechanisms of T cell infection and apoptosis by HSV are still unknown. The present study investigated the molecular mechanisms of apoptosis induction in T cells by HSV The Jurkat T cell line was used as a representative for T cells. Apoptosis detection by Annexin Vassay demonstrated that both HSV-1 and HSV-2 induced apoptosis in Jurkat cells and caspase-3, -8, and -9 inhibitors blocked apoptosis induced by HSV-1 and HSV-2. The data suggested that HSV-1 and HSV-2 induced apoptosis in T lymphocytes by caspase-dependent pathway. However, apoptosis may occur through other mechanism(s) since caspase inhibitors used in the present study could not completely inhibit apoptosis induced by HSV infection. In addition, the data demonstrated that the number of apoptotic cells induced by HSV-2 was significantly higher than byHSV-1 at 12 hour post-infection (h p.i.) (p = 0.003). Further studies in peripheral blood T cells and the proteins of viruses involved in apoptosis induction should be further performed in order to elucidate the molecular mechanisms of apoptosis induced by these viruses.

Searching for virulence Burkholderia pseudomallei genes by immunoscreening the lambda ZAPII expressed genomic library.

The lambdaZAP II expressed genomic library of B. pseudomallei was screened with pooled melioidosis serum preabsorbed with E. coli host cell. The positive clones were detected by using protein A-CDP-star chemiluminescence. All of 14 positive clones reacted with only the pooled absorbed melioidosis serum and not the pooled absorbed normal serum when tested with the plaque dot blot analysis. The expressed genes were detected by using a combination of immunoscreening, bioinformatics and molecular biology. At least six in vivo expressed genes were identified by this approach. Two were well known virulent genes, gmhA (a capsule biosynthetic gene) and bipD (type III secretion protein gene). Another two were genes coded for conserved hypothetical protein. The last two isolated genes were groEL (a chaperonine protein gene), and a gene encoding transmembrane protein.

Situation of laboratory service and instruments in Thailand: a descriptive study from questionnaires.

Laboratory instruments are one of the main items in laboratory investment. To establish data for the situation of laboratory service and instruments in Thailand, questionnaires were randomly sent to one hundred and twenty laboratories. Sixty-three filled questionnaires from eleven university and affiliated hospitals, thirty-four government hospitals, and eighteen private hospital laboratories were sent back to the authors to be analyzed. Only the number of samples and instruments used during office hours were analyzed in this study by descriptive method. From the data it was found that the average number of personnel and tests of the university and affiliated hospital laboratories was the highest. To analyze the efficiency of the instruments used in the laboratories, the authors compared the average service number of samples or tests to the average number of samples or tests that was calculated from the instruments. The ratio of the average number of samples or tests that were calculated from the instruments and the average service number of samples or tests for chemistry and CBC were 2.13, 3.41, 5.24 and 2.33, 2.76, 3.71 in university and affiliated hospital laboratories, government hospital laboratories, and the private hospital laboratories, respectively. From the data, it was concluded that the instrument situation in laboratories of the university and affiliated hospitals was more appropriate than government hospital and private hospital laboratories. To improve the efficiency of using laboratory instruments, more concern must be given to the management of laboratory instruments and cooperation between hospitals could increase the efficiency of the instrument investment.

Detection of antibody in serum and secretion for the diagnosis of Helicobacter pylori infection.

Helicobacter pylori plays a major role in chronic gastritis and peptic ulcer. In addition, it has been shown to be associated with gastric carcinoma. In this study, the authors compared the detection of IgG antibodies specific to H. pylori by enzyme-immunoassay with culture, histology and a CLO test as tools for diagnosis of H. pylori infection. If the criteria that patients will be considered as H. pylori infected only when their samples are positive by culture or CLO test and histology were used, the sensitivity and specificity of detecting IgG in sera were 96.84 and 72.04 per cent respectively. The use of serological test will be useful as a screening test for H. pylori infection without the need of endoscopy. The authors also performed the assay for detecting IgA antibodies in saliva and gastric juice. The sensitivity and specificity of IgA detection in saliva were 26.79 per oent and 75.00 per cent. As for the assay in gastric juice, although the specificity was as high as 91.67 per cent, the sensitivity is very low (22.22%).

Replication of herpes simplex virus in T lymphocytes.

HSV is known to cause infection at various parts in the human body such as skin, mouth, eyes, genital area, and brain. In this study, the authors showed the possibility of HSV replication in Jurkat, a human leukemic T lymphocytes. Although the yield production was very low when compared to the other 2 epithelial cells, Vero and HEp-2 cells, the yield production could enhance after PHA activation. Delayed viral protein expression was observed in Jurkat cells. This might be the reason for low production. However, the exactly mechanism is unknown. Replication of viruses have been examined in a number of cell systems and the duration of successive steps in the replication cycle depends upon the types of cells, the virus strain, and the multiplicity of infection.