Leptospiral Proteins Recognized during the Humoral Immune Response to Leptospirosis in Humans

Leptospirosis is an emerging zoonosis caused by pathogenic spirochetes belonging to the genus Leptospira. An understanding of leptospiral protein expression regulation is needed to develop new immunoprotective and serodiagnostic strategies. We used the humoral immune response during human leptospirosis as a reporter of protein antigens expressed during infection. Qualitative and quantitative immunoblot analysis was performed using sera from 105 patients from Brazil and Barbados. Sera from patients with other diseases and healthy individuals were evaluated as controls. Seven proteins, p76, p62, p48, p45, p41, p37, and p32, were identified as targets of the humoral response during natural infection. In both acute and convalescent phases of illness, antibodies to lipopolysaccharide were predominantly immunoglobulin M (IgM) while antibodies to proteins were exclusively IgG. Anti-p32 reactivity had the greatest sensitivity and specificity: positive reactions were observed in 37 and 84% of acute- and convalescent-phase sera, respectively, while only 5% of community control individuals demonstrated positive reactions. Six immunodominant antigens were expressed by all pathogenic leptospiral strains tested; only p37 was inconsistently expressed. Two-dimensional immunoblots identified four of the seven infection-associated antigens as being previously characterized proteins: LipL32 (the major outer membrane lipoprotein), LipL41 (a surface-exposed outer membrane lipoprotein), and heat shock proteins GroEL and DnaK. Fractionation studies demonstrated LipL32 and LipL41 reactivity in the outer membrane fraction and GroEL and DnaK in the cytoplasmic fraction, while p37 appeared to be a soluble periplasmic protein. Most of the other immunodominant proteins, including p48 and p45, were localized to the inner membrane. These findings indicate that leptospiral proteins recognized during natural infection are potentially useful for serodiagnosis and may serve as targets for vaccine design.     

Threat to Humans from Virus Infections of Non-human Primates

Several hundred distinct non human primate species are recognised, and they are likely to harbour a similar range of viruses to humans. Simians such as cynomolgus and rhesus macaques, African green monkeys, and marmosets are widely used for biomedical research, but despite this extensive close contact very few simian viruses have been shown to pose a threat of infection or illness to humans. Herpesvirus Simiae is the best recognised zoonotic hazard of simians. It is an alphaherpes virus of Asiatic macaques, which causes a mild or subclinical primary infection followed by latency in its natural host. It can be acquired by humans following a bite and causes an ascending meningoencephalitis. Less than 40 human cases have been described and the mortality rate in untreated human infections is 70%. The infection is treatable with acyclovir and extensive guidelines for managing simians and potential exposures have been developed. Ebola virus and Marburg virus have caused epizootics in cynomolgus macaques and vervet monkeys respectively, which have resulted in human infection and fatalities. However, non human primates are unlikely to be their natural host. More recently simian immunodeficiency virus and simian foamy virus have infected researchers, but infection has not been linked to illness. Simian viruses also pose a direct threat to humans through the use of primary monkey tissue cultures in laboratory work and vaccine manufacture, indeed a significant exposure of the human population occurred when cells contaminated with SV40 a polyomavirus of rhesus monkeys were used for polio vaccine production. New medical interventions such as xenotransplantation using primate organs pose a potential risk which requires careful assessment. © 1997 by John Wiley & Sons Ltd.     

Cluster of Infections Caused by Methicillin-Resistant Staphylococcus pseudintermedius in Humans in a Tertiary Hospital

The dog-associated Staphylococcus pseudintermedius is a rare pathogen in humans. Here we describe a cluster of infections caused by the methicillin-resistant S. pseudintermedius clone ST71-J-t02-II–III. It involved four elderly patients at a tertiary hospital. Three patients had wound infections, and the strain had a tendency to cause bullous skin lesions.     

Diagnosis of Mycobacterium microti Infections among Humans by Using Novel Genetic Markers

As a result of DNA typing of Mycobacterium microti isolates from animals in the United Kingdom and The Netherlands, we diagnosed four human M. microti infections. These are the first M. microti infections among humans to be reported. Three of the patients were immunocompromised and suffered from generalized forms of tuberculosis. The fourth patient was a 34-year-old immunocompetent male with a persistent cough and undefined X-ray abnormalities. Two of the M. microti infections were recognized by their IS6110 restriction fragment length polymorphism (RFLP) patterns, which showed a high degree of similarity with those of M. microti strains isolated from a pig and a ferret in The Netherlands. The two other human M. microti infections were recognized by using the recently developed DNA fingerprinting method, “spoligotyping,” directly on clinical material. All M. microti isolates from the United Kingdom and The Netherlands were found to contain an exceptionally short genomic direct repeat region, resulting in identical two-spacer sequence reactions in spoligotyping. In contrast, the highly similar IS6110 RFLP patterns of the vole strains from the United Kingdom differed considerably from the RFLPs of all M. microti strains isolated in The Netherlands, suggesting that geographic isolation led to divergent strains in the United Kingdom and on the continent.     

Molecular Characterization and Antimicrobial Susceptibility of Salmonella Isolates from Infections in Humans in Henan Province, China

We characterized 208 human Salmonella isolates from 2006 to 2007 and 27 human Salmonella enterica serovar Typhimurium isolates from 1987 to 1993 from Henan Province, China, by serotyping, by antimicrobial susceptibility testing, and, for the most common serovars, by pulsed-field gel electrophoresis (PFGE). The most common serovars among the 2006-2007 isolates were S. enterica serovar Typhimurium (27%), S. enterica serovar Enteritidis (17%), S. enterica serovar Derby (10%), S. enterica serovar Indiana (6%), and S. enterica serovar Litchfield (6%). A high percentage of the isolates were multiple-drug resistant, and 54% were resistant to both nalidixic acid and ciprofloxacin. Of these, 42% were resistant to a high level of ciprofloxacin (MIC > 4 μg/ml), whereas for the remaining isolates, the MICs ranged from 0.125 to 2 μg/ml. Five isolates (2%) were ceftiofur resistant and harbored bla_CTX-M14 or bla_CTX-M15. With the possible exception of the quinolones and cephalosporins, the 1987-1993 S. enterica serovar Typhimurium isolates were almost as resistant as the recent isolates. PFGE typing of S. enterica serovar Typhimurium showed that the most common cluster predominated over time. Two other clusters have emerged, and another cluster has disappeared.     

Evaluation of an Epitope-Blocking Enzyme-Linked Immunosorbent Assay for the Diagnosis of West Nile Virus Infections in Humans

An epitope-blocking enzyme-linked immunosorbent assay (b-ELISA) was evaluated for the diagnosis of West Nile virus (WNV) infections in humans. Sera from patients diagnosed with WNV infections from an outbreak in 2003 in Colorado and from patients diagnosed with dengue virus infections from Mexico and Thailand were tested with the b-ELISA. The b-ELISAs were performed using the WNV-specific monoclonal antibody (MAb) 3.1112G and the flavivirus-specific MAb 6B6C-1. Although the WNV-specific b-ELISA was effective in diagnosing WNV infections in humans from Colorado, it was not efficacious for diagnosing WNV infections in serum specimens from Mexico and Thailand. In serum specimens from patients from Colorado, the WNV b-ELISA and the WNV plaque reduction neutralization test showed an overall agreement of 91%. The sensitivity and specificity of the WNV b-ELISA were 89% and 92%, respectively, with a false-positive rate of 5%, based on receiver operating characteristic analysis. In contrast, false-positive rate results in specimens from the countries of Mexico and Thailand, where flaviviruses are endemic, were 79% and 80%, presumably due to the presence of antibodies resulting from previous dengue virus infections in Mexico and/or Japanese encephalitis virus infections or vaccination in Thailand. Thus, in regions where people have experienced previous or multiple flavivirus infections, the use of the b-ELISA for WNV diagnosis is contraindicated.The most medically important flaviviruses include dengue virus (DENV), Japanese encephalitis virus (JEV), West Nile virus (WNV), yellow fever virus (YFV), tick-borne encephalitis virus (TBEV), and Saint Louis encephalitis virus (SLEV) (16, 31, 38). Flaviviruses are positive-strand RNA viruses with genomes of approximately 11 kb that encode three structural and seven nonstructural (NS) proteins in the gene order C (capsid), M (membrane), E (envelope), NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5. WNV is a member of the JEV serocomplex within the genus Flavivirus, family Flaviviridae. The virus has been isolated in Africa, Australia, Eastern Europe, the Middle East, North America, and South America (7, 20, 24). WNV was first detected in the United States in July 1999 and spread rapidly throughout the country, causing large numbers of infections in humans, horses, and birds (19, 31).Prior to 1999, flavivirus infections in humans in the United States were infrequent, and most were attributed to sporadic cases of SLEV and travel-associated cases of DENV (41). In Thailand, all four DENV serotypes and JEV circulate (39), resulting in very high flavivirus transmission and seroprevalence rates. In the Yucatán Peninsula of Mexico, all four DENV serotypes circulate and seroprevalence rates are very high (8). Serological diagnosis of WNV infections is complicated by the high rates of both primary DENV infections and secondary DENV infections in inhabitants of Thailand and Yucatan, Mexico, with seroprevalence rates of >85% in Thailand (1) and 72% in the Yucatán in 1985 (12, 28). WNV introduction into the Yucatán in 2002 was revealed by detection of antibodies in horses (29) and then later in migratory and resident birds (10) and in zoo animals (11). However, no WNV infections of humans have been diagnosed in the Yucatán.The immunoglobulin M (IgM) capture enzyme-linked immunosorbent assay (ELISA) is the preferred test used for diagnosis of WNV in humans in the United States (32). The test is used to detect antibodies to WNV in serum and/or cerebrospinal fluid. The plaque reduction neutralization test (PRNT) is the gold standard for serodiagnosis of flavivirus infections and for identifying the infecting agent (2). However, both of these tests can be confounded if patients have had previous flavivirus infections. Indeed, diagnosis of flavivirus infections in humans is very difficult in geographic areas where multiple flaviviruses are circulating and cause sequential infections. Because of “original antigenic sin” the highest antibody titer may be due to a previous flavivirus infection rather than to the etiologic agent (18, 26). Serological diagnosis of WNV, SLEV, and YFV infections is extremely difficult in patients from areas where DENV is hyperendemic.Previously, we exploited an epitope-blocking ELISA (b-ELISA) to detect antibodies to WNV in diverse species of birds and domestic mammals (3, 4). The WNV b-ELISA measures the ability of antibodies present in sera to block the binding of a monoclonal antibody (MAb) to a WNV-specific epitope on the NS1 protein (17). The WNV b-ELISA had not been previously evaluated for use in humans. In this study, a WNV-specific and a flavivirus broadly reactive b-ELISAs were evaluated for their abilities to detect antibodies against WNV in human serum specimens from countries with differing levels of flavivirus endemicity: the United States, Thailand, and Mexico. The objectives of this study were (i) to determine the ability of the b-ELISA to detect antibodies to WNV in human serum samples and (ii) to determine the effects of previous flavivirus infections of patients (e.g., DENV and JEV) on the diagnostic efficacy of the WNV b-ELISA.     

CX3CR1 Is Dispensable for Control of Mucosal Candida albicans Infections in Mice and Humans

Candida albicans is part of the normal commensal microbiota of mucosal surfaces in a large percentage of the human population. However, perturbations of the host''s immune response or bacterial microbiota have been shown to predispose individuals to the development of opportunistic Candida infections. It was recently discovered that a defect in the chemokine receptor CX₃CR1 increases susceptibility of mice and humans to systemic candidiasis. However, whether CX₃CR1 confers protection against mucosal C. albicans infection has not been investigated. Using two different mouse models, we found that Cx3cr1 is dispensable for the induction of interleukin 17A (IL-17A), IL-22, and IL-23 in the tongue after infection, as well as for the clearance of mucosal candidiasis from the tongue or lower gastrointestinal (GI) tract colonization. Furthermore, the dysfunctional human CX₃CR1 allele CX₃CR1-M280 was not associated with development of recurrent vulvovaginal candidiasis (RVVC) in women. Taken together, these data indicate that CX₃CR1 is not essential for protection of the host against mucosal candidiasis, underscoring the dependence on different mammalian immune factors for control of mucosal versus systemic Candida infections.     

Auto-Antibodies to Tumour Necrosis Factor a in Healthy Humans and Patients with Inflammatory Diseases and Gram-Negative Bacterial Infections

A semi-quantitative immunoblotting method was developed to screen for serum auto-antibodies against tumour necrosis factor alpha (TNF alpha). Forty nitrocellulose strips containing identical amounts of human recombinant TNF alpha (rTNF alpha) were prepared for each set-up, and the anti-TNF alpha antibody immunoreactivities were scored according to the density of the resulting colour reaction. A significant number of sera from apparently healthy donors contained detectable auto-antibodies to TNF alpha (40%), while the strongest reaction was observed in 8%. A higher prevalence of anti-TNF alpha antibodies was found in sera from patients with Gram-negative bacterial septicaemia (66%), cystic fibrosis with chronic Pseudomonas aeruginosa lung infection (72%), and various rheumatic diseases (61%). The antibodies in sera from these patients belonged primarily to the IgG and IgM classes, the latter exhibiting the strongest response. Longitudinally collected serum samples from patients in septic endotoxin shock revealed that the anti-TNF alpha antibodies were induced initially during septicaemia, reaching maximum reactivities within the first week and returning to low or undetectable levels on days 9-20.     

Nonstructural Protein 1-Specific Immunoglobulin M and G Antibody Capture Enzyme-Linked Immunosorbent Assays in Diagnosis of Flaviviral Infections in Humans

IgM antibody- and IgG antibody-capture enzyme-linked immunosorbent assays (MAC/GAC-ELISAs) targeted at envelope protein (E) of dengue viruses (DENV), West Nile virus, and Japanese encephalitis virus (JEV) are widely used as serodiagnostic tests for presumptive confirmation of viral infection. Antibodies directed against the flavivirus nonstructural protein 1 (NS1) have been proposed as serological markers of natural infections among vaccinated populations. The aim of the current study is to optimize an IgM and IgG antibody-capture ELISA (MAC/GAC-ELISA) to detect anti-NS1 antibodies and compare it with anti-E MAC/GAC-ELISA. Plasmids to express premembrane/envelope (prM/E) or NS1 proteins of six medically important flaviviruses, including dengue viruses (DENV-1 to DENV-4), West Nile virus (WNV), and Japanese encephalitis virus (JEV), were constructed. These plasmids were used for the production of prM/E-containing virus-like particles (VLPs) and secreted NS1 (sNS1) from COS-1 cells. Archived clinical specimens from patients with confirmed DENV, JEV, and WNV infections, along with naive sera, were subjected to NS1-MAC/GAC-ELISAs before or after depletion of anti-prM/E antibodies by preabsorption with or without VLPs. Human serum specimens from previously confirmed DENV infections showed significantly enhanced positive-to-negative (P/N) ratios for NS1-MAC/GAC-ELISAs after the depletion of anti-prM/E antibodies. No statistical differences in sensitivities and specificities were found between the newly developed NS1- and VLP-MAC/GAC-ELISAs. Further application of the assays to WNV- and JEV-infected serum panels showed similar results. A novel approach to perform MAC/GAC-ELISAs for NS1 antibody detection was successfully developed with great potential to differentiate antibodies elicited by the tetravalent chimeric yellow fever-17D/dengue vaccine or DENV infection.     

钩端螺旋体内毒素的实验研究 Ⅴ 钩端螺旋体脂多糖增强机体特异性抗感染能力的作用

波摩那群波摩那型56608株钩端螺旋体脂多糖(L-LPS)免疫的豚鼠能抵抗同株钩端螺旋体的攻击而免于发病和死亡,旦其心血和肾脏培养阳性率也明显下降。L-LPS免疫动物后可产生以IgM为主的群特异性抗体。该抗体当有补体存在时,能在体外裂解钩端旋螺体。此外,L-LPS尚能特异性地抑制豚鼠腹腔巨噬细胞的粘附作用。实验结果提示L-LPS为一种保护性抗原,可特异性地增强机体抗感染的能力。     

Cell-Mediated Immunity in Humans During Viral Infection: Dermal Hypersensitivity and In Vitro Lymphocyte Proliferation During Mild Viral Respiratory Infections

In vitro lymphocyte proliferation in response to phytohemagglutinin and streptokinase-streptodornase and delayed dermal hypersensitivity to several antigens were assessed in patients with mild viral upper respiratory infections. The response to phytohemagglutinin in 18 patients was not diminished during the viral infection. Deoxyribonucleic acid synthesis induced by streptokinase-streptodornase remained unaffected by the viral illness in five patients, and skin test reactivity was not depressed during the viral infection. Thus, it appeared that localized viral upper respiratory infections were not associated with suppression of systemic cell-mediated immunity.     

Cross-Neutralization of Leptospiral Hemolysins from Different Serotypes

Cross-neutralization studies on leptospiral hemolysins from strains of two antigenically different serotypes, pomona and canicola, were conducted in sheep. A third strain of serotype hardjo that does not produce hemolysin and is antigenically distinct was included for control purposes. Concentrated hemolysins, prepared from supernatant fluids of canicola or pomona cultures, produced hemolytic anemia in sheep after intravenous injection. Sheep previously infected with hemolysin-producing strains were refractory to effects of homologous or heterologous hemolysins. On the other hand, infection with hardjo did not confer immunity to the action of hemolysins. Hemolysin-neutralizing antibodies were demonstrable in sheep previously infected with pomona or canicola only after challenge with homologous or heterologous hemolysins. Cross-neutralization between two hemolysins were demonstrable in vitro. Hemolysin-neutralizing antibody titers did not correlate with agglutinin titers. Concentrated supernatant fluid of the hardjo culture provoked toxic reactions predominantly in sheep previously infected with pomona or canicola. The causes of these untoward reactions were not determined.     

Isolation and Characterization of a Leptospiral Type-Specific Antigen

The type-specific main (TM) antigen was extracted from the leptospiral cells of strain Kyoto of the Hebdomadis group by 90% phenol and was purified mainly by ethanol precipitation. The TM antigen was specific, forming one precipitin band by immunodiffusion and reacting by complement fixation test with anti-strain Kyoto serum; so far as has been tested, it does not appear to react with antisera of other serotypes. The sedimentation velocity of the TM antigen was 216S. Morphologically the TM antigen was filamentous in shape with a uniform width of 25 to 30 nm and various lengths from 50 nm to 1 μm. The infrared absorption pattern of the TM antigen generally resembled that of gram-negative bacterial endotoxin, and this fact suggests that the antigen is composed mainly of lipopolysaccharides. Galactose, rhamnose, xylose, arabinose, glucosamine, 13 kinds of acidic and neutral amino acids, lipids, and phosphorus were found in the TM antigen.     

Interferon-Induced NK Augmentation in Humans

By combining a single-cell cytotoxicity assay in agarose with estimations of the maximal natural killer (NK) cell potential (Vmax) by 51Cr release, the mechanism behind interferon augmentation of human NK cells were analysed. The number of target-binding cells (TBCs) the fraction of active TBCs and NK cell recycling were studied after short-term interferon treatment. The results demonstrate a dual effect of interferon on human NK cells: effector cell recruitment and increased effector cell recycling. Both of these variables were increased when NK cells were tested against the standard target K-562 and against Daudi and BJAB cells, derived from B-type lymphomas. However, when T cell lines derived from acute lymphocytic leukaemia (Molt-4 and 1310) were used as targets, a larger fraction of active NK cells were found among untreated TBCs, whereas interferon treatment only resulted in increased effector cell recycling and not in effector cell recruitment. No increase in TBCs after interferon treatment could be detected with any cell line tested. The difference seen between T and non-T cell lines with regard to interferon-induced effector cell recruitment is discussed in relation to known characteristics of the human NK system.     

Leptospiral lipopolysaccharide activates cells through a TLR2-dependent mechanism

Leptospira interrogans are zoonotic pathogens that have been linked to a recent increased incidence of morbidity and mortality in highly populated tropical urban centers. They are unique among invasive spirochetes in that they contain outer membrane lipopolysaccharide (LPS) as well as lipoproteins. Here we show that both these leptospiral outer membrane constituents activate macrophages through CD14 and the Toll-like receptor 2 (TLR2). Conversely, it seems that TLR4, a central component for recognition of Gram-negative LPS, is not involved in cellular responses to L. interrogans. We also show that for intact L. interrogans, it is LPS, not lipoprotein, that constitutes the predominant signaling component for macrophages through a TLR2 pathway. These data provide a basis for understanding the innate immune response caused by leptospirosis and demonstrate a new ligand specificity for TLR2.