Matrix metalloproteinases contribute to the regulation of chemokine expression and pulmonary inflammation in Cryptococcus infection.

Matrix metalloproteinases (MMPs) are a family of extracellular proteases that play roles in regulating the immune response in inflammatory processes. Previous studies indicated that different MMPs were involved in the host defence and tissue damage in response to different pathogens. However, the contributions of MMPs during Cryptococcus infection have not been addressed clearly. Here, we examined the expression and activity of MMPs during Cryptococcus infection. Among MMP family members, we found significant increases of MMP-3 and MMP-12 mRNA levels and MMP12 zymographic activities in response to C. neoformans but not C. gattii infection. The expression of MMP12 was induced in RAW cells after C. neoformans treatment and in alveolar macrophages purified from C. neoformans-infected mice. Interestingly, administration of MMP inhibitor GM6001 into C. neoformans-infected mice resulted in a significantly increased pulmonary fungal burden with attenuated inflammatory cell infiltration. Corresponding to this finding, the expression of the macrophage- and neutrophil-attracting chemokines CCL2 and CXCL1 was inhibited in the GM6001-treated group and MMP12 levels were found to be correlated strongly with CCL2 mRNA expression. Thus, our data suggest that the induction of MMPs by C. neoformans infection potentiates inflammatory cell infiltration by modulating pulmonary chemokines, thereby promoting effective host immunity to pulmonary Cryptococcus infection.

Hybrid & El Tor variant biotypes of Vibrio cholerae O1 in Thailand.

BACKGROUND & OBJECTIVES: El Tor Vibrio cholerae O1 carrying ctxB C trait, so-called El Tor variant that causes more severe symptoms than the prototype El Tor strain, first detected in Bangladesh was later shown to have emerged in India in 1992. Subsequently, similar V. cholerae strains were isolated in other countries in Asia and Africa. Thus, it was of interest to investigate the characteristics of V. cholerae O1 strains isolated chronologically (from 1986 to 2009) in Thailand. METHODS: A total of 330 V. cholerae O1 Thailand strains from hospitalized patients with cholera isolated during 1986 to 2009 were subjected to conventional biotyping i.e., susceptibility to polymyxin B, chicken erythrocyte agglutination (CCA) and Voges-Proskauer (VP) test. The presence of ctxA, ctxB, zot, ace, toxR, tcpA C , tcpA E, hlyA C and hlyA E were examined by PCR. Mismatch amplification mutation assay (MAMA) - and conventional- PCRs were used for differentiating ctxB and rstR alleles. RESULTS: All 330 strains carried the El Tor virulence gene signature. Among these, 266 strains were typical El Tor (resistant to 50 units of polymyxin B and positive for CCA and VP test) while 64 had mixed classical and El Tor phenotypes (hybrid biotype). Combined MAMA-PCR and the conventional biotyping methods revealed that 36 strains of 1986-1992 were either typical El Tor, hybrid, El Tor variant or unclassified biotype. The hybrid strains were present during 1986-2004. El Tor variant strains were found in 1992, the same year when the typical El Tor strains disappeared. All 294 strains of 1993-2009 carried ctxBC ; 237 were El Tor variant and 57 were hybrid. INTERPRETATION & CONCLUSIONS: In Thailand, hybrid V. cholerae O1 (mixed biotypes), was found since 1986. Circulating strains, however, are predominantly El Tor variant (El Tor biotype with ctxB C).

Trichinella spiralis-specific monoclonal antibodies and affinity-purified antigen-based diagnosis.

Hybridomas secreting monoclonal antibodies (MAbs) to Trichinella spiralis were produced. Myeloma cells were fused with splenocytes of a mouse immunized with excretory-secretory (E-S) antigen of infective larvae. A large percentage of growing hybrids secreted antibodies cross-reactive to many of 23 heterologous parasites tested. Only 6 monoclones (designated 3F2, 5D1, 10F6, 11E4, 13D6 and 14D11) secreted MAbs specific to the E-S antigen and/or a crude extract (CE) of T. spiralis infective larvae. The 6 monoclones secreted IgM, IgG3, IgM, IgG3, IgG3 and IgG3, respectively. Clone 5D1 was selected to mass produce MAbs which were then coupled to CNBr-activated Sepharose CL-4B to prepare an affinity-purified antigen. Dot-blot ELISA with either purified antigen or CE was evaluated. There were 17 patients with acute trichinellosis and 76 individuals convalescing from T. spiralis infection (group 1). Controls were 170 patients with parasitic infections other than trichinellosis (group 2) and 35 healthy parasite-free controls (group 3). CE-ELISA was positive in all group 1 patients. However, sera from many group 2 patients also were reactive (opisthorchiasis-44.2%, schistosomiasis-44%, gnathostomiasis-30%, paragonimiasis-28.6%, taeniasis-27.3%, strongyloidiasis-23.1% and hookworm infections-20%). Affinity-purified antigen was 100% specific, all sera from group 2 and group 3 individuals tested negative. Although 74 of 76 patients (97.4%) with convalescing trichinellosis tested positive, sera from only 3 of 17 patients (17.6%) with acute T. spiralis were reactive. Thus, CE antigen is appropriate when sensitivity is needed, while purified antigen should be used when specificity is required. Dot-blot ELISA is easier to perform, more rapid and less expensive than indirect ELISA. Many samples can be assayed simultaneously, special equipment is not required, and results can be preserved for retrospective analysis. Dot-blot ELISA is therefore the method of choice for the rapid diagnosis of trichinellosis, particularly when more complex laboratory tests are unavailable.

Detection of Salmonella contamination in food samples by dot-ELISA, DNA amplification and bacterial culture.

A dot-blot enzyme-linked immunosorbent assay (dot-ELISA) employing a genus Salmonella specific monoclonal antibody (MAb) was used for detection of the bacteria in food samples in comparison with the conventional culture method and the DNA amplification. Among the 200 chicken and pork samples (100 each) tested, 9% and 33%, 7% and 20% and 7 and 23% were positive for salmonellae by the dot-ELISA, the culture method and the DNA amplification, respectively. Statistical analyses revealed that the sensitivity, specificity, efficacy, and positive and negative predictive values of the detection of Salmonella in the food samples by dot-ELISA compared with the culture method were 93.33%, 91.76%, 92%, 66.66% and 98.73%, respectively. Comparison of the DNA amplification and the culture method revealed the sensitivity, specificity, efficacy, and positive and negative predictive values of 100%, 91.58%, 92%, 65.21% and 100%, respectively. The dot-ELISA and the DNA amplification results were in a better agreement when the two assays were compared. The sensitivity, specificity, efficacy, positive and negative predictive values of the dot-ELISA compared to the DNA amplification were 91.3%, 100%, 98%, 100% and 97.5%, respectively. From this study, the dot-ELISA is rapid, simple, sensitive, specific at low cost with limited amount of infectious waste to be disposed and offers another advantage in that it detects only the smooth LPS of Salmonella which implies the possible presence of the virulent organisms.

Diagnosis of enteric fever caused by Salmonella spp. in Vietnam by a monoclonal antibody-based dot-blot ELISA.

Enteric fever caused by Salmonella spp. is prevalent in Vietnam. None of the currently available diagnostic methods meets the ideal criteria on rapidity, simplicity, sensitivity, specificity, cost-effectiveness and practicality for developing areas. In this study, a recently developed monoclonal antibody-based dot-blot ELISA was used in comparison with the hemoculture method and the classical Widal test for diagnosis of salmonellosis in 171 Vietnamese patients presenting with clinical features of enteric fever. Urine samples of 50 healthy counterparts were used as negative controls. Salmonella spp. were isolated from 77 of 171 patients (45%) while 98 and 111 patients were positive by dot-blot ELISA and Widal test, respectively. The diagnostic sensitivity, specificity, accuracy, positive predictive value and negative predictive value of the ELISA performed on three serial urine samples collected at 2 hour intervals of the 171 patients were 92.2%, 71.3%, 80.7%, 72.4% and 91.8%, respectively when compared with the culture method. The Widal test performed on acute and convalescence serum samples showed 87.0%, 46.8%, 68.4%, 60.4% and 83.3% diagnostic sensitivity, specificity, accuracy, and positive and negative predictive values, respectively when compared with the bacterial culture method. Kappa coefficience revealed very good agreement beyond chance between the MAb-based ELISA and the culture method. The ELISA was not reactive when tested on urine samples of 50 healthy individuals which indicates 100% specificity. The Salmonella antigenuria of the patients as detected by ELISA lasted 10.3+/-3.9 days after initiating antibiotic treatment. The MAb-based dot-blot ELISA is easy to perform. It is rapid, sensitive, specific, inexpensive, and non-invasive and does not require equipment, thus is suitable for developing areas. It can detect acute/recent infection and can be used for evaluation of the efficacy of the treatment.

Virulence genes and genetic diversity of Streptococcus suis serotype 2 isolates from Thailand.

Isolates of Streptococcus suis from different Western countries as well as those from China and Vietnam have been previously well characterized. So far, the genetic characteristics and relationship between S. suis strains isolated from both humans and pigs in Thailand are unknown. In this study, a total of 245 S. suis isolates were collected from both human cases (epidemic and sporadic) and pigs (diseased and asymptomatic) in Thailand. Bacterial strains were identified by biochemical tests and PCR targeting both, the 16S rRNA and gdh genes. Thirty-six isolates were identified as serotype 2 based on serotyping and the cps2-PCR. These isolates were tested for the presence of six virulence-associated genes: an arginine deiminase (arcA), a 38-kDa protein and protective antigen (bay046), an extracellular factor (epf), an hyaluronidase (hyl), a muramidase-released protein (mrp) and a suilysin (sly). In addition, the genetic diversities of these isolates were studied by RAPD PCR and multilocus sequence typing (MLST) analysis. Four virulence-associated gene patterns (VAGP 1 to 4) were obtained, and the majority of isolates (32/36) carried all genes tested (VAGP1). Each of the three OPB primers used provided 4 patterns designated RAPD-A to RAPD-D. Furthermore, MLST analysis could also distinguish the 36 isolates into four sequence types (STs): ST1 (n = 32), ST104 (n = 2), ST233 (n = 1) and a newly identified ST, ST336 (n = 1). Dendrogram constructions based on RAPD patterns indicated that S. suis serotype 2 isolates from Thailand could be divided into four groups and that the characteristics of the individual groups were in complete agreement with the virulence gene profiles and STs. The majority (32/36) of isolates recovered from diseased pigs, slaughterhouse pigs or human patients could be classified into a single group (VAGP1, RAPD-A and ST1). This genetic information strongly suggests the transmission of S. suis isolates from pigs to humans in Thailand. Our findings are the first to report genetic characteristics of strains from Thailand and to elucidate the genetic relationship among S. suis isolates from human and pig origins.

Immunogenicity and protective role of three formulations of oral cholera vaccine.

Three formulations of oral cholera vaccine were compared with respect to their immunogenicity and protective ability in a rat ileal loop model. Eight-week-old Wistar rats were divided into five groups. The first group received orally vaccine A consisting of liposome-associated V. cholera lipopolysaccharide, fimbriae and procholeragenoid, whereas the rats of groups 2 and 3 received orally vaccines B and C consisting of heatkilled fimbriated and non-fimbriated whole cell V. cholerae, respectively. Rats of groups 4 and 5 were controls that received orally liposomes alone and normal saline solution, respectively. It was found that vaccine A elicited stronger immune responses to all three V. cholerae antigens. The antibody responses were detected in both serum and intestinal lavage samples. Vaccine B elicited only modest serum and intestinal responses to V. cholerae fimbriae (anti-F). No detectable immune response was found in rats of group 3 immunized with vaccine C. Rats immunized with vaccines A and B had a similar order of magnitude of numbers of vibrios adhered to their intestinal mucosa. These numbers were less than those associated with the intestinal tissues of control rats of groups 4 and 5 by about two orders of magnitude. Although without any detectable immune response, rats of group 3 that were immunized with vaccine C showed some reduction in numbers of vibrios associated with their intestinal mucosa. The numbers of vibrios recovered from the intestinal segments of rats of all treatment groups were in the order group 1 = 2 < 4 = 5. Electron micrography also revealed patches of vibrio colonization on the mucosa of rats of groups 3, 4 and 5. These features were not found in the groups vaccinated with vaccines A and B. The inhibition of vibrio colonization afforded by the vaccines was biotype- and serotype-non-specific. The results suggest that the heat-killed whole cell fimbriated V. cholerae may be an alternative vaccine preparation to the liposome-associated refined antigen vaccine at a lower cost.

Characterization of Saa, a novel autoagglutinating adhesin produced by locus of enterocyte effacement-negative Shiga-toxigenic Escherichia coli strains that are virulent for humans.

The capacity of Shiga toxigenic Escherichia coli (STEC) to adhere to the intestinal mucosa undoubtedly contributes to pathogenesis of human disease. The majority of STEC strains isolated from severe cases produce attaching and effacing lesions on the intestinal mucosa, a property mediated by the locus of enterocyte effacement (LEE) pathogenicity island. This element is not essential for pathogenesis, as some cases of severe disease, including hemolytic uremic syndrome (HUS), are caused by LEE-negative STEC strains, but the mechanism whereby these adhere to the intestinal mucosa is not understood. We have isolated a gene from the megaplasmid of a LEE-negative O113:H21 STEC strain (98NK2) responsible for an outbreak of HUS, which encodes an auto-agglutinating adhesin designated Saa (STEC autoagglutinating adhesin). Introduction of saa cloned in pBC results in a 9.7-fold increase in adherence of E. coli JM109 to HEp-2 cells and a semilocalized adherence pattern. Mutagenesis of saa in 98NK2, or curing the wild-type strain of its megaplasmid, resulted in a significant reduction in adherence. Homologues of saa were found in several unrelated LEE-negative STEC serotypes, including O48:H21 (strain 94CR) and O91:H21 (strain B2F1), which were also isolated from patients with HUS. Saa exhibits a low degree of similarity (25% amino acid [aa] identity) with YadA of Yersinia enterocolitica and Eib, a recently described phage-encoded immunoglobulin binding protein from E. coli. Saa produced by 98NK2 is 516 aa long and includes four copies of a 37-aa direct repeat sequence. Interestingly, Saa produced by other STEC strains ranges in size from 460 to 534 aa as a consequence of variation in the number of repeats and/or other insertions or deletions immediately proximal to the repeat domain.

Immunogenicity of liposome-associated and refined antigen oral cholera vaccines in Thai volunteers.

A mixture of Vibrio cholerae antigens made up of crude fimbrial extract, lipopolysaccharide and procholeragenoid was administered orally to Thai volunteers either as free antigen or associated with liposomes. All vaccines and controls were administered in three doses given at 14 day intervals. Nine volunteers received liposome-associated vaccine and seven received free vaccine. Liposomes without antigens were given to eight volunteers and seven volunteers received 5% NaHCO3 solution alone. Both vaccines had 100% immunogenicity as determined by serum vibriocidal antibody responses. Liposomes were shown by indirect ELISA to localize the immune response against lipopolysaccharide and fimbriae to the intestinal mucosa. Vaccines given liposome-associated antigens had a higher rate of antigen-specific antibody response than did individuals who had received free antigens. The vaccines induced intestinal antibodies of IgM and/or IgA isotypes, but not IgG antibody.

Rapid diagnosis of cholera caused by Vibrio cholerae O139.

Hybridomas secreting specific monoclonal antibodies (MAbs) to Vibrio cholerae serogroup O139 were produced. Six monoclones (hybridomas) secreting MAbs specific only to lipopolysaccharide of V. cholerae O139 strains and which did not cross-react to 137 strains of other enteric microorganisms were obtained. These clones were designated 12F5-G11, 12F5-G2, 15F5-H5, 5B9-F8, 14C9-D2, and 6D2-D8. The immunoglobulin (Ig) heavy chain isotypes secreted by these clones were IgG2b, IgG2b, IgG2b, IgM, IgG2b, and IgG3, respectively. Clone 12F5-G11 was selected for mass production of MAb, which was used as a detection reagent in the antigen detection assay for diagnosis of cholera caused by V. cholerae O139, and this assay was compared to the conventional bacterial isolation method. Five batches of rectal swab cultures in alkaline-peptone water were collected from 6,497 patients with watery diarrhea. These were 6,310 patients admitted to Bamrasnaradura Infectious Diseases Hospital, 16 patients from Krung Thon Hospital, 78 patients from Bangkok Children's Hospital, 19 patients from Karen refugee camps, and 74 Indian patients from the National Institute of Cholera and Enteric Diseases, Calcutta, India. The V. cholerae O139 isolations from the rectal swab cultures and the antigen detection assays (i.e., the MAb-based dot-blot ELISA) were performed by different persons of different laboratories, and the results were revealed after all specimens had been tested. Of the 6,497 samples tested, the dot-blot ELISA correctly identified 42 of 42 V. cholerae O139-positive samples and gave a result of positive for three samples which were culture negative for V. cholerae O139. The diagnostic sensitivity, specificity, and efficacy of the dot-blot ELISA were 100, 99.95, and 99.26%, respectively. The ELISA is easy to perform and relatively inexpensive. It can test multiple samples at a single time, does not require special equipment, and does not produce great quantities of contaminated waste. Most of all, it reduces the diagnostic time from at least 2 days for the bacterial isolation to less than 90 min. The assay is recommended as a rapid screening test of cholera cases caused by V. cholerae O139.