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.

Evaluation of the safety and immunogenicity of Plasmodium falciparum apical membrane antigen 1, merozoite surface protein 1 or RTS,S vaccines with adjuvant system AS02A administered alone or concurrently in rhesus monkeys.

In an effort to broaden the immune response induced by the RTS,S/AS02(A),vaccine, we have evaluated the immunogenicity of the RTS,S antigen when combined with MSP1(42) and with AMA1, antigens derived from the asexual blood stage. The objectives of this study were (i) to determine whether MSP1(42) and AMA1 vaccines formulated with the AS02(A) Adjuvant System were safe and immunogenic in the rhesus monkey model; (ii) to investigate whether MSP1(42) or AMA1 induced immune interference to each other, or to RTS,S, when added singly or in combinations at a single injection site; (iii) in the event of immune interference, to determine if this could be reduced when antigens were administered at separate sites. We found that MSP1(42) and AMA1 were safe and immunogenic, eliciting antibodies, and Th1 and Th2 responses using IFN-gamma and IL-5 as markers. When malaria antigens were delivered together in one formulation, MSP1(42) and RTS,S reduced AMA1-specific antibody responses as measured by ELISA however, only MSP1(42) lowered parasite growth inhibitory activity of anti-AMA1 antibodies as measured by in vitro growth inhibition assay. Unlike RTS,S, MSP1(42) significantly reduced AMA1 IFN-gamma and IL-5 responses. MSP1(42) suppression of AMA1 IFN-gamma responses was not seen in animals receiving RTS,S+AMA1+MSP1(42) suggesting that RTS,S restored IFN-gamma responses. Conversely, AMA1 had no effect on MSP1(42) antibody and IFN-gamma and IL-5 responses. Neither AMA1 alone or combined with MSP1(42) affected RTS,S antibody or IFN-gamma and IL-5 responses. Immune interference by MSP1(42) on AMA1 antibody responses was also evident when AMA1, MSP1(42) and RTS,S were administered concurrently at separate sites. These results suggest that immune interference may be complex and should be considered for the design of multi-antigen, multi-stage vaccines against malaria.

Genotypes and phenotypes of Shiga toxin producing-Escherichia coli isolated from healthy cattle in Thailand.

Shiga toxin producing-Escherichia coli (STEC) has not yet been identified as an important aetiologic agent of human disease in Thailand. To evaluate the potential for STEC to contribute to human disease in Thailand, 139 fecal samples were collected from healthy cattle from five different provinces and analysed by genotypic and phenotypic methods for STEC. Of 139 samples, 27 (19.4%) were positive for stx1 and/or stx2 by multiplex polymerase chain reaction, or for O157 lipopolysaccharide (LPS) by immunoassay. Isolates positive for stx and/or O157 were subdivided into 49 strains that varied in the presence of the virulence determinants stx1+/stx2+ (22 strains), stx2+ (22 strains), stx1+ (4 strains), and O157 LPS (1 strain). Within these 49 distinguishable strains, other virulence determinants varied as follows: hlyA+ (77.6%), eae+ and tir+ (4.1%), and katP+ (6.12%). The most predominant profile (22 isolates) was stx1+/stx2+, eae-, tir-, etpD-, hlyA+, katP-. For further characterization of the isolated strains by two molecular typing assays, plasmid profiles and ERIC PCR were performed. The results suggest that the genetic and phenotypic profiles of STEC associated with human disease are not prevalent at this time in cattle in Thailand.

Localization of Shiga toxins of enterohaemorrhagic Escherichia coli in kidneys of paediatric and geriatric patients with fatal haemolytic uraemic syndrome.

Haemolytic uraemic syndrome (HUS) is characterized by haemolytic anaemia, thrombocytopenia and renal failure. Infection with enterohaemorrhagic Escherichia coli (EHEC), mainly O157:H7, has been strongly implicated as the major cause of HUS in children. The pathogenesis of HUS caused by the infection is not well understood and the defined sites of Stx in kidney of EHEC-infected humans has not been clearly demonstrated. The aim of this study was to investigate and compare the locations of Stx deposition in kidneys of paediatric and geriatric patients who died from enterohaemorrhagic E. coli O157 (EHEC) associated HUS, using an immunoperoxidase staining of the tissues. The study revealed that binding of Stx was relatively less and limited only to the renal tubules of an adult case (81 years old), while more binding was found at both renal tubules and glomeruli of an infant case (21 months old). The Stx binding in the infant's glomeruli was at podocytes, mesangial and endothelial cells. It has been known that young children are more susceptible than adults to HUS. One possibility for this is that the more extensive binding of the Stx to the kidney tissue of the paediatric patient might be due to the higher synthesis and expression of Stx receptors, i.e. Gb(3), in infants and less so in the aged individuals. However, other alternatives are possible, for example, the difference in stage of HUS in individual patients. Thus it is too early to draw any conclusion on this enigma and further investigation is required.

Lymphocyte activation after non-thermal trauma.

BACKGROUND: Severe injury causes immunological changes that may contribute to a poor outcome. Longitudinal characterization of lymphocyte response patterns may provide further insight into the basis of these immunological alterations. METHODS: Venous blood obtained seven times over 2 weeks from 61 patients with injury severity scores above 20 was assessed for lymphocyte phenotypic and activation markers together with serum levels of interleukin (IL) 2, IL-4, soluble IL-2 receptor (sIL-2R), soluble CD4 (sCD4), soluble CD8 (sCD8) and interferon gamma. RESULTS: Severe injury was associated with profound changes in the phenotypic and activation profile of circulating lymphocytes. Activation was indicated by increased numbers of T cells expressing CD25, CD69 and CD71, and raised serum levels of IL-2, sIL-2R, sCD4 and sCD8. Relatively higher levels of sIL-2R and sCD4 were found in patients with sepsis syndrome. CONCLUSION: Polytrauma is associated with dramatic alterations in the phenotypic and activation profile of circulating lymphocytes which are generally independent of clinical course. In contrast, several lymphocyte soluble factors, including sCD4 and sIL-2R, paralleled the clinical course. These data provide new insight into lymphocyte responses after injury and suggest that further assessment of soluble factors as clinical correlates, including those related to lymphocyte activation or generalized inflammation, may be warranted.

Impairment of Plasmodium falciparum growth in thalassemic red blood cells: further evidence by using biotin labeling and flow cytometry.

Certain red blood cell (RBC) disorders, including thalassemia, have been associated with an innate protection against malaria infection. However, many in vitro correlative studies have been inconclusive. To better understand the relationship between human RBCs with thalassemia hemoglobinopathies and susceptibility to in vitro infection, we used an in vitro coculture system that involved biotin labeling and flow cytometry to study the ability of normal and variant RBC populations in supporting the growth of Plasmodium falciparum malaria parasites. Results showed that both normal and thalassemic RBCs were susceptible to P falciparum invasion, but the parasite multiplication rates were significantly reduced in the thalassemic RBC populations. The growth inhibition was especially marked in RBCs from alpha-thalassemia patients (both alpha-thalassemia1/alpha-thalassemia2 and alpha-thalassemia1 heterozygote). Our observations support the contention that thalassemia confers protection against malaria and may explain why it is more prevalent in malaria endemic areas.

Culture of malaria parasites in two different red blood cell populations using biotin and flow cytometry.

A novel culture system using biotin/streptavidin and flow cytometry was developed to compare maturation and growth rates in Plasmodium falciparum malaria parasites in two distinct red blood cell (RBC) populations. Biotin was used to label a selected RBC population which was then mixed with another distinct unbiotinylated RBC population. P. falciparum-infected RBCs were used to initiate co-cultures followed over 2-3 schizogonic growth cycles. Co-cultures were harvested and stained with streptavidin-fluorescein isothiocyanate (FITC) followed by fixation and staining of parasite DNA. The combination of biotin/streptavidin-FITC and DNA fluorochrome enabled simultaneous flow cytometric analysis of the two different RBC populations and of the parasitemias in each RBC population. We then used this system to study the in vitro susceptibility of RBCs from individuals with hemoglobin H (Hb H) disease to infection and growth of P. falciparum. Significant reduction in parasite multiplication was found in Hb H RBCs as compared with that in normal RBCs. This novel malaria culture system offers two major innovations: a method to compare directly the relative ability of any two red blood cell populations to support malaria parasite invasion and development under identical conditions, and a critical reduction in the volume of blood and reagents needed to assess parasite growth. The application of biotin-labeled RBCs in the flow cytometric analysis of parasite development may offer new insights in studies of the relationship between RBC defects and susceptibility to malaria parasites.

Interleukin-10 inhibits tumor necrosis factor production but not antigen-specific lymphoproliferation in acute Plasmodium falciparum malaria.

In vivo interleukin (IL)-2, IL-4, IL-10, and interferon (IFN)-gamma production was measured at the mRNA transcript and protein levels in patients acutely infected with Plasmodium falciparum and during convalescence. Both IL-10 and IFN-gamma but not IL-2 were produced regardless of the patients' clinical severity. IL-4 production was variable. Circulating IFN-gamma and IL-10 were significantly higher in patients with severe disease (P < .01 and .001, respectively). In vitro stimulation of peripheral blood mononuclear cells (PBMC) by malarial antigens during acute infection showed that although there was no lymphoproliferation, the cells could produce IL-10 and IFN-gamma. Recombinant human IL-10 completely abolished in vitro tumor necrosis factor (TNF)-alpha production in response to malarial antigens, as well as the antigen-specific proliferative response of convalescent patients. However, anti-IL-10 was insufficient to restore proliferation of PBMC from acutely infected patients. These findings suggest that IL-10 may have an important negative feedback action on the production of inflammatory cytokines in acute falciparum malaria without contributing to the defect in antigen-specific proliferation.

Flow cytometric immunophenotyping of lymphocyte subsets in samples that contain a high proportion of non-lymphoid cells.

Flow cytometric (FCM) immunophenotyping of peripheral blood from thalassemia patients presents technical difficulties because of the high proportion of immature red cells. The combination of forward scatter (FSC) and side scatter (SSC) with fluorescence associated with human leukocyte antigen/monocyte antigen (CD45/CD14) was unable to identify the lymphocyte population in thalassemia patients; therefore, it was necessary to exclude immature red cells to analyze lymphocyte subsets in these patients. A simultaneous three-color FCM method was developed, with the basis that transferrin receptor (CD71) or glycophorin A (glyco A) is present on all immature red cells, but is not expressed on CD45 positive leukocytes. In this study, the lymphocyte population was identified by gating out unwanted cell populations using the FSC/CD71-fluorescein isothiocyanate (FITC), FSC/glyco A-FITC, or FSC/CD45-peridinin chlorophyll protein (PerCP) profiles. The CD71-FITC negative cells, glyco A-FITC negative cells, or CD45-PerCP positive cells were identified, then analyzed on the basis of FSC/SSC to allow any remaining non-lymphocyte cells in FSC/SSC gate to be excluded. The cells in FSC/SSC gate were then analyzed using other irrelevant two-color antibodies. Of the three gating strategies, CD45-PerCP and glyco A-FITC methods are better than the CD71-FITC gating method. Both methods markedly increase the purity of lymphocytes in the analysis gate. Either method is easy, straightforward, requires a six-tube set of reagent tubes, and provides a reliable method for immunophenotyping lymphocyte subsets in preparations containing a large percentage of non-lymphoid cells.

Polyclonal expansion of peripheral gamma delta T cells in human Plasmodium falciparum malaria.

Plasmodium falciparum malaria in humans is associated with an increase in the percentage and absolute number of gamma delta T cells in the peripheral blood. This increase begins during the acute infection phase and persists for at least 4 weeks during convalescence. In the present study, 25 to 30% of the gamma delta T cells expressed HLA-DR antigens in vivo and in some patients they proliferated in response to further stimulation by purified human interleukin 2 in vitro. However, there was no in vitro proliferative response to various malarial antigens, including a 75-kDa heat shock protein and a 72-kDa glucose-regulated protein of P. falciparum during the acute infection phase. Cytofluorographic studies showed that although an increase of V delta 1- gamma delta T cells was largely responsible for the expansion of the total number of gamma delta T cells, there was also a proportional increase in V delta 1+ cells. These results were confirmed with anchored PCR and by DNA sequencing to characterize at the molecular level the set of T-cell receptor (TCR) delta mRNAs expressed in the peripheral blood of two patients with high levels of gamma delta T cells. In each case, most of the TCR delta mRNA transcripts corresponded to nonproductively rearranged delta genes (unrearranged J delta or near J delta spliced to C delta). In those sequences which did represent productively rearranged genes, most of the transcripts originated from a V delta 2/J delta 1 joining, as in normal individuals. A minority of transcripts originated from a V delta 1/J delta 1 rearrangement, and one originated from a V alpha 4/J delta 1 rearrangement. Polyclonal activation of gamma delta T cells was inferred from the extensive junctional diversity seen in the delta mRNAs analyzed. Expansion of a heterogeneous set of both V delta 1(-)- and V delta 1(+)-bearing T cells suggests that the elevated levels of gamma delta T cells seen during acute P. falciparum malaria arose from immune responses to multiple distinct parasite antigens or unidentified host factors.

Effect of orally active hydroxypyridinone iron chelators on human lymphocyte function.

Several iron chelators, 3-hydroxypyridin-4-ones (CP) and desferrioxamine (DF) were compared for their effect on DNA synthesis, cell viability and expression of cell proliferation markers. Short-term (4 h) exposure of human peripheral blood mononuclear cells to CP or DF inhibited the proliferative response of cells to concanavalin A (Con A). Inhibition by CP and DF showed a dose-dependent effect with CP compounds more active than DF. Increased inhibitory activity of CP over DF was partly due to the lipophilic properties of CP. Pre-saturation of CP and DF with exogenous ferric ion either diminished or prevented the inhibitory effect. At high chelator concentrations or prolonged (72 h) exposure of the cells to chelators, inhibition occurred but poor cell viability was observed. In contrast to their inhibition of DNA synthesis, these iron chelators showed little effect on protein synthesis and the expression of transferrin receptors and interleukin-2 (IL-2) receptors. These findings suggest that both DF and CP compounds exert their effect by chelation of ferric ion with subsequent inhibition of DNA synthesis.

Increased gamma delta T cells in acute Plasmodium falciparum malaria.

The T cell receptor of gamma delta is normally expressed on a small percentage of peripheral lymphocytes. Although the role of gamma delta T cells in the physiologic immune response is still unknown, there is accumulating evidence that gamma delta T cells may participate in the immune response to mycobacterial and other infectious organisms. In this study, we have quantitated the number of circulating gamma delta T cells during acute Plasmodium falciparum malaria. The results indicate that gamma delta T cells are elevated during the acute infection and remain elevated for at least 4 weeks during convalescence. T cells may participate in the immune response against P. falciparum by functioning as non-MHC restricted cytotoxic cells against intraerythrocytic parasites. Alternatively, lymphokines may be produced on antigen stimulation which may have antiparasitic activity.

Lymphocyte subpopulations during acute and convalescence phases of malaria.

Lymphocytes of normal healthy persons were separated from blood by Ficoll-Hypaque gradient centrifugation and iron-magnet application. peripheral blood lymphocytes (PBL) were stained by various dye-labeled monoclonal antibodies. Cells positive for specific surface markers were enumerated by a fluorescence activated cell sorter (FACS) and fluorescence microscope (FM). The results revealed that the percentages of cells positive with one monoclonal antibody counted by these two techniques were similar while the percentages of cells with double staining were higher when counted by FACS than by FM. Lymphocyte subpopulations of 18 patients infected with Plasmodium falciparum during acute and convalescence period were studied. Lymphocytopenia occurred during the acute infection while total white blood cell counts were normal. PBL of the patients were stained with OKT3, OKT4, OKT8, Leu-11 and a combination of Leu-7, Leu-1 monoclonal antibodies. The absolute numbers of all lymphocyte subpopulations were decreased during the acute infection while T8 positive cells were decreased in both percentage and absolute number. Thus T4:T8 ratio (1.7:1) became higher than normal (1.3:1) at this period. During convalescence phase, absolute numbers and percentages of Leu-7+, Leu-1+ and perhaps Leu-7+, Leu-11- cells which had low NK cell activity were significantly higher than during acute illness. The finding might explain why the NK cell activity was low during the convalescence period.