Identification of Peptides that Mimic N. meningitidis LOS Epitopes Via the Use of Combinatorial Phage-Display Libraries.

Although capsular polysaccharide-based vaccines are effective at reducing the incidence of meningococcal disease caused by serogroups A, C, Y, and W135 (1-3), immunization against serogroup B disease using similar strategies has proven unsuccessful (4,5). The primary reason for this is that the α2,8-linked N-acetylneuraminic acid homopolymer expressed by serogroup B strains is poorly immunogenic in humans (6). Consequently, considerable effort has been devoted towards the development of alternative strategies for vaccination against serogroup B disease. Many of these newer strategies include the use of lipooligosaccharide (LOS) as a protective antigen (7). One of the approaches that we are currently pursuing involves the use of synthetic oligopeptides to stimulate antibody responses that are cross-reactive with LOS antigens expressed by serogroup B Neisseria meningitidis strains. An integral part of these studies has been the application of combinatorial phage-display technology. Described here is an overview of the methods that we have utilized to identify peptide mimics of LOS epitopes.

Pathogenesis of and immunity to melioidosis.

While Burkholderia pseudomallei, the causative agent of melioidosis, is becoming increasingly recognized as a significant cause of morbidity and mortality in regions to which it is endemic, no licensed vaccine preparation currently exists for immunization against the disease. Therefore, one of the primary goals of our research has been to identify and characterize antigens expressed by B. pseudomallei isolates for the intended purpose of developing a vaccine construct that can be used to actively immunize specific high risk populations against the disease. By utilizing a combination of biochemical, immunological and molecular approaches, our studies now indicate that some of the most promising candidates for this task include flagellin proteins and the endotoxin derived O-polysaccharide (PS) antigens expressed by the organism. In this review, we have attempted to summarize the current status of B. pseudomallei research while endeavoring to provide a rationale for our approach towards the development of a melioidosis vaccine.

Antilipopolysaccharide II: an antibody protective against fatal melioidosis.

This was a study of IgG antibody responses to two S-type lipopolysaccharides (LPS I and LPS II) and flagellin of Burkholderia pseudomallei in patients with melioidosis. The specificity of these antibodies was 91.7%, 90.3%, and 93.8%, respectively, when compared to responses in a population where the organism is not endemic. Only the level of antibody to LPS II (anti-LPS II) was significantly higher in patients who survived than in those who died, as well as in patients with nonsepticemic vs. septicemic melioidosis. Results of logistic regression analysis, controlled for confounding factors such as duration of illness before treatment and bacteremic status, confirmed that a high level of anti-LPS II was a significant factor protective against fatal melioidosis. Thus, LPS II of B. pseudomallei would be a potentially useful component of a vaccine developed against fatal melioidosis. Further studies are in progress to determine the level of this antibody among those with asymptomatic infection in areas where melioidosis is endemic.

Current studies on the pathogenesis of melioidosis.

Burkholderia pseudomallei is a major cause of bacterial septicemias in many parts of the world, particularly Thailand; the known geographic range of the organism appears to be enlarging as awareness of the organism and the disease it causes--melioidosis--increases. B. pseudomallei is intrinsically resistant to most antibiotics, and our knowledge of B. pseudomallei pathogenesis is lacking. Thus, the long-term objective of our research is to define at a molecular level the pathogenesis by combining genetic, immunologic, and biochemical approaches with animal model studies. Basic studies on B. pseudomallei pathogenesis are acutely needed to provide a knowledge base to rationally design new modes of therapy directed against this organism.

Molecular characterization of genetic loci required for secretion of exoproducts in Burkholderia pseudomallei.

Previous studies have demonstrated that Burkholderia pseudomallei secretes protease, lipase, and phospholipase C (PLC) into the extracellular milieu, but their mechanisms of secretion and roles in pathogenesis have not been elucidated. In this study, we isolated and characterized 29 transposon mutants unable to secrete protease, lipase, and PLC.

Burkholderia thailandensis sp. nov., a Burkholderia pseudomallei-like species.

The presence of a Burkholderia pseudomallei-like species based upon the significant genotypic and phenotypic dissimilarities exhibited between these organisms and true B. pseudomallei strains has been reported previously. In this study, a comprehensive 16S rDNA-based phylogenetic analysis further supports the existence of this newly described Burkholderia species for which the name Burkholderia thailandensis sp. nov. is proposed.

The type II O-antigenic polysaccharide moiety of Burkholderia pseudomallei lipopolysaccharide is required for serum resistance and virulence.

Melioidosis, an infection caused by the gram-negative bacterial pathogen Burkholderia pseudomallei, is endemic in south-east Asia and northern Australia. Acute septicaemic melioidosis is a major cause of morbidity and mortality, especially in north-east Thailand. B. pseudomallei is highly resistant to the bactericidal activity of normal human serum (NHS), and we have found that B. pseudomallei 1026b multiplies in 10-30% NHS. We developed a simple screen for the identification of serum-sensitive mutants based on this novel phenotype. Approximately 1200 Tn5-OT182 mutants were screened, and three serum-sensitive mutants were identified. The type II O-antigenic polysaccharide (O-PS) moiety of lipopolysaccharide was not present in the serum-sensitive mutants. A representative serum-sensitive mutant, SRM117, was killed by the alternative pathway of complement and was less virulent than 1026b in three animal models of melioidosis. The Tn5-OT182 integrations in the serum-sensitive mutants were physically linked on the B. pseudomallei chromosome, and further genetic analysis of this locus revealed a cluster of 15 genes required for type II O-PS production. The proteins encoded by these genes were similar to proteins involved in bacterial polysaccharide biosynthesis. The results presented here demonstrate that type II O-PS is essential for B. pseudomallei serum resistance and virulence.

Specificity and functional activity of anti-Burkholderia pseudomallei polysaccharide antibodies.

The lipopolysaccharide (LPS) of Burkholderia pseudomallei, the causative agent of melioidosis, consists of two O-antigenic polysaccharides designated O-PS I and O-PS II. In this study, the O-PS specificity and functional activity of a protective polyclonal antiserum and an immunoglobulin M (IgM) monoclonal antibody were determined. The polyclonal antiserum recognized both O-PS I and O-PS II, while the monoclonal antibody was O-PS II specific. Both mediated phagocytic killing of B. pseudomallei by polymorphonuclear leukocytes. Patients acutely infected with B. pseudomallei also produced antibodies to the two O-PSs, but these antibodies were not produced by asymptomatic individuals from an area of endemicity who were seropositive by an indirect hemagglutination test using sonicated heat-killed whole organisms as antigen. IgM antibodies were detected only in patients with localized infection. IgG antibodies were detected in all acutely infected patients, but there was no significant difference in antibody levels among patients with localized infection, patients who survived septicemic illness, and patients who died from septicemic illness. Further analysis of the IgG response revealed production of IgG1 and IgG2 antibodies by all patient groups, while an IgG3 response was seen only in survivors of septicemic infection. IgG4 was not detectable even when a fivefold-lower serum dilution was used. Patient sera also mediated phagocytic killing by polymorphonuclear leukocytes, and the killing effect was enhanced by complement. These results suggest that antibodies to the LPS O-polysaccharides of B. pseudomallei are protective by promoting phagocytic killing. The antibodies develop during human infection and may facilitate clearance of the organisms, as seen in a diabetic rat model of B. pseudomallei infection.

Characterization of Burkholderia pseudomallei and Burkholderia pseudomallei-like strains.

Previous reports in the literature suggest that Burkholderia pseudomallei strains can be differentiated on the basis of animal virulence. Twenty environmentally and clinically derived isolates of Burkholderia pseudomallei were examined for the production of exoenzymes, morphological and biochemical phenotypes and virulence for Syrian golden hamsters. The partial sequence of the 16S ribosomal RNA [rRNA] genes from a number of these strains was also determined. Based upon these observations, it is suggested that highly virulent Burkholderia pseudomallei strains are true Burkholderia pseudomallei strains. The DNA sequences of the 16S rRNA genes of the true Burkholderia pseudomallei strains were identical to the published sequences for Burkholderia pseudomallei while differences were revealed between the published sequences and those of the lowly virulent strains. Thus, these latter strains have been designated as Burkholderia pseudomallei-like organisms since they demonstrate significant differences in exoenzyme production, hamster virulence and 16S rRNA gene sequences.

Mutagenesis of Burkholderia pseudomallei with Tn5-OT182: isolation of motility mutants and molecular characterization of the flagellin structural gene.

Burkholderia pseudomallei is a human and animal pathogen in tropical regions, especially Southeast Asia and northern Australia. Currently little is known about the genetics and molecular biology of this organism. In this report, we describe the mutagenesis of B. pseudomallei with the transposon Tn5-OT182. B. pseudomallei 1026b transposon mutants were obtained at a frequency of 4.6 x 10(-4) per initial donor cell, and the transposon inserted randomly into the chromosome. We used Tn5-OT182 to identify the flagellin structural gene, fliC. We screened 3,500 transposon mutants and identified 28 motility mutants. Tn5-OT182 integrated into 19 unique genetic loci encoding proteins with homology to Escherichia coli and Salmonella typhimurium flagellar and chemotaxis proteins. Two mutants, MM35 and MM36, contained Tn5-OT182 integrations in fliC. We cloned and sequenced fliC and used it to complement MM35 and MM36 in trans. The fliC transcriptional start site and a sigmaF-like promoter were identified by primer extension analysis. We observed a significant difference in the expression of two distinct fliC-lacZ transcriptional fusions during bacterial growth, suggesting the presence of a latent intragenic transcriptional terminator in fliC. There was no significant difference in the virulence of 1026b compared to that of MM36 in diabetic rats or Syrian hamsters, suggesting that flagella and/or motility are probably not virulence determinants in these animal models of B. pseudomallei infection. A phylogenetic analysis based on the flagellins from a variety of bacterial species supported the recent transfer of B. pseudomallei from the genus Pseudomonas to Burkholderia.

A controlled field trial of group versus individual cognitive-behavioural training for relapse prevention.

Results are presented of a randomized field trial comparing two aftercare regimes, namely individual versus group delivery of a structured relapse prevention approach. Two addictions treatment programs (one a 12-Step 26-day residential program, the other an evening group counselling program) implemented structured relapse prevention in either group or individual format as part of the first three months of aftercare. Process measures (e.g. attendance, client satisfaction) indicated that both group and individual formats were delivered very successfully at both sites. Follow-up rate at 12 months across both programs was 74%, and drinking and drug use at the 12-month follow-up was substantially less than use at entry into treatment. However, there were no significant differences in outcomes between individual and group delivery on any of the alcohol or drug use measures. Only one psychosocial outcome measure (social support from friends at 12-month follow-up) showed a significant difference for format and it favored the group format. These findings suggest some important directions for future research.

Structural and immunological characterization of Burkholderia pseudomallei O-polysaccharide-flagellin protein conjugates.

The O-polysaccharide moiety of Burkholderia pseudomallei 319a lipopolysaccharide was covalently linked to flagellin protein isolated from the same strain. A glycoconjugate incorporating adipic acid dihydrazide as a spacer molecule elicited high-titer immunoglobulin G responses to both the protein and carbohydrate components of the construct. This immunoglobulin G was capable of protecting diabetic rats from challenge with a heterologous B. pseudomallei strain.

Development of the Woman Abuse Screening Tool for use in family practice.

BACKGROUND: This study developed a screening tool for use by family physicians to identify and assess women patients experiencing emotional and/or physical abuse by their partner. METHODS: An initial set of eight questions developed for the Woman Abuse Screening Tool (WAST) was completed by both abused and non-abused women. Participants were also asked to indicate their comfort in answering the questions in both research and family practice contexts. They also completed the Abuse Risk Inventory and a demographic questionnaire. Analysis of the WAST included 1) standard assessment of the validity and reliability of the measure and 2) examination of the efficacy of further reducing the number of questions on the WAST for screening purposes. RESULTS: The final samples of abused (n = 24) and non-abused women (n = 24) differed significantly on a number of demographic and abuse variables. After eliminating one of the original items, a strong single factor structure was identified for the WAST that accounted for 85% of the total variance in responses to the WAST items. The WAST was found to be a highly reliable measure; coefficient alpha was estimated at.95. The scale also demonstrated construct and discriminant validity. The abused women reported being less comfortable responding to the WAST questions, in both the research and family practice contexts, than the non-abused women. The two WAST questions the abused women reported being most comfortable with were used to construct the WAST-Short for initial screening purposes. The WAST-Short correctly classified 100% of the non-abused women and 91.7% of the abused women. CONCLUSIONS: The WAST demonstrated good reliability and validity and discriminated between abused and non-abused women. Development of the WAST-Short provides physicians with a relatively unobtrusive screening tool for assessing abuse. The use of additional WAST questions can be used to further explore the possibility that a woman patient is experiencing abuse by her partner. Further study includes field testing the WAST in the family practice setting.

An analysis of specialty journals on alcohol, drugs and addictive behaviors for sex bias in research methods and reporting.

OBJECTIVE: Several studies have considered the extent to which gender bias has characterized addictions treatment research over the past 20 years. Sex bias in this literature has been shown to be most apparent in a reliance on male subjects and representation of male experience as normative. The current study was undertaken to assess the current status of some basic aspects of gender bias in addictions research generally. METHODS: Articles appearing in 1990 in 17 specialty journals on alcohol, drugs and addictive behaviors were coded with respect to type of article and the gender of the primary investigator. Studies on human subjects also were evaluated with regard to subject selection and reporting practices. RESULTS: Although the proportion of females represented in addictions research was found to have increased over earlier historical periods, studies using only male subjects were still common in 1990. Moreover, in studies using subjects of mostly one sex, investigators were likely to provide legitimate reasons for studying females, but provide no rationale for using male samples. Other subtle forms of bias were identified. For example, many single-sex studies (especially those using all male subjects) did not indicate the gender of the sample in the title or abstract of the study, and did not indicate that results of the study could be generalized to only one gender. CONCLUSIONS: Male biased sampling and misleading reporting of findings continue to be evident in addictions research. The need for gender-sensitive research in the field of addictions is discussed, and suggestions for change are offered.

Isolation and characterization of Pseudomonas pseudomallei flagellin proteins.

Flagellin proteins from several different strains of Pseudomonas pseudomallei have been isolated and purified to homogeneity by mechanical shearing and differential centrifugation techniques. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis yielded flagellin monomer protein bands with an estimated M(r) of 43,400. No lipopolysaccharide contamination of the purified protein preparations was detectable by silver staining of flagellin displayed on polyacrylamide gels and by Western immunoblotting with P. pseudomallei antilipopolysaccharide monoclonal antibody. NH2-terminal amino acid sequence analysis of the flagellin protein of P. pseudomallei 319a revealed significant homology with flagellins from Proteus mirabilis, Bordetella bronchiseptica, and Pseudomonas aeruginosa PAK. Rabbit polyclonal antiserum raised against the 319a flagellin protein reacted with 64 of 65 P. pseudomallei strains tested. The polyclonal antiserum proved effective in inhibiting the motility of these organisms in motility agar plates. Passive immunization studies demonstrated that 319a flagellin-specific antiserum was capable of protecting diabetic rats from challenge with a heterologous P. pseudomallei strain.