Search for potential vaccine candidate open reading frames in the Bacillus anthracis virulence plasmid pXO1: in silico and in vitro screening

A genomic analysis of the Bacillus anthracis virulence plasmid pXO1, aimed at identifying potential vaccine candidates and virulence-related genes, was carried out. The 143 previously defined open reading frames (ORFs) (R. T. Okinaka, K. Cloud, O. Hampton, A. R. Hoffmaster, K. K. Hill, P. Keim, T. M. Koehler, G. Lamke, S. Kumano, J. Mahillon, D. Manter, Y. Martinez, D. Ricke, R. Svensson, and P. J. Jackson, J. Bacteriol. 181:6509-6515, 1999) were subjected to extensive sequence similarity searches (with the nonredundant and unfinished microbial genome databases), as well as motif, cellular location, and domain analyses. A comparative genomics analysis was conducted with the related genomes of Bacillus subtilis, Bacillus halodurans, and Bacillus cereus and the pBtoxis plasmid of Bacillus thuringiensis var. israeliensis. As a result, the percentage of ORFs with clues about their functions increased from approximately 30% (as previously reported) to more than 60%. The bioinformatics analysis permitted identification of novel genes with putative relevance for pathogenesis and virulence. Based on our analyses, 11 putative proteins were chosen as targets for functional genomics studies. A rapid and efficient functional screening method was developed, in which PCR-amplified full-length linear DNA products of the selected ORFs were transcribed and directly translated in vitro and their immunogenicities were assessed on the basis of their reactivities with hyperimmune anti-B. anthracis antisera. Of the 11 ORFs selected for analysis, 9 were successfully expressed as full-length polypeptides, and 3 of these were found to be antigenic and to have immunogenic potential. The latter ORFs are currently being evaluated to determine their vaccine potential.     

Increased median nerve latency at the carpal tunnel of patients with "trigger finger": Comparison of 62 patients and 13 controls

An association between symptomatic compression neuropathy of the median nerve at the carpal tunnel and "trigger finger" has been reported in endocrine and metabolic disorders. We assessed the incidence of increased median nerve latency in subjects with "trigger finger". 62 consecutive patients with "trigger finger" and no signs or symptoms of median nerve compression underwent nerve conduction studies of the median nerve. 13 healthy adults served as controls. 39/62 patients had increased distal motor latency in the median nerve. Only 1 of 13 subjects in the control group had a borderline value of distal motor latency.     

Major motives in non-acceptance of A/H1N1 flu vaccination: the weight of rational assessment

Recent efforts of health authorities to promote vaccination against influenza A/H1N1 were met with low compliance rates in most industrialized countries. Here we analyzed the attitudes of the Israeli public towards A/H1N1 vaccination based on a telephone survey conducted several months after the peak of the outbreak. The findings attest to the low uptake of the A/H1N1 vaccine (17%) in Israel, and identify the socio-demographic characteristics associated with non-compliance. In addition, the survey reveals passiveness, fear and distrust as motives leading to non-compliance. Most importantly, the study identified the substantial weight of reflective assessment in the attitude of lay individuals towards the A/H1N1 vaccine. As many as 30% of the non-vaccinated responders provided reasoned arguments for rejecting the vaccine, based mainly on assessment of threat versus actual risk. These observations highlight the need to consider the opinion of the lay public when implementing new vaccination programs.     

Pyogenic osteomyelitis of the spine in the elderly: three cases of a synchronous non-axial infection by a different pathogen

STUDY DESIGN: A retrospective chart review of patients over 65 years of age treated at the Spine Care Unit for pyogenic vertebral osteomyelitis. OBJECTIVES: To assess the reliability of peripheral blood, urine and sputum cultures in the treatment of pyogenic vertebral osteomyelitis in the elderly. SETTING: Study performed at the Spine Care Unit, Meir Hospital, Kfar-Saba, Israel. METHODS: The Meir Hospital records were searched for patients over 65 years of age, treated at the Spine Care Unit for pyogenic vertebral osteomyelitis. Charts, culture results and imaging studies were reviewed. A medline literature search was performed to survey the literature regarding pyogenic vertebral osteomyelitis in the elderly with emphasis on diagnostic imaging modalities and surgical treatment. RESULTS: Three patients were identified with concurrent peripheral infection by a different organism than the organism causing the vertebral osteomyelitis. Delay in correct diagnosis led to neurologic impairment in all patients and surgical treatment was performed in all three to drain the epidural abscess, decompress the spinal cord and obtain direct tissue culture. Following decompression and epidural abscess evacuation, one patient has functionally recovered and was ambulating with a cane, two patients did not recover and remained paraparetic and ambulate in a wheelchair. CONCLUSIONS: Pyogenic vertebral osteomyelitis in the elderly can be caused by a different pathogen than that isolated from blood, sputum or urine cultures. In the elderly, a biopsy of the vertebral lesion should be obtained for susceptibility studies prior to conservative treatment with bracing and intravenous antibiotics.     

Search for correlates of protective immunity conferred by anthrax vaccine

Vaccination by anthrax protective antigen (PA)-based vaccines requires multiple immunization, underlying the need to develop more efficacious vaccines or alternative vaccination regimens. In spite of the vast use of PA-based vaccines, the definition of a marker for protective immunity is still lacking. Here we describe studies designed to help define such markers. To this end we have immunized guinea pigs by different methods and monitored the immune response and the corresponding extent of protection against a lethal challenge with anthrax spores. Active immunization was performed by a single injection using one of two methods: (i) vaccination with decreasing amounts of PA and (ii) vaccination with constant amounts of PA that had been thermally inactivated for increasing periods. In both studies a direct correlation between survival and neutralizing-antibody titer was found (r(2) = 0.92 and 0.95, respectively). Most significantly, in the two protocols a similar neutralizing-antibody titer range provided 50% protection. Furthermore, in a complementary study involving passive transfer of PA hyperimmune sera to naive animals, a similar correlation between neutralizing-antibody titers and protection was found. In all three immunization studies, neutralization titers of at least 300 were sufficient to confer protection against a dose of 40 50% lethal doses (LD(50)) of virulent anthrax spores of the Vollum strain. Such consistency in the correlation of protective immunity with anti-PA antibody titers was not observed for antibody titers determined by an enzyme-linked immunosorbent assay. Taken together, these results clearly demonstrate that neutralizing antibodies to PA constitute a major component of the protective immunity against anthrax and suggest that this parameter could be used as a surrogate marker for protection.     

Dependence of colorectal cancer risk on the parent-of-origin of mutations in DNA mismatch repair genes

Genetic diseases associated with dynamic mutations in microsatellite DNA often display parent-of-origin effects (POEs) in which the risk of disease depends on the sex of the parent from whom the disease allele was inherited. Carriers of germline mutations in mismatch repair (MMR) genes have high risks of colorectal carcinoma (CRC). We investigated whether these risks depend on the parent-of-origin of the mutation. We studied 422 subjects, including 89 MMR gene mutation carriers, from 17 population-based families who were each recruited via a CRC case diagnosed before age 45 years and found to carry a MMR gene mutation. The POE hazard ratio (HR(POE)), defined to be the CRC incidence for carriers with maternally derived mutations divided by the corresponding paternal incidence, was estimated using a novel application of modified segregation analysis. HR(POE) (95% confidence interval) was estimated to be 3.2 (1.1-9.8) for males (P = 0.03) and 0.8 (0.2-2.8) for females (P = 0.5) and the corresponding cumulative risks to age 80 years were 88% (54%-100%) for male carriers with maternally derived mutations and 38-48% for all other carriers. If confirmed by larger studies, these results will have important implications for the etiology of CRC and for the clinical management of MMR gene mutation carriers.     

Discordance in the effects of Yersinia pestis on the dendritic cell functions manifested by induction of maturation and paralysis of migration

The encounter between invading microorganisms and dendritic cells (DC) triggers a series of events which include uptake and degradation of the microorganism, induction of a maturation process, and enhancement of DC migration to the draining lymph nodes. Various pathogens have developed strategies to counteract these events as a measure to evade the host defense. In the present study we found that interaction of the Yersinia pestis EV76 strain with DC has no effect on cell viability and is characterized by compliance with effective maturation, which is manifested by surface display of major histocompatibility complex class II, of costimulatory markers, and of the chemokine receptor CCR7. This is in contrast to maturation inhibition and cell death induction exerted by the related species Yersinia enterocolitica WA O:8. Y. pestis interactions with DC were found, however, to impair functions related to cytoskeleton rearrangement. DC pulsed with Y. pestis failed to adhere to solid surfaces and to migrate toward the chemokine CCL19 in an in vitro transmembrane assay. Both effects were dependent on the presence of the pCD1 virulence plasmid and on a bacterial growth shift to 37 degrees C prior to infection. Moreover, while instillation of a pCD1-cured Y. pestis strain into mouse airways triggered effective transport of alveolar DC to the mediastinal lymph node, instillation of Y. pestis harboring the plasmid failed to do so. Taken together, these results suggest that virulence plasmid-dependent impairment of DC migration is the major mechanism utilized by Y. pestis to subvert DC function.