Comparison of bipA alleles within and across Bordetella species

The Bordetella BvgAS signal transduction system controls the expression of at least three phenotypic phases, the Bvg(+) or virulent phase, the Bvg(-) or avirulent phase, and the Bvg(i) or Bvg intermediate phase, which has been hypothesized to be important for transmission. bipA, the first identified Bvg(i)-phase gene, encodes a protein with similarity to the well-characterized bacterial adhesins intimin and invasin. Proteins encoded by the bipA genes present in Bordetella pertussis Tohama I and Bordetella bronchiseptica RB50 differ in the number of 90-amino-acid repeats which they possess and in the sequence of the C-terminal domain. To investigate the possibility that bipA alleles segregate according to host specificity and to gain insight into the role of BipA and the Bvg(i) phase in the Bordetella infectious cycle, we compared bipA alleles across members of the B. bronchiseptica cluster, which includes both human-infective (B. pertussis and B. parapertussis(hu)) and non-human-infective (B. bronchiseptica and B. parapertussis(ov)) strains. bipA genes were present in most, but not all, strains. All bipA genes present in B. bronchiseptica strains were identical to bipA of RB50 (at least with regard to the DNA sequence of the 3' C-terminal-domain-encoding region, the number of 90-amino-acid repeats encoded, and expression patterns). Although all bipA genes present in the other Bordetella strains were identical in the 3' C-terminal-domain-encoding region to bipA of B. pertussis Tohama I, they varied in the number of 90-amino-acid repeats that they encoded and in expression level. Notably, the genes present in B. parapertussis(hu) strains were pseudogenes, and the genes present in B. parapertussis(ov) strains were expressed at significantly reduced levels compared with the levels in B. pertussis and B. bronchiseptica strains. Our results indicate that there is a correlation between specific bipA alleles and specific hosts. They also support the hypothesis that both horizontal gene transfer and fine-tuning of gene expression patterns contribute to the evolution of host adaptation in lineages of the B. bronchiseptica cluster.     

Detection of Chlamydia trachomatis in general practice urine samples.

BACKGROUND. Chlamydia trachomatis is frequently overlooked as a cause of dysuria and urinary frequency in general practice patients. AIM. This study set out to determine the impact of performing chlamydial antigen detection on sterile pyuria samples from patients aged 16-65 years and which were submitted to a hospital microbiology laboratory by general practitioners in the Winchester health district for routine microbiological investigations. METHOD. Chlamydial antigen detection was performed by enzyme immunoassay and direct immunofluorescence. The cost of performing the test was estimated. In the first year of the study (1991) questionnaires were sent to general practitioners whose patients had a positive test result. RESULTS. A total of 1025 samples of sterile pyuria were received at the laboratory between January 1991 and March 1993. Chlamydial antigen was detected in 54 samples (5%); 22 men and 32 women aged between 16 and 57 years (mean 25 years). The detection rate was highest in the 16-20 years age group (22% of men had a positive sample and 7% of women). Completed questionnaires from 27 general practitioners revealed that 59% of their patients were referred to the genitourinary clinic for treatment and contact tracing. The others were treated by the general practitioner. The cost of the screening programme per cure in this population was estimated to be 246 pounds. CONCLUSION. C trachomatis is a significant pathogen which may go unrecognized and untreated. The cost, medically and financially, of screening for this pathogen and treating infected patients and contacts is likely to be less than ignoring it, particularly if screening is confined to the 16-30 years age group. General practitioners should consider the diagnosis of chlamydial infection in young adult patients with sterile pyuria, and microbiology laboratories should screen sterile pyuria samples for chlamydial antigen.     

Synergy between the genes for butyrylcholinesterase K variant and apolipoprotein E4 in late-onset confirmed Alzheimer's disease

The allelic frequency of the gene for the K variant of butyrylcholinesterase (BCHE-K) was 0.17 in 74 subjects with late-onset (age > 65 years) histopathologically diagnosed Alzheimer's disease (AD), which was higher than the frequencies in 104 elderly control subjects (0.09), in 14 early-onset cases of confirmed AD (0.07) and in 29 confirmed cases of other dementia (0.10). The association of BCHE-K with late-onset AD was limited to carriers of the epsilon 4 allele of the apolipoprotein E gene (APOE), among whom the presence of BCHE-K gave an odds ratio of confirmed late-onset AD of 6.9 (95% C.I. 1.65-29) in subjects > 65 years and of 12.8 (1.9-86) in subjects > 75 years. In APOE epsilon 4 carriers over 75 years, only 1/22 controls, compared with 10/24 confirmed late-onset AD cases, had BCHE-K. We suggest that BCHE-K, or a nearby gene on chromosome 3, acts in synergy with APOE epsilon 4 as a susceptibility gene for late-onset AD.      

Human growth hormone 1 (GH1) gene expression: complex haplotype-dependent influence of polymorphic variation in the proximal promoter and locus control region

The proximal promoter region of the human pituitary expressed growth hormone (GH1) gene is highly polymorphic, containing at least 15 single nucleotide polymorphisms (SNPs). This variation is manifest in 40 different haplotypes, the high diversity being explicable in terms of gene conversion, recurrent mutation, and selection. Functional analysis showed that 12 haplotypes were associated with a significantly reduced level of reporter gene expression whereas 10 haplotypes were associated with a significantly increased level. The former tend to be more prevalent in the general population than the latter (p<0.01), possibly as a consequence of selection. Although individual SNPs contributed to promoter strength in a highly interactive and non-additive fashion, haplotype partitioning was successful in identifying six SNPs as major determinants of GH1 gene expression. The prediction and functional testing of hitherto unobserved super-maximal and sub-minimal promoter haplotypes was then used to test the efficacy of the haplotype partitioning approach. Electrophoretic mobility shift assays demonstrated that five SNP sites exhibit allele-specific protein binding. An association was noted between adult height and the mean in vitro expression value corresponding to an individual's GH1 promoter haplotype combination (p=0.028) although only 3.3% of the variance of adult height was found to be explicable by reference to this parameter. Three additional SNPs, identified within sites I and II of the upstream locus control region (LCR), were ascribed to three distinct LCR haplotypes. A series of LCR-GH1 proximal promoter constructs were used to demonstrate that 1) the LCR enhanced proximal promoter activity by up to 2.8-fold depending upon proximal promoter haplotype, and that 2) the activity of a given proximal promoter haplotype was also differentially enhanced by different LCR haplotypes. The genetic basis of inter-individual differences in GH1 gene expression thus appears to be extremely complex.     

Viral hemagglutinin augments peptide-specific cytotoxic T cell responses

In attempt to increase the induction of peptide-specific cytolytic T cells (CTL) we investigated the effect of the Newcastle disease virus (NDV) hemagglutinin-neuraminidase (HN) gene product on the activation of peptide-specific CTL. Spleen cells of CH3 mice immunized against the influenza nucleoprotein peptide 50–63 (NP 50–63) were restimulated in vitro (i) with peptide-pulsed syngeneic fibroblast cells (Ltk^?) as antigen-presenting cells, which were in addition (ii) infected with NDV or (iii) stably transfected with the HN cDN A of NDV. A greater than sixfold increase in peptide-specific CTL responses was observed in cultures restimulated with peptide-pulsed Ltk^? cells which co-expressed viral hemagglutinin due to either infection or transfection. A similar augmentation was seen in CTL responses against other types of antigen (major histocompatibility complex alloantigens, minor histocompatibility antigens or tumor antigens) when suboptimal cultures were stimulated with the respective antigen-presenting cells modified by NDV infection. These findings suggest that NDV or viral HN expressed on antigen-presenting cells or tumor cells can exert a T cell co-stimulatory function.     

Culture-negative Bartonella endocarditis in a patient with renal failure: the value of molecular methods in diagnosis

Members of the genus Bartonella are increasingly recognised as a cause of culture-negative endocarditis, particularly in those patients with underlying risk factors (e.g., homelessness and alcoholism (B. quintana) or valvulopathy and cat ownership (B. henselae). The aortic and mitral-valves are most commonly involved. Here, a case is reported of culture-negative right-sided endocarditis, without any of the above risk factors, due to Bartonella sp. in a 69-year-old man who presented with acute renal failure. The diagnosis was made using a broad-range 16S rRNA polymerase chain reaction (PCR) technique and direct automated sequencing on a peripheral blood sample, which was subsequently confirmed serologically. A review of the literature on Bartonella endocarditis is also presented. Molecular laboratory methods using peripheral blood or blood cultures may be very useful in the diagnosis of causal agents in culture-negative endocarditis and add further support to the recently inclusion of molecular (PCR) diagnosis, as a major Duke's criterion, for the diagnosis of infective endocarditis.     

Rapid discrimination of Mycobacterium tuberculosis strains by random amplified polymorphic DNA analysis.

Investigations of the epidemiology of tuberculosis have been hampered by the lack of strain-specific markers that can be used to differentiate isolates of Mycobacterium tuberculosis. We report the development of a rapid protocol for random amplified polymorphic DNA analysis which included the use of a commercially available DNA extraction kit (GeneReleaser). This was applied to 14 strains of M. tuberculosis, including strains associated with temporal and geographical clusters of tuberculosis in the United Kingdom and those from India, Africa, and Saudi Arabia. Strains of M. tuberculosis could be discriminated in about 8 h by this method, which is therefore a rapid and simple alternative to restriction fragment length polymorphism analysis.