Two-year evaluation of Borrelia burgdorferi culture and supplemental tests for definitive diagnosis of Lyme disease

Lyme disease is usually diagnosed and treated based on clinical manifestations. However, laboratory testing is useful for patients with confusing presentations and for validation of disease in clinical studies. Although cultivation of Borrelia burgdorferi is definitive, prior investigations have shown that no single test is optimal for Lyme disease diagnosis. We applied high-volume blood culture, skin biopsy culture, PCR, and serodiagnosis to a cohort of patients with suspected Lyme disease acquired in Maryland and southern Pennsylvania. The study was performed to confirm the relative utility of culture and to identify laboratory testing algorithms that will supplement clinical diagnosis. Overall, 30 of 86 patients (35%) were culture positive, whereas an additional 15 of 84 (18%) were seropositive only (51% total sero- and culture positive), and PCR on skin biopsy identified 4 additional patients who were neither culture nor seropositive. Among 49 laboratory test-positive patients, the highest sensitivity (100%) for diagnosis was obtained when culture, skin PCR, and serologic tests were used, although serologic testing with skin PCR was almost as sensitive (92%). Plasma PCR was infrequently positive and provided no additional diagnostic value. Although culture is definitive and has a relatively high sensitivity, the results required a mean of 3.5 weeks to recovery. The combination of acute-phase serology and skin PCR was 75% sensitive, offering a practical and relatively rapid alternative for confirming clinical impression. The full battery of tests could be useful for patients with confusing clinical signs or for providing strong laboratory support for clinical studies of Lyme disease.     

Application of mycobacterial proteomics to vaccine design: improved protection by Mycobacterium bovis BCG prime-Rv3407 DNA boost vaccination against tuberculosis

Information from comparative proteome analysis of Mycobacterium tuberculosis and Mycobacterium bovis bacillus Calmette-Guerin (BCG) principally allows prediction of potential vaccine candidates. Thirty-six M. tuberculosis DNA vaccine candidates identified by comparative proteome analysis were evaluated in the mouse model for protection against low-dose aerosol M. tuberculosis infection. We identified the DNA vaccine candidate Rv3407 as a protective antigen and analyzed putative major histocompatibility complex class I epitopes by computational predictions and gamma interferon Elispot assays. Importantly, we discovered that the DNA vaccine Rv3407 improved the efficacy of BCG vaccination in a heterologous prime-boost vaccination protocol. Our data demonstrate the rationale of a combination of proteomics, epitope prediction, and broad screening of putative antigens for identification of novel DNA vaccine candidates. Furthermore, our experiments show that heterologous prime-boost vaccination with a defined antigen boost "on top" of a BCG primer provides superior protection against tuberculosis over vaccination with BCG alone.     

Characterization of the filamentous hemagglutinin-like protein FhaS in Bordetella bronchiseptica

Filamentous hemagglutinin (FHA) is a large (>200 kDa), rod-shaped protein expressed by bordetellae that is both surface-associated and secreted. FHA mediates bacterial adherence to epithelial cells and macrophages in vitro and is absolutely required for tracheal colonization in vivo. The recently sequenced Bordetella bronchiseptica genome revealed the presence of a gene, fhaS, that is nearly identical to fhaB, the FHA structural gene. We show that although fhaS expression requires the BvgAS virulence control system, it is maximal only under a subset of conditions in which BvgAS is active, suggesting an additional level of regulation. We also show that, like FHA, FhaS undergoes a C-terminal proteolytic processing event and is both surface-associated and secreted and that export across the outer membrane requires the channel-forming protein FhaC. Unlike FHA, however, FhaS was unable to mediate adherence of B. bronchiseptica to epithelial cell lines in vitro and was not required for respiratory tract colonization in vivo. In a coinfection experiment, a DeltafhaS strain was out-competed by wild-type B. bronchiseptica, indicating that fhaS is expressed in vivo and that FhaS contributes to bacterial fitness in a manner revealed when the mutant must compete with wild-type bacteria. These data suggest that FHA and FhaS perform distinct functions during the Bordetella infectious cycle. A survey of various Bordetella strains revealed two distinct fhaS alleles that segregate according to pathogen host range and that B. parapertussis(hu) most likely acquired its fhaS allele from B. pertussis horizontally, suggesting fhaS may contribute to host-species specificity.     

Outer membrane protein A renders dendritic cells and macrophages responsive to CCL21 and triggers dendritic cell migration to secondary lymphoid organs

Outer membrane protein A (OmpA) is a class of bacterial cell wall protein that is immunogenic without adjuvant. As specific immune responses are initiated in the lymph nodes (LN, we analyzed the effect of the OmpA from Klebsiella pneumoniae (KpOmpA) onchemokine/ chemokine receptor expression by APC and on cell migration to the LN. Upon contact with KpOmpA, human immature DC and macrophages acquire CCR7 expression and responsiveness to CCL21. In parallel, CCR1 and CCR5 expression is down-regulated and CXCL8, CCL2, CCL3 and CCL5 production is up-regulated. Mice injected subcutaneously with KpOmpA present a transient inflammatory reaction at the site of injection accompanied by an enlargement of the draining LN with a higher proportion of DC and macrophages. Lastly, when exposed to KpOmpA prior injection, DC but not macrophages migrate to the draining LN. In conclusion, KpOmpA confers a migratory phenotype to DC and triggers their migration to the regional LN. This property contributes to explain how innate cells initiate adaptive immune response upon recognition of conserved bacterial components and also why OmpA is immunogenic in the absence of adjuvant.     

At least two distinct epigenetic mechanisms are correlated with high-frequency “switching” for aprt phenotypic expression in mouse embryonal carcinoma stem cells

A series of clones displaying high frequency switching phenotypes for expression of the adenine phosphoribosyltransferase (aprt) gene were previously isolated from the P19 mouse embryonal carcinoma stem cell line. Most clones contained only oneaprt allele. We report here the characterization of each of these clones with regards to enzymatic activity, mRNA steady state levels, DNA methylation, and chromatin conformation. When clones were selected for resistance to the purine analog 2,6-diaminopurine, which requires markedly reduced levels of APRT enzymatic activity, two distinct classes were observed. The first class was associated with reduced or undetectable levels ofaprt mRNA, hypermethylation of the 5 CpG island, and a closed chromatin conformation within this region. When clones of this class were selected for reacquisition of APRT enzymatic activity they were found to have increased mRNA levels, a hypomethylated CpG island, and an open chromatin conformation. In contrast, the second class of clones displayed wild-type levels of mRNA, CpG island hypomethylation, and an open chromatin conformation regardless of whether they were selected for the presence or absence of APRT enzymatic activity. The implications of these results for general mechanisms of epigenetic change in somatic cells and the possibility that expression of the mouseaprt gene may be developmentally regulated are discussed.     

An outbreak of amebiasis spread by colonic irrigation at a chiropractic clinic

From June 1978 through December 1980, at least 36 cases of amebiasis occurred in persons who had had colonic-irrigation therapy at a chiropractic clinic in western Colorado. Of 10 persons who required colectomy, six did. Of 176 persons who had been to the clinic in the last four months of 1980, 80 had received other forms of treatment. Twenty-one per cent of the colonic-irrigation group had bloody diarrhea, as compared with 1 per cent of the non-irrigation group (P = 0.00013). Thirty-seven per cent of the colonic-irrigation group who submitted specimens had evidence of amebic infection on either stool examination or serum titer, as compared with 2.4 per cent in the non-irrigation group (P = 0.00012). Persons who were given colonic irrigation immediately after a person with bloody diarrhea received it were at the highest risk for the development of amebiasis. Tests of the colonic-irrigation machine after routine cleaning showed heavy contamination with fecal coliform bacteria. The severity of disease in this outbreak may have been related to the route of inoculation.     

Ultrastructural Morphometric Study of Follicular Center Lymphocytes: II. Analyses of Cleaved-Cell Populations Do Not Support the Lukes-Collins' Concept

Ultrastructural morphometric analysis was carried out on six cases of lymph node biopsies with reactive hyperplasia to establish the frequency and depth of invaginations in nuclear profiles situated in the mantle zones and follicular centers. The frequency distribution of the depth of invaginations was similar in nuclear profiles whether in the small lymphocytes of mantle zones or the small, partially transformed (centrocytes) and fully transformed (centroblasts) lymphocytes of follicular centers. Invaginated and cleaved lymphocytes were not confined to the partially transformed (centrocytic) lymphocytes of follicular centers, and nuclear profiles with invaginations bore no resemblance to those depicted in the Lukes-Collins model. A considerable proportion of mantle zone lymphocyte nuclear profiles had invaginations (ranging from 7.5% to 53.6%) and there was no difference between the frequency of deep indentations or clefts in mantle zone lymphocytes (8.1 ± 5.4%) and the small unstimulated (9.3 ± 5.3%) and partially transformed (8.4 ± 1.4%) lymphocytes in follicular centers. Computer modeling of stylized nuclei with conical indentations indicated that all lymphocytic nuclei likely have multiple invaginations or groove-like creases.