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.     

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.     

Alexithymia in young adulthood: a risk factor for pathological gambling

BACKGROUND: Pathological gambling is more prevalent among postsecondary students than among the general adult population. While the prevalence of pathological gambling in this group has risen over the past decade, factors underlying the development of problem gambling among university students remain largely unexplored. One early study found alexithymia to be associated with pathological gambling. The aim of the present study was to further examine the relationship between alexithymia and gambling among postsecondary students. METHODS: The relationship between alexithymia and pathological gambling was examined in 562 postsecondary students who completed the South Oaks Gambling Screen (SOGS) and the 20-item Toronto Alexithymia Scale (TAS-20). RESULTS: Approximately 12% of the sample was classified as alexithymic according to the TAS-20. These individuals were found to have significantly more gambling problems, as measured by the SOGS, than nonalexithymic individuals. Approximately 9% of the sample was classified as pathological gamblers according to the SOGS. These individuals were found to have significantly higher levels of alexithymia, as measured by the TAS-20, than nonproblem gamblers. CONCLUSIONS: Alexithymia is associated with pathological gambling and may be a risk factor among postsecondary students for developing severe gambling problems.     

Thymidine kinase-negative bovine herpesvirus type 1 mutant is stable and highly attenuated in calves

Summary Calves were vaccinated intranasally (IN) or intravenously (IV) with a thymidine kinase-negative (tk^–) BHV-1 mutant. Vaccinated calves developed neutralizing antibodies but did not show clinical signs of infectious bovine rhinotracheitis (IBR). Vaccination also prevented clinical signs of IBR disease following IN challenge exposure of the calves to parental Los Angeles (LA) and USDA Cooper strains of tk⁺ BHV-1. Nasal swabs were collected for 10 days after the vaccination and the challenge exposures to monitor BHV-1 multiplication. At both 91 and 121 days post vaccination (PV), calves were also stressed for 5 days with dexamethasone (DEX) to induce reactivation of BHV-1 and nasal swabs were obtained. tk^– BHV-1 multiplied in the nasal mucosa of IN vaccinated calves and was also recovered after DEX treatment. Likewise, tk^– BHV-1 was isolated from the buffy coat fraction of IV vaccinated calves, but not from nasal swabs. tk^– BHV-1 vaccination reduced the multiplication of tk⁺ BHV-1 in the nasal mucosa, but did not completely prevent development of a persistent infection by the challenge virus. The phenotypes of viruses isolated from the nasal swabs and buffy coats were analyzed by enzyme assays of extracts from virus-infected cells and by plaque autoradiography. These assays showed that tk^– BHV-1 can persist for at least 3 months in vaccinated calves and may also be transmitted from vaccinated to control calves without revertingin vivo to tk⁺. The results demonstrate that the tk^– BHV mutant is attenuated and efficacious as a vaccine.With 1 Figure     

Modulation of sympathetic and somatomotor function by the ventromedial medulla

The ventromedial medulla is implicated in a variety of functions including nociceptive and cardiovascular modulation and the control of thermoregulation. To determine whether single microinjections into the ventromedial medulla elicit changes in one or multiple functional systems, the GABA(A) receptor antagonist bicuculline was microinjected (70 nl, 5-50 ng) into the ventromedial medulla of lightly anesthetized rats, and cardiovascular, respiratory, and nociceptive measures were recorded. Bicuculline microinjection into either the midline raphe or the laterally adjacent reticular nucleus simultaneously increased interscapular brown adipose tissue temperature, heart rate, blood pressure, expired [CO(2)], and respiration rate and elicited shivering. Bicuculline microinjection also decreased the noxious stimulus-evoked changes in heart rate and blood pressure, decreased the frequency of heat-evoked sighs, and suppressed the cortical desynchronization evoked by noxious stimulation. Although bicuculline suppressed the motor withdrawal evoked by noxious tail heat, it enhanced the motor withdrawal evoked by noxious paw heat, evidence for specifically patterned nociceptive modulation. Saline microinjections into midline or lateral sites had no effect on any measured variable. All bicuculline microinjections, midline or lateral, evoked the same set of physiological effects, consistent with the lack of a topographical organization within the ventromedial medulla. Furthermore, as predicted by the isodendritic morphology of cells in the ventromedial medulla, midline bicuculline microinjection increased the number of c-fos immunoreactive cells in both midline raphe and lateral reticular nuclei. In summary, 70-nl microinjections into ventromedial medulla activate cells in multiple nuclei and elicit increases in sympathetic and somatomotor tone and a novel pattern of nociceptive modulation.