CD25+ T cells induce Helicobacter pylori‐specific CD25− T‐cell anergy but are not required to maintain persistent hyporesponsiveness

The gastric pathogen Helicobacter pylori infects over half the world's population. The lifelong infection induces gastric inflammation but the host fails to generate protective immunity. To study the lack of protective H. pylori immunity, CD4⁺CD25⁺ T_reg cells were investigated for their ability to down‐regulate H. pylori‐specific CD4⁺CD25⁻ cells in a murine model. CD25⁻ lymphocytes from infected mice were hyporesponsive to antigenic stimulation in vitro even in the absence of CD25⁺ T_reg cells unless treated with high‐dose IL‐2. Transfer of CD45RB^hi naïve CD25⁻ cells from infected mice into rag1^−/− mice challenged with H. pylori resulted in severe gastritis and reduced bacterial loads, whereas transfer of CD45RB^lo memory CD25⁻ cells from H. pylori‐infected mice resulted in only mild gastritis and persistent infection. CD25⁻ cells stimulated in the absence of CD25⁺ cells in rag1^−/− mice promoted bacterial clearance, but lost this ability when subsequently transferred to WT mice harboring CD25⁺ cells. These results demonstrate that CD25⁺ cells induce anergy in CD25⁻ cells in response to H. pylori infection but are not required to maintain hyporesponsiveness. In addition, CD25⁺ cells are able to suppress previously activated CD25⁻ cells when responding to H. pylori challenge in vivo.     

Additive Effects of Older Brothers and Homosexual Brothers in the Prediction of Marriage and Cohabitation

Research has shown that male homosexuality tends to cluster in families and that homosexual males have, on average, a greater number of older brothers than do heterosexual males. This study investigated whether the former, between-families effect and the latter, within-families effect are additive. The subjects were 717 full siblings over age 40 reported by 343 heterosexual and homosexual male probands examined in Southern Ontario in 1994–1995. The sibling's history of legal marriage or cohabitation in a heterosexual relationship was taken as a proxy variable for sexual orientation. There were no significant findings for the female siblings. As expected, the never-married male siblings were more likely to come from the sibships of the homosexual probands, and they had a greater average number of older brothers. A bootstrapped logistic regression analysis showed that an additive model best explained the male siblings' data. The results suggest that the familial aggregation of male homosexuality cannot be explained by the birth order effect and that older brothers and family membership reflect separate influences on sexual orientation or sexual orientation-correlated behavior.     

In vivo autofluorescence imaging of early cancers in the human tracheobronchial tree with a spectrally optimized system

The changes in the autofluorescence characteristics of the bronchial tissue is of crucial interest as a cancer diagnostic tool. Evidence exists that this native fluorescence or autofluorescence of bronchial tissues changes when they turn dysplastic and to carcinoma in situ. There is good agreement that the lesions display a decrease of autofluorescence in the green region of the spectrum under illumination with violet-light, and a relative increase in the red region of the spectrum is often reported. Imaging devices rely on this principle to detect early cancerous lesions in the bronchi. Based on a spectroscopic study, an industrial imaging prototype is developed to detect early cancerous lesions in collaboration with the firm Richard Wolf Endoskope GmbH, Germany. A preliminary clinical trial involving 20 patients with this spectrally optimized system shows that the autofluorescence can help to detect most lesions that would otherwise have remained invisible to an experienced endoscopist under white light illumination. A systematic off line analysis of the autofluorescence images pointed out that real-time decisional functions can be defined to reduce the number of false positive results. Using this method, a positive predictive value (PPV) of 75% is reached using autofluorescence only. Moreover, a PPV of 100% is obtained, when combining the white light (WL) mode and the autofluorescence (AF) mode, at the applied conditions. Furthermore, the sensitivity is estimated to be twice higher in the AF mode than in WL mode.     

Myosin VIIA gene: heterogeneity of the mutations responsible for Usher syndrome type IB

Usher syndrome is recognized as the most frequent cause of hereditary deaf-blindness. Usher syndrome type I (USH1), the most severe form of the disease, is characterized by profound congenital sensorineural deafness, constant vestibular dysfunction, and retinitis pigmentosa of prepubertal onset. This form is genetically heterogeneous and five loci (USH1A-E) have been mapped thusfar. However, only the gene responsible for USH1 B (which accounts for approximately 75% of USH1 cases) has been characterized. It encodes a long-tailed unconventional myosin, myosin VIIA, with a predicted 2215 amino acid sequence. Primers covering the complete myosin VIIA coding sequence as well as the 3' non coding sequence were designed, allowing direct sequence analysis of each of the 48 coding exons and flanking splice sites in seven patients affected by USH1. Four novel mutations were thereby identified. The possibility should now be considered of a sequence-based prenatal diagnosis in some of the families affected by this very severe form of Usher syndrome.      

A role for glucose and insulin preprandial profiles to differentiate meals and snacks

A physiological distinction between eating occasions may help account for contradictory findings on the role of eating frequency in energy homeostasis. We assessed this issue using a midafternoon eating occasion known in France as the goûter that often consists of snack foods. Among the 24 male subjects, 8 habitually consumed four meals per day, i.e., were usual goûter eaters (GE) and 16 habitually took 3 meals per day, i.e., usual non-goûter non-snack eaters (NGNSE). All subjects were time blinded from lunchtime and had to request subsequent meals. Blood was continuously withdrawn and collected with a change of tube every 10 min until dinner request. During the session, 8 of the non-goûter eaters (NGE) were offered a snack 210 min after lunch and were designated as non-goûter snack eaters (NGSE) if they ate. Results showed that the goûter was preceded by high hunger scores and a linear decline in plasma glucose (−9.0±3.0%, P<.05) and insulin concentrations (−22.9±6.0%, P<.05). These profiles were not observed before the snack. The dinner of GE was requested later and was smaller compared to NGNSE, whereas the snack altered neither time of request nor energy intake (EI) at dinner. Among blood variables, leptin at the onset of eating was the only factor that was predictive of both intermeal interval and EI. The glucose and insulin profiles indicate that snacks should not be considered as meals in studies on the role of eating frequency in energy homeostasis.     

Priming B cell-mediated anti-HIV envelope responses by vaccination allows for the long-term control of infection in macaques exposed to a R5-tropic SHIV

The potential of vaccine-elicited anti-HIV envelope antibodies to control HIV-infection was evaluated by immunizing macaques with the HIV envelope protein and transiently depleting them of their CD8+ cells before intravenous challenge with the pathogenic CCR5-tropic SIV/HIV chimeric virus, SHIV(SF162P4). Although sterilizing immunity was not achieved, all vaccinated animals effectively controlled infection and remained free of disease for the duration of observation (over 3 years). In contrast, during the same period, the control animals progressed to disease. Both the vaccinees and the controls developed robust cell-mediated antiviral and neutralizing antibody responses following infection. A comparative analysis of these responses suggests that the more effective long-term control of infection by the vaccinated animals is due to the more rapid development of anti-HIV envelope antibodies. These studies suggest that priming by vaccination of B cell anti-HIV envelope responses maybe crucial for the long-term control of HIV infection.     

Auto-protective redox buffering systems in stimulated macrophages

Background

Macrophages, upon encounter with micro-organisms or stimulated by cytokines, produce various effector molecules aimed at destroying the foreign agents and protecting the organism. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are front line molecules exerting strong cytotoxic activities against micro-organisms and many cells, including macrophages themselves. Using cells of the murine macrophage cell line (RAW 264.7) stimulated in vitro with lipopolysaccharide (LPS) and/or interferon (IFN-γ), which induce strong endogenous NO production, we examined by which mechanisms a fraction of activated macrophages protect themselves from nitrosative stress and manage to escape destruction?

Results

We observed that survivors (10–50% depending on the experiments) had acquired a resistant phenotype being capable to survive when further exposed in vitro to an apoptosis inducing dose of the NO donor compound DETA-NO. These cells expressed an increased steady-state levels of Mn SOD, CuZn SOD and catalase mRNA (130–200%), together with an increased activity of the corresponding enzymes. Intracellular concentration of glutathione was also increased (× 3.5 fold at 6 hours, still maintained × 5.2 fold at 48 hours). Neither mRNA for glutathione peroxydase, γ-glutamylcysteine synthase and glutathione reductase, nor thioredoxine and thioredoxine reductase, were significantly modified. Additional experiments in which RAW 264.7 cells were stimulated with LPS and/or IFN-γ in the presence of relatively specific inhibitors of both Mn and Cu/Zn SOD, aminotriazol (ATZ) catalase inhibitor and buthionine sulfoximine (BSO) glutathione inhibitor, showed that inhibiting LPS-induced up-regulation of intracellular redox buffering systems also prevented acquisition of the resistant phenotype.

Conclusions

Our data suggest a direct causal relationship between survival of a fraction of macrophages and a up-regulation of key sets of auto-protective intracellular redox buffering systems, occurring simultaneously with modulation of expression of apoptotic molecules of the Bcl₂-Bcl-_XL/Bax-Bad family.     

Cartilage engineering from ovine umbilical cord blood mesenchymal progenitor cells

We aimed to determine whether three-dimensional (3D) cartilage could be engineered from umbilical cord blood (CB) cells and compare it with both engineered fetal cartilage and native tissue. Ovine mesenchymal progenitor cells were isolated from CB samples (n=4) harvested at 80-120 days of gestation by low-density fractionation, expanded, and seeded onto polyglycolic acid scaffolds. Constructs (n=28) were maintained in a rotating bioreactor with serum-free medium supplemented with transforming growth factor-beta1 for 4-12 weeks. Similar constructs seeded with fetal chondrocytes (n=13) were cultured in parallel for 8 weeks. All specimens were analyzed and compared with native fetal cartilage samples (n=10). Statistical analysis was by analysis of variance and Student's t-test (p<.01). At 12 weeks, CB constructs exhibited chondrogenic differentiation by both standard and matrix-specific staining. In the CB constructs, there was a significant time-dependent increase in extracellular matrix levels of glycosaminoglycans (GAGs) and type-II collagen (C-II) but not of elastin (EL). Fetal chondrocyte and CB constructs had similar GAG and C-II contents, but CB constructs had less EL. Compared with both hyaline and elastic native fetal cartilage, C-II and EL levels were, respectively, similar and lower in the CB constructs, which had correspondingly lower and similar GAG levels than native hyaline and elastic fetal cartilage. We conclude that CB mesenchymal progenitor cells can be successfully used for the engineering of 3D cartilaginous tissue in vitro, displaying select histological and functional properties of both native and engineered fetal cartilage. Cartilage engineered from CB may prove useful for the treatment of select congenital anomalies.     

Molecular identification of a novel deltaproteobacterium as the etiologic agent of epizootic bovine abortion (foothill abortion)

Epizootic bovine abortion (EBA) is endemic in California's coastal range and the foothill regions of the Sierra Nevada, where it has been the primary diagnosed cause of abortion in beef cattle for >50 years. Investigation of these losses has defined a specific fetal syndrome characterized by late-term abortion or birth of weak or dead calves. Although the unusual clinical presentation and unique fetal pathology associated with EBA have been recognized since the 1950s, the identity of the etiologic agent is unknown. In this study, suppression-hybridization PCR was used to identify a fragment of the 16S rRNA gene of a previously undescribed bacterium in thymus tissue derived from affected fetuses. Phylogenetic analysis revealed that this pathogen was a deltaproteobacterium closely related to members of the order Myxococcales. A specific PCR was subsequently developed to detect the presence of this bacterium in DNA extracted from fetal thymuses. Using histopathology as the definitive diagnosis for EBA, this PCR demonstrated 100% specificity and 88% sensitivity. The bacterium was also detected in the argasid tick Ornithodoros coriaceus, which is the recognized vector of EBA. These data imply a close association between this novel agent and the etiology of EBA.