Regulatory T (T(reg)) cells are a critical sub-population of CD4+ T cells that are essential for maintaining self tolerance and preventing autoimmunity, for limiting chronic inflammatory diseases, such as asthma and inflammatory bowel disease, and for regulating homeostatic lymphocyte expansion. However, they also suppress natural immune responses to parasites and viruses as well as anti-tumour immunity induced by therapeutic vaccines. Although the manipulation of T(reg) function is an important goal of immunotherapy, the molecules that mediate their suppressive activity remain largely unknown. Here we demonstrate that Epstein-Barr-virus-induced gene 3 (Ebi3, which encodes IL-27beta) and interleukin-12 alpha (Il12a, which encodes IL-12alpha/p35) are highly expressed by mouse Foxp3+ (forkhead box P3) T(reg) cells but not by resting or activated effector CD4+ T (T(eff)) cells, and that an Ebi3-IL-12alpha heterodimer is constitutively secreted by T(reg) but not T(eff) cells. Both Ebi3 and Il12a messenger RNA are markedly upregulated in T(reg) cells co-cultured with T(eff) cells, thereby boosting Ebi3 and IL-12alpha production in trans. T(reg)-cell restriction of this cytokine occurs because Ebi3 is a downstream target of Foxp3, a transcription factor that is required for T(reg)-cell development and function. Ebi3-/- and Il12a-/- T(reg) cells have significantly reduced regulatory activity in vitro and fail to control homeostatic proliferation and to cure inflammatory bowel disease in vivo. Because these phenotypic characteristics are distinct from those of other IL-12 family members, this novel Ebi3-IL-12alpha heterodimeric cytokine has been designated interleukin-35 (IL-35). Ectopic expression of IL-35 confers regulatory activity on naive T cells, whereas recombinant IL-35 suppresses T-cell proliferation. Taken together, these data identify IL-35 as a novel inhibitory cytokine that may be specifically produced by T(reg) cells and is required for maximal suppressive activity.

The dct locus of Rhodobacter capsulatus encodes a high-affinity transport system for the C 4 -dicarboxylates malate, succinate, and fumarate. The nucleotide sequence of the region downstream of the previously sequenced dctP gene (encoding a periplasmic C 4 -dicarboxylate-binding protein) was determined. Two open reading frames (ORFs) of 681 bp (dctQ) and 1,320 bp (dctM) were identified as additional dct genes by insertional mutagenesis and complementation studies. DctQ (24,763 Da) and DctM (46,827 Da) had hydropathic profiles consistent with the presence of 4 and 12 potential transmembrane segments, respectively, and were localized in the cytoplasmic membrane fraction after heterologous expression of the dctQM ORFs in Escherichia coli. DctP, DctQ, and DctM were found to be unrelated to known transport proteins in the ABC (ATP-binding cassette) superfamily but were shown to be homologous with the products of previously unidentified ORFs in a number of gram-negative bacteria, including Bordetella pertussis, E. coli, Salmonella typhimurium, Haemophilus influenzae, and Synechocystis sp. strain PCC6803. An additional ORF (rypA) downstream of dctM encodes a protein with sequence similarity to eukaryotic protein-tyrosine phosphatases, but interposon mutagenesis of this ORF did not result in a Dct ؊ phenotype. Complementation of a Rhizobium meliloti dctABD deletion mutant by heterologous expression of the dctPQM genes from R. capsulatus demonstrated that no additional structural genes were required to form a functional transport system. Transport via the Dct system was vanadate insensitive, and in uncoupler titrations with intact cells, the decrease in the rate of succinate transport correlated closely with the fall in membrane potential but not with the cellular ATP concentration, implying that the proton motive force, rather than ATP hydrolysis, drives uptake. It is concluded that the R. capsulatus Dct system is a new type of periplasmic secondary transporter and that similar, hitherto-unrecognized systems are widespread in gram-negative bacteria. The name TRAP (for tripartite ATP-independent periplasmic) transporters is proposed for this new group.Bacterial binding-protein-dependent solute transport systems are structurally complex, consisting of both periplasmic and membrane-bound components (3). To date, all such systems which have been sequenced and characterized functionally possess, in addition to the periplasmic binding protein and one or two integral membrane proteins, a highly conserved energy-coupling protein containing an ATP-binding cassette (the ABC protein) (28). In contrast, secondary transport systems, which are coupled to an electrochemical ion gradient rather than ATP hydrolysis, are much simpler and usually consist of a single integral membrane protein only. A number of families of related symporters, antiporters, and uniporters have been identified by primary sequence comparisons (18,23,40). Periplasmic binding proteins always appear to be a component of uptake, but not efflux, systems in the ABC t...

The spectrum of TCR V beta usage is compared for primary and recall CD8(+)D(b)PA(224)(+) T cell responses in mice with influenza pneumonia. Single-cell RT-PCR established that the same clonotypes were present in the lymphoid tissue and in the virus-infected lung. Longitudinal analysis indicated that the memory TCR repertoire reflects the primary response, with no decrease in diversity prior to (or after) secondary challenge. The re-engagement of memory T cells looked to be stochastic in this localized, transient infection. Analysis of clonotypes from the blood, spleen, regional lymph nodes, bone marrow, lung, and liver over a 200 day interval showed no evidence of selective localization or loss. The long-term distribution of memory T cells seemed to be essentially random.

In the rat lung, primary saccules are transformed into alveoli from postnatal Days 4 to 13, after which time there is a 20% reduction in the number of lung fibroblasts as the interstitial volume of the alveolar walls decreases. Our objective was to determine whether apoptosis is a factor in the observed decrease in the number of interstitial lung fibroblasts beyond Day 13. We used both histologic and flow cytometric assays to detect in lung fibroblasts the DNA fragmentation and condensation that are characteristic of apoptosis. In addition, we evaluated levels of bcl-2 and BAX messenger RNAs (mRNAs) using a reverse transcriptase-polymerase chain reaction (RT-PCR) assay. Apoptotic cells were quantitated in glycol methacrylate-embedded sections of neonatal rat lungs using the terminal transferase dUTP-digoxygenin nick end-labeling (TUNEL) method. Although TUNEL-positive interstitial cells were observed in the lungs of rats ranging in age from 10 to 16 d, a dramatic increase in apoptotic cells was seen on Day 17. Although diminished in number, TUNEL-positive cells were still present on Day 28. Hoechst-stained apoptotic bodies were observed in isolated lung cells that were vimentin-positive and factor VIII-negative, which identified the apoptotic cells as fibroblasts as opposed to endothelial cells. Flow cytometric analysis of freshly isolated lung fibroblasts stained with Hoechst 33342 indicated a 24% increase in chromatin condensation in cells from 17-d versus 16-d rats. DNA fragmentation was also quantitated by flow cytometry in freshly isolated fibroblasts labeled with BODIPY-conjugated dUTP in the presence of terminal deoxynucleotidyl transferase. The percentage of lung fibroblasts containing fragmented DNA was 51.4 +/- 13.4 in 17-d, 36.9 +/- 8.6 in 18-d, and 13.8 +/- 5.4 in 19-d rat pups. Finally, evaluation by RT-PCR indicated that on postnatal Day 17, mRNA for bcl-2, which inhibits apoptosis, was decreased to 73.5 +/- 11.4% (P < 0.001) of Day 5 controls; whereas mRNA for BAX, which enhances apoptosis, was increased to 243.0 +/- 102.0% (P < 0.001) of Day 5 values. These results demonstrate that rat lung fibroblasts undergo apoptosis after the completion of alveolarization, and suggest that this decrease in fibroblast number plays an important role in the thinning and remodeling of the alveolar walls of the lung.

Obstructive sleep apnea (OSA) occurs in at least 10% of the population, and leads to higher morbidity and mortality; however, relationships between OSA severity and sleep or psychological symptoms are unclear. Existing studies include samples with wide-ranging comorbidities, so we assessed relationships between severity of OSA and common sleep and psychological disturbances in recently diagnosed OSA patients with minimal co-morbidities. We studied 49 newly diagnosed, untreated OSA patients without major co-morbidities such as mental illness, cardiovascular disease, or stroke; subjects were not using psychoactive medications or tobacco (mean ± std age: 46.8±9.1 years; apnea/hyponea index [AHI]: 32.1±20.5 events/hour; female/male: 12/37; weight <125 kg). We evaluated relationships between the AHI and daytime sleepiness (Epworth Sleepiness Scale; ESS), sleep quality (Pittsburg Sleep Quality Index; PSQI), depressive symptoms (Beck Depression Inventory-II; BDI), and anxiety symptoms (Beck Anxiety Inventory; BAI), as well as sex and body mass index (BMI). AHI was similar in females and males. Mean levels of all symptoms were above normal thresholds, but AHI was not correlated with age, ESS, PSQI, BDI, or BAI; only BMI was correlated with OSA severity. No differences in mean AHI appeared when subjects were grouped by normal versus elevated values of ESS, PSQI, BDI, or BAI. Consistent with other studies, a strong link between OSA severity and psychological symptoms did not appear in these newly diagnosed patients, suggesting that mechanisms additional to the number and frequency of hypoxic events and arousals occurring with apneas contribute to adverse health effects in OSA. OSA patients presenting with mild or moderate severity, and no major co-morbidities will not necessarily have low levels of sleep or psychological disturbances.

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Background Neuropsychological comorbidities, including anxiety symptoms, accompany obstructive sleep apnea (OSA); structural and functional brain alterations also occur in the syndrome. The objective was to determine if OSA patients expressing anxiety symptoms show injury in specific brain sites. Methods Magnetic resonance T2-relaxometry was performed in 46 OSA and 66 control subjects. Anxiety symptoms were evaluated using the Beck Anxiety Inventory (BAI); subjects with BAI scores > 9 were classified anxious. Whole-brain T2-relaxation maps were compared between anxious and non-anxious groups using analysis of covariance (covariates; age and gender). Results Sixteen OSA and seven control subjects showed anxiety symptoms, and 30 OSA and 59 controls were non-anxious. Significantly higher T2-relaxation values, indicating tissue injury, appeared in anxious OSA vs non-anxious OSA subjects in subgenu, anterior, and mid-cingulate, ventral medial prefrontal and bilateral insular cortices, hippocampus extending to amygdala, and temporal, and bilateral parietal cortices. Brain injury emerged in anxious OSA vs non-anxious controls in bilateral insular cortices, caudate nuclei, anterior fornix, anterior thalamus, internal capsule, mid-hippocampus, dorsotemporal, dorsofrontal, ventral medial prefrontal and parietal cortices. Conclusions Anxious OSA subjects showed injury in brain areas regulating emotion, with several regions lying outside structures affected by OSA alone, suggesting additional injurious processes in anxious OSA subjects.

The aim of this study was to determine the in-season micro-cycle scheduling strategies used in professional team sport with particular reference to the reasoning and perceptions that underpin current practice. An online survey was completed by 35 practitioners from professional collision (C; Australian rules football: n = 9; rugby league: n = 6; rugby union: n = 2) and noncollision (NC; soccer; n = 18) sports. Respondents identified a common 48 h post-match recovery period, with few scheduling training stimuli during this period (NC: range: 0-17%; C: range: 0-47%). The majority (C: 78%; NC: 89%) of resistance training sessions were concurrent in nature, often delivering resistance training in the afternoon following field-based training (C: 72%; NC: 92%). NC practitioners were less satisfied with the maintenance of strength, power and hypertrophy (33-44%) versus C sports (59-76%), reflected by the lower frequency of prescription reported (C: 2.4 ± 0.6; NC: 1.5 ± 0.9; p = .003). Cold water immersion following both matches and daily training was highly prevalent, as was the adoption of self-report tools to monitor recovery. The congested and concurrent nature of training prescription during the micro-cycle, together with the universal adoption of routine cold water immersion warrants further investigation to inform evidence-based prescription and scheduling of training and recovery modalities in professional team sports.