Deletion of Tbc1d4/As160 abrogates cardiac glucose uptake and increases myocardial damage after ischemia/reperfusion.

BACKGROUND: Type 2 Diabetes mellitus (T2DM) is a major risk factor for cardiovascular disease and associated with poor outcome after myocardial infarction (MI). In T2DM, cardiac metabolic flexibility, i.e. the switch between carbohydrates and lipids as energy source, is disturbed. The RabGTPase-activating protein TBC1D4 represents a crucial regulator of insulin-stimulated glucose uptake in skeletal muscle by controlling glucose transporter GLUT4 translocation. A human loss-of-function mutation in TBC1D4 is associated with impaired glycemic control and elevated T2DM risk. The study's aim was to investigate TBC1D4 function in cardiac substrate metabolism and adaptation to MI. METHODS: Cardiac glucose metabolism of male Tbc1d4-deficient (D4KO) and wild type (WT) mice was characterized using in vivo [18F]-FDG PET imaging after glucose injection and ex vivo basal/insulin-stimulated [3H]-2-deoxyglucose uptake in left ventricular (LV) papillary muscle. Mice were subjected to cardiac ischemia/reperfusion (I/R). Heart structure and function were analyzed until 3 weeks post-MI using echocardiography, morphometric and ultrastructural analysis of heart sections, complemented by whole heart transcriptome and protein measurements. RESULTS: Tbc1d4-knockout abolished insulin-stimulated glucose uptake in ex vivo LV papillary muscle and in vivo cardiac glucose uptake after glucose injection, accompanied by a marked reduction of GLUT4. Basal cardiac glucose uptake and GLUT1 abundance were not changed compared to WT controls. D4KO mice showed mild impairments in glycemia but normal cardiac function. However, after I/R D4KO mice showed progressively increased LV endsystolic volume and substantially increased infarction area compared to WT controls. Cardiac transcriptome analysis revealed upregulation of the unfolded protein response via ATF4/eIF2α in D4KO mice at baseline. Transmission electron microscopy revealed largely increased extracellular matrix (ECM) area, in line with decreased cardiac expression of matrix metalloproteinases of D4KO mice. CONCLUSIONS: TBC1D4 is essential for insulin-stimulated cardiac glucose uptake and metabolic flexibility. Tbc1d4-deficiency results in elevated cardiac endoplasmic reticulum (ER)-stress response, increased deposition of ECM and aggravated cardiac damage following MI. Hence, impaired TBC1D4 signaling contributes to poor outcome after MI.

Psychiatric diagnostic dilemmas among people with intellectual and developmental disabilities.

BACKGROUND: Research regarding the accuracy of co-morbid psychiatric diagnoses in individuals with intellectual and developmental disabilities (IDD) is sparse. Yet correct diagnostic assignment is vital so that effective and appropriate treatment can be implemented, especially for the large numbers of individuals requiring expensive and restrictive behavioural health crisis services. METHOD: A retrospective review of de-identified data from multidisciplinary specialty team assessments completed for 50 individuals with ID (IntellectualDisability) with and without ASD and unresolved behavioural health challenges was conducted. The accuracy and reliability of the psychiatric diagnoses upon referral were compared with the diagnoses after the comprehensive team evaluation, and within-individual diagnostic agreement was calculated. The agreement between the Mood and Anxiety Semi-Structured interview tool (MASS) and the full team evaluation was also calculated. The influence of demographic and clinical characteristics on diagnostic agreement was explored. RESULTS: The most common chief complaints upon referral were aggression to others and self-injurious behaviour. Individuals were taking a median of six medications (interquartile range: 5 to 7); 80% were taking an antipsychotic medication. The most common medical conditions were constipation (70%) and gastroesophageal reflux disease (52%). Measures of interrater reliability of the referral diagnoses with the team assessment were below 0.5 (kappa range: -0.04 to 0.39), with the exception of ruling out dementia (kappa = 0.85). The interrater reliability estimates for the MASS evaluations for depression and anxiety were higher (kappa = 0.69 and 0.64) and reflected higher sensitivity and PPV. The odds of any referral diagnosis being confirmed by team evaluation were low: 0.25 (range: 0 to 0.67). The level of diagnostic agreement for each patient was not significantly attributable to demographic or clinical characteristics, although effect sizes indicate a possible positive relationship to age and the number of prescribed psychotropic medications at referral. CONCLUSION: Individuals in the current study had serious psychiatric and behavioural problems despite psychiatric care in their communities. The majority of psychiatric diagnoses provided upon referral were not supported by the multidisciplinary specialty team's assessment. In addition to possible diagnostic inaccuracy, the group in the study suffered from multiple medical co-morbidities and were exposed to polypharmacy. Results emphasise the importance of multidisciplinary evaluation by clinicians with expertise in neurodevelopmental disabilities when people with ID with and without ASD have complex behavioural health needs that are unresponsive to usual care. In addition, based on agreement with the full team evaluation, the MASS shows promise as an assessment tool, especially with regards to identifying anxiety and depression.

E-cigarettes and health risks: more to the flavor than just the name.

The growing interest in regulating flavored E-liquids must incorporate understanding of the "flavoring profile" of each E-liquid-which flavorings (flavoring chemicals) are present and at what concentrations not just focusing on the flavor on the label. We investigated the flavoring profile of 10 different flavored E-liquids. We assessed bronchial epithelial cell viability and apoptosis, phagocytosis of bacteria and apoptotic cells by macrophages after exposure to E-cigarette vapor extract (EVE). We validated our data in normal human bronchial epithelial cells (NHBE) and alveolar macrophages (AM) from healthy donors. We also assessed cytokine release and validated in the saliva from E-cigarette users. Increased necrosis/apoptosis (16.1-64.5% apoptosis) in 16HBE cells was flavor dependent, and NHBEs showed an increased susceptibility to flavors. In THP-1 differentiated macrophages phagocytosis was also flavor dependent, with AM also showing increased susceptibility to flavors. Further, Banana and Chocolate were shown to reduce surface expression of phagocytic target recognition receptors on alveolar macrophages. Banana and Chocolate increased IL-8 secretion by NHBE, whereas all 4 flavors reduced AM IL-1ß secretion, which was also reduced in the saliva of E-cigarette users compared with healthy controls. Flavorant profiles of E-liquids varied from simple 2 compound mixtures to complex mixtures containing over a dozen flavorants. E-liquids with high benzene content, complex flavoring profiles, high chemical concentration had the greatest impacts. The Flavorant profile of E-liquids is key to disruption of the airway status quo by increasing bronchial epithelial cell apoptosis, causing alveolar macrophage phagocytic dysfunction, and altering airway cytokines.

BOS is associated with decreased HDAC2 from steroid resistant lymphocytes in the small airways.

Immunosuppression therapies including corticosteroids fail to prevent bronchiolitis obliterans syndrome (BOS), primarily a disease of the small airways, following lung transplantation. We reported increases in steroid-resistant proinflammatory lymphocytes and their loss of histone deacetylase 2 (HDAC2), an important mediator of steroid action, in the blood of stable lung transplant recipients. We noted similar increases in the steroid-resistant lymphocytes in both the blood and small airways in BOS compared with the large airways. We hypothesized that these small airway cells would also exhibit a loss of HDAC2, and that these changes could be reversed by treatment with theophylline (HDAC2 activator). Blood, bronchoalveolar lavage and large and small airway brushings were collected from lung transplant patients with BOS (n = 12) or stable lung function (n = 18) and healthy aged-matched controls (n = 13). Intracellular proinflammatory cytokines [interferon (IFN-γ) and tumour necrosis factor (TNF)-α and HDAC2 were measured in CD8+ T, natural killer (NK) T-like and NK cells from cultured small airway brushings ± 5 mg/l theophylline ± 1 µM prednisolone using flow cytometry. Increased small airway CD8 T, NK T-like and NK cells were identified in BOS versus stable transplant and controls. In BOS, these cells exhibited increased IFN-γ/TNF-α and a loss of HDAC2. HDAC2 expression by small airway CD8+ T cells correlated with forced expiratory volume in 1 s (FEV1 ) (R = 0·880, P = 0·031). Theophylline and prednisolone synergistically up-regulated HDAC2 in CD8+ T cells. BOS is associated with loss of HDAC2 from steroid-resistant proinflammatory CD8+ T, NK T-like and NK cells in the small airways. Therapeutically increasing HDAC2 in these lymphocytes may reduce steroid resistance and improve graft survival.

Bronchiolitis obliterans syndrome is associated with increased p-glycoprotein expression and loss of glucocorticoid receptor from steroid-resistant proinflammatory CD8+ T cells.

Immunosuppressive therapy fails to suppress the production of proinflammatory cytokines, particularly by CD8+ T cells, in stable lung transplant recipients and those undergoing chronic rejection, suggesting that some patients may become relatively resistant to immunosuppressants such as glucocorticoids (GC). We have shown loss of GC receptor (GCR) from the CD8+ cells, and we hypothesized that the drug membrane efflux pump, p-glycoprotein-1 (Pgp), may also be involved in lymphocyte steroid resistance following lung transplant. Pgp/GCR expression and interferon (IFN)-γ/tumour necrosis factor (TNF)-α proinflammatory cytokine production was measured in blood lymphocytes from 15 stable lung transplant patients, 10 patients with bronchiolitis obliterans syndrome (BOS) and 10 healthy aged-matched controls (± prednisolone ± Pgp inhibitor, cyclosporin A ± GCR activator, Compound A) using flow cytometry. Both Pgp+ and Pgp- lymphocyte subsets from all subjects produced IFN-γ/TNF-α proinflammatory cytokines. Pgp expression was increased in CD8+ Pgp+ T cells and correlated with IFN-γ/TNF-α expression and BOS grade. Reduced GCR was observed in CD8+ Pgp- T, natural killer (NK) T-like and NK cells from stable patients compared with controls, and reduced further in CD8+ Pgp- T cells in BOS. The addition of 2·5 ng/ml cyclosporin A and 1 µM prednisolone inhibit IFN-γ/TNF-α production significantly by CD8+ Pgp+ T cells from BOS patients. The addition of 10 µM Compound A and 1 µM prednisolone inhibit IFN-γ/TNF-α production significantly by CD8+ Pgp- T cells from BOS patients. BOS is associated with increased Pgp expression and loss of GCR from steroid-resistant proinflammatory CD8+ T cells. Treatments that inhibit Pgp and up-regulate GCR in CD8+ T cells may improve graft survival.

Nurse provision of support to help inpatients quit smoking.

Issue addressed Identification of the factors that facilitate nurses to provide smoking cessation advice to hospitalised patients. Method Six semistructured focus groups with 26 nurses were conducted in June 2015. Participants completed a structured survey to collect patient demographic data and assess attitudes towards their role in addressing smoking cessation among inpatients. Results Important themes that emerged from the qualitative data were: nurses' negative perceptions of smokers, nurses' confidence in their knowledge of smoking cessation care and nurses' uncertainty around whose role it is to provide smoking cessation care. Conclusion Nurses require training in order to confidently and competently address smoking among inpatients as part of routine care. Formal ways to document the smoking status of inpatients and the offer of smoking cessation support from a nurse to an inpatient would enhance the communication between nurses around which inpatients had been asked about their smoking status and which had not. For patients who are resistant to conventional cessation strategies, innovative ways are needed to reduce the harm caused to them by tobacco use. So what? Nurses need to be provided with education and training around smoking cessation to increase their confidence and skills to provide smoking cessation care to inpatients.

Blood cytotoxic/inflammatory mediators in non-eosinophilic asthma.

BACKGROUND: Non-eosinophilic asthma (NEA) is a distinct, often corticosteroid-resistant inflammatory asthma phenotype. NK and NKT-like cells are effector lymphocytes that we have shown, like CD28null T cells, to be relatively resistant to steroids and major sources of pro-inflammatory/cytotoxic mediators. We hypothesized that these cells and mediators would be increased in peripheral blood in NEA. METHODS: Adults with severe asthma and variable airflow obstruction, poorly controlled despite maintenance therapy with inhaled glucocorticosteroids and long-acting bronchodilators, were recruited. Blood was assessed in those with eosinophilic asthma (n = 12), NEA (n = 25) and healthy non-smoking controls (n = 30). We applied flow cytometry to measure T, CD28null, NK and NKT-like cells and their expression of granzyme B, perforin, and killer inhibitory/activating receptors CD94(Kp43), CD158b and CD107A. Intracellular pro-inflammatory cytokine production (IFN-γ and TNF-α) was assessed in 18 controls and 10 patients with asthma/group. RESULTS: In NEA, there was increased expression of granzyme B by CD8+ T cells vs. CONTROLS: There was increased expression of granzyme B and CD158 and decreased CD94 on NK cells, vs. healthy controls and those with eosinophilic asthma. IFN-γ production by NK cells and TNF-α production by NKT-like cells in NEA were significantly increased vs. CONTROLS: In both eosinophilic and NEA phenotypes, there were significant increases in CD4+28null T cells (72% and 81% increases, respectively, vs. controls) and their expression of pro-inflammatory cytokines. Significant correlations were noted between blood CD4+28null T cells and neutrophil numbers in induced sputum, and between corticosteroid dose and blood NKT-like cells, and their production of granzyme B and TNF-α and NK IFN-γ. CONCLUSION AND CLINICAL RELEVANCE: In poorly controlled asthma, altered expression of cytotoxic/pro-inflammatory mediators can be seen on a variety of lymphocyte subsets in the peripheral blood; these changes are most apparent in NEA. Whether this pattern of expression is a marker of treatment responsiveness and future risk of exacerbations remains to be determined.

Lung cancer is associated with decreased expression of perforin, granzyme B and interferon (IFN)-γ by infiltrating lung tissue T cells, natural killer (NK) T-like and NK cells.

There is a limited understanding how of lung cancer cells evade cytotoxic attack. Previously, we have shown reduced production of the cytotoxic mediator granzyme B by CD8(+) T cells in lung cancer tissue. We hypothesized that lung cancer would be further associated with decreased production of granzyme B, perforin and proinflammatory cytokines by other cytotoxic lymphocytes, natural killer (NK) T-like and NK cells, and that this would result from soluble mediators released by the cancer cells. Lung cancer and non-cancer tissue from five patients was identified by experienced pathologists. Tumour necrosis factor (TNF)-α, interferon (IFN)-γ, granzyme B and perforin were measured in CD4 and CD8(+) T, NK T-like cells and NK cells by flow cytometry. Correlation between cancer stage and granzyme B was analysed retrospectively for 21 patients. The effects of soluble factors released by lung cancer cells on production of cytotoxic mediators and cytokines was assessed, and the role of prostaglandin E2 (PGE)2 /COX investigated using indomethacin inhibition. There were significantly decreased percentages of T, NK T-like and NK cells expressing perforin, TNF-α and IFN-γ in cancer versus non-cancer tissue, and of CD8(+) T cells and CD8(+) NK T-like cells expressing granzyme B (e.g. NK T-like cells: non-cancer 30% ± 7 versus cancer 6% ± 2·5). Cancer cells released soluble factors that inhibited granzyme B, perforin and IFN-γ production that was partially associated with the PGE2 /COX2 pathway. Thus, lung cancer is associated with decreased expression of granzyme B, perforin and IFN-γ by infiltrating T cells, NK T-like and NK cells, possibly as a result of soluble factors produced by the cancer cells including PGE2 . This may be an important immune evasion mechanism.

Full blood count parameters for the detection of asthma inflammatory phenotypes.

BACKGROUND: In asthma, the airway inflammatory phenotype influences clinical characteristics and treatment response. Although induced sputum is the gold standard test for phenotyping asthma, a more accessible method is needed for clinical practice. OBJECTIVE: To investigate whether white blood cell counts and/or their derived ratios can predict sputum eosinophils or neutrophils in uncontrolled asthma. METHODS: This cross-sectional study evaluated 164 treated but uncontrolled asthmatic patients with sputum induction and blood collection. Receiver-operating characteristic (ROC) curves were used to assess the relationship between blood and sputum parameters. RESULTS: There was a significant positive relationship between blood eosinophil parameters and the percentage of sputum eosinophil count. A weak but significant correlation was found between sputum neutrophil percentage and blood neutrophil percentage (r = 0.219, P = 0.005). ROC curve analysis identified that blood eosinophil percentage count was the best predictor for eosinophilic asthma, with an area under the curve (AUC) of 0.907 (P < 0.001). The optimum cut-point for blood eosinophil percentage was 2.7%, and this yielded a sensitivity of 92.2% and a specificity of 75.8%. The absolute blood eosinophil count was also highly predictive with an AUC of 0.898 (P < 0.0001) at a blood eosinophil cut-off of 0.26 × 10(9) /L. The blood eosinophil/lymphocyte ratio (ELR) and eosinophil/neutrophil ratio (ENR) were increased in eosinophilic asthma, and the neutrophil/lymphocyte ratio (NLR) was increased in neutrophilic asthma. Neutrophilic asthma could also be detected by blood neutrophil percentages and NLR, but with less accuracy. CONCLUSIONS AND CLINICAL RELEVANCE: Blood eosinophil counts and derived ratios (ELR and ENR) can accurately predict eosinophilic asthma in patients with persistent uncontrolled asthma despite treatment. Blood neutrophil parameters are poor surrogates for the proportion of sputum neutrophils. Blood counts may be a useful aid in the monitoring of uncontrolled asthma.

Genetic variation in the ovine uncoupling protein 1 gene: association with carcass traits in New Zealand (NZ) Romney sheep, but no association with growth traits in either NZ Romney or NZ Suffolk sheep.

The uncoupling protein 1 (UCP1) plays an important role in the regulation of lipolysis and thermogenesis in adipose tissues. Genetic variation within three regions (the promoter, intron 2 and exon 5) of the ovine UCP1 gene (UCP1) was investigated using polymerase chain reaction-single-strand conformational polymorphism (PCR-SSCP) analyses. These revealed three promoter variants (designated A, B and C) and two intron 2 variants (a and b). The association of this genetic variation with variation in lamb carcass traits and postweaning growth was investigated in New Zealand (NZ) Romney and Suffolk sheep. The presence of B in a lamb's genotype was associated with decreased subcutaneous carcass fat depth (V-GR) (p = 0.004) and proportion of total lean meat yield of loin meat (p = 0.005), and an increased proportion of total lean meat yield of hind-leg meat (p = 0.018). In contrast, having two copies of C was associated with increased V-GR (p < 0.001) and proportion of total lean meat yield of shoulder meat (p = 0.009), and a decreased hind-leg yield (p = 0.032). No associations were found with postweaning growth. These results suggest that ovine UCP1 is a potential gene marker for carcass traits.

Up-regulation of alternate co-stimulatory molecules on proinflammatory CD28null T cells in bronchiolitis obliterans syndrome.

Bronchiolitis obliterans syndrome (BOS) is associated with lack of immunosuppression of T cell proinflammatory cytokines and increased T cell granzyme B. Repeated antigen-driven proliferation down-regulates T cell CD28. We hypothesized that down-regulation of CD28 and up-regulation of alternate co-stimulatory molecules (CD134, CD137, CD152 and CD154) on T cells may be associated with BOS. Co-stimulatory molecules, granzyme B, perforin and intracellular cytokines were measured by flow cytometry on T cells from stable lung transplant patients (n = 38), patients with BOS (n = 20) and healthy controls (n = 10). There was a significant increase in the percentage of CD4/28(null) and CD8/28(null) T cells producing granzyme B, interferon (IFN)-γ and tumour necrosis factor (TNF)-α in BOS compared with stable patients. Down-regulation of CD28 was associated with steroid resistance and up-regulation of CD134, CD137, CD152 and CD154 on CD4(+) T cells and CD137 and CD152 on CD8(+) T cells. There was a significant correlation between increased CD28(null) /CD137 T cells producing IFN-γ, TNF-α with BOS grade (r = 0·861, P < 0·001 for CD28(null) /CD137 IFN-γ/CD8) and time post-transplant (r = 0·698, P < 0·001 for CD28(null) /CD137 IFN-γ/CD8). BOS is associated with down-regulation of CD28 and up-regulation of alternate co-stimulatory molecules on steroid-resistant peripheral blood proinflammatory CD4(+) and CD8(+) T cells. Therapeutic targeting of alternate co-stimulatory molecules on peripheral blood CD28(null) T cells and monitoring response using these assays may help in the management of patients with BOS.

Impaired macrophage phagocytosis in non-eosinophilic asthma.

BACKGROUND: Many patients with non-eosinophilic asthma have increased numbers of neutrophils in the airways. The explanation for this chronic inflammation remains unclear, but may result from an impaired ability of alveolar macrophages to phagocytose apoptotic cells (a process termed 'efferocytosis'), as we have shown in chronic obstructive pulmonary disease (COPD). OBJECTIVES: To examine induced sputum as a non-invasive technique to characterize efferocytosis in chronic lung diseases and to compare efferocytosis in patients with non-eosinophilic asthma, eosinophilic asthma and COPD. METHODS: Participants with stable asthma (20 with eosinophilic and 30 with non-eosinophilic) and COPD (n = 11) underwent clinical assessment including allergy skin tests, saline challenge and sputum induction. Sputum cells were dispersed using dithiothreitol and resuspended in culture medium. Efferocytosis of apoptotic bronchial epithelial cells by sputum-derived macrophages was determined using flow cytometry. RESULTS: There were no significant differences in efferocytosis between paired sputum and bronchoalveolar lavage macrophages from three subjects. Efferocytosis was significantly impaired in patients with non-eosinophilic asthma [mean (SD) 0.95 (0.24)] compared with eosinophilic asthma [1.17 (0.19)] and to a similar degree as patients with COPD [1.04 (0.16)]. Sputum neutrophils were significantly higher in patients with COPD and non-eosinophilic asthma compared with eosinophilic asthma. CONCLUSION AND CLINICAL RELEVANCE: Induced sputum provides a reliable and non-invasive method for studying macrophage efferocytosis in chronic lung disease. Macrophage efferocytosis is impaired in non-eosinophilic asthma to a similar degree as that in COPD and may explain the persistent airway neutrophilia and chronic inflammation that characterizes this asthma subtype.

The cost of dominance: suppressing subordinate reproduction affects the reproductive success of dominant female banded mongooses.

Social species show considerable variation in the extent to which dominant females suppress subordinate reproduction. Much of this variation may be influenced by the cost of active suppression to dominants, who may be selected to balance the need to maximize the resources available for their own offspring against the costs of interfering with subordinate reproduction. To date, the cost of reproductive suppression has received little attention, despite its potential to influence the outcome of conflict over the distribution of reproduction in social species. Here, we investigate possible costs of reproductive suppression in banded mongooses, where dominant females evict subordinates from their groups, thereby inducing subordinate abortion. We show that evicting subordinate females is associated with substantial costs to dominant females: pups born to females who evicted subordinates while pregnant were lighter than those born after undisturbed gestations; pups whose dependent period was disrupted by an eviction attained a lower weight at independence; and the proportion of a litter that survived to independence was reduced if there was an eviction during the dependent period. To our knowledge, this is the first empirical study indicating a possible cost to dominants in attempting to suppress subordinate breeding, and we argue that much of the variation in reproductive skew both within and between social species may be influenced by adaptive variation in the effort invested in suppression by dominants.

Intrasexual competition and sexual selection in cooperative mammals.

In most animals, the sex that invests least in its offspring competes more intensely for access to the opposite sex and shows greater development of secondary sexual characters than the sex that invests most. However, in some mammals where females are the primary care-givers, females compete more frequently or intensely with each other than males. A possible explanation is that, in these species, the resources necessary for successful female reproduction are heavily concentrated and intrasexual competition for breeding opportunities is more intense among females than among males. Intrasexual competition between females is likely to be particularly intense in cooperative breeders where a single female monopolizes reproduction in each group. Here, we use data from a twelve-year study of wild meerkats (Suricata suricatta), where females show high levels of reproductive skew, to show that females gain greater benefits from acquiring dominant status than males and traits that increase competitive ability exert a stronger influence on their breeding success. Females that acquire dominant status also develop a suite of morphological, physiological and behavioural characteristics that help them to control other group members. Our results show that sex differences in parental investment are not the only mechanism capable of generating sex differences in reproductive competition and emphasize the extent to which competition for breeding opportunities between females can affect the evolution of sex differences and the operation of sexual selection.

Role of increased CD8/CD28(null) T cells and alternative co-stimulatory molecules in chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory lung disease; it is a leading cause of death and existing treatments have no proven disease-modifying effect. The mechanisms underlying this resistance are largely unknown, but suggest the presence of some self-maintaining pathogenic process, possibly initiated by cigarette smoking, that prevents the normal resolution of inflammation. We have previously reported increased production of proinflammatory cytokines and granzyme b by CD8(+) T cells in COPD; costimulatory receptor/ligand interactions required include CD80:86/CD28, B7-1/CTLA4, 4-1BB/1BBL and OX40/OX40L. We hypothesized that a dysregulated expression/function of these molecules may play a role in inflammatory/autoimmune components of COPD. We analysed T cell co-stimulatory molecules in blood from 34 controls, 15 smokers and 48 COPD subjects. We assessed the potential functional relevance of CD8/CD28(null) cells in COPD by measuring their production of proinflammatory cytokines, co-stimulatory molecules, granzyme and perforin. A smoke-exposed murine model was applied to investigate the relative expression of CD8/CD28(null) T cells in blood, lung tissue and airway. CD8/CD28(null) cells were increased in both current- and ex-smoker COPD groups; these cells expressed significantly more interferon (IFN)-γ, OX40, 4-1BB, CTLA4, granzyme and perforin when stimulated than CD8/CD28(+) T cells. There were no changes in CD4/CD28(null) T cells. In mice exposed to cigarette smoke for 12 weeks, CD8/CD28(null) T cells were significantly increased in the airway with a trend for an increase in lung tissue and blood. Increased production of proinflammatory cytokines and expression of alternative co-stimulatory molecules by CD8/CD28(null) T cells may play a role in inflammatory or autoimmune responses in COPD and identify therapeutic targets.

Increased T regulatory cells and decreased Th1 pro-inflammatory cytokines correlate with culture-positive infection in febrile neutropenia childhood oncology patients.

BACKGROUND: Paediatric oncology patients with febrile neutropenia are usually hospitalised and treated with empirical broad-spectrum antibiotic therapy to counter the risk of infection. However, there is currently no method available to rapidly identify bacteremia in these patients. T-helper-type-1 (Th1) cytokines are required for effective immune response to many pathogenic organisms and T regulatory cells are known suppressors of Th1 cells. We hypothesized that characterization of reduced intracellular Th1 cytokines and increased T regulatory cells (Tregs) may prove useful in identifying infection in childhood oncology patients with febrile neutropenia. METHODS: Intracellular Th 1 cytokines and Tregs were enumerated in peripheral blood from a group of childhood oncology patients with febrile neutropenia using multiparameter flow cytometry. RESULTS: There was a significant increase in the percentage of CD25(+) CD127(-) CD8(-) CD3(+) Tregs and a significant decrease in Th1 intracellular cytokines IFNγ, IL-2 and TNFα in the blood of culture positive patients compared with culture negative patients. CONCLUSIONS: Enumeration of Tregs and intracellular Th1 cytokines may provide a sensitive, specific test for determining infection in childhood oncology patients before blood culture results become available.

Panic disorder is associated with the serotonin transporter gene (SLC6A4) but not the promoter region (5-HTTLPR).

Panic disorder (PD) and social anxiety disorder (SAD) are moderately heritable anxiety disorders. We analyzed five genes, derived from pharmacological or translational mouse models, in a new case-control study of PD and SAD in European Americans: (1) the serotonin transporter (SLC6A4), (2) the serotonin receptor 1A, (3) catechol-O-methyltransferase, (4) a regulator of g-protein signaling and (5) the gastrin-releasing peptide receptor. Cases were interviewed using the schedule for affective disorders and schizophrenia and were required to have a probable or definite lifetime diagnosis of PD (N=179), SAD (161) or both (140), with first onset by age 31 and a family history of anxiety. Final diagnoses were determined using the best estimate procedure, blind to genotyping data. Controls were obtained from the National Institute of Mental Health Human Genetics Initiative; only subjects above 25 years of age who screened negative for all psychiatric symptoms were included (N=470). A total of 45 single nucleotide polymorphisms were successfully genotyped over the five selected genes using Applied Biosystems SNPlex protocol. SLC6A4 provided strong and consistent evidence of association with the PD and PD+SAD groups, with the most significant association in both groups being at rs140701 (chi(2)=10.72, P=0.001 with PD and chi(2)=8.59, P=0.003 in the PD+SAD group). This association remained significant after multiple test correction. Those carrying at least one copy of the haplotype A-A-G constructed from rs3794808, rs140701 and rs4583306 have 1.7 times the odds of PD than those without the haplotype (95% confidence interval: 1.2-2.3). The SAD only group did not provide evidence of association, suggesting a PD-driven association. The findings remained after adjustment for age and sex, and there was no evidence that the association was due to population stratification. The promoter region of the gene, 5-HTTLPR, did not provide any evidence of association, regardless of whether analyzed as a triallelic or biallelic locus, nor did any of the other four candidate genes tested. Our findings suggest that the serotonin transporter gene may play a role in PD; however, the findings require replication. Future studies should attend to the entire genetic region rather than the promoter.

Reproductive competition and the evolution of extreme birth synchrony in a cooperative mammal.

Reproductive events in animal societies often show a high degree of temporal clustering, but the evolutionary causes of this synchronization are poorly understood. Here, we suggest that selection to avoid the negative effects of competition with other females has given rise to a remarkable degree of birth synchrony in the communal-breeding banded mongoose (Mungos mungo). Within banded mongoose groups, births are highly synchronous, with 64 per cent of females giving birth on exactly the same night. Our results indicate that this extreme synchrony arises because offspring suffer an increased risk of infanticide if their mother gives birth before other females, but suffer in competition with older littermates if their mother gives birth after them. These findings highlight the important influence that reproductive competition can have for the evolution of reproductive synchrony.

Time post-lung transplant correlates with increasing peripheral blood T cell granzyme B and proinflammatory cytokines.

Immunosuppression therapy following lung transplant fails to prevent chronic rejection/bronchiolitis obliterans syndrome, which we have shown is associated with lack of suppression of peripheral blood T cell granzyme B, interferon (IFN)-γ and tumour necrosis factor (TNF)-α. We hypothesized that these proinflammatory mediators may increase with time post-transplant in otherwise stable patients before clinical signs of declining lung function, and patients experiencing declining lung function would show a further increase in these mediators. Intracellular cytokine profiles and granzyme B were investigated in T cells in whole blood and airways from lung transplant patients using flow cytometry. There was a significant negative correlation between forced expiratory volume in 1 s (FEV(1) ), drug dose and time post-transplant. A significant correlation between increased granzyme B, IFN-γ, interleukin (IL)-2 and TNF-α and time post-transplant was noted in peripheral blood T cells but not lung T cells from stable patients. Patients with similar drug dose but experiencing declining FEV(1) showed a further increase in peripheral blood T cell IFN-γ, IL-2 and TNF-α. Time post-lung transplant correlates with increasing peripheral blood T cell granzyme B and proinflammatory cytokines. Declining FEV(1) is associated with a further increase in these proinflammatory mediators. Drugs that reduce these inflammatory mediators effectively may reduce the incidence of chronic graft rejection.

Posttransplant bronchiolitis obliterans syndrome is associated with bronchial epithelial to mesenchymal transition.

Bronchiolitis obliterans syndrome (BOS) compromises lung transplant outcomes and is characterised by airway epithelial damage and fibrosis. The process whereby the normal epithelial configuration is replaced by fibroblastic scar tissue is poorly understood, but recent studies have implicated epithelial mesenchymal transition (EMT). The primary aim of this study was to assess the utility of flow cytometry in detecting and quantifying EMT in bronchial epithelial cells. Large airway brushings were obtained at 33 bronchoscopies in 16 BOS-free and 6 BOS grade 1-3 patients at 2-120 months posttransplant. Flow cytometry was used to assess expression of the mesenchymal markers alphaSMA, S100A4 and ED-A FN and HLA-DR. TGF beta 1 and HGF were measured in Bronchoalveolar lavage (BAL). Expression of all three mesenchymal markers was increased in BOS, as was HLA-DR. BAL HGF, but not TGF beta 1 was increased in BOS. Longitudinal investigation of one patient revealed a 100% increase in EMT markers concurrent with a 6-fold increase in BAL TGF beta 1 and the diagnosis of BOS at 17 months posttransplant. Flow cytometric evaluation of bronchial epithelium may provide a novel and rapid means to assess lung allografts at risk of BOS.