Insights into GABA receptor signalling in TM3 Leydig cells

gamma-Aminobutyric acid (GABA) is an emerging signalling molecule in endocrine organs, since it is produced by endocrine cells and acts via GABA(A) receptors in a paracrine/autocrine fashion. Testicular Leydig cells are producers and targets for GABA. These cells express GABA(A) receptor subunits and in the murine Leydig cell line TM3 pharmacological activation leads to increased proliferation. The signalling pathway of GABA in these cells is not known in this study. We therefore attempted to elucidate details of GABA(A) signalling in TM3 and adult mouse Leydig cells using several experimental approaches. TM3 cells not only express GABA(A) receptor subunits, but also bind the GABA agonist {[}H-3] muscimol with a binding affinity in the range reported for other endocrine cells (K-d = 2.740 +/- 0.721 nM). However, they exhibit a low B-max value of 28.08 fmol/mg protein. Typical GABA(A) receptor-associated events, including Cl- currents, changes in resting membrane potential, intracellular Ca2+ or cAMP, were not measurable with the methods employed in TM3 cells, or, as studied in part, in primary mouse Leydig cells. GABA or GABA(A) agonist isoguvacine treatment resulted in increased or decreased levels of several mRNAs, including transcription factors (c-fos, hsf-1, egr-1) and cell cycle-associated genes (Cdk2, cyclin D1). In an attempt to verify the cDNA array results and because egr-1 was recently implied in Leydig cell development, we further studied this factor. RT-PCR and Western blotting confirmed a time-dependent regulation of egr-1 in TM3. In the postnatal testis egr-1 was seen in cytoplasmic and nuclear locations of developing Leydig cells, which bear GABA(A) receptors and correspond well to TM3 cells. Thus, GABA acts via an untypical novel signalling pathway in TM3 cells. Further details of this pathway remain to be elucidated. Copyright (c) 2005 S. Karger AG, Base

Regional microbial signatures positively correlate with differential wine phenotypes: evidence for a microbial aspect to terroir

Many crops display differential geographic phenotypes and sensorial signatures, encapsulated by the concept of terroir. The drivers behind these differences remain elusive, and the potential contribution of microbes has been ignored until recently. Significant genetic differentiation between microbial communities and populations from different geographic locations has been demonstrated, but crucially it has not been shown whether this correlates with differential agricultural phenotypes or not. Using wine as a model system, we utilize the regionally genetically differentiated population of Saccharomyces cerevisiae in New Zealand and objectively demonstrate that these populations differentially affect wine phenotype, which is driven by a complex mix of chemicals. These findings reveal the importance of microbial populations for the regional identity of wine, and potentially extend to other important agricultural commodities. Moreover, this suggests that long-term implementation of methods maintaining differential biodiversity may have tangible economic imperatives as well as being desirable in terms of employing agricultural practices that increase responsible environmental stewardship

Dual Hypocretin Receptor Antagonism Is More Effective for Sleep Promotion than Antagonism of Either Receptor Alone

The hypocretin (orexin) system is involved in sleep/wake regulation, and antagonists of both hypocretin receptor type 1 (HCRTR1) and/or HCRTR2 are considered to be potential hypnotic medications. It is currently unclear whether blockade of either or both receptors is more effective for promoting sleep with minimal side effects. Accordingly, we compared the properties of selective HCRTR1 (SB-408124 and SB-334867) and HCRTR2 (EMPA) antagonists with that of the dual HCRTR1/R2 antagonist almorexant in the rat. All 4 antagonists bound to their respective receptors with high affinity and selectivity in vitro. Since in vivo pharmacokinetic experiments revealed poor brain penetration for SB-408124, SB-334867 was selected for subsequent in vivo studies. When injected in the mid-active phase, SB-334867 produced small increases in rapid-eye-movement (REM) and non-REM (NR) sleep. EMPA produced a significant increase in NR only at the highest dose studied. In contrast, almorexant decreased NR latency and increased both NR and REM proportionally throughout the subsequent 6 h without rebound wakefulness. The increased NR was due to a greater number of NR bouts; NR bout duration was unchanged. At the highest dose tested (100 mg/kg), almorexant fragmented sleep architecture by increasing the number of waking and REM bouts. No evidence of cataplexy was observed. HCRTR1 occupancy by almorexant declined 4–6 h post-administration while HCRTR2 occupancy was still elevated after 12 h, revealing a complex relationship between occupancy of HCRT receptors and sleep promotion. We conclude that dual HCRTR1/R2 blockade is more effective in promoting sleep than blockade of either HCRTR alone. In contrast to GABA receptor agonists which induce sleep by generalized inhibition, HCRTR antagonists seem to facilitate sleep by reducing waking “drive”

Site of Allergic Airway Narrowing and the Influence of Exogenous Surfactant in the Brown Norway Rat

Background: The parameters RN (Newtonian resistance), G (tissue damping), and H (tissue elastance) of the constant phase model of respiratory mechanics provide information concerning the site of altered mechanical properties of the lung. The aims of this study were to compare the site of allergic airway narrowing implied from respiratory mechanics to a direct assessment by morphometry and to evaluate the effects of exogenous surfactant administration on the site and magnitude of airway narrowing. Methods: We induced airway narrowing by ovalbumin sensitization and challenge and we tested the effects of a natural surfactant lacking surfactant proteins A and D (InfasurfH) on airway responses. Sensitized, mechanically ventilated Brown Norway rats underwent an aerosol challenge with 5 % ovalbumin or vehicle. Other animals received nebulized surfactant prior to challenge. Three or 20 minutes after ovalbumin challenge, airway luminal areas were assessed on snap-frozen lungs by morphometry. Results: At 3 minutes, RN and G detected large airway narrowing whereas at 20 minutes G and H detected small airway narrowing. Surfactant inhibited RN at the peak of the early allergic response and ovalbumin-induced increase in bronchoalveolar lavage fluid cysteinyl leukotrienes and amphiregulin but not IgE-induced mast cell activation in vitro. Conclusion: Allergen challenge triggers the rapid onset of large airway narrowing, detected by RN and G, and subsequen

Transcriptional profiles predict treatment outcome in patients with tuberculosis and diabetes at diagnosis and at two weeks after initiation of anti-tuberculosis treatment

Data sharing statement: RNA sequence data have been submitted to NCBI Gene Expression Omnibus (GEO) under accession number GSE193979. dcRT-MLPA data can be found in Supplementary Table S6.Copyright © 2022 The Authors. Background: Globally, the tuberculosis (TB) treatment success rate is approximately 85%, with treatment failure, relapse and death occurring in a significant proportion of pulmonary TB patients. Treatment success is lower among people with diabetes mellitus (DM). Predicting treatment outcome early after diagnosis, especially in TB-DM patients, would allow early treatment adaptation for individuals and may improve global TB control. Methods: Samples were collected in a longitudinal cohort study of adult TB patients from South Africa (n = 94) and Indonesia (n = 81), who had concomitant DM (n = 59), intermediate hyperglycaemia (n = 79) or normal glycaemia/no DM (n = 37). Treatment outcome was monitored, and patients were categorized as having a good (cured) or poor (failed, recurrence, died) outcome during treatment and 12 months follow-up. Whole blood transcriptional profiles before, during and at the end of TB treatment were characterized using unbiased RNA-Seq and targeted gene dcRT-MLPA. Findings: We report differences in whole blood transcriptome profiles, which were observed before initiation of treatment and throughout treatment, between patients with a good versus poor TB treatment outcome. An eight-gene and a 22-gene blood transcriptional signature distinguished patients with a good TB treatment outcome from patients with a poor TB treatment outcome at diagnosis (AUC = 0·815) or two weeks (AUC = 0·834) after initiation of TB treatment, respectively. High accuracy was obtained by cross-validating this signature in an external cohort (AUC = 0·749). Interpretation: These findings suggest that transcriptional profiles can be used as a prognostic biomarker for treatment failure and success, even in patients with concomitant DM.TANDEM Consortium, received funding from the European Community's Seventh Framework Programme (FP7/2007-2013 Grant Agreement No. 305279) and the Netherlands Organization for Scientific Research (NWO-TOP Grant Agreement No. 91214038). The research leading to the results presented in the Indian validation cohort was supported by Research Council of Norway Global Health and Vaccination Research (GLOBVAC) projects: RCN 179342, 192534, and 248042, the University of Bergen (Norway)

Transcriptional profiles predict treatment outcome in patients with tuberculosis and diabetes at diagnosis and at two weeks after initiation of anti-tuberculosis treatment.

BACKGROUND: Globally, the tuberculosis (TB) treatment success rate is approximately 85%, with treatment failure, relapse and death occurring in a significant proportion of pulmonary TB patients. Treatment success is lower among people with diabetes mellitus (DM). Predicting treatment outcome early after diagnosis, especially in TB-DM patients, would allow early treatment adaptation for individuals and may improve global TB control. METHODS: Samples were collected in a longitudinal cohort study of adult TB patients from South Africa (n  =  94) and Indonesia (n  =  81), who had concomitant DM (n  =  59), intermediate hyperglycaemia (n  =  79) or normal glycaemia/no DM (n  =  37). Treatment outcome was monitored, and patients were categorized as having a good (cured) or poor (failed, recurrence, died) outcome during treatment and 12 months follow-up. Whole blood transcriptional profiles before, during and at the end of TB treatment were characterized using unbiased RNA-Seq and targeted gene dcRT-MLPA. FINDINGS: We report differences in whole blood transcriptome profiles, which were observed before initiation of treatment and throughout treatment, between patients with a good versus poor TB treatment outcome. An eight-gene and a 22-gene blood transcriptional signature distinguished patients with a good TB treatment outcome from patients with a poor TB treatment outcome at diagnosis (AUC = 0·815) or two weeks (AUC = 0·834) after initiation of TB treatment, respectively. High accuracy was obtained by cross-validating this signature in an external cohort (AUC = 0·749). INTERPRETATION: These findings suggest that transcriptional profiles can be used as a prognostic biomarker for treatment failure and success, even in patients with concomitant DM. FUNDING: The research leading to these results, as part of the TANDEM Consortium, received funding from the European Community's Seventh Framework Programme (FP7/2007-2013 Grant Agreement No. 305279) and the Netherlands Organization for Scientific Research (NWO-TOP Grant Agreement No. 91214038). The research leading to the results presented in the Indian validation cohort was supported by Research Council of Norway Global Health and Vaccination Research (GLOBVAC) projects: RCN 179342, 192534, and 248042, the University of Bergen (Norway)

Impaired resolution of blood transcriptomes through tuberculosis treatment with diabetes comorbidity

Clare Eckold and Cassandra L.R. van Doorn contributed equally to this manuscript. Eleonora Vianello and Jacqueline M. Cliff contributed equally to this manuscript.Data availability statement: The data that support the RNA-Seq findings of this study are openly available in NCBI-GEO at https://www.ncbi.nlm.nih.gov/geo/, accession number GSE193978. The data that support the dcRT-MLPA findings of this study are available in the supplementary material of this article.Copyright © 2023 The Authors. Background: People with diabetes are more likely to develop tuberculosis (TB) and to have poor TB-treatment outcomes than those without. We previously showed that blood transcriptomes in people with TB-diabetes (TB-DM) co-morbidity have excessive inflammatory and reduced interferon responses at diagnosis. It is unknown whether this persists through treatment and contributes to the adverse outcomes. Methods: Pulmonary TB patients recruited in South Africa, Indonesia and Romania were classified as having TB-DM, TB with prediabetes, TB-related hyperglycaemia or TB-only, based on glycated haemoglobin concentration at TB diagnosis and after 6 months of TB treatment. Gene expression in blood at diagnosis and intervals throughout treatment was measured by unbiased RNA-Seq and targeted Multiplex Ligation-dependent Probe Amplification. Transcriptomic data were analysed by longitudinal mixed-model regression to identify whether genes were differentially expressed between clinical groups through time. Predictive models of TB-treatment response across groups were developed and cross-tested. Results: Gene expression differed between TB and TB-DM patients at diagnosis and was modulated by TB treatment in all clinical groups but to different extents, such that differences remained in TB-DM relative to TB-only throughout. Expression of some genes increased through TB treatment, whereas others decreased: some were persistently more highly expressed in TB-DM and others in TB-only patients. Genes involved in innate immune responses, anti-microbial immunity and inflammation were significantly upregulated in people with TB-DM throughout treatment. The overall pattern of change was similar across clinical groups irrespective of diabetes status, permitting models predictive of TB treatment to be developed. Conclusions: Exacerbated transcriptome changes in TB-DM take longer to resolve during TB treatment, meaning they remain different from those in uncomplicated TB after treatment completion. This may indicate a prolonged inflammatory response in TB-DM, requiring prolonged treatment or host-directed therapy for complete cure. Development of transcriptome-based biomarker signatures of TB-treatment response should include people with diabetes for use across populations.European Community's Seventh Framework Programme. Grant Number: 305279; Netherlands Organization for Scientific Research. Grant Number: 9121403

Impaired resolution of blood transcriptomes through tuberculosis treatment with diabetes comorbidity

Background People with diabetes are more likely to develop tuberculosis (TB) and to have poor TB-treatment outcomes than those without. We previously showed that blood transcriptomes in people with TB-diabetes (TB-DM) co-morbidity have excessive inflammatory and reduced interferon responses at diagnosis. It is unknown whether this persists through treatment and contributes to the adverse outcomes. Methods Pulmonary TB patients recruited in South Africa, Indonesia and Romania were classified as having TB-DM, TB with prediabetes, TB-related hyperglycaemia or TB-only, based on glycated haemoglobin concentration at TB diagnosis and after 6 months of TB treatment. Gene expression in blood at diagnosis and intervals throughout treatment was measured by unbiased RNA-Seq and targeted Multiplex Ligation-dependent Probe Amplification. Transcriptomic data were analysed by longitudinal mixed-model regression to identify whether genes were differentially expressed between clinical groups through time. Predictive models of TB-treatment response across groups were developed and cross-tested. Results Gene expression differed between TB and TB-DM patients at diagnosis and was modulated by TB treatment in all clinical groups but to different extents, such that differences remained in TB-DM relative to TB-only throughout. Expression of some genes increased through TB treatment, whereas others decreased: some were persistently more highly expressed in TB-DM and others in TB-only patients. Genes involved in innate immune responses, anti-microbial immunity and inflammation were significantly upregulated in people with TB-DM throughout treatment. The overall pattern of change was similar across clinical groups irrespective of diabetes status, permitting models predictive of TB treatment to be developed. Conclusions Exacerbated transcriptome changes in TB-DM take longer to resolve during TB treatment, meaning they remain different from those in uncomplicated TB after treatment completion. This may indicate a prolonged inflammatory response in TB-DM, requiring prolonged treatment or host-directed therapy for complete cure. Development of transcriptome-based biomarker signatures of TB-treatment response should include people with diabetes for use across populations

An autologous dendritic cell vaccine polarizes a Th-1 response which is tumoricidal to patient-derived breast cancer cells.

Breast cancer remains one of the leading causes of cancer-associated death worldwide. Conventional treatment is associated with substantial toxicity and suboptimal efficacy. We, therefore, developed and evaluated the in vitro efficacy of an autologous dendritic cell (DC) vaccine to treat breast cancer. We recruited 12 female patients with stage 1, 2, or 3 breast cancer and matured their DCs with autologous tumour-specific lysate, a toll-like receptor (TLR)-3 and 7/8 agonist, and an interferon-containing cocktail. The efficacy of the vaccine was evaluated by its ability to elicit a cytotoxic T-lymphocyte response to autologous breast cancer cells in vitro. Matured DCs (≥ 60% upregulation of CD80, CD86, CD83, and CCR7) produced high levels of the Th1 effector cytokine, IL12-p70 (1.2 ng/ml; p < 0.0001), compared to DCs pulsed with tumour lysate, or matured with an interferon-containing cocktail alone. We further showed that matured DCs enhance antigen-specific CD8 + T-cell responses to HER-2 (4.5%; p < 0.005) and MUC-1 (19%; p < 0.05) tetramers. The mature DCs could elicit a robust and dose-dependent antigen-specific cytotoxic T-lymphocyte response (65%) which was tumoricidal to autologous breast cancer cells in vitro compared to T-lymphocytes that were primed with autologous lysate loaded-DCs (p < 0.005). Lastly, we showed that the mature DCs post-cryopreservation maintained high viability, maintained their mature phenotype, and remained free of endotoxins or mycoplasma. We have developed a DC vaccine that is cytotoxic to autologous breast cancer cells in vitro. The tools and technology generated here will now be applied to a phase I/IIa clinical trial