Impact of Age and Estimated Glomerular Filtration Rate on the Glycemic Efficacy and Safety of Canagliflozin: A Pooled Analysis of Clinical Studies.

AbstractObjectiveReduced efficacy has been reported in the elderly; it may be a consequence of an age-dependent decline in estimated glomerular filtration rate (eGFR) rather than ageing per se. We sought to determine the impact of these 2 parameters, as well as sex and baseline body mass index (BMI), on the efficacy and safety of canagliflozin, a sodium glucose co-transporter 2 inhibitor, in people with type 2 diabetes.MethodsData were pooled from 6 randomized, double-blind, placebo-controlled studies (18 or 26 weeks; N=4053). Changes in glycated hemoglobin (A1C) and systolic blood pressure (BP) from baseline with canagliflozin 100 mg and 300 mg and placebo were evaluated in subgroups by sex, baseline BMI, baseline age and baseline eGFR. Safety was assessed by reports of adverse events.ResultsPlacebo-subtracted reductions in A1C with canagliflozin 100 mg and 300 mg were similar in men and women. A1C reductions with canagliflozin were seen across BMI subgroups and in participants aged <65 years and ≥65 years. Significantly greater placebo-subtracted reductions in A1C were seen with both canagliflozin doses in participants with higher baseline eGFR (≥90 mL/min/1.73 m2). Reductions in systolic BP were seen with canagliflozin across subgroups of sex, BMI, age and eGFR. A1C reductions with canagliflozin were similar for participants aged <65 or ≥65 years who had baseline eGFR ≥60 mL/min/1.73 m2 and were smaller in older than in younger participants with baseline eGFR 45 to <60 mL/min/1.73 m2. The overall incidence of adverse events was similar across treatment groups regardless of sex, baseline BMI, baseline age or baseline eGFR.ConclusionsCanagliflozin improved glycemic control, reduced BP and was generally well tolerated in people with type 2 diabetes across a range of ages, BMIs and renal functions

Investigating Skeletal Muscle Metabolic Adaptations underlying Aerobic Fitness Gains following High Intensity Interval Training in a Rat Model of Pulmonary Arterial Hypertension

poster abstractRationale: In patients with pulmonary arterial hypertension (PAH) a shift from oxidative to a less efficient non-oxidative (glycolytic) metabolism in skeletal muscle is believed to contribute to the reduced exercise tolerance hallmark of the disease. As seen for other cardiopulmonary diseases, exercise training (ExT) may ameliorate this “glycolytic switch” in PAH and improve exercise capacity. Previous studies in this lab showed an improved metabolic profile of skeletal muscle in PAH rats following an ExT protocol of continuous running at moderate relative intensity, 60 minutes at 75% of maximal aerobic capacity (VO2 Max). This study tests the hypothesis in a PAH rat model that HIIT will also result in preserved aerobic capacity and attenuation of skeletal muscle glycolytic shift. Methods: Male Sprague-Dawley rats received either monocrotaline (MCT, 40 mg/kg) to induce mild PAH (n= 14), or saline, for healthy controls (n=9). After 2 wks, a 6 wkprogram of treadmill HIIT was initiated for a subset of PAH (n= 8) and healthy controls (n=6). The 30 min HIIT sessions alternated between 2 minutes at 85% VO2 max and 3 minutes at ~30% VO2 max. VO2 max was assessed at baseline, and in pre-training and post-training via analysis of expired gases. Preliminary results: MCT-induced decrement in VO2 max was attenuated by HIIT (p0.05). Western blotting of soleus homogenates for cytochromes I-V of the electron transport chain (OXPHOS), and for PGC1α, a potent stimulus for mitochondrial biogenesis, is being performed at present to further investigate potential training-induced adaptations in skeletal muscle metabolis

The acute-to-chronic workload ratio:An inaccurate scaling index for an unnecessary normalisation process?

BACKGROUND: Problematic use of alcohol and other drugs (AOD) is highly prevalent among people living with the human immunodeficiency virus (PLWH), and untreated AOD use disorders have particularly detrimental effects on human immunodeficiency virus (HIV) outcomes. The Healthcare Effectiveness Data and Information Set (HEDIS) measures of treatment initiation and engagement are important benchmarks for access to AOD use disorder treatment. To inform improved patient care, we compared HEDIS measures of AOD use disorder treatment initiation and engagement and health care utilization among PLWH and patients without an HIV diagnosis. METHODS: Patients with a new AOD use disorder diagnosis documented between October 1, 2014, and August 15, 2015, were identified using electronic health records (EHR) and insurance claims data from 7 health care systems in the United States. Demographic characteristics, clinical diagnoses, and health care utilization data were also obtained. AOD use disorder treatment initiation and engagement rates were calculated using HEDIS measure criteria. Factors associated with treatment initiation and engagement were examined using multivariable logistic regression models. RESULTS: There were 469 PLWH (93% male) and 86,096 patients without an HIV diagnosis (60% male) in the study cohort. AOD use disorder treatment initiation was similar in PLWH and patients without an HIV diagnosis (10% vs. 11%, respectively). Among those who initiated treatment, few engaged in treatment in both groups (9% PLWH vs. 12% patients without an HIV diagnosis). In multivariable analysis, HIV status was not significantly associated with either AOD use disorder treatment initiation or engagement. CONCLUSIONS: AOD use disorder treatment initiation and engagement rates were low in both PLWH and patients without an HIV diagnosis. Future studies need to focus on developing strategies to efficiently integrate AOD use disorder treatment with medical care for HIV

High Intensity Interval Training Benefits Right Heart Function in a Rat Model of Pulmonary Arterial Hypertension

poster abstractPulmonary Arterial Hypertension (PAH) is a disease of progressive remodeling in pulmonary arteries that elevates pulmonary pressures and eventually leads to right ventricular (RV) failure and death. The purpose of this study was to examine the benefit and detriment of high intensity interval training (HIIT) to the RV in a monocrotaline (MCT) PAH rat model. It is hypothesized that HIIT will improve indicators of RV function without increasing myocardial inflammation or apoptosis. Male Sprague Dawley rats were injected with either MCT (40 mg/kg, n=14)) to induce mild PAH or saline for healthy controls (CON, n=9). A subgroup of MCT (n= 8) and CON rats (n=6) performed a 6 week treadmill HIIT program 5x/week using short bouts of alternating high intensity (2 min, 85-90%VO2max) and low intensity (3 min, ~30%VO2max) running for 30 min/session. Histochemistry/immunohistochemistry was performed on cryofixed or formalin-fixed/paraffin-embedded RV sections to assess indicators of inflammation (CD45+ cells), apoptosis (TUNEL), fibrosis (trichrome) and was imaged using epifluorescence or brightfield microscopy. Image quantification was performed using ImageJ. For the HIIT rats, a reduction in MCTinduced RV hypertrophy was observed, as measured echocardiographically, and by the calculated ratio of RV mass relative to LV+Septum mass. RV function was better preserved for HIIT vs. sedentary MCT, as indicated by stroke volume and cardiac index (cardiac output normalized by body weight) in echocardiography. MCT-induced RV fibrosis as measured by trichrome staining was lower for HIIT, also indicating a healthier myocardium. HIIT did not prompt greater counts per field of CD45+ cells and TUNEL+ cells in HIIT vs. sedentary MCT RV myocardium. In conclusion, in the monocrotaline rat model of PAH, HIIT appears to be a beneficial exercise approach that improves RV function without exacerbating RV inflammation or apoptosis. Future work will examine effects in other PAH models and ultimately in patients with disease

High-intensity interval training, but not continuous training, reverses right ventricular hypertrophy and dysfunction in a rat model of pulmonary hypertension

Exercise is beneficial in pulmonary arterial hypertension (PAH), although studies to date indicate little effect on the elevated pulmonary pressures or maladaptive right ventricle (RV) hypertrophy associated with the disease. For chronic left ventricle failure, high-intensity interval training (HIIT) promotes greater endothelial stimulation and superior benefit than customary continuous exercise training (CExT); however, HIIT has not been tested for PAH. Therefore, here we investigated acute and chronic responses to HIIT vs. CExT in a rat model of monocrotaline (MCT)-induced mild PAH. Six weeks of treadmill training (5 times/wk) were performed, as either 30 min HIIT or 60 min low-intensity CExT. To characterize acute hemodynamic responses to the two approaches, novel recordings of simultaneous pulmonary and systemic pressures during running were obtained at pre- and 2, 4, 6, and 8 wk post-MCT using long-term implantable telemetry. MCT-induced decrement in maximal aerobic capacity was ameliorated by both HIIT and CExT, with less pronounced pulmonary vascular remodeling and no increase in RV inflammation or apoptosis observed. Most importantly, only HIIT lowered RV systolic pressure, RV hypertrophy, and total pulmonary resistance, and prompted higher cardiac index that was complemented by a RV increase in the positive inotrope apelin and reduced fibrosis. HIIT prompted a markedly pulsatile pulmonary pressure during running and was associated with greater lung endothelial nitric oxide synthase after 6 wk. We conclude that HIIT may be superior to CExT for improving hemodynamics and maladaptive RV hypertrophy in PAH. HIIT’s superior outcomes may be explained by more favorable pulmonary vascular endothelial adaptation to the pulsatile HIIT stimulus

De novo mutations in histone modifying genes in congenital heart disease

Congenital heart disease (CHD) is the most frequent birth defect, affecting 0.8% of live births1. Many cases occur sporadically and impair reproductive fitness, suggesting a role for de novo mutations. By analysis of exome sequencing of parent-offspring trios, we compared the incidence of de novo mutations in 362 severe CHD cases and 264 controls. CHD cases showed a significant excess of protein-altering de novo mutations in genes expressed in the developing heart, with an odds ratio of 7.5 for damaging mutations. Similar odds ratios were seen across major classes of severe CHD. We found a marked excess of de novo mutations in genes involved in production, removal or reading of H3K4 methylation (H3K4me), or ubiquitination of H2BK120, which is required for H3K4 methylation2–4. There were also two de novo mutations in SMAD2; SMAD2 signaling in the embryonic left-right organizer induces demethylation of H3K27me5. H3K4me and H3K27me mark `poised' promoters and enhancers that regulate expression of key developmental genes6. These findings implicate de novo point mutations in several hundred genes that collectively contribute to ~10% of severe CHD

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Planktonic interference and biofilm alliance between aggregation substance and endocarditis- and biofilm-associated pili in Enterococcus faecalis

Like many bacteria, Enterococcus faecalis encodes a number of adhesins involved in colonization or infection of different niches. Two well-studied E. faecalis adhesins, aggregation substance (AS) and endocarditis- and biofilm-associated pili (Ebp), both contribute to biofilm formation on abiotic surfaces and in endocarditis, suggesting that they may be expressed at the same time. Because different regulatory pathways have been reported for AS and Ebp, here, we examined if they are coexpressed on the same cells and what is the functional impact of coexpression on individual cells and within a population. We found that while Ebp are only expressed on a subset of cells, when Ebp and AS are expressed on the same cells, pili interfere with AS-mediated clumping and impede AS-mediated conjugative plasmid transfer during planktonic growth. However, when the population density increases, horizontal gene transfer rates normalize and are no longer affected by pilus expression. Instead, at higher cell densities during biofilm formation, Ebp and AS differentially contribute to biofilm development and structure, synergizing to promote maximal biofilm formation.NRF (Natl Research Foundation, S’pore)MOE (Min. of Education, S’pore)Published versio

Multiplex CRISPRi System Enables the Study of Stage-Specific Biofilm Genetic Requirements in Enterococcus faecalis

© 2020 Afonina et al. Enterococcus faecalis is an opportunistic pathogen, which can cause multidrug-resistant life-threatening infections. Gaining a complete understanding of enterococcal pathogenesis is a crucial step in identifying a strategy to effectively treat enterococcal infections. However, bacterial pathogenesis is a complex process often involving a combination of genes and multilevel regulation. Compared to established knockout methodologies, CRISPR interference (CRISPRi) approaches enable the rapid and efficient silencing of genes to interrogate gene products and pathways involved in pathogenesis. As opposed to traditional gene inactivation ap-proaches, CRISPRi can also be quickly repurposed for multiplexing or used to study essential genes. Here, we have developed a novel dual-vector nisin-inducible CRISPRi system in E. faecalis that can efficiently silence via both nontemplate and template strand targeting. Since the nisin-controlled gene expression system is functional in various Gram-positive bacteria, the developed CRISPRi tool can be extended to other genera. This system can be applied to study essential genes, genes involved in anti-microbial resistance, and genes involved in biofilm formation and persistence. The system is robust and can be scaled up for high-throughput screens or combinatorial targeting. This tool substantially enhances our ability to study enterococcal biology and pathogenesis, host-bacterium interactions, and interspecies communication. IMPORTANCE Enterococcus faecalis causes multidrug-resistant life-threatening infections and is often coisolated with other pathogenic bacteria from polymicrobial biofilm-associated infections. Genetic tools to dissect complex interactions in mixed microbial communities are largely limited to transposon mutagenesis and traditional time-and labor-intensive allelic-exchange methods. Built upon streptococcal dCas9, we developed an easily modifiable, inducible CRISPRi system for E. faecalis that can efficiently silence single and multiple genes. This system can silence genes involved in biofilm formation and antibiotic resistance and can be used to interrogate gene essentiality. Uniquely, this tool is optimized to study genes important for biofilm ini-tiation, maturation, and maintenance and can be used to perturb preformed bio-films. This system will be valuable to rapidly and efficiently investigate a wide range of aspects of complex enterococcal biology