#36 - The Effect of Chondroitin Sulfate Glycosaminoglycan Substrates on the Immunosuppressive Capacity of Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are multipotent stem cells that possess promising therapeutic potential for immunomodulation, tumor targeting, and regenerative medicine. The therapeutic potential of MSCs is largely regulated through paracrine signaling mechanisms including binding interactions with the extracellular matrix (ECM). Since sulfated glycosaminoglycans are an important ECM constituent in the bone marrow stem cell niche with binding affinities reliant on sulfation patterns, we hypothesized that collagen bound chondroitin sulfate glycosaminoglycans (CS-GAGs) possessing different sulfation patterns, will induce changes in MSC immunosuppressive potency and morphology. Collagen provides distinct binding sites for CS-GAGs, hence we employed a layer-by-layer strategy to immobilize CS-GAGs to the underlying collagen substrates, and assessed the effects of mono-sulfated CS-GAGs (CS-A) and over-sulfated CS-GAGs (CS-E) on MSC Indoleamine 2,3-dioxygenase (IDO) activity. Standard tissue culture plates were coated with collagen 50ug/mL overnight prior to CS-GAG coating. High (2mg/mL) and low (1mg/mL) concentrations of CS-GAGs diluted in PBS were overlaid on top of the collagen coating overnight. Then, MSCs were cultured on CS-GAG immobilized culture plates for 24 hours in culture media supplemented with 50ng/ml INF-γ. MSCs from multiple donors were used to detect any donor-to-donor variability. IDO assay was used to analyze how much immunomodulated activity took place through the detection of kynurenine (KYN) production.MSC morphology on collagen bound CS-GAG substrates was imaged using Array Scan VTI HCS Reader and analyzed using Partial Least Squares Discriminant analysis (PLS-DA). Our study demonstrated that IFN-γstimulated MSCs cultured on collagen bound over-sulfated CS-GAG substrates displayed distinct morphology and expressed higher IDO activity indicating higher immunosuppressive capacity. With this study, the aim is to enhance the biomanufacturing of MSCs through the manipulation of the CS-GAG surface. Further information about the adhesion, migration, and proliferation of MSCs can be applied towards broad industry and clinical use

General Attitudes Towards and Barriers to Integrating Tobacco Interventions into Substance Abuse Facilities: A Study of R.J. Blackley in Butner, North Carolina

Patients in clinical settings for alcohol and drug addiction treatment are likely to be smokers and over their lifetime are more likely to die from tobacco-related causes rather than their addictions. A compelling case also exists for incorporating tobacco cessation interventions into these settings to enhance patient health outcomes, but it has not been common to do so partially due to staff and patient resistance. The following study provides a qualitative analysis of data gathered from the Alcohol and Drug Abuse Treatment Center in Butner, North Carolina regarding staff attitudes towards the introduction of a tobacco cessation program as the center is in the process of becoming a smoke-free campus and employees are helping design the transition. Twenty conversational semi-structured interviews were performed with members of the leadership team, design team, and other front-line staff members to gain a better understanding of existing perceptions about tobacco cessation, the process in place for the transition to a tobacco free campus, and any barriers to success. The interviews were analyzed and general attitudes about tobacco cessation programs in clinical behavioral health settings were assessed. Finally, using the appraised qualitative data, further recommendations are provided to potentially allow other addiction treatment centers to implement similar programs to address tobacco usage amongst their own patients.Master of Public Healt

Changes in Mitochondrial Dynamics and iNOS in the Dorsal Vagal Complex of the Brain affects Insulin Sensing, Feeding Behaviours and Body Weight Gain in Rats

Worldwide obesity has nearly tripled since 1975, with over 650 million cases in 2016. Obesity can lead to many adverse metabolic effects in the cardiovascular, brain and endocrine systems. It has been previously shown, the dorsal vagal complex (DVC) of the brain regulates glucose homeostasis and controls food intake through insulin signalling in rats. In these rats, a 3-day high fat diet (HFD) has been shown to induce insulin resistance and diminishes the DVC’s ability to regulate glucose metabolism and food intake, though exact mechanistic effects of this are still unknown. HFD-feeding is associated with an increase in mitochondrial fission in the DVC. Mitochondrial fission is regulated by dynamin related protein 1 (Drp1), and an increase in Drp1 activity in the DVC modulates the insulin signalling pathway. Activation of Drp1 has been correlated with increased levels of inducible nitric oxide synthase (iNOS), increased endoplasmic reticulum (ER) stress and insulin resistance in the DVC. This study has shown that activation of Drp1 in the DVC leads to an increase in body weight gain, cumulative food intake and adipose tissue, these rats are also insulin resistant compared to regular chow (RC)- fed littermates and displayed higher levels of iNOS in the DVC. Conversely inhibiting Drp1, or knocking down iNOS, in the DVC in HFD-fed rats decreased body weight gain, cumulative food intake and adipose tissue, and prevented the development of insulin resistance. In addition to this, selective inhibition of Drp1 in astrocytes of the DVC in HFD-fed rats resulted in a decrease in food intake and body weight and prevented HFD-induced insulin resistance. Finally, in diet-induced obese, insulin resistant rats, inhibition of mitochondrial fission or knocking down iNOS in the DVC restored insulin sensitivity and decreased fat deposition. This study has determined the integral role Drp1 and iNOS in the DVC and the effect this has on feeding behaviours and body weight gain, in particular I demonstrated that it is sufficient to target astrocytes in the DVC to affect insulin sensing, feeding behaviours and fat deposition

Age at antiretroviral therapy initiation and cell-associated HIV-1 DNA levels in HIV-1-infected children

Background The latent viral reservoir is the major obstacle to achieving HIV remission and necessitates life-long antiretroviral therapy (ART) for HIV-infected individuals. Studies in adults and children have found that initiating ART soon after infection is associated with a reduction in the size of the HIV-1 reservoir. Here we quantified cell-associated HIV-1 DNA in early-treated but currently older HIV-infected children suppressed on ART. Methods The study participants comprised of a cohort of 146 early-treated children with HIV-1 RNA <50 copies/ml enrolled as part of a clinical trial in Johannesburg, South Africa. A stored buffy coat sample collected after a median 4.3 years on ART and where HIV-1 RNA was <50 copies/ml was tested for cell-associated HIV-1 DNA levels. An in-house, semi-nested real-time quantitative hydrolysis probe PCR assay to detect total HIV-1 subtype C proviral DNA was used. Children were followed prospectively for up to 3 years after this measurement to investigate subsequent HIV-1 RNA rebound/failure while remaining on ART. Age at ART initiation, HIV-1 RNA decline prior to HIV-1 DNA measurement and other factors were investigated. Results A gradient between age at ART initiation and later HIV-1 DNA levels was observed. When ART was started 50 copies/ml whilst on ART within 3 years after the DNA measurement was 2.07 (95% CI: 1.352–3.167) times greater if the HIV-1 DNA level was above the median of 55 copies/106 cells. Conclusions Cell-associated HIV-1 DNA levels measured after more than 4 years on ART were lower the younger the age of the child when ART was initiated. This marker of the size of the viral reservoir also predicted subsequent viral rebound/treatment failure while ART was sustained. The results provide additional evidence of the benefits of prompt diagnosis and early ART initiation in newborns and infants

Neuroactivational and Behavioral Correlates of Psychosocial Stress-Induced Cocaine Seeking in Rats

A prominent feature of cocaine abuse is a high risk of relapse even despite prolonged periods of abstinence. Psychosocial stress is thought to be a major contributor to the onset of cocaine craving and relapse in human substance abusers, yet most preclinical models of stress-induced relapse employ physical stressors (e.g., unpredictable footshock) or pharmacological stressors (e.g., yohimbine to elicit a drug seeking response) and do not rely upon psychosocial stress per se. Importantly, social stressors are well known to activate distinct neural circuits within the brain as compared to other stressors. It is therefore possible that currently available animal models of stress-induced drug relapse do not fully engage the neuroanatomical, neurochemical, and/or molecular substrates that are recruited specifically by psychosocial stressors to produce drug-seeking behavior. Social defeat stress has been proposed as an ethologically valid psychosocial stressor in rodents that more closely models the forms of psychosocial stress that precede relapse episodes in drug abusers. We previously developed a model of psychosocial stress-induced reinstatement in rats in which cocaine seeking is elicited via exposure to a cue signaling impending social defeat stress. Using this model, we discovered that predilection towards displaying active coping behaviors during prior social defeat stress exposures was positively correlated with levels of psychosocial stress-induced cocaine seeking. The present study aimed to expand upon these initial findings by assessing and comparing patterns of neural activation in key brain areas during stress induced cocaine seeking that is triggered by psychosocial or footshock stress predictive cues

Male obesity associated gonadal dysfunction and the role of bariatric surgery

Obesity is an ever growing pandemic and a prevalent problem among men of reproductive age that can both cause and exacerbate male-factor infertility by means of endocrine abnormalities, associated comorbidities, and direct effects on the precision and throughput of spermatogenesis. Robust epidemiologic, clinical, genetic, epigenetic, and preclinical data support these findings. Clinical studies on the impact of medically induced weight loss on serum testosterone concentrations and spermatogenesis is promising but may show differential and unsustainable results. In contrast, literature has demonstrated that weight loss after bariatric surgery is correlated with an increase in serum testosterone concentrations that is superior than that obtained with only lifestyle modifications, supporting a further metabolic benefit from surgery that may be specific to the male reproductive system. The data on sperm and semen parameters is controversial to date. Emerging evidence in the burgeoning field of genetics and epigenetics has demonstrated that paternal obesity can affect offspring metabolic and reproductive phenotypes by means of epigenetic reprogramming of spermatogonial stem cells. Understanding the impact of this reprogramming is critical to a comprehensive view of the impact of obesity on subsequent generations. Furthermore, conveying the potential impact of these lifestyle changes on future progeny can serve as a powerful tool for obese men to modify their behavior. Healthcare professionals treating male infertility and obesity need to adapt their practice to assimilate these new findings to better counsel men about the importance of paternal preconception health and the impact of novel non-medical therapeutic interventions. Herein, we summarize the pathophysiology of obesity on the male reproductive system and emerging evidence regarding the potential role of bariatric surgery as treatment of male obesity-associated gonadal dysfunction

Weight Change and the Onset of Cardiovascular Diseases: Emulating Trials Using Electronic Health Records.

BACKGROUND: Cross-sectional measures of body mass index (BMI) are associated with cardiovascular disease (CVD) incidence, but less is known about whether weight change affects the risk of CVD. METHODS: We estimated the effect of 2-y weight change interventions on 7-y risk of CVD (CVD death, myocardial infarction, stroke, hospitalization from coronary heart disease, and heart failure) by emulating hypothetical interventions using electronic health records. We identified 138,567 individuals with 45-69 years of age without chronic disease in England from 1998 to 2016. We performed pooled logistic regression, using inverse-probability weighting to adjust for baseline and time-varying confounders. We categorized each individual into a weight loss, maintenance, or gain group. RESULTS: Among those of normal weight, both weight loss [risk difference (RD) vs. weight maintenance = 1.5% (0.3% to 3.0%)] and gain [RD = 1.3% (0.5% to 2.2%)] were associated with increased risk for CVD compared with weight maintenance. Among overweight individuals, we observed moderately higher risk of CVD in both the weight loss [RD = 0.7% (-0.2% to 1.7%)] and the weight gain group [RD = 0.7% (-0.1% to 1.7%)], compared with maintenance. In the obese, those losing weight showed lower risk of coronary heart disease [RD = -1.4% (-2.4% to -0.6%)] but not of stroke. When we assumed that chronic disease occurred 1-3 years before the recorded date, estimates for weight loss and gain were attenuated among overweight individuals; estimates for loss were lower among obese individuals. CONCLUSION: Among individuals with obesity, the weight-loss group had a lower risk of coronary heart disease but not of stroke. Weight gain was associated with increased risk of CVD across BMI groups. See video abstract at, http://links.lww.com/EDE/B838

ORMA: a tool for identification of species-specific variations in 16S rRNA gene and oligonucleotides design

16S rRNA gene is one of the preferred targets for resolving species phylogenesis issues in microbiological-related contexts. However, the identification of single-nucleotide variations capable of distinguishing a sequence among a set of homologous ones can be problematic. Here we present ORMA (Oligonucleotide Retrieving for Molecular Applications), a set of scripts for discriminating positions search and for performing the selection of high-quality oligonucleotide probes to be used in molecular applications. Two assays based on Ligase Detection Reaction (LDR) are presented. First, a new set of probe pairs on cyanobacteria 16S rRNA sequences of 18 different species was compared to that of a previous study. Then, a set of LDR probe pairs for the discrimination of 13 pathogens contaminating bovine milk was evaluated. The software determined more than 100 candidate probe pairs per dataset, from more than 300 16S rRNA sequences, in less than 5 min. Results demonstrated how ORMA improved the performance of the LDR assay on cyanobacteria, correctly identifying 12 out of 14 samples, and allowed the perfect discrimination among the 13 milk pathogenic-related species. ORMA represents a significant improvement from other contexts where enzyme-based techniques have been employed on already known mutations of a single base or on entire subsequences

Pan-Cancer Analysis of lncRNA Regulation Supports Their Targeting of Cancer Genes in Each Tumor Context

Long noncoding RNAs (lncRNAs) are commonly dys-regulated in tumors, but only a handful are known toplay pathophysiological roles in cancer. We inferredlncRNAs that dysregulate cancer pathways, onco-genes, and tumor suppressors (cancer genes) bymodeling their effects on the activity of transcriptionfactors, RNA-binding proteins, and microRNAs in5,185 TCGA tumors and 1,019 ENCODE assays.Our predictions included hundreds of candidateonco- and tumor-suppressor lncRNAs (cancerlncRNAs) whose somatic alterations account for thedysregulation of dozens of cancer genes and path-ways in each of 14 tumor contexts. To demonstrateproof of concept, we showed that perturbations tar-geting OIP5-AS1 (an inferred tumor suppressor) andTUG1 and WT1-AS (inferred onco-lncRNAs) dysre-gulated cancer genes and altered proliferation ofbreast and gynecologic cancer cells. Our analysis in-dicates that, although most lncRNAs are dysregu-lated in a tumor-specific manner, some, includingOIP5-AS1, TUG1, NEAT1, MEG3, and TSIX, synergis-tically dysregulate cancer pathways in multiple tumorcontexts

Pan-cancer Alterations of the MYC Oncogene and Its Proximal Network across the Cancer Genome Atlas

Although theMYConcogene has been implicated incancer, a systematic assessment of alterations ofMYC, related transcription factors, and co-regulatoryproteins, forming the proximal MYC network (PMN),across human cancers is lacking. Using computa-tional approaches, we define genomic and proteo-mic features associated with MYC and the PMNacross the 33 cancers of The Cancer Genome Atlas.Pan-cancer, 28% of all samples had at least one ofthe MYC paralogs amplified. In contrast, the MYCantagonists MGA and MNT were the most frequentlymutated or deleted members, proposing a roleas tumor suppressors.MYCalterations were mutu-ally exclusive withPIK3CA,PTEN,APC,orBRAFalterations, suggesting that MYC is a distinct onco-genic driver. Expression analysis revealed MYC-associated pathways in tumor subtypes, such asimmune response and growth factor signaling; chro-matin, translation, and DNA replication/repair wereconserved pan-cancer. This analysis reveals insightsinto MYC biology and is a reference for biomarkersand therapeutics for cancers with alterations ofMYC or the PMN