Publications by authors named "Sabita Roy"

87 Publications

Brief Hydromorphone Exposure During Pregnancy Sufficient to Induce Maternal and Neonatal Microbial Dysbiosis.

J Neuroimmune Pharmacol 2021 Sep 25. Epub 2021 Sep 25.

Department of Microbiology and Immunology, University of Miami Miller School of Medicine, Miami, FL, 33136, USA.

Prenatal opioid exposure is associated with significantly adverse medical, developmental, and behavioral outcomes in offspring, though the underlying mechanisms driving these impairments are still unclear. Accumulating evidence implicates gut microbial dysbiosis as a potential modulator of these adverse effects. However, how opioid exposure during pregnancy alters the maternal and neonatal microbiome remain to be elucidated. Here, we utilize a murine model of brief hydromorphone exposure during pregnancy (gestation day 11-13; i.p.; 10 mg/kg) to examine its impact on the maternal and neonatal microbiome. Fecal samples were collected at various timepoints in dams (4 days post hydromorphone exposure, birth, and weaning) and offspring (2, 3, and 5 weeks) to interrogate longitudinal changes in the microbiome. Stomach contents at 2 weeks were also collected as a surrogate for breastmilk and microbial analysis was performed using 16S rRNA sequencing. Alongside alterations in the maternal gut microbial composition, offspring gut microbiota exhibited distinct communities at 2 and 3 weeks. Furthermore, functional profiling of microbial communities revealed significant differences in microbial community-level phenotypes gram-negative, gram-positive, and potentially pathogenic in maternal and/or neonatal hydromorphone exposed groups compared with controls. We also observed differences in stomach microbiota in opioid-exposed vs non-exposed offspring, which suggests breast milk may also play a role in shaping the development of the neonatal gut microbiota. Together, we provide evidence of maternal and neonatal microbial dysbiosis provoked even with brief hydromorphone exposure during pregnancy.
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http://dx.doi.org/10.1007/s11481-021-10019-2DOI Listing
September 2021

HIV Tat-Mediated Induction of Monocyte Transmigration Across the Blood-Brain Barrier: Role of Chemokine Receptor CXCR3.

Front Cell Dev Biol 2021 30;9:724970. Epub 2021 Aug 30.

Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE, United States.

HIV trans-activator of transcription (Tat), one of the cytotoxic proteins secreted from HIV-infected cells, is also known to facilitate chemokine-mediated transmigration of monocytes into the brain leading, in turn, to neuroinflammation and thereby contributing to the development of HIV-associated neurocognitive disorders (HAND). The mechanism(s) underlying HIV Tat-mediated enhancement of monocyte transmigration, however, remain largely unknown. CXC chemokine receptor 3 (CXCR3) that is expressed by the peripheral monocytes is known to play a role in the monocyte influx and accumulation. In the present study, we demonstrate for the first time that exposure of human monocytes to HIV Tat protein resulted in upregulated expression of CXCR3 leading, in turn, to increased monocyte transmigration across the blood-brain barrier (BBB) both in the and model systems. This process involved activation of toll-like receptor 4 (TLR4), with downstream phosphorylation and activation of TANK-binding kinase 1 (TBK1), and subsequent phosphorylation and nuclear translocation of interferon regulatory factor 3 (IRF3), ultimately leading to enhanced expression of CXCR3 in human monocytes. These findings imply a novel molecular mechanism underlying HIV Tat-mediated increase of monocyte transmigration across the BBB, while also implicating a novel role of CXCR3-dependent monocyte transmigration in HIV Tat-mediated neuroinflammation.
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http://dx.doi.org/10.3389/fcell.2021.724970DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8435688PMC
August 2021

Housing conditions and microbial environment do not affect the efficacy of vaccines for treatment of opioid use disorders in mice and rats.

Hum Vaccin Immunother 2021 Aug 19:1-10. Epub 2021 Aug 19.

Department of Pharmacology, University of Minnesota Medical School, Minneapolis, MN, USA.

Vaccines offer a promising prophylactic and therapeutic intervention to counteract opioid use disorders (OUD) and fatal overdoses. Vaccines generate opioid-specific antibodies that bind the target opioid, reducing drug distribution to the brain and preventing drug-induced behavioral and pharmacological effects. Due to their selectivity, anti-opioid vaccines can be administered in combination with FDA-approved medications. Because patients with OUD or other substance use disorders may be affected by other multifactorial co-morbidities, such as infection or depression, it is important to test whether vaccine efficacy is modified by factors that may impact individual innate or adaptive immunity. To that end, this study tested whether housing conditions would affect the efficacy of two lead vaccine formulations targeting oxycodone and fentanyl in male mice and rats, and further analyzed whether differences in the gastrointestinal (GI) microbiome would be correlated with either vaccine efficacy or housing conditions. Results showed that housing mice and rats in either conventional (non-controlled) or specific pathogen-free (SPF, sterile barrier maintained) environment did not affect vaccine-induced antibody responses against oxycodone and fentanyl, nor their efficacy against oxycodone- and fentanyl-induced antinociception, respiratory depression, and bradycardia. Differences in the GI microbiome detected via 16S rRNA gene sequencing were related to the housing environment. This study supports use of anti-opioid vaccines in clinical populations that may display deficits in microbiome function.
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http://dx.doi.org/10.1080/21645515.2021.1954442DOI Listing
August 2021

Gut permeability and cognitive decline: A pilot investigation in the Northern Manhattan Study.

Brain Behav Immun Health 2021 Mar 29;12. Epub 2021 Jan 29.

Department of Neurology, University of Miami Miller School of Medicine, Miami, FL, USA.

Background: Gut microbiota may impact cognitive function and decline, though data are limited. This pilot study examines the associations between gut dysbiosis products, plasma lipopolysaccharide (LPS) and soluble CD14 (sCD14), with cognitive decline and immune molecule activation among 40 participants in the longitudinal population-based Northern Manhattan Study.

Methods: We selected stroke- and dementia-free participants at baseline with high activation levels of core components of the immune signaling pathways underlying microbiota metabolite-cognitive associations (IL-1, IL-17, TNF). Participants were followed with up to three complete neuropsychological assessments, at least 5 years apart.

Results: Elevated sCD14 was associated with high levels of IL-1 pathway activation (p < 0.05), whereas in samples where only those molecules within the IL-17 and TNF pathways were increased, LPS and sCD14 levels were not elevated. LPS was associated with decline in global cognitive performance over 2-3 assessments (adjusted β = -0.023 per SD per year, 95% CI:-0.036, -0.010). The association between sCD14 and cognitive decline was marginal (adjusted β = -0.018 per SD per year, 95% CI:-0.040, 0.004).

Conclusions: These preliminary data support the hypothesis that gut dysbiosis leads to systemic and neuro-inflammation, and subsequently cognitive decline. Further large targeted and untargeted gut microbiota-derived metabolomic studies are needed.
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http://dx.doi.org/10.1016/j.bbih.2021.100214DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8186438PMC
March 2021

Minnelide, a prodrug, inhibits cervical cancer growth by blocking HPV-induced changes in p53 and pRb.

Am J Cancer Res 2021 15;11(5):2202-2214. Epub 2021 May 15.

Department of Surgery, Miller School of Medicine, University of Miami FL, USA.

HPV-induced cervical cancer is one of the prevalent gynecological cancers world-wide. In the present study, we determined the efficacy of Minnelide, a prodrug which is converted to its active form (Triptolide) in vivo against cervical cancer cells. Our studies show that Triptolide inhibited HPV-16 and HPV-18 positive cells at nanomolar concentrations. Tumor cells treated with Triptolide failed to grow in 3-D cultures in a concentration-dependent manner. Triptolide markedly reduced E6 and E7 transcript levels. Further studies revealed that exposure to Triptolide increased the levels of p53 and pRb. As a consequence, Caspase-3/7 activation and apoptosis was induced in cervical cancer cells by Triptolide. Subsequently, we evaluated the efficacy of Minnelide in xenotransplantation models of cervical cancer. Minnelide at very low doses effectively inhibited the growth of established cervical cancers in all the three animal models tested. Furthermore, Minnelide treatment was more effective when combined with platinum-based chemotherapy. These studies show that Minnelide can be used to inhibit the growth of cervical cancer.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8167699PMC
May 2021

Immune modulation mediated by extracellular vesicles of intestinal organoids is disrupted by opioids.

Mucosal Immunol 2021 07 14;14(4):887-898. Epub 2021 Apr 14.

Department of Surgery, Miller School of Medicine, University of Miami, Miami, FL, USA.

Extracellular vesicles (EVs) are effective mediators of intercellular communications between enterocytes and immune cells. The current study showed that EVs isolated from mouse and human intestinal organoids modulated inflammatory responses of various immune cells including mouse bone-marrow derived-macrophages, dendritic cells, microglia cells, and human monocytes. EVs suppressed LPS-elicited cytokine production in these cells while morphine abolished EVs' immune modulatory effects. Microarray analysis showed that various microRNAs, especially Let-7, contributed to EV-mediated immune modulation. Using murine models, we showed that injection of EVs derived from intestinal organoids reduced endotoxin-induced systemic inflammation and alleviated the symptoms of DSS-induced colitis. EVs derived from morphine-treated organoids failed to suppress the immune response in both these models. Our study suggests that EVs derived from intestinal crypt cells play crucial roles in maintaining host homeostasis and opioid use is a risk factor for exacerbating inflammation in patients with inflammatory diseases such as sepsis and colitis.
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http://dx.doi.org/10.1038/s41385-021-00392-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8225561PMC
July 2021

Viral vector-mediated gene therapy for opioid use disorders.

Exp Neurol 2021 07 26;341:113710. Epub 2021 Mar 26.

Department of Anesthesiology, Perioperative Medicine & Pain Management, University of Miami Miller School of Medicine, Miami, FL, United States of America. Electronic address:

Chronic exposure to opioids typically results in adverse consequences. Opioid use disorder (OUD) is a disease of the CNS with behavioral, psychological, neurobiological, and medical manifestations. OUD induces a variety of changes of neurotransmitters/neuropeptides in the nervous system. Existing pharmacotherapy, such as opioid maintenance therapy (OMT) is the mainstay for the treatment of OUD, however, current opioid replacement therapy is far from effective for the majority of patients. Pharmacological therapy for OUD has been challenging for many reasons including debilitating side-effects. Therefore, developing an effective, non-pharmacological approach would be a critical advancement in improving and expanding treatment for OUD. Viral vector mediated gene therapy provides a potential new approach for treating opioid abused patients. Gene therapy can supply targeting gene products directly linked to the mechanisms of OUD to restore neurotransmitter and/or neuropeptides imbalance, and avoid the off-target effects of systemic administration of drugs. The most commonly used viral vectors in rodent studies of treatment of opioid-used disorder are based on recombinant adenovirus (AV), adeno-associated virus (AAV), lentiviral (LV) vectors, and herpes simplex virus (HSV) vectors. In this review, we will focus on the recent progress of viral vector mediated gene therapy in OUD, especially morphine tolerance and withdrawal.
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http://dx.doi.org/10.1016/j.expneurol.2021.113710DOI Listing
July 2021

Prenatal opioid exposure and vulnerability to future substance use disorders in offspring.

Authors:
Yaa Abu Sabita Roy

Exp Neurol 2021 05 29;339:113621. Epub 2021 Jan 29.

Department of Microbiology and Immunology, University of Miami Miller School of Medicine, Miami, FL 33136, USA; Department of Surgery, University of Miami Miller School of Medicine, Miami, FL 33136, USA. Electronic address:

The heightened incidence of opioid use during pregnancy has resulted in unprecedented rates of neonates prenatally exposed to opioids. Prenatal opioid exposure (POE) results in significantly adverse medical, developmental, and behavioral outcomes in offspring. Of growing interest is whether POE contributes to future vulnerability to substance use disorders. The effects of POE on brain development is difficult to assess in humans, as the timing, dose, and route of drug exposure together with complex genetic and environmental factors affect susceptibility to addiction. Preclinical models of POE have allowed us to avoid methodological difficulties and confounding factors of POE in humans. Here, we review the effects of maternal opioid exposure on the developing brain with an emphasis on the neurobiological basis of drug addiction and on preclinical models of POE and their limitations. These studies have indicated that POE increases self-administration of drugs, reward-driven behaviors in the conditioned place paradigm, and locomotor sensitization. While addiction is multifaceted and vulnerability to drug addiction is still inconclusive in human studies of prenatally exposed infants, animal studies do provide a noteworthy corroboration of negative behavioral outcomes.
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http://dx.doi.org/10.1016/j.expneurol.2021.113621DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8012222PMC
May 2021

Glycogen synthase kinase-3 inhibition rescues sex-dependent contextual fear memory deficit in human immunodeficiency virus-1 transgenic mice.

Br J Pharmacol 2020 12 23;177(24):5658-5676. Epub 2020 Nov 23.

Department of Surgery, University of Miami Miller School of Medicine, Miami, FL, USA.

Background And Purpose: A significant number of HIV-1 patients on antiretroviral therapy develop HIV-associated neurocognitive disorders (HAND). Evidence indicate that biological sex may regulate HAND pathogenesis, but the mechanisms remain unknown. We investigated synaptic mechanisms associated with sex differences in HAND, using the HIV-1-transgenic 26 (Tg26) mouse model.

Experimental Approach: Contextual- and cue-dependent memories of male and female Tg26 mice and littermate wild type mice were assessed in a fear conditioning paradigm. Hippocampal electrophysiology, immunohistochemistry, western blot, qRT-PCR and ELISA techniques were used to investigate cellular, synaptic and molecular impairments.

Key Results: Cue-dependent memory was unaltered in male and female Tg26 mice, when compared to wild type mice. Male, but not female, Tg26 mice showed deficits in contextual fear memory. Consistently, only male Tg26 mice showed depressed hippocampal basal synaptic transmission and impaired LTP induction in area CA1. These deficits in male Tg26 mice were independent of hippocampal neuronal loss and microglial activation but were associated with increased HIV-1 long terminal repeat mRNA expression, reduced hippocampal synapsin-1 protein, reduced BDNF mRNA and protein, reduced AMPA glutamate receptor (GluA1) phosphorylation levels and increased glycogen synthase kinase 3 (GSK3) activity. Importantly, selective GSK3 inhibition using 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione increased levels of synapsin-1, BDNF and phosphorylated-GluA1 proteins, restored hippocampal basal synaptic transmission and LTP, and improved contextual fear memory in male Tg26 mice.

Conclusion And Implications: Sex-dependent impairments in contextual fear memory and synaptic plasticity in Tg26 mice are associated with increased GSK3 activity. This implicates GSK3 inhibition as a potential therapeutic strategy to improve cognition in HIV-1 patients.
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http://dx.doi.org/10.1111/bph.15288DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7707089PMC
December 2020

Opioid Modulation of the Gut-Brain Axis in Opioid-Associated Comorbidities.

Authors:
Li Zhang Sabita Roy

Cold Spring Harb Perspect Med 2021 Sep 1;11(9). Epub 2021 Sep 1.

Department of Pharmacology, University of Minnesota, Minnesota McGuire Translational Research Facility, Minneapolis, Minnesota 55455, USA.

Growing evidence from animal and human studies show that opioids have a major impact on the composition and function of gut microbiota. This leads to disruption in gut permeability and altered microbial metabolites, driving both systemic and neuroinflammation, which in turn impacts central nervous system (CNS) homeostasis. Tolerance and dependence are the major comorbidities associated with prolonged opioid use. Inflammatory mediators and signaling pathways have been implicated in both opioid tolerance and dependence. We provide evidence that targeting the gut microbiome during opioid use through prebiotics, probiotics, antibiotics, and fecal microbial transplantation holds the greatest promise for novel treatments for opioid abuse. Basic research and clinical trials are required to examine what is more efficacious to yield new insights into the role of the gut-brain axis in opioid abuse.
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http://dx.doi.org/10.1101/cshperspect.a040485DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8415294PMC
September 2021

STING differentially regulates experimental GVHD mediated by CD8 versus CD4 T cell subsets.

Sci Transl Med 2020 07;12(552)

Department of Microbiology and Immunology, University of Miami Miller School of Medicine, Miami, FL 33136, USA.

The stimulator of interferon genes (STING) pathway has been proposed as a key regulator of gastrointestinal homeostasis and inflammatory responses. Although STING reportedly protects against gut barrier damage and graft-versus-host disease (GVHD) after major histocompatibility complex (MHC)-mismatched allogeneic hematopoietic stem cell transplantation (aHSCT), its effect in clinically relevant MHC-matched aHSCT is unknown. Studies here demonstrate that STING signaling in nonhematopoietic cells promoted MHC-matched aHSCT-induced GVHD and that STING agonists increased type I interferon and MHC I expression in nonhematopoietic mouse intestinal organoid cultures. Moreover, mice expressing a human STING allele containing three single-nucleotide polymorphisms associated with decreased STING activity also developed reduced MHC-matched GVHD, demonstrating STING's potential clinical importance. STING recipients experienced reduced GVHD with transplant of purified donor CD8 T cells in both MHC-matched and MHC-mismatched models, reconciling the seemingly disparate results. Further examination revealed that STING deficiency reduced the activation of donor CD8 T cells early after transplant and promoted recipient MHC class II antigen-presenting cell (APC) survival. Therefore, APC persistence in STING pathway absence may account for the increased GVHD mediated by CD4 T cells in completely mismatched recipients. In total, our findings have important implications for regulating clinical GVHD by targeting STING early after aHSCT and demonstrate that an innate immune pathway has opposing effects on the outcome of aHSCT, depending on the donor/recipient MHC disparity.
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http://dx.doi.org/10.1126/scitranslmed.aay5006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7392054PMC
July 2020

Viral Vector-Mediated Gene Transfer of Glutamic Acid Decarboxylase for Chronic Pain Treatment: A Literature Review.

Hum Gene Ther 2020 04 24;31(7-8):405-414. Epub 2020 Mar 24.

Department of Anesthesiology, University of Miami Miller School of Medicine, Miami, Florida.

Chronic pain is long-lasting nociceptive state, impairing the patient's quality of life. Existing analgesics are generally not effective in the treatment of chronic pain, some of which such as opioids have the risk of tolerance/dependence and overdose death with higher daily opioid doses for increasing analgesic effect. Opioid use disorders have already reached an epidemic level in the United States; therefore, nonopioid analgesic approach and/or use of nonpharmacologic interventions will be employed with increasing frequency. Viral vector-mediated gene therapy is promising in clinical trials in the nervous system diseases. Glutamic acid decarboxylase (GAD) enzyme, a key enzyme in biosynthesis of γ-aminobutyric acid (GABA), plays an important role in analgesic mechanism. In the literature review, we used PubMed and bioRxiv to search the studies, and the eligible criteria include (1) article written in English, (2) use of viral vectors expressing GAD67 or GAD65, and (3) preclinical pain models. We identified 13 eligible original research articles, in which the pain models include nerve injury, HIV-related pain, painful diabetic neuropathy, and formalin test. GAD expressed by the viral vectors from all the reports produced antinociceptive effects. Restoring GABA systems is a promising therapeutic strategy for chronic pain, which provides evidence for the clinical trial of gene therapy for pain in the near future.
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http://dx.doi.org/10.1089/hum.2019.359DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7194328PMC
April 2020

Opioids Impair Intestinal Epithelial Repair in HIV-Infected Humanized Mice.

Front Immunol 2019 17;10:2999. Epub 2020 Jan 17.

Department of Surgery, University of Miami, Miami, FL, United States.

Intestinal barrier dysfunction and subsequent microbial translocation play crucial roles in persistent immune activation leading to HIV disease progression. Opioid use is associated with worse outcome in HIV-infected patients. The exacerbated disease progression by opioids is mainly driven by excessive intestinal inflammation and increased gut permeability. The objective of this study is to investigate how opioids potentiate HIV disease progression by compromising intestinal barrier function and impairing intestinal epithelial self-repair mechanism. In the present study, abnormal intestinal morphology and reduced epithelial proliferation were observed in bone marrow-liver-thymus humanized mice and in HIV-infected patients who were exposed to opioids. In bone marrow-liver-thymus mice, HIV, and morphine independently, and additively induced gut dysbiosis, especially depletion of Lachnospiraceae, Ruminococcaceae, and Muribaculaceae. We also observed that the abundance of Lachnospiraceae, Ruminococcaceae, and Muribaculaceae negatively correlated with apoptosis of epithelial cells, and intestinal IL-6 levels. Previous studies have shown that these bacterial families play crucial roles in maintaining intestinal homeostasis because they include most short-chain fatty acid-producing members. Short-chain fatty acids have been shown to maintain stem cell populations and suppress inflammation in the gut by inhibiting histone deacetylases (HDAC). In addition, we demonstrate that morphine exposure inhibited growth of intestinal organoids derived from HIV transgenic mice by suppressing Notch signaling in an HDAC-dependent manner. These studies implicate an important role for HDAC in intestinal homeostasis and supports HDAC modulation as a therapeutic intervention in improving care of HIV patients, especially in opioid-abusing population.
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http://dx.doi.org/10.3389/fimmu.2019.02999DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6978907PMC
December 2020

Prescription Opioids induce Gut Dysbiosis and Exacerbate Colitis in a Murine Model of Inflammatory Bowel Disease.

J Crohns Colitis 2020 Jul;14(6):801-817

Department of Surgery, Miller School of Medicine, University of Miami, Miami, FL, USA.

Background And Aims: Opioids are the most prescribed analgesics for pain in inflammatory bowel diseases [IBD]; however, the consequences of opioid use on IBD severity are not well defined. This is the first study investigating consequences of hydromorphone in both dextran sodium sulphate [DSS]-induced colitis and spontaneous colitis (IL-10 knockout [IL-10-/-]) mouse models of IBD.

Methods: To determine the consequences of opioids on IBD pathogenesis, wild-type [WT] mice were treated with clinically relevant doses of hydromorphone and colitis was induced via 3% DSS in drinking water for 5 days. In parallel we also determined the consequences of opioids in a spontaneous colitis model.

Results: Hydromorphone and DSS independently induced barrier dysfunction, bacterial translocation, disruption of tight junction organisation and increased intestinal and systemic inflammation, which were exacerbated in mice receiving hydromorphone in combination with DSS. Hydromorphone + DSS-treated mice exhibited significant microbial dysbiosis. Predictive metagenomic analysis of the gut microbiota revealed high abundance in the bacterial communities associated with virulence, antibiotic resistance, toxin production, and inflammatory properties. Hydromorphone modulates tight junction organisation in a myosin light chain kinase [MLCK]-dependent manner. Treatment with MLCK inhibitor ML-7 ameliorates the detrimental effects of hydromorphone on DSS-induced colitis and thus decreases severity of IBD. Similarly, we demonstrated that hydromorphone treatment in IL-10-/- mice resulted in accelerated clinical manifestations of colitis compared with control mice.

Conclusions: Opioids used for pain management in IBD accelerate IBD progression by dysregulation of the gut microbiota, leading to expansion of pathogenic bacteria, translocation of bacteria, immune deregulation and sustained inflammation.
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http://dx.doi.org/10.1093/ecco-jcc/jjz188DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7346895PMC
July 2020

Acetylcholinesterase Inhibitor Pyridostigmine Bromide Attenuates Gut Pathology and Bacterial Dysbiosis in a Murine Model of Ulcerative Colitis.

Dig Dis Sci 2020 01 23;65(1):141-149. Epub 2019 Oct 23.

Lovelace Respiratory Research Institute, 2425 Ridgecrest Dr SE, Albuquerque, NM, 87108, USA.

Background: Ulcerative colitis (UC) is a Th2 inflammatory bowel disease characterized by increased IL-5 and IL-13 expression, eosinophilic/neutrophilic infiltration, decreased mucus production, impaired epithelial barrier, and bacterial dysbiosis of the colon. Acetylcholine and nicotine stimulate mucus production and suppress Th2 inflammation through nicotinic receptors in lungs but UC is rarely observed in smokers and the mechanism of the protection is unclear.

Methods: In order to evaluate whether acetylcholine can ameliorate UC-associated pathologies, we employed a mouse model of dextran sodium sulfate (DSS)-induced UC-like conditions, and a group of mice were treated with Pyridostigmine bromide (PB) to increase acetylcholine availability. The effects on colonic tissue morphology, Th2 inflammatory factors, MUC2 mucin, and gut microbiota were analyzed.

Results: DSS challenge damaged the murine colonic architecture, reduced the MUC2 mucin and the tight-junction protein ZO-1. The PB treatment significantly attenuated these DSS-induced responses along with the eosinophilic infiltration and the pro-Th2 inflammatory factors. Moreover, PB inhibited the DSS-induced loss of commensal Clostridia and Flavobacteria, and the gain of pathogenic Erysipelotrichia and Fusobacteria.

Conclusions: Together, these data suggest that in colons of a murine model, PB promotes MUC2 synthesis, suppresses Th2 inflammation and attenuates bacterial dysbiosis therefore, PB has a therapeutic potential in UC.
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http://dx.doi.org/10.1007/s10620-019-05838-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6943409PMC
January 2020

Blood Pressure and Marijuana Use: Results from a Decade of NHANES Data.

Am J Health Behav 2019 09;43(5):887-897

Sarah E. Messiah, Professor, University of Texas School of Public Health, Dallas, TX.

After 14 years of no change, new blood pressure (BP) guidelines were released; yet, the impact of marijuana on BP remains unclear. Our objective was to examine the association between marijuana use and BP. We analyzed data for adults (N = 10,709; mean age 44.8 years; 50.3% men) who completed 2005-2014 National Health and Nutrition Examination Surveys. Marijuana use was defined as never (no lifetime use), past (lifetime, not in past 30 days), and current (≥ 1 in past 30 days). Frequency of use was categorized based past 30-day use. BP was categorized as elevated BP, Stage 1 hypertension (HTN-I), or Stage 2 hypertension (HTN-II) based on updated guidelines. Current users had a higher prevalence of elevated BP (19.4%), HTN-I (22.7%), HTN-II (12.9%) than never users (16.1%, 21.4%, and 11.99%) respectively; p = .03). After covariate adjustment, heavy users had 1.80 higher odds of elevated BP than never users (95% CI: 1.13-2.88). There were no statistically significant differences in BP in any other marijuana use category. Driven by heavy use, current users had a higher prevalence of elevated BP than never users. Patients at risk for abnormal BP should use caution when engaging in heavy marijuana use.
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http://dx.doi.org/10.5993/AJHB.43.5.2DOI Listing
September 2019

Cocaine Induces Inflammatory Gut Milieu by Compromising the Mucosal Barrier Integrity and Altering the Gut Microbiota Colonization.

Sci Rep 2019 08 21;9(1):12187. Epub 2019 Aug 21.

Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE, 68198, USA.

Cocaine use disorder (CUD), a major health crisis, has traditionally been considered a complication of the CNS; however, it is also closely associated with malnourishment and deteriorating gut health. In light of emerging studies on the potential role of gut microbiota in neurological disorders, we sought to understand the causal association between CUD and gut dysbiosis. Using a comprehensive approach, we confirmed that cocaine administration in mice resulted in alterations of the gut microbiota. Furthermore, cocaine-mediated gut dysbiosis was associated with upregulation of proinflammatory mediators including NF-κB and IL-1β. In vivo and in vitro analyses confirmed that cocaine altered gut-barrier composition of the tight junction proteins while also impairing epithelial permeability by potentially involving the MAPK/ERK1/2 signaling. Taken together, our findings unravel a causal link between CUD, gut-barrier dysfunction and dysbiosis and set a stage for future development of supplemental strategies for the management of CUD-associated gut complications.
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http://dx.doi.org/10.1038/s41598-019-48428-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6704112PMC
August 2019

Opioid use potentiates the virulence of hospital-acquired infection, increases systemic bacterial dissemination and exacerbates gut dysbiosis in a murine model of infection.

Gut Microbes 2020 5;11(2):172-190. Epub 2019 Aug 5.

Department of Veterinary Population Medicine, University of Minnesota, Minneapolis, MN, USA.

Opioid analgesics are frequently prescribed in the United States and worldwide. However, serious side effects such as addiction, immunosuppression and gastrointestinal symptoms limit their use. It was recently demonstrated that morphine treatment results in a significant disruption in gut barrier function, leading to an increased translocation of gut commensal bacteria. Further studies have indicated distinct alterations in the gut microbiome and metabolome following morphine treatment, contributing to the negative consequences that are associated with opioid use. However, it is unclear how opioids modulate gut homeostasis in the context of a hospital-acquired bacterial infection. is an ideal murine model of human infections with enteropathogenic (EPEC) and enterohemorrhagic (EHEC). In the current study, a mouse model of infection was used to investigate the role of morphine in the modulation of gut homeostasis in the context of a hospital-acquired bacterial infection. Morphine treatment resulted in 1) the promotion of systemic dissemination, 2) an increase in the expression of the virulence factors of colonization in intestinal contents, 3) altered gut microbiome, 4) damaged integrity of gut epithelial barrier function, 5) inhibition of the -induced increase in goblet cells, and 6) dysregulated IL-17A immune response. This study demonstrates and further validates a positive correlation between opioid drug use/abuse and an increased risk of infections, suggesting that the overprescription of opioids may increase the susceptibility to hospital-acquired infection.
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http://dx.doi.org/10.1080/19490976.2019.1629237DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7053978PMC
September 2020

Extracellular Vesicles with Exosome-like Features Transfer TLRs between Dendritic Cells.

Immunohorizons 2019 06 4;3(6):186-193. Epub 2019 Jun 4.

Department of Surgery, Miller School of Medicine, University of Miami, Miami, FL 33101; and

Accumulating evidence shows that extracellular vesicles (EVs) secreted by immune cells play an important role in intercellular communication. In the current report, we show that EVs released from wild-type bone marrow-derived dendritic cells (BMDCs) transfer TLRs to TLR4-knockout (TLR4KO) BMDCs and increase cellular responsiveness to LPS in recipient cells. The transferred EVs have exosomal characteristics and induce the activation of NF-κB signaling pathways in recipient cells. We further show that BMDC-derived EVs can promote LPS-induced inflammation in TLR4KO mice in vivo. These results indicate that functional TLR4 can be transferred from wild-type to TLR4KO BMDCs through exosome-like EVs.
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http://dx.doi.org/10.4049/immunohorizons.1900016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8011940PMC
June 2019

Morphine tolerance is attenuated in germfree mice and reversed by probiotics, implicating the role of gut microbiome.

Proc Natl Acad Sci U S A 2019 07 17;116(27):13523-13532. Epub 2019 Jun 17.

Department of Surgery, University of Miami, Miami, FL 33136;

Prolonged exposure to opioids results in analgesic tolerance, drug overdose, and death. The mechanism underlying morphine analgesic tolerance still remains unresolved. We show that morphine analgesic tolerance was significantly attenuated in germfree (GF) and in pan-antibiotic-treated mice. Reconstitution of GF mice with naïve fecal microbiota reinstated morphine analgesic tolerance. We further demonstrated that tolerance was associated with microbial dysbiosis with selective depletion in and Probiotics, enriched with these bacterial communities, attenuated analgesic tolerance in morphine-treated mice. These results suggest that probiotic therapy during morphine administration may be a promising, safe, and inexpensive treatment to prolong morphine's efficacy and attenuate analgesic tolerance. We hypothesize a vicious cycle of chronic morphine tolerance: morphine-induced gut dysbiosis leads to gut barrier disruption and bacterial translocation, initiating local gut inflammation through TLR2/4 activation, resulting in the activation of proinflammatory cytokines, which drives morphine tolerance.
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http://dx.doi.org/10.1073/pnas.1901182116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6613141PMC
July 2019

Morphine Potentiates Dysbiotic Microbial and Metabolic Shifts in Acute SIV Infection.

J Neuroimmune Pharmacol 2019 06 21;14(2):200-214. Epub 2018 Sep 21.

Department of Pharmacology, University of Nebraska, Omaha, NE, USA.

Human Immunodeficiency Virus (HIV) pathogenesis has been closely linked with microbial translocation, which is believed to drive inflammation and HIV replication. Opioid drugs have been shown to worsen this symptom, leading to a faster progression of HIV infection to Acquired Immunodeficiency Syndrome (AIDS). The interaction of HIV and opioid drugs has not been studied at early stages of HIV, particularly in the gut microbiome where changes may precede translocation events. This study modeled early HIV infection by examining Simian Immunodeficiency Virus (SIV)-infected primates at 21 days or less both independently and in the context of opioid use. Fecal samples were analyzed both for 16S analysis of microbial populations as well as metabolite profiles via mass spectrometry. Our results indicate that changes are minor in SIV treated animals in the time points examined, however animals treated with morphine and SIV had significant changes in their microbial communities and metabolic profiles. This occurred in a time-independent fashion with morphine regardless of how long the animal had morphine in its system. Globally, the observed changes support that microbial dysbiosis is occurring in these animals at an early time, which likely contributes to the translocation events observed later in SIV/HIV pathogenesis. Additionally, metabolic changes were predictive of specific treatment groups, which could be further developed as a diagnostic tool or future intervention target to overcome and slow the progression of HIV infection to AIDS.
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http://dx.doi.org/10.1007/s11481-018-9805-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6917429PMC
June 2019

Gut Microbiota Promotes Tumor Growth in Mice by Modulating Immune Response.

Gastroenterology 2018 07 7;155(1):33-37.e6. Epub 2018 Apr 7.

Department of Surgery at Sylvester Comprehensive Cancer Center and University of Miami Miller School of Medicine, Miami, Florida. Electronic address:

We studied the effects of gut microbiome depletion by oral antibiotics on tumor growth in subcutaneous and liver metastases models of pancreatic cancer, colon cancer, and melanoma. Gut microbiome depletion significantly reduced tumor burden in all the models tested. However, depletion of gut microbiome did not reduce tumor growth in Rag1-knockout mice, which lack mature T and B cells. Flow cytometry analyses demonstrated that gut microbiome depletion led to significant increase in interferon gamma-producing T cells with corresponding decrease in interleukin 17A and interleukin 10-producing T cells. Our results suggest that gut microbiome modulation could emerge as a novel immunotherapeutic strategy.
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http://dx.doi.org/10.1053/j.gastro.2018.04.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6035070PMC
July 2018

Morphine induces changes in the gut microbiome and metabolome in a morphine dependence model.

Sci Rep 2018 02 26;8(1):3596. Epub 2018 Feb 26.

Department of Veterinary Population Medicine, University of Minnesota, 225 VMC 1365 Gortner Ave., St Paul, MN, 55108, USA.

Opioid analgesics are frequently prescribed in the United States and worldwide. However, serious comorbidities, such as dependence, tolerance, immunosuppression and gastrointestinal disorders limit their long-term use. In the current study, a morphine-murine model was used to investigate the role of the gut microbiome and metabolome as a potential mechanism contributing to the negative consequences associated with opioid use. Results reveal a significant shift in the gut microbiome and metabolome within one day following morphine treatment compared to that observed after placebo. Morphine-induced gut microbial dysbiosis exhibited distinct characteristic signatures, including significant increase in communities associated with pathogenic function, decrease in communities associated with stress tolerance and significant impairment in bile acids and morphine-3-glucuronide/morphine biotransformation in the gut. Moreover, expansion of Enterococcus faecalis was strongly correlated with gut dysbiosis following morphine treatment, and alterations in deoxycholic acid (DCA) and phosphatidylethanolamines (PEs) were associated with opioid-induced metabolomic changes. Collectively, these results indicate that morphine induced distinct alterations in the gut microbiome and metabolome, contributing to negative consequences associated with opioid use. Therapeutics directed at maintaining microbiome homeostasis during opioid use may reduce the comorbidities associated with opioid use for pain management.
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http://dx.doi.org/10.1038/s41598-018-21915-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5827657PMC
February 2018

Substance-associated elevations in monocyte activation among methamphetamine users with treated HIV infection.

AIDS 2018 03;32(6):767-771

University of Miami School of Medicine, Miami, Florida.

Objective: Microbial translocation and monocyte activation predict mortality in treated HIV. We examined whether substance use independently contributes to these pathophysiologic processes.

Design: Cross-sectional study at baseline for a randomized controlled trial.

Methods: HIV-positive, methamphetamine-using MSM with undetectable HIV viral load (less than 40 copies/ml) were enrolled. We examined if plasma biomarkers of monocyte activation and intestinal barrier integrity were associated with the following: reactive urine toxicology results (Tox+) for stimulants (i.e., methamphetamine or cocaine) and substance use severity measured by the Addiction Severity Index. Multiple linear regression models adjusted for age, antiretroviral therapy regimen, CD4 T-cell count, interleukin-6, and alcohol use severity.

Results: The sample of 84 virally suppressed MSM had a median CD4 T-cell count of 645 cells/μl. Those who were Tox+ for stimulants displayed higher soluble CD14 (sCD14) levels (2087 versus 1801 ng/ml; P = 0.009), and this difference remained significant after adjusting for covariates (standardized beta = 0.23, P = 0.026). Greater substance use severity was also independently associated with higher sCD14 after adjusting for covariates (standardized beta = 0.29, P = 0.013). Being Tox+ for stimulants and substance use severity were not associated with soluble CD163 (sCD163) or intestinal fatty acid binding protein (iFABP) levels (P > 0.05).

Conclusions: Monocyte activation is one plausible mechanism by which stimulant use may increase clinical HIV progression.
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http://dx.doi.org/10.1097/QAD.0000000000001751DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5912167PMC
March 2018

Prescription opioids are associated with higher mortality in patients diagnosed with sepsis: A retrospective cohort study using electronic health records.

PLoS One 2018 2;13(1):e0190362. Epub 2018 Jan 2.

Department of Surgery, University of Minnesota, Minneapolis, MN, United States of America.

Sepsis continues to be a major problem for hospitalized patients. Opioids are widely used medications for pain management despite recent evidence revealing their adverse effects. The present study evaluates survival differences between opioid-treated patients and non-opioid-treated patients hospitalized with a diagnosis of sepsis. Clinical data was extracted from the University of Minnesota's Clinical Data Repository, which includes Electronic Health Records (EHRs) of the patients seen at 8 hospitals. Among 5,994 patients diagnosed with sepsis, 4,540 opioid-treated patients and 1,454 non-opioid patients were included based on whether they are exposed to prescription opioids during their hospitalization. Cox proportional hazards regression showed that after adjustments for demographics, clinical comorbidities, severity of illness, and types of infection, opioid-treated patients had a significantly higher risk of death at 28 days.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0190362PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5749778PMC
February 2018

Enteric glial-mediated enhancement of intestinal barrier integrity is compromised by morphine.

J Surg Res 2017 11 30;219:214-221. Epub 2017 Jun 30.

Department of Surgery and Pediatrics, University of Minnesota, Minneapolis, Minnesota. Electronic address:

Background: The opioid epidemic is a growing concern, and emerging evidence suggests that morphine use may be associated with sepsis. Enteric glial cells (EGCs) are the most numerous cell type in the enteric nervous system and regulate gastrointestinal function through the production of trophic factors, including glial-derived neurotrophic factor (GDNF). We sought to determine the effect of morphine on enteric glia and hypothesized that morphine contributes to EGC dysfunction and increased gut permeability.

Materials And Methods: Rat intestinal epithelial cells (IECs) and EGC lines were purchased from ATCC. Immunocytochemistry was used to evaluate the impact of EGCs on IEC barrier proteins and detect the μ-opioid receptor. Co-culture assays were used to determine the effect of EGCs, GDNF, and morphine on barrier integrity. Quantitative polymerase chain reaction and western blotting were performed to determine the impact of morphine in GDNF production. Transepithelial resistance of IEC-6 cell monolayers was measured in the presence of EGC-conditioned media (EGC-CM) and morphine treated EGC-CM using electrical cell impedance sensing.

Results: EGC-CM enhanced tight junction organization in IECs. IEC barrier integrity was enhanced when co-cultured with unstimulated EGCs or with GDNF alone; this barrier protective effect was lost with morphine-treated EGCs. GDNF RNA and protein expression were decreased by morphine treatment. Transepithelial resistance was decreased in IEC confluent monolayers when exposed to morphine-treated EGC-CM compared with control.

Conclusions: Morphine compromises intestinal epithelial cell barrier function through a mechanism which appears to involve GDNF. Further studies are warranted to delineate the role of enteric glial cell function in opioid signaling and sepsis.
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http://dx.doi.org/10.1016/j.jss.2017.05.099DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5708166PMC
November 2017

Morphine worsens the severity and prevents pancreatic regeneration in mouse models of acute pancreatitis.

Gut 2018 04 22;67(4):600-602. Epub 2017 Jun 22.

Sylvester Comprehensive Cancer Center Department of Surgery, University of Miami, Miami, Florida, USA.

Background: Opioids such as morphine are widely used for the management of pain associated with acute pancreatitis. Interestingly, opioids are also known to affect the immune system and modulate inflammatory pathways in non-pancreatic diseases. However, the impact of morphine on the progression of acute pancreatitis has never been evaluated. In the current study, we evaluated the impact of morphine on the progression and severity of acute pancreatitis.

Methods: Effect of morphine treatment on acute pancreatitis in caerulein, L-arginine and ethanol-palmitoleic acid models was evaluated after induction of the disease. Inflammatory response, gut permeability and bacterial translocation were compared. Experiments were repeated in mu (µ) opioid receptor knockout mice (MORKO) and in wild-type mice in the presence of opioid receptor antagonist naltrexone to evaluate the role of µ-opioid receptors in morphine's effect on acute pancreatitis. Effect of morphine treatment on pathways activated during pancreatic regeneration like sonic Hedgehog and activation of embryonic transcription factors like pdx-1 and ptf-1 were measured by immunofluorescence and quantitative PCR.

Results: Histological data show that treatment with morphine after induction of acute pancreatitis exacerbates the disease with increased pancreatic neutrophilic infiltration and necrosis in all three models of acute pancreatitis. Morphine also exacerbated acute pancreatitis-induced gut permeabilisation and bacteraemia. These effects were antagonised in the MORKO mice or in the presence of naltrexone suggesting that morphine's effect on severity of acute pancreatitis are mediated through the µ-opioid receptors. Morphine treatment delayed macrophage infiltration, sonic Hedgehog pathway activation and expression of pdx-1 and ptf-1.

Conclusion: Morphine treatment worsens the severity of acute pancreatitis and delays resolution and regeneration. Considering our results, the safety of morphine for analgesia during acute pancreatitis should be re-evaluated in future human studies.
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http://dx.doi.org/10.1136/gutjnl-2017-313717DOI Listing
April 2018

Pirfenidone ameliorates murine chronic GVHD through inhibition of macrophage infiltration and TGF-β production.

Blood 2017 05 2;129(18):2570-2580. Epub 2017 Mar 2.

Division of Blood and Marrow Transplantation, Department of Pediatrics, Masonic Cancer Center, University of Minnesota, Minneapolis, MN.

Allogeneic hematopoietic stem cell transplantation is hampered by chronic graft-versus-host disease (cGVHD), resulting in multiorgan fibrosis and diminished function. Fibrosis in lung and skin leads to progressive bronchiolitis obliterans (BO) and scleroderma, respectively, for which new treatments are needed. We evaluated pirfenidone, a Food and Drug Administration (FDA)-approved drug for idiopathic pulmonary fibrosis, for its therapeutic effect in cGVHD mouse models with distinct pathophysiology. In a full major histocompatibility complex (MHC)-mismatched, multiorgan system model with BO, donor T-cell responses that support pathogenic antibody production are required for cGVHD development. Pirfenidone treatment beginning one month post-transplant restored pulmonary function and reversed lung fibrosis, which was associated with reduced macrophage infiltration and transforming growth factor-β production. Pirfenidone dampened splenic germinal center B-cell and T-follicular helper cell frequencies that collaborate to produce antibody. In both a minor histocompatibility antigen-mismatched as well as a MHC-haploidentical model of sclerodermatous cGVHD, pirfenidone significantly reduced macrophages in the skin, although clinical improvement of scleroderma was only seen in one model. In vitro chemotaxis assays demonstrated that pirfenidone impaired macrophage migration to monocyte chemoattractant protein-1 (MCP-1) as well as IL-17A, which has been linked to cGVHD generation. Taken together, our data suggest that pirfenidone is a potential therapeutic agent to ameliorate fibrosis in cGVHD.
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http://dx.doi.org/10.1182/blood-2017-01-758854DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5418639PMC
May 2017

Gut Homeostasis, Microbial Dysbiosis, and Opioids.

Toxicol Pathol 2017 01 28;45(1):150-156. Epub 2016 Nov 28.

1 Department of Veterinary Population Medicine, College of Veterinary Medicine, University of Minnesota, Saint Paul, Minnesota, USA.

Gut homeostasis plays an important role in maintaining animal and human health. The disruption of gut homeostasis has been shown to be associated with multiple diseases. The mutually beneficial relationship between the gut microbiota and the host has been demonstrated to maintain homeostasis of the mucosal immunity and preserve the integrity of the gut epithelial barrier. Currently, rapid progress in the understanding of the host-microbial interaction has redefined toxicological pathology of opioids and their pharmacokinetics. However, it is unclear how opioids modulate the gut microbiome and metabolome. Our study, showing opioid modulation of gut homeostasis in mice, suggests that medical interventions to ameliorate the consequences of drug use/abuse will provide potential therapeutic and diagnostic strategies for opioid-modulated intestinal infections. The study of morphine's modulation of the gut microbiome and metabolome will shed light on the toxicological pathology of opioids and its role in the susceptibility to infectious diseases.
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http://dx.doi.org/10.1177/0192623316679898DOI Listing
January 2017

Inhibition of NF-kappa B pathway leads to deregulation of epithelial-mesenchymal transition and neural invasion in pancreatic cancer.

Lab Invest 2016 12 24;96(12):1268-1278. Epub 2016 Oct 24.

Division of Surgical Oncology, Department of Surgery Sylvester Comprehensive Cancer Center, University of Miami, Miami, FL, USA.

NF-κB has an essential role in the initiation and progression of pancreatic cancer and specifically mediates the induction of epithelial-mesenchymal transition and invasiveness. In this study, we demonstrate the importance of activated NF-κB signaling in EMT induction, lymphovascular metastasis, and neural invasion. Modulation of NF-κB activity was accomplished through the specific NF-κB inhibitor (BAY 11-7085), triptolide, and Minnelide treatment, as well as overexpression of IKBα repressor and IKK activator plasmids. In the classical lymphovascular metastatic cascade, inhibition of NF-κB decreased the expression of several EMT transcription factors (SNAI1, SNAI2, and ZEB1) and mesenchymal markers (VIM and CDH2) and decreased in vitro invasion, which was rescued by IKK activation. This was further demonstrated in vivo via BAY 11-7085 treatment in a orthotopic model of pancreatic cancer. In vivo NF-κB inhibition decreased tumor volume; decreased tumor EMT gene expression, while restoring cell-cell junctions; and decreasing overall metastasis. Furthermore, we demonstrate the importance of active NF-κB signaling in neural invasion. Triptolide treatment inhibits Nerve Growth Factor (NGF) mediated, neural-tumor co-culture in vitro invasion, and dorsal root ganglia (DRG) neural outgrowth through a disruption in tumor-neural cross talk. In vivo, Minnelide treatment decreased neurotrophin expression, nerve density, and sciatic nerve invasion. Taken together, this study demonstrates the importance of NF-κB signaling in the progression of pancreatic cancer through the modulation of EMT induction, lymphovascular invasion, and neural invasion.
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http://dx.doi.org/10.1038/labinvest.2016.109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5121017PMC
December 2016
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