Regulation of Toll-like Receptor 4 Signaling and Expression by Endogenous Heat Shock Protein 70 in the Newborn Intestinal Epithelium

Necrotizing enterocolitis (NEC) is the leading cause of gastrointestinal-related mortality in premature infants, and it develops under conditions of exaggerated TLR4 signaling in the newborn intestinal epithelium. Because NEC does not develop spontaneously, despite the presence of seemingly tonic stimulation of intestinal TLR4, we hypothesized that mechanisms must exist to constrain TLR4 signaling that become diminished during NEC pathogenesis and focused on the intracellular stress response protein and chaperone heat shock protein-70 (Hsp70). We demonstrate that the induction of intracellular Hsp70 in enterocytes dramatically reduced TLR4 signaling, as assessed by LPS-induced NF-κB translocation, cytokine expression, and apoptosis. These findings were confirmed in vivo, using mice that either globally lacked Hsp70 or overexpressed Hsp70 within the intestinal epithelium. TLR4 activation itself significantly increased Hsp70 expression in enterocytes, which provided a mechanism of autoinhibition of TLR4 signaling in enterocytes. In seeking to define the mechanisms involved, intracellular Hsp70-mediated inhibition of TLR4 signaling required both its substrate-binding EEVD domain and association with the cochaperone CHIP, resulting in ubiquitination and proteasomal degradation of TLR4. The expression of Hsp70 in the intestinal epithelium was significantly decreased in murine and human NEC compared with healthy controls, suggesting that loss of Hsp70 protection from TLR4 could lead to NEC. In support of this, intestinal Hsp70 overexpression in mice and pharmacologic upregulation of Hsp70 reversed TLR4-induced cytokines and enterocyte apoptosis, as well as prevented and treated experimental NEC. Thus, a novel TLR4 regulatory pathway exists within the newborn gut involving Hsp70 that may be pharmacologically activated to limit NEC severity

Demographic and health profiles of people with severe mental illness in general practice in Australia: a cross-sectional study

Background. People with severe mental illness have a higher rate of premature death than the general population, largely due to primary care preventable diseases. There has been little research on the health profile of this population attending Australian general practices. Methods. In this nationwide cross-sectional study, MedicineInsight data for adult patients regularly attending general practices in 2018 were analysed to estimate the prevalence of schizophrenia or bipolar disorders (SBD) and investigate the health profile of people with SBD compared with other patients. Multilevel models clustered by practice (n = 565) and patient, and practice characteristics were created. Results. The prevalence of recorded SBD was 1.91% (95% CI = 1.88%–1.94%) among the 618 849 patients included. Patients with recorded SBD were more likely than other patients to have records of health risk factors, particularly smoking (aOR = 3.8, 95% CI = 3.6–3.9) and substance use (aOR = 5.9, 95% CI = 5.6–6.3), and higher probabilities of comorbidities including cardiovascular diseases (aOR = 1.3, 95% CI = 1.2–1.4), cancer (aOR = 1.1, 95% CI = 1.0–1.2), diabetes mellitus type 2 (aOR = 2.2, 95% CI = 2.0–2.3), chronic kidney diseases (aOR = 1.7, 95% CI = 1.5–2.0), chronic liver diseases (aOR = 3.3, 95% CI = 2.6–4.0) and chronic respiratory diseases (aOR = 1.7, 95% CI = 1.7–1.8). Conclusions. The higher prevalence of health risk factors and comorbidities among patients with recorded SBD underscores the need for proactive health risk monitoring and preventive care to address this health inequity

Modeling the interactions of bacteria and Toll-like receptor-mediated inflammation in necrotizing enterocolitis

Necrotizing enterocolitis (NEC) is a severe disease of the gastrointestinal tract in premature infants, characterized by a disrupted intestinal epithelium and an exaggerated pro-inflammatory response. Since the activation of Toll-like receptor-4 (TLR4) blocks cell migration and proliferation and contributes to an uncontrolled inflammatory response within the intestine, this receptor has been identified as a key contributor to the development of NEC. Toll-like receptor-9 (TLR9) has been shown to sense bacterial genome components (CpG DNA) and to play an anti-inflammatory role in NEC. We present in vitro results demonstrating direct inhibition of TLR4 activation by CpG DNA, and we develop a mathematical model of bacteria-immune interactions within the intestine to investigate how such inhibition of TLR4 signaling might alter inflammation, associated bacterial invasion of tissue, and resulting outcomes. The model predicts that TLR9 can inhibit both the beneficial and detrimental effects of TLR4, and thus a proper balance of action by these two receptors is needed to promote intestinal health. The model results are also used to explore three interventions that could potentially prevent the development of NEC: reducing bacteria in the mucus layer, administering probiotic treatment, and blocking TLR4 activation. While the model shows that these interventions would be successful in most cases, the model is also used to identify situations in which the proposed treatments might be harmful

Discovery and Validation of a New Class of Small Molecule Toll-Like Receptor 4 (TLR4) Inhibitors

Many inflammatory diseases may be linked to pathologically elevated signaling via the receptor for lipopolysaccharide (LPS), toll-like receptor 4 (TLR4). There has thus been great interest in the discovery of TLR4 inhibitors as potential anti-inflammatory agents. Recently, the structure of TLR4 bound to the inhibitor E5564 was solved, raising the possibility that novel TLR4 inhibitors that target the E5564-binding domain could be designed. We utilized a similarity search algorithm in conjunction with a limited screening approach of small molecule libraries to identify compounds that bind to the E5564 site and inhibit TLR4. Our lead compound, C34, is a 2-acetamidopyranoside (MW 389) with the formula C17H27NO9, which inhibited TLR4 in enterocytes and macrophages in vitro, and reduced systemic inflammation in mouse models of endotoxemia and necrotizing enterocolitis. Molecular docking of C34 to the hydrophobic internal pocket of the TLR4 co-receptor MD-2 demonstrated a tight fit, embedding the pyran ring deep inside the pocket. Strikingly, C34 inhibited LPS signaling ex-vivo in human ileum that was resected from infants with necrotizing enterocolitis. These findings identify C34 and the β-anomeric cyclohexyl analog C35 as novel leads for small molecule TLR4 inhibitors that have potential therapeutic benefit for TLR4-mediated inflammatory diseases. © 2013 Neal et al

Innate Immune Signaling in the Pathogenesis of Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) is a challenging disease to treat, and caring for patients afflicted by it remains both frustrating and difficult. While NEC may develop quickly and without warning, it may also develop slowly, insidiously, and appear to take the caregiver by surprise. In seeking to understand the molecular and cellular processes that lead to NEC development, we have identified a critical role for the receptor for bacterial lipopolysaccharide (LPS) toll like receptor 4 (TLR4) in the pathogenesis of NEC, as its activation within the intestinal epithelium of the premature infant leads to mucosal injury and reduced epithelial repair. The expression and function of TLR4 were found to be particularly elevated within the intestinal mucosa of the premature as compared with the full-term infant, predisposing to NEC development. Importantly, factors within both the enterocyte itself, such as heat shock protein 70 (Hsp70), and in the extracellular environment, such as amniotic fluid, can curtail the extent of TLR4 signaling and reduce the propensity for NEC development. This review will highlight the critical TLR4-mediated steps that lead to NEC development, with a focus on the proinflammatory responses of TLR4 signaling that have such devastating consequences in the premature host

Palmitate-induced ER stress and inhibition of protein synthesis in cultured myotubes does not require Toll-like receptor 4

Saturated fatty acids, such as palmitate, are elevated in metabolically dysfunctional condi- tions like type 2 diabetes mellitus. Palmitate has been shown to impair insulin sensitivity and suppress protein synthesis while upregulating proteolytic systems in skeletal muscle. Increased sarco/endoplasmic reticulum (ER) stress and subsequent activation of the unfolded protein response may contribute to the palmitate-induced impairment of muscle protein synthesis. In some cell types, ER stress occurs through activation of the Toll-like receptor 4 (TLR4). Given the link between ER stress and suppression of protein synthesis, we investigated whether palmitate induces markers of ER stress and protein synthesis by activating TLR4 in cultured mouse C2C12 myotubes. Myotubes were treated with vehicle, a TLR4-specific ligand (lipopolysaccharides), palmitate, or a combination of palmitate plus a TLR4-specific inhibitor (TAK-242). Inflammatory indicators of TLR4 activation (IL-6 and TNFα) and markers of ER stress were measured, and protein synthesis was assessed using puromycin incorporation. Palmitate substantially increased the levels of IL-6, TNF-α, CHOP, XBP1s, and ATF 4 mRNAs and augmented the levels of CHOP, XBP1s, phospho- PERK and phospho-eIF2α proteins. The TLR4 antagonist attenuated both acute palmitate and LPS-induced increases in IL-6 and TNFα, but did not reduce ER stress signaling with either 6 h or 24 h palmitate treatment. Similarly, treating myotubes with palmitate for 6 h caused a 43% decline in protein synthesis consistent with an increase in phospho-eIF2α, and the TLR4 antagonist did not alter these responses. These results suggest that palmitate does not induce ER stress through TLR4 in muscle, and that palmitate impairs protein syn- thesis in skeletal muscle in part by induction of ER stress

Understanding the Value of Tumor Markers in Pediatric Ovarian Neoplasms

Purpose The purpose of this study was to determine the diagnostic accuracy of tumor markers for malignancy in girls with ovarian neoplasms. Methods A retrospective review of girls 2–21 years who presented for surgical management of an ovarian neoplasm across 10 children's hospitals between 2010 and 2016 was performed. Patients who had at least one concerning feature on imaging and had tumor marker testing were included in the study. Sensitivity, specificity, and negative and positive predictive values (PPV) of tumor markers were calculated. Results Our cohort included 401 patients; 22.4% had a malignancy. Testing for tumor markers was inconsistent. AFP had high specificity (98%) and low sensitivity (42%) with a PPV of 86%. The sensitivity, specificity, and PPV of beta-hCG was 44%, 76%, and 32%, respectively. LDH had high sensitivity (95%) and Inhibin A and Inhibin B had high specificity (97% and 92%, respectively). Conclusions Tumor marker testing is helpful in preoperative risk stratification of ovarian neoplasms for malignancy. Given the variety of potential tumor types, no single marker provides enough reliability, and therefore a panel of tumor marker testing is recommended if there is concern for malignancy. Prospective studies may help further elucidate the predictive value of tumor markers in a pediatric ovarian neoplasm population

Allopregnanolone suppresses diabetes-induced neuropathic pain and motor deficit through inhibition of GABAA receptor down-regulation in the spinal cord of diabetic rats

Objective(s):Painful diabetic neuropathy is associated with hyperexcitability and hyperactivity of spinal cord neurons. However, its underlying pathophysiological mechanisms have not been fully clarified. Induction of excitatory/inhibitory neurotransmission imbalance at the spinal cord seems to account for the abnormal neuronal activity in diabetes. Protective properties of neurosteroids have been demonstrated in numerous cellular and animal models of neurodegeneration. Materials and Methods: Here, the protective effects of allopregnanolone, a neurosteroid were investigated in an in vivo model of diabetic neuropathy. The tail-flick test was used to assess the nociceptive threshold. Diabetes was induced by injection of 50 mg/kg (IP) streptozotocin. Seven weeks after the induction of diabetes, the dorsal half of the lumbar spinal cord was assayed for the expression of γ2 subunit of GABAA receptor using semiquantitative RT-PCR. Results: The data shows that allopregnanolone (5 and 20 mg/kg) markedly ameliorated diabetes-induced thermal hyperalgesia and motor deficit. The weights of diabetic rats that received 5 and 20 mg/kg allopregnanolone did not significantly reduce during the time course of study. Furthermore, this neurosteroid could inhibit GABAA receptor down-regulation induced by diabetes in the rat spinal cord. Conclusion: The data revealed that allopregnanolone has preventive effects against hyperglycemic-induced neuropathic pain and motor deficit which are related to the inhibition of GABAA receptor down-regulation