Higher Activity of the Inducible Nitric Oxide Synthase Contributes to Very Early Onset Inflammatory Bowel Disease

OBJECTIVES: The NOS2 gene encodes for the inducible nitric oxide synthase (iNOS), responsible for nitric oxide (NO) production, which contributes to antimicrobial and antipathogenic activities. Higher levels of both iNOS and NO-induced damage have been observed in inflammatory bowel disease (IBD) patients. NOS2 may have a role in a specific subset of IBD patients with severe and/or extensive colitis. Therefore, the aim of this study is to examine the role of NOS2 in such a subset, very early onset IBD (VEO-IBD). METHODS: Seventeen tag single nucleotide polymorphisms (SNPs) in the NOS2 gene were successfully genotyped in VEO-IBD patients. Genetic associations were replicated in an independent VEO-IBD cohort. Functional analysis for iNOS activity was performed on the most significantly associated functional variant. RESULTS: The NOS2 rs2297518 SNP was found to be associated in VEO-IBD in two independent cohorts. Upon combined analysis, a coding variant (S608L) showed the strongest association with VEO-IBD (Pcombined=1.13 × 10−6, OR (odds ratio)=3.398 (95% CI (confidence interval) 2.02–5.717)) as well as associations with VEO-Crohn's disease and VEO-ulcerative colitis (UC). This variant also showed an association with UC diagnosed between 11 and 17 years of age but not with adult-onset IBD (>17 years). B-cell lymphoblastoid cell lines genotyped for the risk variant as well as Henle-407 cells transfected with a plasmid construct with the risk variant showed higher NO production. Colonic biopsies of VEO-IBD patients showed higher immunohistochemical staining of nitrotyrosine, indicating more nitrosative stress and tissue damage. CONCLUSIONS: These studies suggest the importance of iNOS in genetic susceptibility to younger IBD presentation due to higher NO production

Generation of Immortal Cell Lines from the Adult Pituitary: Role of cAMP on Differentiation of SOX2-Expressing Progenitor Cells to Mature Gonadotropes

The pituitary is a complex endocrine tissue composed of a number of unique cell types distinguished by the expression and secretion of specific hormones, which in turn control critical components of overall physiology. The basic function of these cells is understood; however, the molecular events involved in their hormonal regulation are not yet fully defined. While previously established cell lines have provided much insight into these regulatory mechanisms, the availability of representative cell lines from each cell lineage is limited, and currently none are derived from adult pituitary. We have therefore used retroviral transfer of SV40 T-antigen to mass immortalize primary pituitary cell culture from an adult mouse. We have generated 19 mixed cell cultures that contain cells from pituitary cell lineages, as determined by RT-PCR analysis and immunocytochemistry for specific hormones. Some lines expressed markers associated with multipotent adult progenitor cells or transit-amplifying cells, including SOX2, nestin, S100, and SOX9. The progenitor lines were exposed to an adenylate cyclase activator, forskolin, over 7 days and were induced to differentiate to a more mature gonadotrope cell, expressing significant levels of α-subunit, LHβ, and FSHβ mRNAs. Additionally, clonal populations of differentiated gonadotropes were exposed to 30 nM gonadotropin-releasing hormone and responded appropriately with a significant increase in α-subunit and LHβ transcription. Further, exposure of the lines to a pulse paradigm of GnRH, in combination with 17β-estradiol and dexamethasone, significantly increased GnRH receptor mRNA levels. This array of adult-derived pituitary cell models will be valuable for both studies of progenitor cell characteristics and modulation, and the molecular analysis of individual pituitary cell lineages

The Hormonal Contol of Neuropeptide Y and Gonadotropin-releasing Hormone Hypothalamic Neurons

The physiological mechanisms that control energy homeostasis are reciprocally linked to reproduction. However, the neuroendocrine circuitry that registers endocrine cues to direct homeostatic responses in energy balance and reproduction remain unknown. Neuropeptide Y (NPY) neurons have emerged as a key central target of estrogen and leptin that are capable of modulating both reproduction and energy balance. The hypothesis was generated that NPY neuronal subpopulations act as an integration centre to regulate the effects of estrogen and leptin on these important physiological processes through specific signaling pathways. Using hypothalamic cell lines that express the leptin receptor (Ob-R), estrogen receptor (ER) and NPY, this hypothesis was tested in three aims. 17β-estradiol (E2) was previously demonstrated to biphasically regulate NPY mRNA in the mHypoE-38 neuronal cell line; where 24 h E2 exposure induced NPY gene expression that our group proposed may be involved in the gonadotropin-releasing hormone (GnRH) preovulatory surge. E2 also acts as an anorexigenic hormone through unknown hypothalamic targets. E2 directly decreased NPY secretion in the mHypoE-42 and mHypoA-2/12 neuronal cell lines through ER-α. The anorexigenic action of E2 was mediated through the energy sensing 5’ AMP-activated protein kinase (AMPK) and the phosphoinositide-3-kinase (PI3K) pathway. NPY secretion was also decreased by leptin in mHypoA-59 and NPY-GFP cell models through AMPK- and PI3K-dependent mechanisms. Prolonged exposure to leptin in NPY-GFP cell lines prevented AMPK signaling and the leptin-mediated reduction in NPY secretion, indicating NPY neuronal resistance with prolonged leptin exposure. Leptin also stimulated NPY secretion in mHypoE-38 neurons, which was blocked by pharmacological inhibitors of the mitogen-activated protein kinase (MAPK) and PI3K pathways. Importantly, conditioned medium from the mHypoE-38 NPY neuronal cells induced GnRH transcripts in GT1-7 neurons, which was inhibited by Y1-receptor antagonists. Pharmacological inhibitors of the MAPK and PKA signal transduction pathways attenuated the NPY-mediated increase in GnRH transcription. Based upon these findings, I propose NPY neurons in the hypothalamus consist of a heterogeneous population of neurons, and provide the first evidence of intrinsically different responses to function as physiological integrators for two different systems: NPY secretion can be suppressed to decrease food intake and induced to stimulate GnRH neurons.Ph

PRP in OA knee – update, current confusions and future options

Positive results have been uniformly observed by various researchers for platelet-rich plasma (PRP) in early osteoarthritis (OA) knee in the past few years. PRP has clearly demonstrated its supremacy in comparison to hyaluronic acid (HA) and placebo in various clinical trials and is undoubtedly the best option available for symptomatic treatment in early OA. The release of growth factors from PRP occurs immediately and lasts for around three weeks and the clinical effect tends to wane down by the end of the year. Prolonged and sustained release of growth factors from platelets could possibly help in much better biological healing and sustained clinical effects. PRP in combination with biocompatible carriers could be one way of achieving this. Gelatin hydrogel PRP and chitosan PRP seem to be promising based on early in vitro studies and animal studies. PRP in combination with hyaluronic acid also seems to be additive. This article intends to discuss the present status of the PRP, confusions surrounding its use, upcoming trends and ideas for improvising PRP for use early OA knees based on available evidence

Neuronal suppressor of cytokine signaling-3 deficiency enhances hypothalamic leptin-dependent phosphatidylinositol 3-kinase signaling

Suppressor of cytokine signaling-3 (SOCS3) is thought to be involved in the development of central leptin resistance and obesity by inhibiting STAT3 pathway. Because phosphatidylinositol 3-kinase (PI3K) pathway plays an important role in transducing leptin action in the hypothalamus, we examined whether SOCS3 exerted an inhibition on this pathway. We first determined whether leptin sensitivity in the hypothalamic PI3K pathway was increased in brain-specific Socs3-deficient (NesKO) mice. In NesKO mice, hypothalamic insulin receptor substrate-1 (IRS1)-associated PI3K activity was significantly increased at 30 min and remained elevated up to 2 h after leptin intraperitoneal injection, but in wild-type (WT) littermates, the significant increase was only at 30 min. Hypothalamic p-STAT3 levels were increased up to 5 h in NesKO as opposed to 2 h in WT mice. In food-restricted WT mice with reduced body weight, leptin increased hypothalamic PI3K activity only at 30 min, and p-STAT3 levels at 30–120 min postinjection. These results suggest increased leptin sensitivity in both PI3K and STAT3 pathways in the hypothalamus of NesKO mice, which was not due to a lean phenotype. In the next experiment with a clonal hypothalamic neuronal cell line expressing proopiomelanocortin, we observed that whereas leptin significantly increased IRS1-associated PI3K activity and p-JAK2 levels in cells transfected with control vector, it failed to do so in SOCS3-overexpressed cells. Altogether, these results imply a SOCS3 inhibition of the PI3K pathway of leptin signaling in the hypothalamus, which may be one of the mechanisms behind the development of central leptin resistance and obesity

Protocol for a quasi-experimental study to ascertain the effectiveness of using eKnee School approach to impart self-care education to patients suffering from knee osteoarthritis during COVID-19 pandemic

BACKGROUND: Knee osteoarthritis (KOA) patients seek improvement in their quality of life by attaining independence in activities of daily living. Literature recommends nonpharmacological intervention as first-line treatment for KOA. The study aims to ascertain the effectiveness of online supervised nonpharmacological intervention sessions of virtual knee school (eKS) training among mild and moderate KOA patients in comparison to routine care during COVID-19 pandemic and assessment of cost-effectiveness of eKS against the routine care for KOA patients during COVID-19 pandemic.MATERIALS AND METHODS: A quasi-experimental pre-post with control group, enrolling 50 participants each in two groups: usual/routine KOA care or usual care plus KS interventions via virtual mode. Our primary outcome measures are pain, quality of life, and incremental cost-effectiveness ratio. Secondary outcomes include performance-based tests (30-second chair test, timed up and go test, 6-minute walk test) and patient satisfaction. Intervention fidelity will be assessed with a priori checklist tailored to eKS assessing adherence, dose, quality, and user engagement in the key components. Quantitative data collection will be conducted at baseline and 6 months. Descriptive data analysis will be carried for quantitative data. For qualitative data, the thematic analysis will be performed; we propose to undertake a deterministic and probabilistic sensitivity analysis to address the issue of uncertainty in the present cost-effectiveness analysis model.CONCLUSION: The management of KOA through virtual mode emphasizes the concepts of patient-as-person, family-centered, with socially interactive approach. The study will provide information on the effectiveness of nonpharmacological interventions for improving the quality of life of patients suffering from KOA through virtual knee school. Nevertheless, pitfalls in running eKS will be noted, which will help improve all aspects of online medical communications in the future

Talar Neck Malunions: Evaluation of Kinematics, Pedobarographic Changes and Patient Reported Outcome Measures

Category: Trauma; Hindfoot Introduction/Purpose: Malunion is a disabling complication of talar neck fractures and is prevalent in approximately 17% of cases. The impact of talar neck malunions (TNM) on foot biomechanics and functional outcomes is not well established. The available evidence is primarily derived from cadaveric studies which have demonstrated that TNMs result in reduced motion and significant alterations in contact characteristics of the subtalar joint. Owing to the paucity of literature on this subject, we conducted this study to evaluate the kinematic and pedobarographic changes and functional outcomes associated with TNMs. Methods: In this study, adult patients with talar neck malunions (TNM) without ankle arthrosis were prospectively enrolled over a 5-year period. The Rammelt and Zwipp classification was utilized to categorize the deformities. Demographic data and ankle and subtalar range of motion were assessed. Weight-bearing anteroposterior (AP), lateral, and long axial radiographs, as well as CT scans of both feet, were obtained. Dynamic pedobarography was performed to evaluate gait kinematics and plantar pressure distribution. Functional outcomes were evaluated using the Manchester Oxford Foot Questionnaire (MOxFQ), Visual Analog Score, and the EQ5D questionnaire. The t-test was utilized to compare the range of motion, pedobarographic and kinematic parameters between the normal and pathologic foot. Furthermore, correlation coefficients were calculated to determine the strength of the association between changes in talar neck geometry, plantar pressures, kinematics, and functional outcomes. Results: A total of 10 patients, 6 males, and 4 females, with a mean age of 32.4 years were enrolled. On the TNM side, significant increases were observed in step length and step time, while significant decreases were noted in the single limb support time and single limb support center of pressure line. Moreover, midfoot forces were significantly increased, whereas the forefoot and hindfoot forces were significantly decreased on the TNM side. A strong positive correlation was found between midfoot force and the talar torsion angle, and a moderate negative correlation was observed between hindfoot and midfoot forces and the inclination angle. A strong positive correlation was also noted between high midfoot pressures and VAS Scores, MOxFQ scores, and the EQ5D walking and usual activities domains. Conclusion: This study demonstrates that TNMs are associated with decreased single limb support time, increased step length and time, increased midfoot pressures, and decreased forefoot and hindfoot pressures. Additionally, an increase in talar neck torsion after TNM is linked with higher midfoot pressures, which can lead to higher levels of pain and poorer function. Our findings provide valuable insights into the altered foot biomechanics after TNMs, which can assist surgeons in offering optimal management strategies for these patients