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Comparison of Lysis and Amplification Methodologies for Optimal 16S rRNA Gene Profiling for Human and Mouse Microbiome Studies.
When conducting sequence-based analysis of microbiome samples, it is important to accurately represent the bacterial communities present. The aim of this study was to compare two commercially available DNA isolation and PCR amplification approaches to determine their impact on the taxonomic composition of microbiome samples following 16S rRNA gene sequencing. A well-established 16S rRNA gene profiling approach, which was widely used in the Human Microbiome Project (HMP), was compared with a novel alkaline degenerative technique that utilizes alkaline cell lysis in combination with a degenerate pool of primers for nucleic acid extraction and PCR amplification. When comparing these different approaches for the microbiome profiling of human and mouse fecal samples, we found that the alkaline-based method was able to detect greater taxonomic diversity. An in silico analysis of predicted primer binding against a curated 16S rRNA gene reference database further suggested that this novel approach had the potential to reduce population bias found with traditional methods, thereby offering opportunities for improved microbial community profiling.
Upadacitinib for Induction of Remission in Paediatric Crohn's Disease: An International Multicentre Retrospective Study.
BACKGROUND: There are scarce data available on upadacitinib in children with Crohn's disease (CD). AIM: To evaluate the effectiveness and safety of upadacitinib as an induction therapy in paediatric CD. METHODS: This was a multicentre retrospective study between 2022 and 2024 of children treated with upadacitinib for induction of remission of active CD conducted in 30 centres worldwide affiliated with the IBD Interest and Porto group of the ESPGHAN. We recorded demographic, clinical and laboratory data and adverse events (AEs) at week 8 post-induction. The analysis of the primary outcome was based upon the intention-to-treat (ITT) principle. RESULTS: We included 100 children (median age 15.8 [interquartile range 14.3-17.2]). All were previously treated with biologic therapies including 89 with ≥ 2 biologics. At the end of the 8-week induction period, we observed clinical response, clinical remission and corticosteroid- and exclusive enteral nutrition-free clinical remission (CFR) in 75%, 56% and 52%, respectively. By the end of induction, 68% had achieved normalisation of C-reactive protein, and 58% had faecal calprotectin (FC)
Short-chain fatty acids: linking diet, the microbiome and immunity.
The short-chain fatty acids (SCFAs) butyrate, propionate and acetate are microbial metabolites and their availability in the gut and other organs is determined by environmental factors, such as diet and use of antibiotics, that shape the diversity and metabolism of the microbiota. SCFAs regulate epithelial barrier function as well as mucosal and systemic immunity via evolutionary conserved processes that involve G protein-coupled receptor signalling or histone deacetylase activity. Indicatively, the anti-inflammatory role of butyrate is mediated through direct effects on the differentiation of intestinal epithelial cells, phagocytes, B cells and plasma cells, and regulatory and effector T cells. Intestinally derived SCFAs also directly and indirectly affect immunity at extra-intestinal sites, such as the liver, the lungs, the reproductive tract and the brain, and have been implicated in a range of disorders, including infections, intestinal inflammation, autoimmunity, food allergies, asthma and responses to cancer therapies. An ecological understanding of microbial communities and their interrelated metabolic states, as well as the engineering of butyrogenic bacteria may support SCFA-focused interventions for the prevention and treatment of immune-mediated diseases.
Effectiveness and Safety of Adalimumab in Patients With Very Early-Onset Inflammatory Bowel Disease: A Retrospective Study on Behalf of the Porto Inflammatory Bowel Disease Working Group of European Society for Pediatric Gastroenterology Hepatology and Nutrition.
BACKGROUND AND AIMS: Patients with very early-onset inflammatory bowel disease (VEO-IBD), with an age of onset
Dyspareunia is rarely assessed in rodent models of endometriosis and interstitial cystitis/bladder pain syndrome.
ABSTRACT: Dyspareunia, or pain during sex, is a common and often debilitating symptom in individuals with endometriosis and/or interstitial cystitis/bladder pain syndrome (IC/BPS). Despite its significant impact on quality of life, it is frequently overlooked in research. This review evaluates how dyspareunia has been addressed in preclinical investigations of these conditions. A systematic search was conducted using Embase from 1998 to 2021, identifying original in vivo preclinical studies using female rodents to model (i) endometriosis and (ii) IC/BPS. The search aimed to identify studies that assessed dyspareunia. Study quality and risk of bias were evaluated using a modified CAMARADES checklist. Our analysis found 1,286 studies modelling endometriosis and 674 modelling IC/BPS, but only 18 and 1, respectively, measured dyspareunia. The most common method involved vaginal distention in rats, assessed by either behavioural escape responses or visceromotor reflexes of abdominal muscles. Despite the high prevalence of dyspareunia in these conditions, it is rarely measured in preclinical studies. We identify a significant gap in the literature and offer succinct recommendations for future translational research to address this important symptom. LAY SUMMARY: Dyspareunia describes pain occurring before, during or after sexual intercourse. This poorly understood symptom is particularly common in people suffering from two chronic pain-related conditions: endometriosis and IC/BPS, severely impacting their quality of life. Therefore, effective treatments addressing painful sex in people with these conditions are needed. To see the benefits of medical research at the patient's bedside, it is important to build from basic science research to preclinical animal studies then to human studies. Our study aims to assess the work that has been done so far at the 'preclinical' stage. Developments have been made in the methodology used to investigate this symptom in animals, and a summary of all the key findings may help build a platform to design future studies. Given the urgent need to develop new therapeutic strategies, attention given to painful sex by scientific medical researchers and physicians needs to improve.
The Diverse Pathways for Cell Surface MT1-MMP Localization in Migratory Cells
Controlled cell migration is an essential biological process in health, while uncontrolled cell migration contributes to disease progression. For cells to migrate through tissue, they must first degrade the extracellular matrix (ECM), which acts as a physical barrier to cell migration. A type I transmembrane-type matrix metalloproteinase, MT1-MMP, is the key enzyme involved in this process. It has been extensively shown that MT1-MMP promotes the migration of different cell types in tissue, including fibroblasts, epithelial cells, endothelial cells, macrophages, mesenchymal stem cells, and cancer cells. MT1-MMP is tightly regulated at different levels, and its localization to leading-edge membrane structures is an essential process for MT1-MMP to promote cellular invasion. Different cells display different motility-associated membrane structures, which contribute to their invasive ability, and there are diverse mechanisms of MT1-MMP localization to these structures. In this article, we will discuss the current understanding of MT1-MMP regulation, in particular, localization mechanisms to these different motility-associated membrane structures.
The in£uence of CD25+ cells on the generation of immunity to tumour cell lines in mice
CD25+ regulatory T cells comprise 5-10% of CD4+ T cells in naive mice and have been shown in several in vivo murine models to prevent the induction of autoimmune disease and in£ammatory disease. Since T cells, which mediate autoimmunity, can through recognition of self-antigens also target tumour cells, it was postulated that CD25+ regulatory cells would also inhibit the generation of immune responses to tumours. Depletion of these cells using CD25-specific monoclonal antibodies has indeed been shown to promote rejection of several transplantable murine tumour cell lines including melanoma, leukaemia and colorectal carcinoma. Results obtained using these models indicate that in the absence of regulatory cells, CD4+ T cells mediate tumour immunity, although the precise mechanisms through which these cells result in tumour rejection have not yet been elucidated. The target antigens recognized by these CD4+ T cells have also not yet been identified. Immunization of mice with tumour cells in the absence of CD25+ regulatory cells does, however, induce immunity against a variety of different tumour cell lines indicating that the target antigen(s) are shared amongst tumours of distinct histological origins. Since CD25+ regulatory cells have been identified in humans, the possibility that the cells inhibit immune responses to shared rejection antigens expressed by human tumours is worthy of investigation.
T Cell Resistance: On the Mechanisms of T Cell Non-activation.
Immunological tolerance is a fundamental arm of any functioning immune system. Not only does tolerance mitigate collateral damage from host immune responses, but in doing so permits a robust response sufficient to clear infection as necessary. Yet, despite occupying such a cornerstone, research aiming to unravel the intricacies of tolerance induction is mired by interchangeable and often misused terminologies, with markers and mechanistic pathways that beg the question of redundancy. In this review we aim to define these boarders by providing new perspectives to long-standing theories of tolerance. Given the central role of T cells in enforcing immune cascades, in this review we choose to explore immunological tolerance through the perspective of T cell 'resistance to activation,' to delineate the contexts in which one tolerance mechanism has evolved over the other. By clarifying the important biological markers and cellular players underpinning T cell resistance to activation, we aim to encourage more purposeful and directed research into tolerance and, more-over, potential therapeutic strategies in autoimmune diseases and cancer. The tolerance field is in much need of reclassification and consideration, and in this review, we hope to open that conversation.
Investigations for Suspected Head and Neck Squamous Cell Carcinoma of Unknown Primary (HNSCCUP): A National Cohort Study.
ObjectivesHead and neck squamous cell carcinoma from unknown primary (HNSCCUP) is a rare and challenging condition. This study aimed to investigate the diagnostic pathways of suspected HNSCCUP patients in the United Kingdom.MethodsA retrospective observational cohort study was conducted, over 5 years from January 2015, in UK Head and Neck centres of consecutive adults undergoing 18F-Fluorodeoxyglucose-PET-CT (PET-CT) within 3 months of diagnosis with metastatic cervical squamous cell carcinoma. Patients with no primary site on examination and no previous head and neck cancer were eligible.ResultsData for 965 patients were received from 57 centres; 68.5% were HPV-related disease. Three investigation cycles were observed: ultrasound with biopsy, cross-sectional imaging (MRI and/or CT) and PET-CT, at median times of 17, 29.5 and 46 days from referral. No primary was identified on PET-CT in 49.8% (n = 478/960). Diagnostic tonsillectomy was performed in 58.2% (n = 278/478) and tongue base mucosectomy (TBM) in 21.7% (n = 104/479). Ipsilateral tonsillectomy carried the highest diagnostic yield (18.7%, n = 52/278), followed by TBM (15.4%, n = 16/104). Contralateral tonsillectomy, performed in 49.0% (n = 234/478), carried the lowest yield (0.9%, n = 2/234). PET-CT with concurrent MRI was associated with higher primary site detection than PET-CT with concurrent CT (p = 0.003). A minority of patients undergoing treatment with curative intent received first-definitive-treatment within 62 days of referral (15.2%, n = 77/505, median 92 days, IQR: 71-117).ConclusionsMost patients experienced a protracted diagnostic pathway and waited over 3 months for definitive treatment. Earlier PET-CT with concurrent MRI may expedite diagnosis. TBM appears more productive than contralateral tonsillectomy for primary site detection.
Iberdomide increases innate and adaptive immune cell subsets in the bone marrow of patients with relapsed/refractory multiple myeloma.
Iberdomide is a potent cereblon E3 ligase modulator (CELMoD agent) with promising efficacy and safety as a monotherapy or in combination with other therapies in patients with relapsed/refractory multiple myeloma (RRMM). Using a custom mass cytometry panel designed for large-scale immunophenotyping of the bone marrow tumor microenvironment (TME), we demonstrate significant increases of effector T and natural killer (NK) cells in a cohort of 93 patients with multiple myeloma (MM) treated with iberdomide, correlating findings to disease characteristics, prior therapy, and a peripheral blood immune phenotype. Notably, changes are dose dependent, associated with objective response, and independent of prior refractoriness to MM therapies. This suggests that iberdomide broadly induces innate and adaptive immune activation in the TME, contributing to its antitumor efficacy. Our approach establishes a strategy to study treatment-induced changes in the TME of patients with MM and, more broadly, patients with cancer and establishes rational combination strategies for iberdomide with immune-enhancing therapies to treat MM.
Inflammatory disease microbiomes share a functional pathogenicity predicted by C-reactive protein.
We examine disease-specific and cross-disease functions of the human gut microbiome by colonizing germ-free mice, at risk for inflammatory arthritis, colitis, or neuroinflammation, with over 100 human fecal microbiomes from subjects with rheumatoid arthritis, ankylosing spondylitis, multiple sclerosis, ulcerative colitis, Crohn's disease, or colorectal cancer. We find common inflammatory phenotypes driven by microbiomes from individuals with intestinal inflammation or inflammatory arthritis, as well as distinct functions specific to microbiomes from multiple sclerosis patients. Inflammatory disease in mice colonized with human microbiomes correlated with systemic inflammation, measured by C-reactive protein, in the human donors. These cross-disease patterns of human microbiome pathogenicity mirror features of the inflammatory diseases, including therapeutic targets and the presence or absence of systemic inflammation, suggesting shared and disease-specific mechanisms by which the microbiome is shaped and drives pathogenic inflammatory responses.