TGFbeta primes breast tumors for lung metastasis seeding through angiopoietin-like 4.

Cells released from primary tumors seed metastases to specific organs by a nonrandom process, implying the involvement of biologically selective mechanisms. Based on clinical, functional, and molecular evidence, we show that the cytokine TGFbeta in the breast tumor microenvironment primes cancer cells for metastasis to the lungs. Central to this process is the induction of angiopoietin-like 4 (ANGPTL4) by TGFbeta via the Smad signaling pathway. TGFbeta induction of Angptl4 in cancer cells that are about to enter the circulation enhances their subsequent retention in the lungs, but not in the bone. Tumor cell-derived Angptl4 disrupts vascular endothelial cell-cell junctions, increases the permeability of lung capillaries, and facilitates the trans-endothelial passage of tumor cells. These results suggest a mechanism for metastasis whereby a cytokine in the primary tumor microenvironment induces the expression of another cytokine in departing tumor cells, empowering these cells to disrupt lung capillary walls and seed pulmonary metastases.

The IBD-associated long noncoding RNA IFNG-AS1 regulates the balance between inflammatory and anti-inflammatory cytokine production after T-cell stimulation.

The inflammatory bowel diseases (IBD) are a complex set of chronic gastrointestinal inflammatory conditions arising from the interplay of genetic and environmental factors. This study focuses on noncoding RNA transcripts as potential mediators of IBD pathophysiology. One particular gene, interferon γ-antisense 1 (IFNG-AS1), has been consistently observed to be elevated in the intestinal mucosa of patients with actively inflamed IBD versus healthy controls. This study builds on these observations, demonstrating that the second splice variant is specifically altered, and this alteration even stratifies within inflamed patients. With the use of a CRISPR-based overexpression system, IFNG-AS1 was selectively overexpressed directly from its genomic loci in T cells. An unbiased mRNA array on these cells identified a large increase in many inflammatory cytokines and a decrease in anti-inflammatory cytokines after IFNG-AS1 overexpression. Media from T cells overexpressing IFNG-AS1 elicited an inflammatory signaling cascade in primary human peripheral blood mononuclear cells, suggesting the potential functional importance of IFNG-AS1 in IBD pathophysiology. The significance of these results is amplified by studies suggesting that a single-nucleotide polymorphism in IFNG-AS1, rs7134599, was associated with both subtypes of patients with IBD independently of race.NEW & NOTEWORTHY Long noncoding RNAs are an emerging field of inflammatory bowel disease (IBD) research. This study mechanistically analyzes the role of a commonly upregulated gene in IBD and shows IFNG-AS1 as a mediator of an inflammatory signaling cascade.

miR-24 Is Elevated in Ulcerative Colitis Patients and Regulates Intestinal Epithelial Barrier Function.

Inflammatory bowel disease is characterized by high levels of inflammation and loss of barrier integrity in the colon. The intestinal barrier is a dynamic network of proteins that encircle intestinal epithelial cells. miRNAs regulate protein-coding genes. In this study, miR-24 was found to be elevated in colonic biopsies and blood samples from ulcerative colitis (UC) patients compared with healthy controls. In the colon of UC patients, miR-24 is localized to intestinal epithelial cells, which prompted an investigation of intestinal epithelial barrier function. Two intestinal epithelial cell lines were used to study the effect of miR-24 overexpression on barrier integrity. Overexpression of miR-24 in both cell lines led to diminished transepithelial electrical resistance and increased dextran flux, suggesting an effect on barrier integrity. Overexpression of miR-24 did not induce apoptosis or affect cell proliferation, suggesting that the effect of miR-24 on barrier function was due to an effect on cell-cell junctions. Although the tight junctions in cells overexpressing miR-24 appeared normal, miR-24 overexpression led to a decrease in the tight junction-associated protein cingulin. Loss of cingulin compromised barrier formation; cingulin levels negatively correlated with disease severity in UC patients. Together, these data suggest that miR-24 is a significant regulator of intestinal barrier that may be important in the pathogenesis of UC.

MicroRNA-31-3p Is Involved in Substance P (SP)-Associated Inflammation in Human Colonic Epithelial Cells and Experimental Colitis.

Substance P (SP) mediates colitis. SP signaling regulates the expression of several miRNAs, including miR-31-3p, in human colonocytes. However, the role of miR-31-3p in colitis and the underlying mechanisms has not been elucidated. We performed real-time PCR analysis of miR-31-3p expression in human colonic epithelial cells overexpressing neurokinin-1 receptor (NCM460 NK-1R) in response to SP stimulation and in NCM460 cells after IL-6, IL8, tumor necrosis factor (TNF)-α, and interferon-γ exposure. Functions of miR-31-3p were tested in NCM460-NK-1R cells and the trinitrobenzene sulfonic acid (TNBS) and dextran sodium sulfate (DSS) models of colitis. Targets of miRNA-31-3p were confirmed by Western blot analysis and luciferase reporter assay. Jun N-terminal kinase inhibition decreased SP-induced miR-31-3p expression. miR-31-3p expression was increased in both TNBS- and DSS-induced colitis and human colonic biopsies from ulcerative colitis, compared with controls. Intracolonic administration of a miR-31-3p chemical inhibitor exacerbated TNBS- and DSS-induced colitis and increased colonic TNF-α, CXCL10, and chemokine (C-C motif) ligand 2 (CCL2) mRNA expression. Conversely, overexpression of miR-31-3p ameliorated the severity of DSS-induced colitis. Bioinformatic, luciferase reporter assay, and Western blot analyses identified RhoA as a target of miR-31-3p in NCM460 cells. Constitutive activation of RhoA led to increased expression of CCL2, IL6, TNF-α, and CXCL10 in NCM460-NK-1R cells on SP stimulation. Our results reveal a novel SP-miR-31-3p-RhoA pathway that protects from colitis. The use of miR-31-3p mimics may be a promising approach for colitis treatment.

Identification of novel mRNAs and lncRNAs associated with mouse experimental colitis and human inflammatory bowel disease.

Inflammatory bowel disease (IBD) is a complex disorder that is associated with significant morbidity. While many recent advances have been made with new diagnostic and therapeutic tools, a deeper understanding of its basic pathophysiology is needed to continue this trend toward improving treatments. By utilizing an unbiased, high-throughput transcriptomic analysis of two well-established mouse models of colitis, we set out to uncover novel coding and noncoding RNAs that are differentially expressed in the setting of colonic inflammation. RNA-seq analysis was performed using colonic tissue from two mouse models of colitis, a dextran sodium sulfate-induced model and a genetic-induced model in mice lacking IL-10. We identified 81 coding RNAs that were commonly altered in both experimental models. Of these coding RNAs, 12 of the human orthologs were differentially expressed in a transcriptomic analysis of IBD patients. Interestingly, 5 of the 12 of human differentially expressed genes have not been previously identified as IBD-associated genes, including ubiquitin D. Our analysis also identified 15 noncoding RNAs that were differentially expressed in either mouse model. Surprisingly, only three noncoding RNAs were commonly dysregulated in both of these models. The discovery of these new coding and noncoding RNAs expands our transcriptional knowledge of mouse models of IBD and offers additional targets to deepen our understanding of the pathophysiology of IBD. NEW & NOTEWORTHY Much of the genome is transcribed as non-protein-coding RNAs; however, their role in inflammatory bowel disease is largely unknown. This study represents the first of its kind to analyze the expression of long noncoding RNAs in two mouse models of inflammatory bowel disease and correlate them to human clinical samples. Using high-throughput RNA-seq analysis, we identified new coding and noncoding RNAs that were differentially expressed such as ubiquitin D and 5730437C11Rik.

Role of G protein-coupled receptors-microRNA interactions in gastrointestinal pathophysiology.

G protein-coupled receptors (GPCRs) make up the largest transmembrane receptor superfamily in the human genome and are expressed in nearly all gastrointestinal cell types. Coupling of GPCRs and their respective ligands activates various phosphotransferases in the cytoplasm, and, thus, activation of GPCR signaling in intestine regulates many cellular and physiological processes. Studies in microRNAs (miRNAs) demonstrate that they represent critical epigenetic regulators of different pathophysiological responses in different organs and cell types in humans and animals. Here, we reviewed recent research on GPCR-miRNA interactions related to gastrointestinal pathophysiology, such as inflammatory bowel diseases, irritable bowel syndrome, and gastrointestinal cancers. Given that the presence of different types of cells in the gastrointestinal tract suggests the importance of cell-cell interactions in maintaining gastrointestinal homeostasis, we also discuss how GPCR-miRNA interactions regulate gene expression at the cellular level and subsequently modulate gastrointestinal pathophysiology through molecular regulatory circuits and cell-cell interactions. These studies helped identify novel molecular pathways leading to the discovery of potential biomarkers for gastrointestinal diseases.

Gastrointestinal Manifestations of Hereditary Hemorrhagic Telangiectasia (HHT): A Systematic Review of the Literature.

Hereditary hemorrhagic telangiectasia (HHT), also called Osler-Weber-Rendu syndrome, is an autosomal dominant genetic disease that affects the vasculature of numerous organs. The prevalence of HHT is estimated to be between 1.5 and 2 persons per 10,000. While there is still much to learn about this condition, there is an increasing understanding its underlying pathophysiology, genetic basis, presentations, and management. Recognizing that the clinical manifestations of HHT can involve a number of organ systems will provide clinicians with a higher index of suspicion for the disease. This early diagnosis and genotyping can greatly reduce mortality for a patient with HHT through appropriate screening for complications. This review will focus on the gastrointestinal manifestations of HHT and how these can dictate treatment and prognosis.

A long noncoding RNA signature for ulcerative colitis identifies IFNG-AS1 as an enhancer of inflammation.

High-throughput technologies revealed new categories of genes, including the long noncoding RNAs (lncRNAs), involved in the pathogenesis of human disease; however, the role of lncRNAs in the ulcerative colitis (UC) has not been evaluated. Gene expression profiling was used to develop lncRNA signatures in UC samples. Jurkat T cells were activated by PMA/ionomycin subsequently interferon-γ (IFNG) and tumor necrosis factor (TNF)-α protein levels were assessed by ELISA. Anti-sense molecules were designed to block IFNG-AS1 expression. A unique set of lncRNAs was differentially expressed between UC and control samples. Of these, IFNG-AS1 was among the highest statistically significant lncRNAs (fold change: 5.27, P value: 7.07E-06). Bioinformatic analysis showed that IFNG-AS1 was associated with the IBD susceptibility loci SNP rs7134599 and its genomic location is adjacent to the inflammatory cytokine IFNG. In mouse models of colitis, active colitis samples had increased colonic expression of this lncRNA. Utilizing the Jurkat T cell model, we found IFNG-AS1 to positively regulate IFNG expression. Novel lncRNA signatures differentiate UC patients with active disease, patients in remission, and control subjects. A subset of these lncRNAs was found to be associated with the clinically validated IBD susceptibility loci. IFNG-AS1 was one of these differentially expressed lncRNAs in UC patients and found to regulate the key inflammatory cytokine, IFNG, in CD4 T cells. Taking these findings together, our study revealed novel lncRNA signatures deregulated in UC and identified IFNG-AS1 as a novel regulator of IFNG inflammatory responses, suggesting the potential importance of noncoding RNA mechanisms on regulation of inflammatory bowel disease-related inflammatory responses.

Altered brain-gut axis in autism: comorbidity or causative mechanisms?

The concept that alterated communications between the gut microbiome and the brain may play an important role in human brain disorders has recently received considerable attention. This is the result of provocative preclinical and some clinical evidence supporting early hypotheses about such communication in health and disease. Gastrointestinal symptoms are a common comorbidity in patients with autism spectrum disorders (ASD), even though the underlying mechanisms are largely unknown. In addition, alteration in the composition and metabolic products of the gut microbiome has long been implicated as a possible causative mechanism contributing to ASD pathophysiology, and this hypothesis has been supported by several recently published evidence from rodent models of autism induced by prenatal insults to the mother. Recent evidence in one such model involving maternal infection, that is characterized by alterations in behavior, gut physiology, microbial composition, and related metabolite profile, suggests a possible benefit of probiotic treatment on several of the observed abnormal behaviors.

Endogenous human microRNAs that suppress breast cancer metastasis.

A search for general regulators of cancer metastasis has yielded a set of microRNAs for which expression is specifically lost as human breast cancer cells develop metastatic potential. Here we show that restoring the expression of these microRNAs in malignant cells suppresses lung and bone metastasis by human cancer cells in vivo. Of these microRNAs, miR-126 restoration reduces overall tumour growth and proliferation, whereas miR-335 inhibits metastatic cell invasion. miR-335 regulates a set of genes whose collective expression in a large cohort of human tumours is associated with risk of distal metastasis. miR-335 suppresses metastasis and migration through targeting of the progenitor cell transcription factor SOX4 and extracellular matrix component tenascin C. Expression of miR-126 and miR-335 is lost in the majority of primary breast tumours from patients who relapse, and the loss of expression of either microRNA is associated with poor distal metastasis-free survival. miR-335 and miR-126 are thus identified as metastasis suppressor microRNAs in human breast cancer.

Loss of miR-24-3p promotes epithelial cell apoptosis and impairs the recovery from intestinal inflammation.

While apoptosis plays a significant role in intestinal homeostasis, it can also be pathogenic if overactive during recovery from inflammation. We recently reported that microRNA-24-3p (miR-24-3p) is elevated in the colonic epithelium of ulcerative colitis patients during active inflammation, and that it reduced apoptosis in vitro. However, its function during intestinal restitution following inflammation had not been examined. In this study, we tested the influence of miR-24-3p on mucosal repair by studying recovery from colitis in both novel miR-24-3p knockout and miR-24-3p-inhibited mice. We observed that knockout mice and mice treated with a miR-24-3p inhibitor had significantly worsened recovery based on weight loss, colon length, and double-blinded histological scoring. In vivo and in vitro analysis of miR-24-3p inhibition in colonic epithelial cells revealed that inhibition promotes apoptosis and increases levels of the pro-apoptotic protein BIM. Further experiments determined that silencing of BIM reversed the pro-apoptotic effects of miR-24-3p inhibition. Taken together, these data suggest that miR-24-3p restrains intestinal epithelial cell apoptosis by targeting BIM, and its loss of function is detrimental to epithelial restitution following intestinal inflammation.

Evaluating tofacitinib citrate in the treatment of moderate-to-severe active ulcerative colitis: design, development and positioning of therapy.

The etiology of ulcerative colitis (UC) is complex and involves a host of genetic, epigenetic and environmental factors. Over the last thirty years, signaling pathways like the Janus kinase (JAK) signaling pathway have been implicated in its pathogenesis. Pharmacologic blockade of this pathway is available through several small molecule inhibitors, including tofacitinib. Tofacitinib is an orally administered pan-JAK inhibitor that was first approved by the Food and Drug Administration (FDA) for use in rheumatologic disorders such as rheumatoid arthritis and psoriatic arthritis. The FDA approved its use in moderate-to-severe active ulcerative colitis in 2018. The aim of this review will be to discuss the role of tofacitinib in ulcerative colitis. We will discuss the role of JAK-STAT signaling, clinical data available for tofacitinib, and the safety profile for this therapy. Tofacitinib's place in the UC management algorithm is currently being debated. This effective oral therapy is poised to be a mainstay of UC therapeutics. This review will highlight the key clinical features and detail the UC experience to date.

Linear and circular CDKN2B-AS1 expression is associated with Inflammatory Bowel Disease and participates in intestinal barrier formation.

AIMS: The role of long non-coding RNA's (lncRNA) in the biology of ulcerative colitis (UC) is not well understood. We have previously detected changes in lncRNA's associated with UC. This study aims to characterize one specific lncRNA, CDKN2B-AS1 whose expression was downregulated in UC patients. MAIN METHODS: UC biopsies were used to determine the levels of linear and circular CDKN2B-AS1 relative to healthy controls. In situ hybridization was used to determine the localization of CKDN2B-AS1 in the colon. The intestinal epithelial cell line, Caco-2, was used to study the effects of shRNA mediated loss of CDKN2B-AS1. Transepithelial electrical resistance was used to measure barrier function. An RT-PCR array, immunoblots and immunohistochemistry were used to determine tight junction proteins that CDKN2B-AS1 regulates. KEY FINDINGS: CDKN2B-AS1 is transcribed into not only linear transcripts but also as circular RNA through back-splicing and both forms are decreased in IBD. CDKN2B-AS1 is expressed mainly in colonic epithelial cells. Cells with down-regulated CDKN2B-AS1 exhibited increased proliferation and no alterations in apoptosis. Targeting both the linear and circular transcripts of CDKN2B-AS1 with short hairpin RNAs enhanced barrier function. We subsequently determined that Claudin-2, a "leaky Claudin" known to decrease barrier function, was decreased in CDKN2B-AS1 knockdown cells. SIGNIFICANCE: This study identifies a novel lncRNA with both linear and circular transcripts affecting UC biology.

Overexpressing Long Noncoding RNAs Using Gene-activating CRISPR.

Long noncoding RNA (lncRNA) biology is a new and exciting field of research, with the number of publications from this field growing exponentially since 2007. These studies have confirmed that lncRNAs are altered in almost all diseases. However, studying the functional roles for lncRNAs in the context of disease remains difficult due to the lack of protein products, tissue-specific expression, low expression levels, complexities in splice forms, and lack of conservation among species. Given the species-specific expression, lncRNA studies are often restricted to human research contexts when studying disease processes. Since lncRNAs function at the molecular level, one way to dissect lncRNA biology is to either remove the lncRNA or overexpress the lncRNA and measure cellular effects. In this article, a written and visualized protocol to overexpress lncRNAs in vitro is presented. As a representative experiment, an lncRNA associated with inflammatory bowel disease, Interferon Gamma Antisense 1 (IFNG-AS1), is shown to be overexpressed in a Jurkat T-cell model. To accomplish this, the activating clustered regularly interspaced short palindromic repeats (CRISPR) technique is used to enable overexpression at the endogenous genomic loci. The activating CRISPR technique targets a set of transcription factors to the transcriptional start site of a gene, enabling a robust overexpression of multiple lncRNA splice forms. This procedure will be broken down into three steps, namely (i) guide RNA (gRNA) design and vector construction, (ii) virus generation and transduction, and (iii) colony screening for overexpression. For this representative experiment, a greater than 20-fold enhancement in IFNG-AS1 in Jurkat T cells was observed.

Novel Lipid Long Intervening Noncoding RNA, Oligodendrocyte Maturation-Associated Long Intergenic Noncoding RNA, Regulates the Liver Steatosis Gene Stearoyl-Coenzyme A Desaturase As an Enhancer RNA.

The global obesity epidemic is driving the concomitant rise in nonalcoholic fatty liver disease (NAFLD). To identify new genes involved in central liver functions, we examined liver RNA-sequence data from 259 patients who underwent morbidly obese bariatric surgery. Of these patients, 84 had normal liver histology, 40 simple steatosis, 43 nonalcoholic steatohepatitis, and the remaining 92 patients had varying degrees of NAFLD based on liver histology. We discovered oligodendrocyte maturation-associated long intergenic noncoding RNA (OLMALINC), a long intervening noncoding RNA (lincRNA) in a human liver co-expression network (n = 75 genes) that was strongly associated with statin use and serum triglycerides (TGs). OLMALINC liver expression was highly correlated with the expression of known cholesterol biosynthesis genes and stearoyl-coenzyme A desaturase (SCD). SCD is the rate-limiting enzyme in monounsaturated fatty acids and a key TG gene that is known to be up-regulated in liver steatosis and NAFLD and resides adjacent to OLMALINC on the human chromosome 10q24.31. Next, we functionally demonstrated that OLMALINC regulates SCD as an enhancer-RNA (eRNA), thus describing the first lincRNA that functions as an eRNA to regulate lipid metabolism. Specifically, we show that OLMALINC promotes liver expression of SCD in cis through regional chromosomal DNA-DNA looping interactions. Conclusion: The primate-specific lincRNA OLMALINC is a novel epigenetic regulator of the key TG and NAFLD gene SCD.

A Challenging Case of Severe Ulcerative Colitis following the Initiation of Secukinumab for Ankylosing Spondylitis.

Secukinumab is an interleukin-17 inhibitor used for the treatment of ankylosing spondylitis (AS), psoriasis, and psoriatic arthritis. The risk of exacerbating underlying inflammatory bowel disease (IBD) in patients being treated with secukinumab for other conditions is controversial. We document a patient with AS and previously undiagnosed IBD, found to be in a severe ulcerative colitis flare shortly after receiving the loading dose of secukinumab. There are no guidelines regarding biologic salvage therapy for IBD in the setting of active treatment with another biologic agent. After waiting one half-life of secukinumab, our patient had an excellent response to initiation of infliximab.

Osteoid osteoma of the distal clavicle.

The osteoid osteoma is a bone tumor that accounts for 10% of benign tumors. It was described in 1935 by Jaffe, as a tumor that affects the young adult population, with a predominance of males. This study aims to present a case of late diagnosis of a patient with osteoid osteoma of the distal clavicle region. Female patient, 44 years old, non-professional volleyball player, reported pain in the anterior and superior region of the shoulder girdle, specifically in the acromioclavicular joint, which worsened at night and had been treated for nine months as tendinitis of the rotator cuff and acromioclavicular joint arthritis. After confirming the diagnosis, the patient underwent open surgery with resection of the distal clavicle. At two years of follow-up, the patient presents without local pain. In the radiographic evaluation, coracoclavicular distance is preserved and there are no signs of recurrence. Tumors of the shoulder girdle are rare and are often diagnosed late. A high degree of suspicion for the diagnosis of tumors of the shoulder girdle is needed in order to avoid late diagnosis.

O osteoma osteóide é um tumor ósseo que corresponde a 10% dos tumores benignos. Foi descrito em 1935 por Jaffe, como um tumor que acomete a população adulta jovem, com predominância no sexo masculino. O objetivo do trabalho é apresentar um caso de diagnóstico tardio de uma paciente com osteoma osteóide da região da clavícula distal e relatar seu tratamento. Paciente de 44 anos, jogadora de vôlei não profissional, com dores na região anterior e superior da cintura escapular, mais especificamente na articulação acromioclavicular, as quais pioravam a noite e que era tratada havia nove meses como uma tendinite do manguito rotador e artrite da articulação acromioclavicular. Após confirmação diagnóstica, a paciente foi submetida ao tratamento cirúrgico aberto com ressecção da clavícula distal.Atualmente a paciente encontra-se com dois anos de evolução sem dor local. Na avaliação radiográfica, a distância coracoclavicular encontra-se preservada e não há sinais de recidiva. Tumores ósseos da cintura escapular são raros e frequentemente são diagnosticados tardiamente. Deve-se ter um alto grau de suspeição para o diagnóstico de neoplasias da cintura escapular, a fim de evitar o diagnóstico tardio.

Novel approaches to treating Clostridium difficile-associated colitis.

Clostridium difficile is being recognized as a growing threat to many health-care systems. Epidemiology data shows that infection rates are soaring and the disease burden is increasing. Despite the efficacy of standard treatments, it is becoming evident that novel therapeutics will be required to tackle this disease. These new treatments aim to enhance the intestinal microbial barrier, activate the immune system and neutralize the toxins that mediate this disease. Many of these therapies are still in the beginning stages of investigation, however, in the next few years, more clinical data will become available to help implement many of these exciting new therapeutic approaches.