Mechanical Stress-induced Connective Tissue Growth Factor Plays a Critical Role in Intestinal Fibrosis in Crohn's-like Colitis.

Crohn's disease (CD) is an inflammatory bowel disease characterized by transmural inflammation and intestinal fibrosis. Mechanisms of fibrosis in CD are not well understood. Transmural inflammation is associated with inflammatory cell infiltration, stenosis, and distention, which present mechanical stress (MS) to the bowel wall. We hypothesize that MS induces gene expression of pro-fibrotic mediators such as connective tissue growth factor (CTGF), which may contribute to fibrosis in CD. A rodent model of CD was induced by intracolonic instillation of TNBS to the distal colon. TNBS instillation induced a localized transmural inflammation (site I), with a distended colon segment (site P) proximal to site I. We detected significant fibrosis and collagen content not only in site I, but also in site P in CD rats by day 7. CTGF expression increased significantly in sites P and I, but not in the segment distal to the inflammation site. Increased CTGF expression was detected mainly in the smooth muscle cells (SMC). When rats were fed exclusively with clear liquid diet to prevent mechanical distention in colitis, expression of CTGF in sites P and I was blocked. Direct stretch led to robust expression of CTGF in colonic SMC. Treatment of CD rats with anti-CTGF antibody FG-3149 reduced fibrosis and collagen content in both sites P and I and exhibited consistent trends towards normalizing expression of collagen mRNAs. In conclusion, our studies suggest that mechanical stress, by up-regulating pro-fibrotic mediators i.e. CTGF, may play a critical role in fibrosis in CD.

GPR120 Inhibits Colitis Through Regulation of CD4+ T Cell Interleukin 10 Production.

BACKGROUND & AIMS: G protein-coupled receptor (GPR) 120 has been implicated in regulating metabolic syndromes with anti-inflammatory function. However, the role of GPR120 in intestinal inflammation is unknown. Here, we investigated whether and how GPR120 regulates CD4+ T cell function to inhibit colitis development. METHODS: Dextran sodium sulfate (DSS)-induced colitis model, Citrobacter rodentium infection model, and CD4+ T cell adoptive transfer model were used to analyze the role of GPR120 in regulating colitis development. The effect of GPR120 on CD4+ T cell functions was analyzed by RNA sequencing, flow cytometry, and Seahorse metabolic assays. Mice were administered GPR120 agonist for investigating the potential of GPR120 agonist in preventing and treating colitis. RESULTS: Deficiency of GPR120 in CD4+ T cells resulted in more severe colitis in mice upon dextran sodium sulfate insult and enteric infection. Transfer of GPR120-deficient CD4+CD45Rbhi T cells induced more severe colitis in Rag-/- mice with lower intestinal interleukin (IL) 10+CD4+ T cells. Treatment with the GPR120 agonist CpdA promoted CD4+ T cell production of IL10 by up-regulating Blimp1 and enhancing glycolysis, which was regulated by mTOR. GPR120 agonist-treated wild-type, but not IL10-deficient and Blimp1-deficient, T helper 1 cells induced less severe colitis. Furthermore, oral administration of GPR120 agonist protected mice from intestinal inflammation in both prevention and treatment schemes. Gpr120 expression was positively correlated with Il10 expression in the human colonic mucosa, including patients with inflammatory bowel diseases. CONCLUSIONS: Our findings show the role of GPR120 in regulating intestinal CD4+ T cell production of IL10 to inhibit colitis development, which identifies GPR120 as a potential therapeutic target for treating inflammatory bowel diseases.

Restarting elective endoscopy safely amidst an evolving pandemic and the impact of patient perception.

BACKGROUND: The COVID-19 pandemic has led to disruptions in elective and outpatient procedures. Guidance from the Centers for Medicare and Medicaid Services provided a framework for gradual reopening of outpatient clinical operations. As the infrastructure to restart endoscopy has been more clearly described, patient concerns regarding viral transmission during the procedure have been identified. Moreover, the efficacy of the measures in preventing transmission have not been clearly delineated. METHODS: We identified patients with pandemic-related procedure cancellations from 3/16/2020 to 4/20/2020. Patients were stratified into tier groups (1-4) by urgency. Procedures were performed using our hospital risk mitigation strategies to minimize transmission risk. Patients who subsequently developed symptoms or tested for COVID-19 were recorded. RESULTS: Among patients requiring emergent procedures, 57.14% could be scheduled at their originally intended interval. COVID-19 concerns represented the most common rescheduling barrier. No patients who underwent post-procedure testing were positive for COVID-19. No cases of endoscopy staff transmission were identified. CONCLUSIONS: Non-COVID-19 related patient care during the pandemic is a challenging process that evolved with the spread of infection, requiring dynamic monitoring and protocol optimization. We describe our successful model for reopening endoscopy suites using a tier-based system for safe reintroduction of elective procedures while minimizing transmission to patients and staff. Important barriers included financial and transmission concerns that need to be addressed to enable the return to pre-pandemic utilization of elective endoscopic procedures.

Preoperative Evaluation for Noncardiac Surgery.

This issue provides a clinical overview of preoperative evaluation for noncardiac surgery, focusing on risk factors, elements of evaluation, medication management, and practice improvement. The content of In the Clinic is drawn from the clinical information and education resources of the American College of Physicians (ACP), including MKSAP (Medical Knowledge and Self-Assessment Program). Annals of Internal Medicine editors develop In the Clinic in collaboration with the ACP's Medical Education and Publishing divisions and with the assistance of additional science writers and physician writers.

Multi-mode acquisition (MMA): An MS/MS acquisition strategy for maximizing selectivity, specificity and sensitivity of DIA product ion spectra.

In proteomics studies, it is generally accepted that depth of coverage and dynamic range is limited in data-directed acquisitions. The serial nature of the method limits both sensitivity and the number of precursor ions that can be sampled. To that end, a number of data-independent acquisition (DIA) strategies have been introduced with these methods, for the most part, immune to the sampling issue; nevertheless, some do have other limitations with respect to sensitivity. The major limitation with DIA approaches is interference, i.e., MS/MS spectra are highly chimeric and often incapable of being identified using conventional database search engines. Utilizing each available dimension of separation prior to ion detection, we present a new multi-mode acquisition (MMA) strategy multiplexing both narrowband and wideband DIA acquisitions in a single analytical workflow. The iterative nature of the MMA workflow limits the adverse effects of interference with minimal loss in sensitivity. Qualitative identification can be performed by selected ion chromatograms or conventional database search strategies.

Exclusive Enteral Nutrition Alleviates Th17-Mediated Inflammation via Eliminating Mechanical Stress-Induced Th17-Polarizing Cytokines in Crohn's-like Colitis.

BACKGROUND AND AIMS: Exclusive enteral nutrition (EEN) with a liquid diet is the only established dietary treatment for Crohn's' disease (CD). However, the mechanism of action of EEN in CD is unclear. T helper 17 (Th17) immune response plays a critical role in CD. We hypothesized that EEN alleviates Th17 response by eliminating mechanical stress-induced expression of Th17-polarizing cytokines. METHODS: A rat model of Crohn's-like colitis was established by intracolonic instillation of TNBS (65 mg/kg in 250 µL of 40% ethanol). Control rats were treated with saline. We characterized immunophenotypes and molecular changes of the colon in control and colitis rats with and without EEN treatment. Th17 differentiation was determined using coculture assays. RESULTS: TNBS instillation induced transmural inflammation with stenosis in the inflammation site and a marked increase of Th17-polarizing cytokines interleukin (IL)-6 and osteopontin and the Th17 cell population in the mechanically distended preinflammation site (P-site). EEN treatment eliminated mechanical distention and the increase of IL-6, osteopontin, and Th17 response in the P-site. IL-6 and osteopontin expression was found mainly in the muscularis externa. Mechanical stretch of colonic smooth muscle cells in vitro induced a robust increase of IL-6 and osteopontin. When naïve T cells were cultured with conditioned media from the P-site tissue or stretched cells, Th17 differentiation was significantly increased. Inhibition of IL-6, but not deletion of osteopontin, blocked the increase of Th17 differentiation. CONCLUSIONS: Mechanical stress induces Th17-polarizing cytokines in the colon. EEN attenuates Th17 immune response by eliminating mechanical stress-induced IL-6 in Crohn's-like colitis.

Malignant Tumors Identified in Adult Polycystic Kidney Disease Can Be Derived from Both Proximal Tubular and Distal Tubular Origins.

Adult polycystic kidney disease (APKD) is a genetic disorder leading to premature renal dysfunction and failure. The prevalence of malignant renal tumors occurring in the APKD setting has been rarely reported. OBJECTIVE: To better characterize malignant renal tumors in nephrectomy specimens of APKD and apply modern pathologic evaluation. METHODS: We reviewed our database of APKD specimens over the past 11 years (from 2012 to 2023) for primary malignant tumors within the kidneys of APKD. RESULTS: Of 48 nephrectomy specimens with APKD evaluated, 10 malignant renal tumors were identified, indicating a prevalence of 20.8 % (10/48). These included three clear cell (cc) renal cell carcinomas (RCC) (ranging from 1 mm to 6.7 cm), three papillary RCCs (2.5, 3.5, and 14 cm with lymph node metastasis), two cases of clear cell papillary (CCP) RCC, one acquired cystic disease (ACD) with associated RCC (4 mm), and one urothelial adenocarcinoma. The urothelial adenocarcinoma was found near a tubulovillous adenoma in a collecting duct and stained positively for GATA3 and Uroplakin-2 but was negative for PAX8 & CDX2. The tumor showed extensive invasion into perirenal fatty tissue and the rectum. Next generating sequencing (NGS) analysis of the tumor showed mutations in TERT, RB1, TP53, ERBB2, and TET1 genes, further supporting its urothelial origin. CONCLUSIONS: We found a prevalence of 20.8%, which was higher than in previous reports of malignant renal tumors in patients who underwent resections for APKD. Renal tumors were mostly from damaged proximal tubular origins (clear cell or papillary RCC), but less commonly were from distal tubular or urothelial cells as well (clear cell papillary RCC and urothelial adenocarcinoma).

Primary cilia metaplasia in renal transplant biopsies with acute tubular injury.

Primary cilia are hair-like organelles singly distributed along the apical surface of proximal and distal nephron tubules as mechanosensors. The goal of this study was to use electron microscopy to systemically evaluate cilia changes in acute tubular injury (ATI) from both transplant and native renal biopsies. Three groups of cases were included: control group 1-native biopsies without major changes in renal tubules; study group 2-native biopsies with prominent ATI; and study group 3-renal transplant biopsies with prominent ATI (delayed renal function group). Extensive search for ciliary structures along renal tubules was conducted in each case, focused on proximal tubular areas with injured (diminished) apical microvilli. Singly located cilia were found in 3/19 specimens in control group 1, 4/18 in group 2 (native ATI), and 6/24 in group 3 (transplant ATI). Importantly, there were clusters of cilia in proximal tubules with markedly diminished apical microvilli in 3/24 biopsies from 2 patients in group 3, but none from groups 1 and 2. The clusters of cilia ranged from 6 to 15 individual cilia along the apical surface with diminished apical microvilli. Under high magnifications, the cilia demonstrated 9 pairs of peripheral microtubules without a central pair of microtubules, consistent with primary cilia (9 + 0) rather than motile cilia (9 + 2). In summary, the authors found clusters of cilia in proximal tubules with remarkable apical microvillar injury in 3 renal transplant biopsies with ATI, implying a reactive, or repairing, process following tubular injury, thus they name this finding "cilia metaplasia".

Myeloperoxidase Staining Identifies Endothelial Injury of Peritubular Capillaries and Glomeruli in Renal Antibody-Mediated Rejection.

OBJECTIVE: It remains unclear if C4d staining is related to any peritubular and glomerular injury during antibody mediated rejection (ABMR). The goal of this study was to determine if myeloperoxidase (MPO) staining can highlight endothelial injury in peritubular capillaries (PTC) and glomeruli. METHODS: The study included 12 native negative controls, 19 transplant biopsies with borderline changes (BC) as transplant controls, and one group of renal transplant biopsies with ABMR as the study group (acute/chronic, n=22). All three groups were stained for MPO immunohistochemically, and the MPO expressions in the endothelium of PTC and glomeruli were evaluated and correlated with serum creatinine (SCr). In addition, the ultrastructural layers of the PTC (an index for chronic allograft rejection) were correlated with MPO indices in PTC. RESULTS: The negative control group and the transplant controls showed no MPO expression in the endothelium of glomeruli and PTC. However, in the biopsies with ABMR, there were MPO-positive stains in the endothelial cells of glomeruli (15/21 cases, 71.4 %) and PTC (16/22 cases, 72.7 %). There were significant correlations between the peritubular MPO staining versus SCr (r=0.355 and p=0.0106) and glomerular MPO staining versus SCr (r=0.365 and p=0.0092). Furthermore, the layers of PTC by electron microscopy were significantly correlated with MPO scores in PTC (r=0.696, p=0.0001). CONCLUSION: Our data suggest that the MPO-positive endothelial injuries are most likely the cause leading to renal graft dysfunction following ABMR.

Fibroblast growth factor receptor-3 (FGFR-3) regulates expression of paneth cell lineage-specific genes in intestinal epithelial cells through both TCF4/beta-catenin-dependent and -independent signaling pathways.

Fibroblast growth factor receptor-3 (FGFR-3) expression in the developing intestine is restricted to the undifferentiated epithelial cells within the lower portion of the crypt. We previously showed that mice lacking functional FGFR-3 have a significant decrease in the number of Paneth cells in the small intestine. Here, we used Caco2 cells to investigate whether FGFR-3 signaling can directly modulate expression of Paneth cell differentiation markers through its effects on TCF4/ß-catenin or through other signaling pathways downstream of this receptor. Caco2 cells treated with FGFR-3 ligands or expressing FGFR-3(K650E), a constitutively active mutant, resulted in a significantly increased expression of genes characteristic of mature Paneth cells, including human α-defensins 5 and 6 (HD5 and HD6) and Paneth cell lysozyme, whereas enterocytic differentiation markers were reduced. Activation of FGFR-3 signaling sustained high levels of ß-catenin mRNA expression, leading to increased TCF4/ß-catenin-regulated transcriptional activity in Caco2 cells. Sustained activity of the TCF4/ß-catenin pathway was required for the induction of Paneth cell markers. Activation of the MAPK pathway by FGFR-3 is also required for the induction of Paneth cell markers in addition to and independent of the effect of FGFR-3 on TCF4/ß-catenin activity. These studies suggest that coordinate activation of multiple independent signaling pathways downstream of FGFR-3 is involved in regulation of Paneth cell differentiation.

The primary defect in experimental ileitis originates from a nonhematopoietic source.

The initiating etiologic factor in Crohn's disease (CD) remains unclear. SAMP1/YitFc (SAMP) mice develop chronic ileitis similar to human CD. We used bone marrow chimeras to determine if SAMP ileitis results from a primary immunological defect or from dysregulated mucosal immunity secondary to intrinsic, nonhematopoietic (e.g., epithelial) dysfunction. SAMP mice receiving wild-type (AKR) BM developed severe ileitis, whereas SAMP BM did not confer ileitis to WT recipients. WT lymphocytes from reconstituted SAMP mice resembled native SAMP populations in regard to surface phenotype and cytokine production. Ilea from native SAMP mice and SAMP recipients of wild-type BM displayed decreased epithelial barrier resistance ex vivo and increased epithelial permeability in vivo compared to native WT mice and AKR recipients of SAMP BM. This permeability defect preceded the development of ileal inflammation, was present in the absence of commensal bacteria, and was accompanied by altered ileal mRNA expression of the tight junction proteins claudin-2 and occludin. Our results provide evidence that the primary defect conferring ileitis in SAMP mice originates from a nonhematopoietic source. Generation of pathogenic lymphocytes is a consequence of this defect and does not reflect intrinsic proinflammatory leukocyte properties. Decreased barrier function suggests that defects in the epithelium may represent the primary source of SAMP ileitis susceptibility.

Focal Segmental Glomerulosclerosis (FSGS) Progressing to Collapsing Glomerulopathy in Renal Transplant Recipients With and Without COVID-19 Infection.

BACKGROUND: Collapsing glomerulopathy (CGN) secondary to HIV or COVID-19 infection mainly occurs in patients of African American descent due to APOL-1 gene mutations, but CGN is occasionally reported in white patients. CGNs are rarely reported in renal transplant biopsies and their association with idiopathic focal segmental glomerulosclerosis (FSGS) is unclear. METHODS AND RESULTS: Patient #1 was a 48-year-old Caucasian white man who had a renal transplant 8 years ago and was recently diagnosed with COVID-19 infection. Two weeks post infection, his serum creatinine (SCr) increased to 2.01 mg/dL from a baseline of 1.40 mg/dL, and he developed concomitant nephrotic range proteinuria. The first renal transplant biopsy showed FSGS. Four weeks later, his sCr level increased to 2.65 mg/dL with worsening proteinuria, and a second renal transplant biopsy revealed CGN. Patient #2 was a 32-year-old African American man whose native renal biopsy revealed primary FSGS. He received a renal transplant with initial post-transplant sCr level at 1.17 mg/dL. Four months later, his sCr and protein-to-creatinine ratio began to rise. Sequential biopsies revealed that the patient had developed recurrent FSGS, which progressed to show features of CGN. The CGN was further confirmed in his transplant kidney graft at autopsy later. CONCLUSIONS: This is the first case report of CGN in a white renal recipient with COVID-19 infection. The pathologic presentations of FSGS progressing to collapsing FSGS in our 2 renal transplant recipients suggest that FSGS and GGN may share a common pathophysiologic mechanism of podocytopathy.

Elevated expression of Paneth cell CRS4C in ileitis-prone SAMP1/YitFc mice: regional distribution, subcellular localization, and mechanism of action.

Paneth cells at the base of small intestinal crypts of Lieberkühn secrete host defense peptides and proteins, including alpha-defensins, as mediators of innate immunity. Mouse Paneth cells also express alpha-defensin-related Defcr-rs genes that code for cysteine-rich sequence 4C (CRS4C) peptides that have a unique CPX triplet repeat motif. In ileitis-prone SAMP1/YitFc mice, Paneth cell levels of CRS4C mRNAs and peptides are induced more than a 1000-fold relative to non-prone strains as early as 4 weeks of age, with the mRNA and peptide levels highest in distal ileum and below detection in duodenum. CRS4C-1 peptides are found exclusively in Paneth cells where they occur only in dense core granules and thus are secreted to function in the intestinal lumen. CRS4C bactericidal peptide activity is membrane-disruptive in that it permeabilizes Escherichia coli and induces rapid microbial cell K(+) efflux, but in a manner different from mouse alpha-defensin cryptdin-4. In in vitro studies, inactive pro-CRS4C-1 is converted to bactericidal CRS4C-1 peptide by matrix metalloproteinase-7 (MMP-7) proteolysis of the precursor proregion at the same residue positions that MMP-7 activates mouse pro-alpha-defensins. The absence of processed CRS4C in protein extracts of MMP-7-null mouse ileum demonstrates the in vivo requirement for intracellular MMP-7 in pro-CRS4C processing.

Epithelial cell apoptosis facilitates Entamoeba histolytica infection in the gut.

Entamoeba histolytica is the protozoan parasite that causes amebic colitis. The parasite triggers apoptosis on contact with host cells; however, the biological significance of this event during intestinal infection is unclear. We examined the role of apoptosis in a mouse model of intestinal amebiasis. Histopathology revealed that abundant epithelial cell apoptosis occurred in the vicinity of amoeba in histological specimens. Epithelial cell apoptosis occurred rapidly on co-culture with amoeba in vitro as measured by annexin positivity, DNA degradation, and mitochondrial dysfunction. Administration of the pan caspase inhibitor ZVAD decreased the rate and severity of amebic infection in CBA mice by all measures (cecal culture positivity, parasite enzyme-linked immunosorbent assay, and histological scores). Similarly, caspase 3 knockout mice on the resistant C57BL/6 background exhibited even lower cecal parasite antigen burden and culture positive rates than wild type mice. The permissive effect of apoptosis on infection could be tracked to the epithelium, in that transgenic mice that overexpressed Bcl-2 in epithelial cells were more resistant to infection as measured by cecal parasite enzyme-linked immunosorbent assay and histological scores. We concluded that epithelial cell apoptosis in the intestine facilitates amebic infection in this mouse model. The parasite's strategy for inducing apoptosis may point to key virulence factors, and therapeutic maneuvers to diminish epithelial apoptosis may be useful in amebic colitis.

Update in perioperative cardiac medicine 2021.

Several studies published in the last year have shed light on the preoperative assessment of perioperative cardiovascular risk and on the need for anticoagulation in patients with postoperative atrial fibrillation, which are reviewed here.

The promoter for intestinal cell kinase is head-to-head with F-Box 9 and contains functional sites for TCF7L2 and FOXA factors.

BACKGROUND: Intestinal cell kinase (ICK; GeneID 22858) is a conserved MAPK and CDK-like kinase that is widely expressed in human tissues. Data from the Cancer Genome Anatomy Project indicated ICK mRNA is increased in cancer, and that its expression correlated with expression of mRNA for an uncharacterized F-box protein, FBX9 (GeneID: 26268). ICK and FBX9 genes are arranged head-to-head on opposite strands, with start sites for transcription separated by approximately 3.3 kb. We hypothesized ICK and FBX9 are potentially important genes in cancer controlled by a bidirectional promoter. RESULTS: We assessed promoter activity of the intergenic region in both orientations in cancer cell lines derived from breast (AU565, SKBR3), colon (HCT-15, KM12), and stomach (AGS) cancers, as well as in embryonic human kidney (HEK293T) cells. The intergenic segment was active in both orientations in all of these lines, and ICK promoter activity was greater than FBX9 promoter activity. Results from deletions and truncations defined a minimal promoter for ICK, and revealed that repressors and enhancers differentially regulate ICK versus FBX9 promoter activity. The ICK promoter contains consensus motifs for several FOX-family transcription factors that align when mouse and human are compared using EMBOSS. FOXA1 and FOXA2 increase luciferase activity of a minimal promoter 10-20 fold in HEK293T cells. Consensus sites for TCF7L2 (TCF4) (Gene Id: 6934) are also present in both mouse and human. The expression of beta-catenin increased activity of the minimal promoter approximately 10 fold. ICK reference mRNAs (NM_014920.3, NM_016513) are expressed in low copy number and increased in some breast cancers, using a ten base tag 5'-TCAACCTTAT-3' specific for both ICK transcripts. CONCLUSION: ICK and FBX9 are divergently transcribed from a bidirectional promoter that is GC-rich and contains a CpG island. A minimal promoter for ICK contains functional sites for beta-catenin/TCF7L2 and FOXA. These data are consistent with functions that have been proposed for ICK in development and in proliferation or survival of some breast and colon cancers.