Oncostatin M Induces a Pro-inflammatory Phenotype in Intestinal Subepithelial Myofibroblasts.

BACKGROUND: Oncostatin-M (OSM) is associated with antitumor necrosis factor (anti-TNF)-α resistance in inflammatory bowel disease (IBD) and fibrosis in inflammatory diseases. We studied the expression of OSM and its receptors (OSMR, gp130) on intestinal subepithelial myofibroblasts (SEMFs) and the effect of OSM stimulation on SEMFs. METHODS: The mRNA and protein expression of OSM, OSMR, gp130, and several fibrotic and chemotactic factors were studied in mucosal biopsies and isolated human intestinal SEMFs of patients with IBD and healthy controls (HCs) and in a model of human intestinal organoids (HIOs). Subepithelial myofibroblasts and HIOs were stimulated with OSM and interleukin (IL)-1α/TNF-α. RNAseq data of mucosal biopsies were also analyzed. RESULTS: Oncostatin-M receptors and gp130 were overexpressed in mucosal biopsies of patients with IBD (P < .05), especially in inflamed segments (P < .05). The expression of OSM, OSMR, and gp130 in SEMFs from HCs was increased after stimulation with IL-1α/TNF-α (P < .001; P < .01; P < .01). The expression of CCL2, CXCL9, CXCL10, and CXCL11 was increased in SEMFs from patients with IBD and HCs after stimulation with OSM in a dose-dependent manner (P < .001; P < .05; P < .001; P < .001) and was further increased after prestimulation with IL-1α/TNF-α (P < .01 vs OSM-alone). Similar results were yielded after stimulation of HIOs (P < .01). Oncostatin-M did not induce the expression of collagen I, III, and fibronectin. Oncostatin-M receptor expression was positively correlated with CCL2, CXCL9, CXCL10, and CXCL11 expression in mucosal biopsies (P < .001; P < .001; P = .045; P = .033). CONCLUSIONS: Human SEMFs overexpress OSMR in an inflammatory microenvironment. Oncostatin-M may promote inflammation in IBD via its stimulatory effects on SEMFs, which primarily involve chemoattraction of immune cells to the intestinal mucosa.

Oncostatin-M/OSMR show elevated expression on intestinal fibroblasts that is regulated by IBD-relevant pro-inflammatory stimuli. In turn, OSM induces a pro-inflammatory phenotype on primary intestinal fibroblasts, with prominent overexpression of chemotactic factors, without demonstrating a substantial profibrotic effect.

Intestinal Stromal Cells in the Turmoil of Inflammation and Defective Connective Tissue Remodeling in Inflammatory Bowel Disease.

In steady state, intestinal subepithelial myofibroblasts form a thin layer below the basement membrane. Unlike the rest of the stromal cells in the lamina propria, they express tensile proteins, guide epithelial regeneration, and sense luminal microbiota. Upon inflammation in inflammatory bowel disease (IBD), they express activation markers, accept trophic signaling by infiltrating neutrophils and macrophages, and are activated by cytokines from helper T cells to produce a narrow spectrum of cytokines and a wider spectrum of chemokines, attract cells of innate and adaptive immunity, orchestrate inflammatory responses, and qualitatively and quantitatively modify the extracellular matrix. Thus, beyond being structural tissue components, they assume active roles in the pathogenesis of complicated IBD. Discrimination between myofibroblasts and fibroblasts may be an oversimplification in light of single-cell sequencing data unveiling the complexity of multiple phenotypes of stromal cells with distinct roles and plasticity. Spatial transcriptomics revealed distinct phenotypes by histologic localization and, more intriguingly, the assembly of mucosal neighborhoods that support spatially distinct functions. Current IBD treatments target inflammation but fail in fibrostenotic or fistulizing disease. Baseline and recent findings on stromal cells, molecules, and pathways involved in disrupted extracellular matrix homeostasis are reviewed to provide relevant pharmacologic targets.

Single-cell sequencing and spatial transcriptomics are now dissecting intestinal stromal cells into multiple phenotypes with distinct roles, in crosstalk with neighboring or infiltrating cells. Pathways involved in disrupted extracellular matrix homeostasis are reviewed to provide relevant pharmacologic targets.

Exploring Microbial Metabolite Receptors in Inflammatory Bowel Disease: An In Silico Analysis of Their Potential Role in Inflammation and Fibrosis.

Metabolites produced by dysbiotic intestinal microbiota can influence disease pathophysiology by participating in ligand-receptor interactions. Our aim was to investigate the differential expression of metabolite receptor (MR) genes between inflammatory bowel disease (IBD), healthy individuals (HIs), and disease controls in order to identify possible interactions with inflammatory and fibrotic pathways in the intestine. RNA-sequencing datasets containing 643 Crohn's disease (CD) patients, 467 ulcerative colitis (UC) patients and 295 HIs, and 4 Campylobacter jejuni-infected individuals were retrieved from the Sequence Read Archive, and differential expression was performed using the RaNA-seq online platform. The identified differentially expressed MR genes were used for correlation analysis with up- and downregulated genes in IBD, as well as functional enrichment analysis using a R based pipeline. Overall, 15 MR genes exhibited dysregulated expression in IBD. In inflamed CD, the hydroxycarboxylic acid receptors 2 and 3 (HCAR2, HCAR3) were upregulated and were associated with the recruitment of innate immune cells, while, in the non-inflamed CD ileum, the cannabinoid receptor 1 (CNR1) and the sphingosine-1-phospate receptor 4 (S1PR4) were downregulated and were involved in the regulation of B-cell activation. In inflamed UC, the upregulated receptors HCAR2 and HCAR3 were more closely associated with the process of TH-17 cell differentiation, while the pregnane X receptor (NR1I2) and the transient receptor potential vanilloid 1 (TRPV1) were downregulated and were involved in epithelial barrier maintenance. Our results elucidate the landscape of metabolite receptor expression in IBD, highlighting associations with disease-related functions that could guide the development of new targeted therapies.

Rates of Adverse Events in Patients With Ulcerative Colitis Undergoing Colectomy During Treatment With Tofacitinib vs Biologics: A Multicenter Observational Study.

INTRODUCTION: Patients with ulcerative colitis (UC) receiving immunosuppressive drugs are at substantial risk of colectomy. We aimed to assess the risk of postoperative complications of tofacitinib exposure before colectomy in comparison with biologics. METHODS: A multicenter, retrospective, observational study was conducted in patients with UC who underwent total colectomy for medically refractory disease, exposed to tofacitinib or a biologic before surgery. Primary outcome was the occurrence of any complication within 30 (early) and 90 (late) days after surgery. Secondary outcomes were the occurrence of infections, sepsis, surgical site complications, venous thromboembolic events (VTE), hospital readmissions, and redo surgery within the same timepoints. RESULTS: Three hundred one patients (64 tofacitinib, 162 anti-tumor necrosis factor-α agents, 54 vedolizumab, and 21 ustekinumab) were included. No significant differences were reported in any outcome, except for a higher rate of early VTE with anti-tumor necrosis factor-α agents ( P = 0.047) and of late VTE with vedolizumab ( P = 0.03). In the multivariate analysis, drug class was not associated with a higher risk of any early and late complications. Urgent colectomy increased the risk of any early (odds ratio [OR] 1.92, 95% confidence interval [CI] 1.06-3.48) complications, early hospital readmission (OR 4.79, 95% CI 1.12-20.58), and early redo surgery (OR 7.49, 95% CI 1.17-47.85). A high steroid dose increased the risk of any early complications (OR 1.96, 95% CI 1.08-3.57), early surgical site complications (OR 2.03, 95% CI 1.01-4.09), and early redo surgery (OR 7.52, 95% CI 1.42-39.82). Laparoscopic surgery decreased the risk of any early complications (OR 0.54, 95% CI 0.29-1.00), early infections (OR 0.39, 95% CI 0.18-0.85), and late hospital readmissions (OR 0.34, 95% CI 0.12-1.00). DISCUSSION: Preoperative tofacitinib treatment demonstrated a postoperative safety profile comparable with biologics in patients with UC undergoing colectomy.

Crohn's Disease-Like Features in a Patient With IgE and Selective IgG1 and IgG3 Deficiency.

We report a 19-year-old male with congenital, combined deficiency of immunoglobulin (Ig) E and 2/4 subclasses of IgG (G1, G3) and chronic diarrhea. He presented at six years of age with chronic recurrent diarrhea responsive to immunoglobulin treatment. Initially, it was considered of infectious origin. However, at the age of 14 years, ileocolonoscopy and magnetic resonance enterography (MRE) were performed, and they showed a mild, limited, non-specific, terminal ileitis with increased eosinophil count on histology. A diagnosis of possible eosinophilic gastroenteritis was made, and budesonide was administered with temporary relief. However, at the age of 19 years, repeat ileocolonoscopy showed multiple ulcers in the terminal ileum and aphthous ulcers in the cecum, and repeat MRE demonstrated extensive ileal involvement. Esophagogastroduodenoscopy demonstrated the involvement of the upper GI tract with aphthous ulcers. Subsequently, gastric, ileal, and colonic biopsies revealed Ziehl-Neelsen-negative, non-caseating granulomas. We hereby report the first case of IgE and selective IgG1 and IgG3 deficiency complicated with Crohn's disease-like extensive GI involvement.

The Probiotic Strains Bifidοbacterium lactis, Lactobacillus acidophilus, Lactiplantibacillus plantarum and Saccharomyces boulardii Regulate Wound Healing and Chemokine Responses in Human Intestinal Subepithelial Myofibroblasts

Bifidobacterium lactis, Lactobacillus acidophilus, Lactiplantibacillus plantarum and Saccharomyces boulardii are common probiotic supplements. Colonic subepithelial myofibroblasts (cSEMFs) are actively involved in mucosal wound healing and inflammation. cSEMFs, isolated from healthy individuals, were stimulated with 102 or 104 cfu/mL of these probiotic strains alone and in combination, and their effect on chemokine and wound healing factor expression was assessed by qRT-PCR, ELISA and Sircol Assay, and on cSEMFs migration, by Wound Healing Assay. These strains remained viable and altered cSEMFs' inflammatory and wound healing behavior, depending on the strain and concentration. cSEMFs treated with a combination of the four probiotics had a moderate, but statistically significant, increase in the mRNA and/or protein expression of chemokines CXCL1, CXCL2, CXCL4, CXCL8, CXCL10, CCL2 and CCL5, and healing factors, collagen type I and III, fibronectin and tissue factor. In contrast, when each strain was administered alone, different effects were observed, with greater increase or decrease in chemokine and healing factor expression, which was balanced by the mixture. Overall, this study highlights that the use of multiple probiotic strains can potentially alert the gut mucosal immune system and promote wound healing, having a better effect on mucosal immunity than the use of single probiotics.

Nutritional Assessment of Greek Liver Cirrhosis Patients: Mini Nutritional Assessment Predicts Mortality.

Malnutrition is highly prevalent in liver cirrhosis (LC). It increases as the severity of the disease progresses and it is related to poor survival. The objectives of the study were the nutritional assessment of Greek LC patients, using various nutritional assessment and screening tools, and the comparison of their predictive value for mortality. In total, 137 (77 male) consecutive LC patients (median age: 67 years) were assessed with subjective global assessment (SGA) and mini nutritional assessment (MNA) questionnaires, anthropometrics, handgrip strength (HGS) tests, and bioelectric impedance analysis (BIA), in comparison to a control group of 148 healthy people. Disease severity was assessed using the model for end-stage liver disease (MELD) scores. Patients were followed up for a median of 19 months. Survival curves were calculated using the Kaplan-Meier method. In total, 60% and 43% of patients were of adequate nutritional status by SGA and MNA, respectively, which was confirmed by most anthropometric measurements. MNA and SGA scores correlated significantly with anthropometrics and BIA-derived parameters. Besides the MELD score, mid-arm circumference (MAC), triceps skinfold (TSF), BIA's phase angle (Pha), and MNA predicted mortality in cirrhotic patients. The nutritional assessment demonstrated an unexpectedly high prevalence of well-nourished LC patients. MNA was a strong predictor of mortality.

Co-expression of fibrotic genes in inflammatory bowel disease; A localized event?

Introduction: Extracellular matrix turnover, a ubiquitous dynamic biological process, can be diverted to fibrosis. The latter can affect the intestine as a serious complication of Inflammatory Bowel Diseases (IBD) and is resistant to current pharmacological interventions. It embosses the need for out-of-the-box approaches to identify and target molecular mechanisms of fibrosis. Methods and results: In this study, a novel mRNA sequencing dataset of 22 pairs of intestinal biopsies from the terminal ileum (TI) and the sigmoid of 7 patients with Crohn's disease, 6 with ulcerative colitis and 9 control individuals (CI) served as a validation cohort of a core fibrotic transcriptomic signature (FIBSig), This signature, which was identified in publicly available data (839 samples from patients and healthy individuals) of 5 fibrotic disorders affecting different organs (GI tract, lung, skin, liver, kidney), encompasses 241 genes and the functional pathways which derive from their interactome. These genes were used in further bioinformatics co-expression analyses to elucidate the site-specific molecular background of intestinal fibrosis highlighting their involvement, particularly in the terminal ileum. We also confirmed different transcriptomic profiles of the sigmoid and terminal ileum in our validation cohort. Combining the results of these analyses we highlight 21 core hub genes within a larger single co-expression module, highly enriched in the terminal ileum of CD patients. Further pathway analysis revealed known and novel inflammation-regulated, fibrogenic pathways operating in the TI, such as IL-13 signaling and pyroptosis, respectively. Discussion: These findings provide a rationale for the increased incidence of fibrosis at the terminal ileum of CD patients and highlight operating pathways in intestinal fibrosis for future evaluation with mechanistic and translational studies.

Development of a Human Intestinal Organoid Model for In Vitro Studies on Gut Inflammation and Fibrosis.

Inflammatory Bowel Diseases (IBDs) are characterized by chronic intestinal inflammation and fibrosis, the latter being the predominant denominator for long-term complications. Epithelial and mesenchymal 2D cultures are highly utilized in vitro models for the preclinical evaluation of anti-inflammatory and antifibrotic therapies. More recently, human intestinal organoids (HIOs), a new 3D in vitro model derived from pluripotent stem cells, have the advantage to closely resemble the architecture of the intestinal mucosa. However, the appropriate timing for the study of inflammatory and fibrotic responses, during HIO development, has not been adequately investigated. We developed HIOs from the human embryonic stem cell line, H1, and examined the expression of mesenchymal markers during their maturation process. We also investigated the effect of inflammatory stimuli on the expression of fibrotic and immunological mediators. Serial evaluation of the expression of mesenchymal and extracellular matrix (ECM) markers revealed that HIOs have an adequately developed mesenchymal component, which gradually declines through culture passages. Specifically, CD90, collagen type I, collagen type III, and fibronectin were highly expressed in early passages but gradually diminished in late passages. The proinflammatory cytokines IL-1α and TNF-α induced the mRNA expression of fibronectin, collagen types I and III, tissue factor (TF), and alpha-smooth muscle actin (α-SMA) primarily in early passages. Similarly, HIOs elicited strong mRNA and protein mesenchymal (CXCL10) and epithelial (CXCL1, CCL2, CXCL8, and CCL20) chemokine responses in early but not late passages. In contrast, the epithelial tight junction components, CLDN1 and JAMA, responded to inflammatory stimulation independently of the culture passage. Our findings indicate that this HIO model contains a functional mesenchymal component, during early passages, and underline the significance of the mesenchymal cells' fitness in inflammatory and fibrotic responses. Therefore, we propose that this model is suitable for the study of epithelial-mesenchymal interactions in early passages when the mesenchymal component is active.

Real-life utility and diagnostic accuracy of a home-performed fecal calprotectin test to predict endoscopic activity in patients with inflammatory bowel disease under maintenance treatment with adalimumab.

AIMS: The home-performed fecal calprotectin (FC) test has been proposed for the remote management of inflammatory bowel disease (IBD) patients. We present our real-world experience on the use of FC home testing in IBD patients under maintenance treatment with adalimumab. METHODS: Consecutive IBD patients on maintenance treatment with adalimumab were studied retrospectively on the basis of prospectively recorded data. FC calprotectin home test (IBDoc, Βühlmann Laboratories AG, Schönenbuch, Switzerland) was analyzed alongside sufficient information on baseline characteristics, follow-up data and treatment modifications, as well as serum biomarkers and endoscopic assessment data on the basis of validated endoscopic scores. RESULTS: From a total of 72 IBD patients under maintenance treatment with adalimumab, 65 (90%) showed compliance with performing the home FC test. FC values were significantly higher in patients who finally needed treatment modification (37%) compared with those who were maintained on stable treatment (63%) (761 µg/g [537-1000] vs. 108 [41-335], P < 0.0001). In the logistic regression analysis FC and erythrocyte sedimentation rate (ESR) were independently correlated with endoscopically active disease (odds ratio: 1.003; 95% confidence interval, 1.001-1.006, P < 0.01 and odds ratio: 1.058; 95% confidence interval, 1.013-1.105, P < 0.05). FC identified patients with endoscopically active disease more effectively than other biomarkers with an area under the receiver operating characteristic curve of 0.78. FC levels >413 µg/g had a sensitivity of 75% and a specificity of 76% in predicting active disease in endoscopy. CONCLUSIONS: These first real-life results indicate that in IBD patients under maintenance treatment with adalimumab FC home test is a valuable tool with high compliance rates that performs better than the other biomarkers in predicting disease endoscopic activity.

Prevalence of Clostridium difficile infection among hospitalized inflammatory bowel disease patients in Greece.

BACKGROUND: Inflammatory bowel disease (IBD) is an independent risk factor for Clostridium difficile infection (CDI), which is associated significantly with disease severity. We aimed to determine the rates of CDI among hospitalized IBD patients in major tertiary referral hospitals in Greece. PATIENTS AND METHODS: A retrospective analysis was carried out of stool cultures from hospitalized patients investigated for diarrhea, during 2016, tested for CDI with glutamate dehydrogenase (GDH) and toxins A and B. RESULTS: In total, 6932 patients were tested for CDI; 894 were positive for GDH (12.89%) and 339 were also positive for C. difficile toxin (4.89%). The prevalence of CDI among all hospitalized patients was 1.6/1000 patient-days. Among these, there were 401 IBD patients, and 62 were positive for GDH (15.46%) and 30 were also positive for C. difficile toxin (7.48%). The prevalence of CDI in IBD patients was 2.5/1000 patient-days, significantly higher than in non-IBD hospitalized patients (30/401 vs. 309/6531, P=0.013). Among the 30 IBD patients (ulcerative colitis=18, Crohn's disease=12) with CDI, six were receiving biologics, three were on corticosteroids [one combined with azathioprine (AZA) and one combined with 5-ASA], nine were on AZA monotherapy and 12 were on 5-ASA monotherapy. The prevalence of CDI among patients receiving AZA monotherapy was significantly higher than in patients receiving other medications (9/68 vs. 21/333, P=0.047). Mild CDI (n=28) was treated with metronidazole and/or vancomycin, whereas severe CDI (n=2) was treated with vancomycin. CONCLUSION: The prevalence of CDI is higher in hospitalized IBD patients than those without IBD and AZA monotherapy increases the risk of CDI.

Gut Microbial Signatures Underline Complicated Crohn's Disease but Vary Between Cohorts; An In Silico Approach.

Microflora dysbiosis is implicated in the pathophysiology of Crohn's disease. This work analyzes differences in microbial communities and relevant metabolic pathways among the nonstricturing nonpenetrating (B1), stricturing (B2), and penetrating (B3) subphenotypes of Crohn's disease vs healthy controls. We conducted a bioinformatics analysis using the QIIME pipeline and the Calypso, linear discriminant analysis effect size, Phylogenetic Investigation of Communities by Reconstruction of Unobserved States, and STAMP tools on publicly available 16S bacterial rRNA sequencing data from terminal ileum mucosal biopsies of healthy controls and the 3 subphenotypes of Crohn's disease. We analyzed differences in microbial diversity and taxonomy, inferred active metabolic pathways via relevant genes' abundance, and detected bacterial families that could serve as biomarkers. Microbiota α-diversity was decreased within all 3 Crohn's disease subphenotypes vs control samples, with more significant reductions in B2 and B3 compared with B1. ß-diversity analysis identified similar microbial patterns in B2 and B3 samples, different from those of B1 and from those of healthy controls. Abundance analysis of microbial families in cohorts, beyond altered abundances compared with healthy controls, highlighted significant differences between the B2 and B3 subphenotypes and the B1 subphenotype. A similar pattern was observed in the inference of microbial metabolomics: the B2 and B3 cohorts had different predicted metabolotypes from the B1 cohort, in addition to differences observed in Crohn's disease vs healthy controls. Our findings indicate distinct microbiome signatures in complicated Crohn's disease subphenotypes and provide the basis for further investigation into the role of gut microflora in the natural course of Crohn's disease.

Cytokine Receptor Profiling in Human Colonic Subepithelial Myofibroblasts: A Differential Effect of Th Polarization-Associated Cytokines in Intestinal Fibrosis.

Background: Colonic subepithelial myofibroblasts (cSEMFs) are mesenchymal cells with a pivotal role in the pathophysiology of Crohn's disease (CD) fibrosis. Here, we demonstrate for the first time a complete expression mapping of cytokine receptors, implicated in inflammatory bowel diseases, in primary human cSEMFs and how pro-inflammatory cytokines regulate this expression. Furthermore, we show the effect of Th1-, Th2-, Th17- and Treg-related cytokines on a fibrosis-related phenotype of cSEMFs. Methods: Colonic subepithelial myofibroblasts were isolated from healthy individuals' colonic biopsies. Interleukin (IL)-1α- and/or tumor necrosis factor (TNF)-α-induced mRNA and protein expression of cytokine receptors was assayed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunofluorescence, respectively. Th-related cytokine effects on mRNA and protein profibrotic factor expression were analyzed by qRT-PCR and/or colorimetric assays and on the wound-healing capacity of cSEMFs by scratch test. Results: In cSEMFs, we observed basal cytokine receptor expression, which was modified by IL-1α and TNF-α. Th1-related cytokines upregulated tissue factor (TF), collagen, fibronectin and matrix metalloproteinase (MMP)-1 and downregulated α-smooth muscle actin (α-SMA), MMP-9, and wound healing rate. Th2-related cytokines upregulated collagen, TF, α-SMA, MMP-1, and wound healing rate and downregulated fibronectin and MMP-9. IL-17 and IL-23 upregulated fibronectin, and IL-22 downregulated TF. IL-17 and IL-22 decreased wound healing rate. Similar to TGF-ß, IL-23 upregulated MMP-1, tissue inhibitor of metalloproteinases-1, collagen expression, and wound healing rates. Conclusions: Our results suggest that cSEMFs have a central role in inflammation and fibrosis, as they express a great variety of Th-related cytokine receptors, making them responsive to pro-inflammatory cytokines, abundant in the inflamed mucosa of CD patients.

Stromal and immune cells in gut fibrosis: the myofibroblast and the scarface.

Post-inflammatory scarring is the end-result of excessive extracellular matrix (ECM) accumulation and tissue architectural destruction. It represents a failure to effectively remodel ECM and achieve proper reinstitution and healing during chronic relapsing inflammatory processes. Scarring may affect the functionality of any organ, and in the case of inflammatory bowel disease (IBD)-associated fibrosis leads to stricture formation and often surgery to remove the affected bowel. The activated myofibroblast is the final effector cell that overproduces ECM under the influence of various mediators generated by an intense interplay of classic and non-classic immune cells. This review focuses on how proinflammatory mediators from various sources produced in different stages of intestinal inflammation can form profibrotic pathways that eventually lead to tissue scarring through sustained activation of myofibroblasts.

Protein profiling of isolated uterine AA amyloidosis causing fetal death in goats.

Pathologic amyloid accumulates in the CNS or in peripheral organs, yet the mechanism underlying the targeting of systemic amyloid deposits is unclear. Serum amyloid A (SAA) 1 and 2 are produced predominantly by the liver and form amyloid most commonly in the spleen, liver, and kidney. In contrast, SAA3 is produced primarily extrahepatically and has no causal link to amyloid formation. Here, we identified 8 amyloidosis cases with amyloid composed of SAA3 expanding the uterine wall of goats with near-term fetuses. Uterine amyloid accumulated in the endometrium, only at the site of placental attachment, compromising maternal-fetal gas and nutrient exchange and leading to fetal ischemia and death. No other organ contained amyloid. SAA3 mRNA levels in the uterine endometrium were as high as SAA2 in the liver, yet mass spectrometry of the insoluble uterine peptides identified SAA3 as the predominant protein, and not SAA1 or SAA2. These findings suggest that high local SAA3 production led to deposition at this unusual site. Although amyloid A (AA) amyloid deposits typically consist of an N-terminal fragment of SAA1 or SAA2, here, abundant C-terminal peptides indicated that the uterine amyloid was largely composed of full-length SAA3. The exclusive deposition of SAA3 amyloid in the uterus, together with elevated uterine SAA3 transcripts, suggests that the uterine amyloid deposits were due to locally produced SAA3. This is the first report of SAA3 as a cause of amyloidosis and of AA amyloid deposited exclusively in the uterus.

Extracellular adenosine regulates colitis through effects on lymphoid and nonlymphoid cells.

Adenosine is a purine metabolite that can mediate anti-inflammatory responses in the digestive tract through the A(2A) adenosine receptor (A(2A)AR). We examined the role of this receptor in the control of inflammation in the adoptive transfer model of colitis. Infection of A(2A)AR(-/-) mice with Helicobacter hepaticus increased colonic inflammation scores compared with uninfected A(2A)AR controls. Comparison of T cell subsets in wild-type and A(2A)AR(-/-) mice revealed differences in markers associated with activated helper T (Th) cells and regulatory T (Treg) cells. Previous studies showed that expression of A(2A)AR on CD45RB(HI) and CD45RB(LO) Th cells is essential for the proper regulation of colonic inflammation. Adoptive transfer of CD45RB(HI) with CD45RB(LO) from wild-type mice into RAG1(-/-)/A(2A)AR(-/-) mice induced severe disease within 3 wk, although transfer of the same subsets into RAG1(-/-) mice does not induce colitis. This suggests that the presence of A(2A)AR on recipient cells is also important for controlling colitis. To investigate the role of A(2A)AR in myeloid cells, chimeric recipients were generated by injection of bone marrow from RAG1(-/-) or RAG1(-/-)/A(2A)AR(-/-) mice into irradiated RAG1(-/-) mice. After adoptive transfer, these recipients did not develop colitis, regardless of A(2A)AR expression by the donor. Together, our results suggest that the control of inflammation in vivo is dependent on A(2A)AR signaling through multiple cell types that collaborate in the regulation of colitis by responding to extracellular adenosine.

Effects of octreotide and insulin on colon cancer cellular proliferation and correlation with hTERT activity.

Peptide hormone somatostatin and its receptors have a wide range of physiological functions and play a role in the treatment of numerous human diseases, including colorectal cancer. Octreotide, a synthetic somatostatin-analog peptide, inhibits growth of colonic cancer cells primarily by binding to G-protein coupled receptors and elicits cellular responses through second-messenger systems. Insulin also initiates mitogenic signals in certain cell types. The objective of the present study was to explore the effects of octreotide with or without insulin treatment, on Caco-2 and HT-29 human colon-cancer cell proliferation and to correlate their effects with the activation of telomerase reverse transcriptase (hTERT). The involvement of protein tyrosine phosphatases in the regulation of the anti-proliferative effect of octreotide was also evaluated. Sodium orthovanadate was used to reverse the anti- proliferative effect of octreotide. Telomerase activity was determined for each time point under octreotide and/or insulin treatment. Elevated expression of sst1, sst2 and sst5 was confirmed in both cell lines by RT-PCR. Immunocytochemistry detected sst1, sst2A, sst2B, sst3, sst4 and sst5 protein expression in the membranes of both cell lines. Octreotide inhibited the proliferation of Caco-2 and HT-29 cells in a time and dose-dependent manner. Insulin exerted proliferative effects in Caco-2 cells and octreotide reversed its effect in both cell lines. Sodium orthovanadate suppressed the anti-proliferative effect of octreotide both in Caco-2 and HT-29 cells. Telomerase activity was significantly reduced when Caco-2 cells were exposed to octreotide, under serum-free cultured medium. On the other hand, telomerase attenuation after octreotide treatment could not counteract the actions of insulin on both cells. Our data indicate that the use of octreotide could provide a possible therapeutic approach to the management of certain patients who suffer from colon cancer.

Proinflammatory cytokines induce crosstalk between colonic epithelial cells and subepithelial myofibroblasts: implication in intestinal fibrosis.

BACKGROUND AND AIMS: Colonic epithelial cells and adjacent subepithelial myofibroblasts are important counterparts in the pathogenesis of intestinal inflammation and fibrosis. We investigated the possible crosstalk between them, whilst focusing on the mucosal inflammation pathways that potentially trigger intestinal fibrosis. METHODS: We studied the effects of proinflammatory cytokines (IL-1α, TNF-α, IFN-γ) on human colonic epithelial cell lines and the effects of epithelial cell-conditioned media on primary human colonic subepithelial myofibroblasts isolated from normal controls or patients with inflammatory Crohn's disease along with the corresponding 18CO cell line. Readouts included production of TGF-ß and TIMP-1, total collagen synthesis, matrix metalloproteinases MMP-2 and MMP-9 and myofibroblast migration/mobility. RESULTS: Proinflammatory cytokines upregulated TGF-ß and TIMP-1 in colonic epithelial cells. Conditioned medium from these epithelial cell cultures induced production of MMP-9 and collagen and inhibited the migration/mobility of subepithelial myofibroblasts. MMP-9 production depended on endothelin receptor A signalling on responding myofibroblasts. Collagen up-regulation was independent of TGF-ß, CTGF, TF and endothelin. Subepithelial myofibroblasts isolated from Crohn's disease patients had similar responses to those isolated from normal controls, with the exception of higher basal collagen production. CONCLUSIONS: Our study indicates that colonic epithelial cells may respond to an inflammatory milieu by inducing myofibroblast functions similar to those observed during intestinal fibrosis.

TNF receptors in Kupffer cells.

INTRODUCTION: Somatostatin is a mediator of immune functions and has been used as an antineoplastic agent in animal models and human neoplasias. We have demonstrated that Octreotide inhibits only LPS induced secretion of proinflammatory cytokines including TNFa by Kupffer cells (KC). We, therefore, tested the hypothesis that somatostatin modulates the expression of tumor necrosis factor alpha (TNF?) receptors and apoptosis of KC. METHODS: Rat KC were isolated by centrifugal elutriation. TNFR1 and TNFR2 expression was studied by RT-PCR, quantitative PCR, Western Blot and immunofluorescence before and after Octreotide pre-incubation. Apoptosis was assessed by quantitative measurement of cytoplasmic histone-associated DNA fragments. TNFa mRNA expression was assessed by semiquantitative PCR and TNFa was measured in cell supernatants by ELISA. RESULTS: TNFR1 and TNFR2 mRNA are constitutively expressed in KC. Octreotide incubation increased both receptors expression with a peak at 6?h and return to basal levels at 24?h. TNFR1 was mostly influenced. However, only increase in TNFR2 protein was identified, whereas a band at 90 kD was present instead of a band at 55 kD as expected for TNFR1. TNF? mRNA expression was inhibited by Octreotide and a significant inhibition was observed at 48?h. TNF had no effect on KC apoptosis, whereas Octreotide significantly increased their apoptosis, and this effect was not influenced by co-incubation with TNFa. CONCLUSION: TNFR1 and TNFR2 are constitutively expressed in KC and their expression is strongly increased by somatostatin. Moreover, somatostatin increases KC apoptosis. These findings may in part explain the antineoplasmatic effect of somatostatin.