Caroline B. Appleyard

Gut Microbial Diversity is Reduced by the Probiotic VSL#3 and Correlates with Decreased TNBS-Induced Colitis

Background: Compositional changes within the normal intestinal microbiota have been associated with the development of various intestinal inflammatory disorders such as pouchitis and inflammatory bowel diseases (IBD). Therefore, it has been speculated that manipulation of a dysbiotic intestinal microbiota has the potential to restore microbial homeostasis and attenuate inflammation. Methods: We performed community composition analyses by terminal restriction fragment length polymorphism (T‐RFLP) of the bacterial 16S ribosomal RNA gene to investigate the impact of the probiotic VSL#3 on colonic microbial community composition and development of trinitrobenzene sulfonic acid (TNBS)‐induced colitis in rats. Results: TNBS‐induced chronic colitis was significantly reduced in VSL#3‐fed rats compared to controls (P < 0.05). T‐RFLP analysis revealed distinct microbial communities at luminal versus mucosal sites. Within the luminal microbiota, chronic colitis was associated with an overall decrease in bacterial richness and diversity (Margalefs richness, P < 0.01; Shannon diversity, P < 0.01). This decrease in luminal microbial diversity was enhanced in TNBS‐treated rats fed VSL#3 (Margalefs richness, P < 0.001; Shannon diversity, P < 0.001) and significantly correlated with reduced clinical colitis scores (Pearson correlation P < 0.05). Conclusions: Our data demonstrate that the probiotic VSL#3 alters the composition of the intestinal microbiota and these changes correlate with VSL#3‐induced disease protection. (Inflamm Bowel Dis 2011;)

Basic epidemiology of inflammatory bowel disease in Puerto Rico

BackgroundPrevious epidemiological studies have suggested that the incidence of inflammatory bowel disease (IBD) is lower in Latin American populations. The aim of this study was to estimate the incidence of IBD in Puerto Rico, a predominantly Hispanic population. MethodsA nonconcurrent prospective study was conducted in collaboration with private gastroenterologists in southwest Puerto Rico. Basic medical history and demographics were extracted from the medical records of patients for which a new diagnosis of IBD (Crohn’s disease, CD; ulcerative colitis, UC; or nonspecified IBD) was made during each of the years 1996–2000. ResultsA total of 202 eligible cases of IBD were identified (95 male, 107 female). Forty-eight patients were diagnosed with CD, 102 with UC, and 52 with nonspecified IBD. The total incidence of IBD increased significantly between 1996 and 2000 (3.07/100,000 to 7.74/100,000; p < 0.001), being significantly higher for CD (fourfold increase, p < 0.01) and nonspecified IBD (fourfold increase, p < 0.005), but not UC (1.7-fold increase). The prevalence of CD was higher in males with an earlier age of onset (p < 0.05). ConclusionsThis study demonstrates that the incidence of IBD within the Puerto Rican population is increasing and may be higher than previously reported for other Latin American populations.

Pretreatment with the probiotic VSL#3 delays transition from inflammation to dysplasia in a rat model of colitis-associated cancer

Evidence supports involvement of microflora in the transition of chronic inflammation to neoplasia. We investigated the protective efficacy of the probiotic VSL#3 in a model of colitis-associated colorectal cancer. Chronic colitis was induced in Sprague-Dawley rats by administration of trinitrobenzene sulfonic acid (TNBS), followed 6 wk later by systemic reactivation. To induce colitis-associated dysplasia and cancer, the animals received TNBS (intravenously) twice a week for 10 wk. One group received VSL#3 in drinking water from 1 wk before colitis induction until death. The colons were examined for damage and presence of dysplasia or cancer. Samples were analyzed for cell proliferation and apoptosis, vitamin D receptor (VDR) expression, angiogenic factors, and presence of alkaline sphingomyelinase or phosphatase. Microbial community composition was evaluated by terminal restriction fragment-length polymorphism analysis of the bacterial 16S rRNA gene. None of the probiotic-treated animals developed carcinoma, and no high-grade dysplasia was found in either the proximal or mid colon. In contrast, 29% of the animals in the control group developed carcinoma in one or more regions of the colon. VSL#3-treated animals had significantly less damage than the vehicle treated-controls in all areas of the colon, and this correlated with decreased richness and diversity of the mucosally adherent microbiota. Treatment with the probiotic increased the antiangiogenic factor angiostatin, VDR expression, and alkaline sphingomyelinase. We concluded that pretreatment with the probiotic VSL#3 can attenuate various inflammatory-associated parameters, delaying transition to dysplasia and cancer, thus offering its potential therapeutic use in patients with long-standing colitis.

Prolonged Chronic Inflammation Progresses to Dysplasia in a Novel Rat Model of Colitis-Associated Colon Cancer

Inflammatory bowel disease (IBD) is a gastrointestinal disorder of unknown etiology or cure. One complication of IBD is an increased risk for development of colon cancer. The aims of this study were to use a previously established rat model of colitis to develop a new model of colitis-associated colon cancer and ascertain the involvement of three cancer-related genes: K-ras, adenomatous polyposis coli (APC), and p53. Four groups of rats were used: reactivated 1,2-dimethylhydrazine [DMH; trinitrobenzene sulfonic acid (TNBS) was used to induce colitis followed by a weekly s.c. dose of DMH], prolonged reactivation (inflammation was induced with TNBS, then maintained twice a week), saline-DMH (animals received saline instead of TNBS followed by a weekly dose of DMH), and normal (received no treatment). Animals were sacrificed at 5, 10, or 15 weeks, and colon samples were taken for pathologic analysis and gene mutation detection. No dysplasia was found in the normal group. The highest incidences of dysplasia were as follows: prolonged reactivation group at 5 weeks (60%), reactivated DMH group at 10 weeks (83%), and saline-DMH group at 15 weeks (67%). Carcinoma was found in both the prolonged reactivation and saline-DMH groups. No mutations were found in the K-ras oncogene; however 62% of the APC samples (exon 15 at nucleotide 2778) and 76% of p53 (exon 6 at nucleotide 1327) showed substitutions. The prolonged reactivation group may be considered a new model of colitis-associated colon cancer, offering the potential to study cancer prevention strategies for patients with IBD.

Molecular profiling of a rat model of colitis: Validation of known inflammatory genes and identification of novel disease-associated targets

Background: Inflammatory bowel disease (IBD) is a disease of unknown etiology characterized by acute and chronic relapsing inflammation. The most suitable animal model for studying this disease is still under debate. Microarray transcript profiling has the potential to illuminate the molecular processes that are involved in both the human condition and animal models. Aim: To identify differentially expressed genes in the 2,4,6‐trinitrobenzene sulfonic acid (TNBS) model of experimental colitis and compare gene expression profiles with that reported in patients. Methods: Colitis was induced by TNBS administration (30 mg in 50% ethanol) in female Sprague‐Dawley rats. Controls received the vehicle. Seventy‐two hours later, the animals were killed, the colons were removed and scored for damage, and total RNA was isolated. Gene expression levels were analyzed after hybridizing experimental and control cDNA to PIQOR Toxicology Rat Microarrays containing 1,252 genes. Genes with 2‐fold or more higher or 0.5‐fold or less lower expression levels were selected as significantly differentially expressed. Results were validated using real‐time reverse‐transcription polymerase chain reaction (RT‐PCR). Results: We observed increased expression of genes that have previously been shown to be up‐regulated in IBD patients, including chemokines/cytokines, extracellular matrix/remodeling genes, transcription factors and tumor necrosis factor family members. Using real‐time RT‐PCR, we validated 9 of 10 critical genes identified by DNA microarray. Fibulin 2 and lysyl oxidase are among some of the novel genes not previously associated with IBD that could potentially be related to the pathogenesis of this condition. Conclusion: We provide evidence supporting the TNBS colitis model as an appropriate tool to study the pathology of IBD and identify molecular targets with clinical relevance.

Experimental colitis increases blood-brain barrier permeability in rabbits

Extraintestinal manifestations of inflammatory bowel disease are numerous. This study examined the effects of two models of acute colitis on cerebral blood flow (CBF) and permeability of the blood-brain barrier in rabbits. CBF (measured with radiolabeled microspheres), or the extraction ratio or permeability-surface area (PS) product of the blood-brain barrier to fluorescein and FITC-dextran, was measured 48 h after colitis induction with acetic acid (HAc) or trinitrobenzene sulfonic acid (TNBS). PS product for fluorescein increased (P < 0.05) in TNBS colitis (1.33 x 10(-5) +/- 0.52 x 10(-5) ml/s and 0.48 x 10(-5) +/- 0.13 x 10(-5) ml/s (mean +/- SE) for treated (n = 14) and untreated (n = 10) animals, respectively. PS product for the larger FITC-dextran was not different in TNBS colitis (0.24 x 10(-5) +/- 0.09 x 10(-5) ml/s, n = 7) compared with untreated controls (0.19 x 10(-5) +/- 0.04 x 10(-5) ml/s, n = 8). PS product for fluorescein increased (P < 0.01) in HAc colitis compared with vehicle (2.66 x 10(-5) +/- 1.46 x 10(-5) ml/s and 0.33 x 10(-5) +/- 0.05 x 10(-5) ml/s, respectively; n = 6 in each group). The extraction of fluorescein from the blood to the brain increased by 75% during TNBS colitis when compared with vehicle (P < 0.05). CBF and cerebrovascular resistance did not change from the untreated control after TNBS colitis. Our data suggest that, irrespective of induction method, acute colitis increases the permeability of the blood-brain barrier to small molecules without changing CBF.Extraintestinal manifestations of inflammatory bowel disease are numerous. This study examined the effects of two models of acute colitis on cerebral blood flow (CBF) and permeability of the blood-brain barrier in rabbits. CBF (measured with radiolabeled microspheres), or the extraction ratio or permeability-surface area (PS) product of the blood-brain barrier to fluorescein and FITC-dextran, was measured 48 h after colitis induction with acetic acid (HAc) or trinitrobenzene sulfonic acid (TNBS). PS product for fluorescein increased ( P < 0.05) in TNBS colitis (1.33 × 10-5 ± 0.52 × 10-5 ml/s and 0.48 × 10-5 ± 0.13 × 10-5ml/s (mean ± SE) for treated ( n = 14) and untreated ( n = 10) animals, respectively. PS product for the larger FITC-dextran was not different in TNBS colitis (0.24 × 10-5 ± 0.09 × 10-5ml/s, n = 7) compared with untreated controls (0.19 × 10-5 ± 0.04 × 10-5 ml/s, n = 8). PS product for fluorescein increased ( P < 0.01) in HAc colitis compared with vehicle (2.66 × 10-5 ± 1.46 × 10-5 ml/s and 0.33 × 10-5 ± 0.05 × 10-5ml/s, respectively; n = 6 in each group). The extraction of fluorescein from the blood to the brain increased by 75% during TNBS colitis when compared with vehicle ( P < 0.05). CBF and cerebrovascular resistance did not change from the untreated control after TNBS colitis. Our data suggest that, irrespective of induction method, acute colitis increases the permeability of the blood-brain barrier to small molecules without changing CBF.

Stress Exacerbates Endometriosis Manifestations and Inflammatory Parameters in an Animal Model

Women with endometriosis have significant emotional distress; however, the contribution of stress to the pathophysiology of this disease is unclear. We used a rat model of endometriosis to examine the effects of stress on the development of this condition and its influence on inflammatory parameters. Female Sprague-Dawley rats were subjected to swim stress for 10 consecutive days prior to the surgical induction of endometriosis by suturing uterine horn implants next to the intestinal mesentery (endo-stress). Sham-stress animals had sutures only, and an endo-no stress group was not subjected to the stress protocol. At the time of sacrifice on day 60, endometriotic vesicles were measured and colons assessed for macroscopic and microscopic damage. Colonic tissue and peritoneal fluid were collected for inflammatory cell analysis. Endometriosis, regardless of stress, produced a decrease in central corticotropin-releasing factor immunoreactivity, specifically in the CA3 subregion of the hippocampus. Prior exposure to stress increased both the number and severity of vesicles found in animals with endometriosis. Stress also increased colonic inflammation, motility, myeloperoxidase levels, and numbers of mast cells. In summary, prior stress may contribute to the development and severity of endometriosis in this animal model through mechanisms involving cell recruitment (eg, mast cells), release of inflammatory mediators, and deregulation of hypothalamic–pituitary axis responses in the hippocampus.

Colonic macrophage polarization in homeostasis, inflammation, and cancer.

Our review focuses on the colonic macrophage, a monocyte-derived, tissue-resident macrophage, and the role it plays in health and disease, specifically in inflammatory conditions such as inflammatory bowel disease and cancer of the colon and rectum. We give special emphasis to macrophage polarization, or phenotype, in these different states. We focus on macrophages because they are one of the most numerous leukocytes in the colon, and because they normally contribute to homeostasis through an anti-inflammatory phenotype. However, in conditions such as inflammatory bowel disease, proinflammatory macrophages are increased in the colon and have been linked to disease severity and progression. In colorectal cancer, tumor cells may employ anti-inflammatory macrophages to promote tumor growth and dissemination, whereas proinflammatory macrophages may antagonize tumor growth. Given the key roles that this cell type plays in homeostasis, inflammation, and cancer, the colonic macrophage is an intriguing therapeutic target. As such, potential macrophage-targeting strategies are discussed.

AN ANGIOTENSIN II RECEPTOR ANTAGONIST REDUCES INFLAMMATORY PARAMETERS IN TWO MODELS OF COLITIS

UNLABELLED Little is known about the effects of the pro-inflammatory hormone Angiotensin II (Ang II) in inflammatory bowel disease. The aim of this study was to evaluate the effect of valsartan (Diovan), an Ang II receptor antagonist, in two models of colitis. METHODS Colitis was induced in Sprague-Dawley rats by administration of trinitrobenzene sulfonic acid (TNBS; 30 mg in 50% ETOH i.c.) or 5% Dextran Sulphate Sodium (DSS) in drinking water ad libitum for 5 days. Valsartan was administered orally in drinking water (160 mg/L) during thirty days prior to the induction of the colitis, and for 5 days after. All animals were evaluated for weight change, diarrhea, myeloperoxidase activity, macroscopic and microscopic damage. Cytokine levels in the colon were measured by ELISA, real-time RT-PCR and immunohistochemistry. RESULTS In the TNBS model, valsartan reduced the macroscopic damage score, significantly decreased the microscopic damage (p<0.01), and accelerated weight gain after colitis. In the DSS-colitis model, valsartan-treated animals had less diarrhea and microscopic damage. Valsartan reduced the protein levels of TGFbeta (p<0.05), and IL-18 in the TNBS model, and led to over expression of IL-10 mRNA in the DSS model. CONCLUSION These data demonstrate a possible anti-inflammatory effect for valsartan in colitis.

Experimental Intestinal Endometriosis Is Characterized by Increased Levels of Soluble TNFRSF1B and Downregulation of Tnfrsf1a and Tnfrsf1b Gene Expression

Abstract Endometriosis is commonly associated with symptoms similar to those of gastrointestinal diseases, such as inflammatory bowel disease (IBD), leading to erroneous diagnosis and inappropriate management. The role of tumor necrosis factor alpha (TNF) in IBD is well established, but its role in endometriosis—also characterized by the activation of inflammatory mechanisms—is still under study. Furthermore, little is known about the involvement of TNF receptors. Intestinal endometriosis was surgically induced in female Sprague-Dawley rats (n = 10). Control rats (n = 10) received sutures with no implants. Samples of tissue and fluids were collected 60 days after surgery. Endometriotic implants were classified in grades, and the gastrointestinal tract was examined for damage. A significant increase was observed in protein levels of TNF and soluble TNFRSF1B in the peritoneal fluid of experimental rats compared to controls. Expression of Tnf mRNA was significantly increased both in peritoneal leukocytes and in intestinal segments associated with implants in experimental animals. Bioactivity of TNF in tissues was confirmed by overexpression of Icam1, Sele, Vegfa, Flt1 and Kdr. Gene expression of Tnfrsf1a and Tnfrsf1b was downregulated in colon and small intestine of experimental animals, possibly as a mechanism of protection against TNF cytotoxicity. Significant overexpression of genes encoding TNF receptor-associated factors that have been linked to activation of antiapoptotic pathways also was observed. Overexpression of TNF and target genes, underexpression of TNF-receptor genes, and increased shedding of TNFRSF1B in this animal model provide further evidence for involvement of the TNF system in the pathogenesis of endometriosis.