Laurence D. Jewell

Lactobacillus species prevents colitis in interleukin 10 gene–deficient mice

BACKGROUND & AIMS Intestinal luminal microflora, or their products, are likely an important initiating factor in the pathogenesis of inflammatory bowel disease. The aim of this study was to determine the role of colonic aerobic luminal bacteria and Lactobacillus species in the development of colitis in interleukin (IL)-10 gene-deficient mice. METHODS Intestine from 2-16-week-old mice was scored histologically and cultured for bacteria. Lactobacillus sp. repopulation of the colonic lumen was achieved via daily rectal delivery of Lactobacillus reuteri or oral lactulose therapy. RESULTS At 2 weeks of age, IL-10 gene-deficient mice showed no colonic injury but did display abnormal colonic bacterial colonization with increased colonic mucosal aerobic adherent and translocated bacteria in conjunction with reduced Lactobacillus sp. levels. In association with the abnormal colonic bacterial colonization, colitis developed by 4 weeks of age. Restoring Lactobacillus sp. to normal levels reduced levels of colonic mucosal adherent and translocated bacteria and attenuated the development of the colitis. CONCLUSIONS In the neonatal period, IL-10 gene-deficient mice have decreased levels of colonic Lactobacillus sp. and an increase in colonic mucosal adherent and translocated bacteria. Normalizing Lactobacillus sp. levels reduced colonic mucosal adherent and translocated bacteria and prevented colitis.

The Isolation of Nucleic Acids from Fixed, Paraffin-Embedded Tissues-Which Methods Are Useful When?

Museums and pathology collections around the world represent an archive of genetic material to study populations and diseases. For preservation purposes, a large portion of these collections has been fixed in formalin-containing solutions, a treatment that results in cross-linking of biomolecules. Cross-linking not only complicates isolation of nucleic acid but also introduces polymerase “blocks” during PCR. A wide variety of methods exists for the recovery of DNA and RNA from archival tissues, and although a number of previous studies have qualitatively compared the relative merits of the different techniques, very few have undertaken wide scale quantitative comparisons. To help address this issue, we have undertaken a study that investigates the quality of nucleic acids recovered from a test panel of fixed specimens that have been manipulated following a number of the published protocols. These include methods of pre-treating the samples prior to extraction, extraction and nucleic acid purification methods themselves, and a post-extraction enzymatic repair technique. We find that although many of the published methods have distinct positive effects on some characteristics of the nucleic acids, the benefits often come at a cost. In addition, a number of the previously published techniques appear to have no effect at all. Our findings recommend that the extraction methodology adopted should be chosen carefully. Here we provide a quick reference table that can be used to determine appropriate protocols for particular aims.

Interleukin‐10 gene‐deficient mice develop a primary intestinal permeability defect in response to enteric microflora

The normal intestinal epithelium provides a barrier relatively impermeable to luminal constituents. However, patients with inflammatory bowel disease experience enhanced intestinal permeability that correlates with the degree of injury. IL-10 gene-deficient mice were studied to determine whether increased intestinal permeability occurs as a primary defect before the onset of mucosal inflammation or is secondary to mucosal injury. At 2 weeks of age, IL-10 gene-deficient mice show an increase in ileal and colonic permeability in the absence of any histological injury. This primary permeability defect is associated with increased mucosal secretion of interferon-gamma and tumor necrosis factor-alpha, and does not involve an increase in nitric oxide synthase activity. Colonic permeability remains elevated as inflammation progresses, while ileal permeability normalizes by 6 weeks of age. IL-10 gene-deficient mice raised under germ-free conditions have no inflammation, and demonstrate normal permeability and cytokine levels. This data suggests that the intestinal permeability defect in IL-10 gene-deficient mice occurs due to a dysregulated immune response to normal enteric microflora and, furthermore, this permeability defect exists prior to the development of mucosal inflammation.

Lack of correlation between Lewis antigen expression by Helicobacter pylori and gastric epithelial cells in infected patients

BACKGROUND & AIMS Lewis antigens are expressed by both human gastric epithelial tissue and Helicobacter pylori. We examined Lewis antigens expressed by gastric epithelium and by H. pylori isolated from the corresponding biopsy tissue. METHODS H. pylori Lewis expression was determined by enzyme immunoassays, and immunoelectron microscopy was used to confirm the Lewis antigens on some H. pylori cells and in some biopsy specimens. Histopathology using identical monoclonal antibodies specific for Lewis A, B, X, and Y antigens was used to detect these antigens in 24 gastric biopsy specimens. RESULTS We identified Lewis Y in 100%, Lewis X and Lewis B in 95.8%, and Lewis A in 87.5% of biopsy specimens. In H. pylori, 87.5% expressed Lewis Y, 79.2% Lewis X, and 4.2% (one strain) Lewis B. No Lewis A was detected. Antibody specific for Lewis X labeled the bacteria and associated adhesion pedestal. The cagA gene was present in 92% of strains. CONCLUSIONS There was no direct relationship between Lewis antigen expression by H. pylori and gastric epithelial cells in infected patients. Expression of Lewis X and Lewis Y by H. pylori suggests the possibility of their requirement for establishment and/or maintenance of infection. An immunoelectron micrograph of H. pylori interaction with the gastric epithelial adhesion pedestal suggests a tentative role for Lewis X in the adhesion process.

Cholestatic hepatocellular injury with azathioprine: A case report and review of the mechanisms of hepatotoxicity

Azathioprine is a drug commonly used for the treatment of inflammatory bowel disease, organ transplantation and various autoimmune diseases. Hepatotoxicity is a rare, but important complication of this drug. The cases reported to date can be grouped into three syndromes: hypersensitivity; idiosyncratic cholestatic reaction; and presumed endothelial cell injury with resultant raised portal pressures, venoocclusive disease or peliosis hepatis. The components of azathioprine, 6-mercaptopurine and the imidazole group, may play different roles in the pathogenesis of hepatotoxicity. The strong association with male sex, and perhaps with human leukocyte antigen type, suggests a genetic predisposition of unknown type. Many of the symptoms of hepatotoxicity, such as nausea, abdominal pain and diarrhea, can be nonspecific and can be confused with a flare-up of inflammatory bowel disease. As well, the subtype resulting in portal hypertension can occur without biochemical abnormalities. A 63-year-old man with Crohns disease who is presented developed the rare idiosyncratic form of azathioprine hepatotoxicity, but also had a severe disabling steroid myopathy, peripheral neuropathy, resultant deep venous thrombosis and pulmonary embolism related to immobility, and a nosocomial pneumonia. His jaundice and liver enzyme levels improved markedly on withdrawal of the drug, returning to almost normal in five weeks. Treating inflammatory bowel disease effectively while trying to limit iatrogenic disease is a continuous struggle. Understanding the risks of treatment is the first important step. There must be a low threshold for obtaining liver function tests, especially in men, and alertness to the need to discontinue the drug or perform a liver biopsy should patients on azathioprine develop liver biochemical abnormalities, unexplained hepatomegaly or signs of portal hypertension.

Resuscitation with 100% oxygen causes intestinal glutathione oxidation and reoxygenation injury in asphyxiated newborn piglets.

Objective:To compare mesenteric blood flow, oxidative stress, and mucosal injury in piglet small intestine during hypoxemia and reoxygenation with 21%, 50%, or 100% oxygen. Summary Background Data:Necrotizing enterocolitis is a disease whose pathogenesis likely involves hypoxia-reoxygenation and the generation of oxygen-free radicals, which are known to cause intestinal injury. Resuscitation of asphyxiated newborns with 100% oxygen has been shown to increase oxidative stress, as measured by the glutathione redox ratio, and thus may predispose to free radical-mediated tissue injury. Methods:Newborn piglets subjected to severe hypoxemia for 2 hours were resuscitated with 21%, 50%, or 100% oxygen while superior mesenteric artery (SMA) flow and hemodynamic parameters were continuously measured. Small intestinal tissue samples were analyzed for histologic injury and levels of oxidized and reduced glutathione. Results:SMA blood flow decreased to 34% and mesenteric oxygen delivery decreased to 9% in hypoxemic piglets compared with sham-operated controls. With reoxygenation, SMA blood flow increased to 177%, 157%, and 145% of baseline values in piglets resuscitated with 21%, 50%, and 100% oxygen, respectively. Mesenteric oxygen delivery increased to more than 150% of baseline values in piglets resuscitated with 50% or 100% oxygen, and this correlated significantly with the degree of oxidative stress, as measured by the oxidized-to-reduced glutathione ratio. Two of eight piglets resuscitated with 100% oxygen developed gross and microscopic evidence of pneumatosis intestinalis and severe mucosal injury, while all other piglets were grossly normal. Conclusions:Resuscitation of hypoxemic newborn piglets with 100% oxygen is associated with an increase in oxygen delivery and oxidative stress, and may be associated with the development of small intestinal hypoxia-reoxygenation injury. Resuscitation of asphyxiated newborns with lower oxygen concentrations may help to decrease the risk of necrotizing enterocolitis.

Colon carcinoid tumors. A population-based study.

PURPOSE: The aim of our investigation was to evaluate the clinical presentation of patients with carcinoid tumors of the colon and to estimate the survival and potential prognostic factors of this tumor type. METHODS: A population-based study was performed using data from the Alberta Cancer Registry between 1964 and 1988 (inclusive). The clinical records and the pathologic material of eligible patients were reviewed. Survival was estimated both as crude survival and with the Kaplan-Meier method. RESULTS: During the 25-year study period (1964–1988), 36 true carcinoid tumors of the colon were diagnosed in Alberta. Carcinoids of the ileocecal region and of the rectum were excluded from the study. The average age at time of diagnosis was 68.4 years; there were 20 males and 16 females. Symptoms (abdominal pain, diarrhea, weakness, anorexia) occurred late in the course of the disease: 64 percent of the lesions were in Dukes D stage and 22 percent were Dukes C at diagnosis. Only one patient presented with a malignant carcinoid syndrome. Lesions occurred most frequently in the cecum (39 percent), followed by transverse and sigmoid colon. Most of the patients were managed surgically. The perioperative mortality rate was with 22 percent, which is quite high. The average size of the lesions was 5.8 (range, 2–10) cm, and most tumors (31/36) had invaded the pericolic fat. The most common immunohistochemical pattern was argentaffin/argyrophil negative and neuron-specific enolase positive. Two-year and five-year actuarial (Kaplan-Meier) survival was 34 percent and 26 percent, respectively. Survival for carcinoids of the colon was significantly lower compared with carcinoids of the rectum or appendix, and with colon adenocarcinomas. Size of the tumor and tumor invasion into the muscularis propria—the two major histopathologic prognostic factors for carcinoids of the gastrointestinal tract—were not found to influence survival significantly. Rather, tumor stage, histologic pattern, tumor differentiation, nuclear grade, and mitotic rate were found to significantly influence the survival rate. CONCLUSION: Carcinoid tumors of the colon are extremely rare tumors, diagnosed late in the course of the disease, and they carry a bad prognosis. Prognostic factors are tumor stage, histologic pattern, differentiation, nuclear grade, and mitotic rate of the tumor.

Long-term results of total abdominal colectomy for chronic idiopathic constipation

PURPOSE: A small proportion of patients with chronic idiopathic constipation are incapacitated by the problem. We have assessed 1) the efficacy of total abdominal colectomy, and 2) the predictive value of preoperative testing. METHODS: Preoperative testing included complete history and physical examination, appropriate biochemical and hematologic assessment, psychiatric interview, colon transit studies using ingested radiopaque pellets, anorectal manometry, colonic intraluminal manometry, and measurement of colon diameters and length on barium enema examination. All patients were followed for 65±40 months. RESULTS: Seventy-one percent had excellent or very good results. Twenty-one percent were satisfied, had improved the quality of their life, and felt the operation was worthwhile despite frequent residual or new symptoms. Two (8 percent) patients did not improve. Patients with a psychiatric history or physiologic evidence of an afferent nerve defect had poorer results (P<0.05). CONCLUSIONS: Total abdominal colectomy with ileorectal anastomosis is highly effective in alleviating symptoms in patients with chronic idiopathic constipation.

Defining the role of a tailored luminal solution for small bowel preservation

The mucosal layer is the initial site of small bowel (SB) graft injury sustained during cold storage. Vascular administration of preservation solutions alone is unable to prevent ischemic injury of this layer during clinically relevant storage periods. The SB is unique in that it possesses both a vascular and a luminal route by which preservation solutions can be administered. We hypothesized that addition of a luminal‐delivered solution, formulated on amino acid requirements for energy‐ and non‐energy‐related reactions, would provide site‐specific preservation of mucosal energetics, barrier function and morphology throughout an extended period of cold storage. Of the three luminal solutions containing amino acids which were tested (UWG, AA1, AA2), only the two groups (AA1, AA2), containing glutamine plus 18 other amino acids, ± osmotic agent (lactobionate) and buffer (BES), exhibited significant improvements in energetics, barrier function, and histology compared to the clinical standard of isolated vascular University of Wisconsin (UW) solution. Although the AA1 and AA2 groups preserved barrier function and morphology up to 24 h better than all other solutions tested, AA2 proved to be the only luminal solution with values of permeability, conductance, and short‐circuit current not significantly different from freshly isolated tissues. Furthermore, the greatest reduction in histologic injury was effected by AA2 treatment (median grade 2 compared to control, UW(v), grade 8). This study documents that a luminal‐delivered solution, formulated on physiologic SB requirements, provides targeted preservation of the SB mucosa.

Colonic delivery of dexamethasone from a prodrug accelerates healing of colitis in rats without adrenal suppression

BACKGROUND/AIMS Dexamethasone-beta-D-glucuronide, a colon-specific prodrug of dexamethasone, may be useful in the treatment of ulcerative colitis and Crohns colitis. The aim of this study was to evaluate colonic delivery and efficacy of this prodrug in the rat. METHODS Distribution of dexamethasone in luminal contents and tissues of the gastrointestinal tract and in plasma was measured after oral administration of dexamethasone-beta-D-glucuronide or free dexamethasone. Efficacy of the prodrug and free drug was tested in an acetic acid-induced rat colitis model. Healing of induced colitis was assessed by measuring net intestinal fluid absorption, colonic surface area of ulceration, histology, and myeloperoxidase activity. Glucocorticosteroid toxicity was evaluated with serum corticosterone and plasma adrenocorticotropic hormone levels. RESULTS The drug delivery index (a measure of relative targeting efficiency) was 6.7 and 8.6 in the cecal and colonic mucosa, respectively. The prodrug was significantly more potent than free drug in improving net colonic fluid absorption while significantly reducing surface area of ulceration and histological grade in colitic rats. Treatment with free dexamethasone significantly reduced serum corticosterone levels to subnormal levels, and treatment with the prodrug maintained serum corticosterone and plasma adrenocorticotropic hormone levels near control levels. CONCLUSIONS The prodrug dexamethasone-beta-D-glucuronide delivers efficacious amounts of dexamethasone to the large intestine from lower doses than free dexamethasone.