Stomach gas analyses in canine acute gastric dilatation with volvulus.

BACKGROUND: The origin of the gas in the stomachs of dogs with acute gastric dilatation or gastric dilatation with volvulus (GDV) often is disputed. HYPOTHESIS: We tested the hypothesis that gaseous distention resulted from aerophagia. ANIMALS: Ten cases of GDV that were submitted to an emergency clinic were sampled intraoperatively. METHODS: With the abdomen open, the needle of a vacutainer blood collection set was inserted into the distended stomach, and gas was collected into 10 mL glass vacutainer vials with rubber stoppers. These were stored at room temperature for 1-7 days and then analyzed by gas chromatography and mass spectroscopy. RESULTS: CO2 composition ranged from 13 to 20%. One dog had an H2 concentration of 29%. CONCLUSIONS: Because the CO2 content of atmospheric air is less than 1%, these findings suggest that the gaseous gastric distention in GDV is not the result of aerophagia.

Lymphatics, tertiary lymphoid organs and the granulomas of Crohn's disease: an immunohistochemical study.

BACKGROUND: Case studies in the past repeatedly suggested that the fundamental alteration in Crohn's disease occurs in the regional lymphatics of the intestine. AIM: To evaluate the lymphatic inflammation in Crohn's disease, and to characterise lymphoid aggregates and granulomas in and surrounding lymphatics and blood vasculature. METHODS: Forty-eight tissue blocks from 24 Crohn's disease patients and 23 tissue blocks from 23 control patients were selected. Tissue sections were immunostained with a lymphatic endothelial cell marker (D2-40), a marker for blood vasculature (FVIII), and markers for T cells (CD3), B cells (CD20) and macrophages (CD68). RESULTS: Lymphangiectasia and lymphocytic perilymphangitis were demonstrated in all 24 patients, lymphocyte-obstructed lymphatics in seven patients, granuloma-obstructed lymphatics in nine patients and inflammatory lymphoid follicles in all 24 patients. Free-standing granulomas occurred in 19 patients, and in three further patients granulomas were in or attached to blood vascular units. CONCLUSIONS: This study, employing immunohistochemistry, revealed, better than standard microscopy, the association of inflammation, granulomas and tertiary lymphoid follicles or organs with the lymphatic vasculature in Crohn's disease. Disease in some patients was characterised by perilymphangitis and lymphoid follicular inflammation and in others by granulomas, some of which totally obstructed lymphatics. These findings have aetiological, therapeutic and prognostic implications.

The forgotten role of lymphangitis in Crohn's disease.

Research into Crohn's disease has recently been focused on the genetics of the patient, the gastrointestinal flora, the gut epithelium and mucosal immune responses. For over 60 years pathologists have reported that the fundamental alteration in Crohn's disease occurs in regional lymphatics of the intestine--the disease is a lymphocytic and granulomatous lymphangitis. At an earlier time, experimental sclerosis of regional intestinal lymphatics of the pig produced a chronic segmental enteritis with many features of Crohn's disease, including lymphocytic and granulomatous lymphangitis of the bowel wall and enteroenteric and enterocutaneous fistulas. In Crohn's disease, differences in the anatomic distribution of vasa recta appear to explain long-segment disease of the ileum and short-segment disease of the more proximal intestine. A variety of bacteria and viruses cause lymphangitis, suggesting that microorganisms may be at the centre of the basic changes in Crohn's disease. Dietary antigens and lipids are worthy of consideration as well. Now that antibodies to label lymphatics are available, attention should be directed at defining the initial damage to lymphatic endothelium and agents that might be responsible.

The comparative importance of E. coli antigen in granulomatous colitis of Boxer dogs.

Granulomatous colitis of Boxer dogs is characterized by mucosal and submucosal infiltration by abundant large macrophages and lymphocytes and plasma cells. Involved intestine is thickened, corrugated and ulcerated. The macrophages that occur in colon, cecum and regional lymph nodes are PAS-positive, lipid-rich, contain cholesterol, and some of the time can be seen to hold bacteria. Paraffin tissue blocks of formalin-fixed colon and colic lymph nodes from 10 cases were cut at 5 microm and immunostained by a streptavidin-biotin immunoperoxidase technique, employing primary antibodies against Escherichia coli, E. coli 0157: 2, Campylobacter, C. jejuni-coli, Yersinia pseudotuberculosis, Salmonella, Shigella, Pseudomonas and Lawsonia intracellularis. The macrophages in the lamina propria and submucosa, as well as those in aggregates in regional lymph nodes, showed immunoreactivity with polyclonal E. coli antibody in all 10 cases. Tissues lacking granulomas were negative, as were those reacted with the other eight antibodies, with the exception that there was rare focal staining for Campylobacter, Lawsonia and Salmonella in a few dogs. We believe these results identify the causative agent of this granulomatous disease of Boxer dogs, a disease with great histologic and etiologic similarity to granulomatous leptomeningitis of Beagle dogs, and malacoplakia and xanthogranulomatous cholecystitis of man. Macrophages that are immunopositive for E. coli antigen occur in Crohn's disease as well, where their significance is less well understood.

A clustering of Crohn's disease in Mankato, Minnesota.

Infrequently, clusterings of Crohn's disease (CD) occur that suggest it is transmissible. We studied such a clustering. Graduates of the Mankato West High School Class of 1980 were contacted by mail and asked to respond, by self-addressed postcard, to a six-item questionnaire about inflammatory bowel disease and CD. Responses were followed-up by telephone contact and additional mailings. Two visits were made to Mankato, Minnesota, to interview individuals with CD, to obtain medical records, radiographs, and sera, and to study environmental risk factors. Of the 320 graduates of the class of 1980, 285 were contacted. Seven cases of CD were identified, the equivalent of a prevalence of 2,400/100,000. Concerns were discovered that CD may have emanated from recreational swimming. Fecal coliform counts in excess of 200/dL, the standard above which water is regarded as unsafe for recreational use, had been recorded year after year for the Blue Earth River at Mankato and for the Minnesota River. Recent fecal coliform counts (1993-1995) of Lake Washington, Lake German/Jefferson, and Lake Shetek were greater than 200/dL in 57%, 65%, and 62% of water samples. This clustering, in unrelated individuals, argues against a genetic cause for CD and suggests that environmental transmission occurred.

Gastric intramural hematoma and hemoperitoneum in a captive northern fur seal.

A 16-yr-old adult male northern fur seal (Callorhinus ursinus) was found dead in its outdoor pool in November 1995. The animal was maintained at Mystic Aquarium (Mystic, Connecticut, USA) from March 1980 to November 1995. Gross necropsy findings included hemoperitoneum and locally extensive gastric intramural hemorrhage that involved the posterior fundic, antral, and pyloric regions and extended into the duodenum. The gastric mural thickening grossly resembled hemangioma, and the gastric serosa was ruptured at the site of maximal mural expansion. In histologic sections of the stomach, a cribiform network of fibrin, which encompassed numerous variably-sized aggregates of closely packed erythrocytes, markedly expanded the submucosa. No vascular endothelium was identified in serial histologic sections of the expanded gastric submucosa stained with hematoxylin and eosin or immunohistochemically with antibodies to vimentin and Factor VIII-related antigen, establishing an absence of hemangioma. Carstairs' and Weigert's histochemical stains confirmed that the framework expanding the submucosa was fibrin. Although the appearance of the gastric wall resembled hemangioma, a population of neoplastic endothelial cells was not identified within the submucosal expansion of hemorrhage and fibrin, and microscopic evidence was most consistent with the diagnosis of gastric intramural hematoma. This lesion is a rare pathologic event that has not been reported in marine mammals, but one that should be included in diagnostic considerations of hemoperitoneum and gastric mural expansion.

Virus serology in familial Crohn disease.

BACKGROUND: Recent findings that early-in-life virus infections represent a risk factor for Crohn disease, that exacerbations of disease sometimes appear associated with common viral infections, and, in particular, suggestions that Crohn disease may be the result of persistent infection with measles virus prompted serologic studies for antibody to 19 common viruses, Chlamydia psittaci, and Mycoplasma pneumoniae. METHODS: Sera from 14 affected members of 2 French families with a high frequency of Crohn disease and from age- and sex-matched controls, taken in 1990 and 1992, and from unaffected family members were tested. Complement fixation, enzyme immunoassay, and indirect immunofluorescent tests were used. RESULTS: There were no significant differences between patients and controls or between affected and unaffected family members of family 2, with the exception that affected siblings carried higher antibody titers for mycoplasma one year and for varicella another year. There were no differences in measles IgM and IgG or mumps IgG antibody levels between patients and controls, or between patients and unaffected family members. CONCLUSIONS: Serology failed to find evidence of participation by 19 common viruses, C. psittaci, and M. pneumoniae in Crohn disease. The data do not support the hypothesis that persistent measles virus infection causes Crohn disease.

Lack of support for a common etiology in Johne's disease of animals and Crohn's disease in humans.

The superficial similarity of Johne's disease to Crohn's disease led to the hypothesis that, like the former. Crohn's disease was caused by Mycobacterium paratuberculosis. Detailed pathologic comparisons, however, reveal little similarity between these two entities, including the lack of important extraintestinal manifestations. Attempts to recover M. paratuberculosis by culture have only rarely succeeded and the significance of spheroplasts that appear more frequently on culture is seriously in question. Five immunocytochemistry studies have failed to find mycobacterial antigens in diseased tissues and the five most recent polymerase chain reaction (PCR) attempts to find genomic evidence of M. paratuberculosis were uniformly negative. Numerous serologic studies failed to demonstrate antibody to M. paratuberculosis and attempts to show cell-mediated immunity were also unrewarding. Inoculation of numerous experimental animals with Crohn's disease tissue has failed to induce Johne's disease, and inoculation of various animal species with M. paratuberculosis has equally failed to result in Crohn's disease. Controlled studies of the treatment of Crohn's disease with antimycobacterial agents have generally resulted in no improvement, and most studies that have shown a positive response are either uncontrolled or include broad-spectrum antibiotics that may be acting on pathogens other than mycobacteria. Finally, although Johne's disease is common in farm animals, and infected animals shed M. paratuberculosis in large numbers, no record of zoonotic transmission has been recorded.

The role of Peyer's patches in the age-related incidence of Crohn's disease.

Recently researchers have suggested that clinical subsets of Crohn's disease occur, which are variously described as inflammatory, fibrostenotic, and fistulizing. In addition, it has been observed that within families with multiple cases, often there is concordance of the site and type of disease. The lesions of Crohn's disease occur in segments that suggest that distribution of Peyer's patches. When the age-related incidence of Crohn's disease was plotted for all countries from which such data were available, the peaks of greatest case frequency occurred at ages 15 to 25 years and paralleled a similar peak representing the number of Peyer's patches as a function of age. This correlation suggests that Crohn's disease may develop as an inflammatory process specifically targeting these important lymphoid structures. Similar peaks of activity in the adolescent to early adult years occur for appendicitis and tonsillitis.

Mucosal inflammation in pediatric diversion colitis: a quantitative analysis.

BACKGROUND: Diversion colitis commonly occurs in bypassed segments of colorectum, and has been described qualitatively in Hirschsprung's disease patients with colostomies. The objective of this study was to characterize quantitatively the changes in the inflammatory cell population in the mucosa of children with diversion colitis. METHODS: Paraffin blocks of well-oriented, full-thickness colorectal tissues were obtained from 15 children with diversion colitis (all with Hirschsprung's disease), four pediatric controls and four adult controls. Sections were immunostained for B and T lymphocytes, macrophages, IgG, IgM, and IgA. Measurements were made referent to a standard length of muscularis mucosae. Lymphoid follicles were counted and the areas occupied by B and T cells were determined by image analysis. Cells in the interfollicular lamina propria were counted separately, but IgA-containing plasma cells were too abundant to enumerate. RESULTS: Pediatric diversion colitis was characterized by enlarged and more numerous lymphoid follicles with approximately four times as many B lymphocytes and twice as many T lymphocytes in the follicular compartment of the mucosa when compared to pediatric controls. The interfollicular mucosa was thickened (499 +/- 27 versus 380 +/- 56 microns) and contained approximately six times as many B cells and eight times as many T cells as controls. Macrophages and plasma cells containing IgG and IgM were not significantly increased. CONCLUSIONS: These findings extend the qualitative observations of increased follicular and lamina propria lymphoid tissue in bypassed segments of colon, and are consistent with the hypothesis of persistent antigenic stimulation of the mucosa-associated lymphoid tissue.

Clinicopathological features and histopathology of experimental hepatic capillariasis in muskrats (Ondatra zibethicus).

Ten muskrats (Ondatra zibethicus) each were infected with 17,000 eggs (long-term study) and eight muskrats each were infected with 8,000 eggs (short-term study) of Capillaria hepatica (Nematoda). Food intake, body weight, and selected clinicopathological parameters were measured every 2 days for 28 days in the short-term study and every 14 days for 184 days in the long-term study. Muskrats in the short-term study had moderate to severe necrotizing granulomatous hepatitis associated with mild anorexia and weight loss, varying degrees of leukocytosis with eosinophilia and elevation of serum alanine and aspartate aminotransferases. No significant changes in packed cell volume, hemoglobin, total plasma protein, albumin, blood urea nitrogen, bilirubin, lactate dehydrogenase or alkaline phosphatase were found among animals from the short-term study. Muskrats in the long-term study had severe necrotizing granulomatous hepatitis associated with marked anorexia, weight loss and 60% mortality over 39 days post-inoculation (PI); animals that survived for 184 days did not return to pre-inoculation body weights despite returning to normal food intake. Hepatic lesions at 184 days PI consisted of minimal to severe liver replacement by C. hepatica eggs. No statistically significant differences in values of clinical parameters between inoculated animals and a non-inoculated control group from the long term study were found.

Selective induction of mucosal immune responses to 2-acetylaminofluorene.

Mucosal vaccination with chemical carcinogens coupled to enterotoxins such as cholera toxin (CT) can elicit carcinogen-specific immunoglobulin secretion into the intestinal lumen. The present study examines the ability of several related bacterial enterotoxins and their subunits to act as adjuvants or carrier proteins in stimulating an intestinal secretory IgA (S-IgA) response to 2-acetylaminofluorene (AAF). Using Thiry-Vella loops in rabbits, CT, cholera toxin B subunit (CTB) and the recombinant B subunit of the heat labile enterotoxin from E. coli (rLTB) were all found to be effective carrier proteins and adjuvants for eliciting S-IgA anti-AAF. However, marked differences in the ratio of mucosal S-IgA to serum IgG production were observed. CT elicited the highest luminal S-IgA anti-AAF titers as well as the highest ratio of intestinal S-IgA/serum IgG when used as an adjuvant. Conversely, rLTB elicited a high serum IgG anti-AAF titer but only a modest intestinal S-IgA response. Dialysis studies using monoclonal IgA versus IgG anti-AAF on opposing sides of a semipermeable membrane demonstrated the potential importance of the intestinal S-IgA/serum IgG ratio. A high "intestinal" IgA/"serum" IgG ratio abolished carcinogen transfer to the "serum" side of the membrane, while a low ratio enhanced transfer. Thus, to generate an active mucosal immune response capable of blocking carcinogen absorption, the carrier protein or adjuvant should be selected to optimize the intestinal S-IgA/serum IgG ratio.

The relationship of granulomas to blood vessels in intestinal Crohn's disease.

It has been suggested that granulomatous vasculitis is a primary mechanism in the production of pathologic changes seen in Crohn's disease. We set out to investigate the relationship of granulomas to blood vessels and to confirm or refute previous reports of granulomatous vasculitis in Crohn's disease. Thirty paraffin embedded tissues from 11 patients with Crohn's disease were selected after examination of H&E stained sections for the presence of granulomas. Using an immunohistochemical method, various monoclonal antibodies were applied to sequential sections from each tissue to demonstrate vascular structures and granulomas. In three patients none of the granulomas occurred in association with blood vessels, in five a small proportion of the granulomas affected blood vessels, and in three granulomatous vasculitis appeared occlusive and significant. A total of 232 granulomas were identified, 22% of which were closely associated with blood vessels, which included both arteries and veins; 16% were perivascular, while 6% were intravascular. Perivascular granulomas did not surround blood vessels or invade the medial layers. They were asymmetric, suggesting that they originated by encroachment of nearby lymphatic or connective tissue granulomas. These results indicate that the granulomas of Crohn's disease are usually not associated with blood vessels; however, there is a minority of patients in whom vascular granulomatous inflammation may be important, although probably as a secondary phenomenon.

On the use of antibiotics in Crohn's disease.

It is difficult to understand how a disease process characterized by ulcerations, fissures, abscesses, fistulas, lymphangitis, and granulomas has not attracted greater use of antibiotics, particularly as the sites are constantly contaminated by intestinal bacteria. I have had a favorable experience with broad-spectrum antibiotics in the treatment of a variety of forms of ileocolitis in animals and now advocate that Crohn's disease be treated at length with these drugs. Microbiologic culture of serosa, mesenteric lymph nodes, and fistulas has demonstrated that bacterial species are present in a significant proportion of cases, and serology has shown that patients have elevated antibody levels to many of these same microorganisms. Now immunocytochemistry provides documentation of Escherichia coli and streptococcal antigen within the lesions of a majority of patients. That these bacteria may be secondary invaders should not decrease our need to address them. Several chronic granulomatous diseases that were once thought to be intractable now yield to long-term antibiotic treatment, including Whipple's disease, malakoplakia, and granulomatous colitis of Boxer dogs. Many of the perianal lesions of Crohn's disease respond to short-term metronidazole, and the medium-term (3-6 months) use of broad-spectrum antibiotics, most recently ciprofloxacin, has shown promising results. In view of the increasing evidence of bacterial participation in this disease, it is now important that physicians test some of our newer broad-spectrum antibiotics, in a controlled format, and over an extended time.

Immunocytochemical evidence of Listeria, Escherichia coli, and Streptococcus antigens in Crohn's disease.

BACKGROUND/AIMS: Infectious agents have long been suspected of playing a role in the initiation of Crohn's disease. The objective of this study was to search for likely microbial agents in diseased tissues using immunocytochemical techniques. METHODS: Intestines and mesenteric lymph node specimens of 21 patients from two French families with a high frequency of Crohn's disease and from Connecticut were studied. The microbial agents searched for included Bacteroides vulgatus, Borrelia burgdorferi, Escherichia coli, Listeria monocytogenes, Streptococcus spp., bovine viral diarrhea virus, influenza A virus, measles virus, parainfluenza virus, and respiratory syncytial virus. RESULTS: Seventy-five percent of the patients with Crohn's disease (12 of 16) were positively labeled with the antibody to Listeria. Macrophages and giant cells immunolabeled for this antigen were distributed underneath ulcers, along fissures, around abscesses, within the lamina propria, in granulomas, and in the germinal centers of mesenteric lymph nodes. In addition, 57% (12 of 21) of the cases contained the E. coli antigen, and 44% (7 of 16) contained the streptococcal antigen. The immunolabeling for the latter two agents also occurred within macrophages and giant cells, distributed in a pattern similar to that of Listeria antigen. CONCLUSIONS: The results suggest that Listeria spp., E. coli, and streptococci, but not measles virus, play a role in the pathogenesis of Crohn's disease.

Inflammatory bowel disease in married couples: 10 cases in Nord Pas de Calais region of France and Liège county of Belgium.

Ten pairs of husband-wife couples are reported with inflammatory bowel disease who were seen in the same geographical area in Nord Pas de Calais region of France and in Liège county (Belgium). Among these 10 couples, four were concordant for Crohn's disease, two for ulcerative colitis, and four were discordant. In nine of 10 couples neither spouse had symptoms before marriage but inflammatory bowel disease subsequently developed in both. In one couple, one spouse had Crohn's disease before marriage and the other partner experienced symptoms afterwards. Eighteen children were born to eight of 10 couples. Five of them developed Crohn's disease but four belong to the same family. In all cases the affected children were born to parents who both developed Crohn's disease after they had married and were conceived at a time when parents did not yet have symptoms. It is proposed that this pattern of emergence of inflammatory bowel disease suggests a role for an infectious agent yet to be identified.

An immunocytochemical search for infectious agents in Crohn's disease.

Microorganisms have long been suspected of causing Crohn's disease (CD); however, an etiologic agent has yet to be identified. Few studies have employed immunocytochemistry (ICC) to examine tissue from patients with CD for microbial antigens. We investigated 36 formalin-fixed tissues from 16 patients with CD with ICC. No evidence of adenovirus, Borrelia, Brucella, BVDV, Campylobacter, Campylobacter-like organisms, Chlamydia, coronavirus, CMV, EBV, Legionella, mycobacteria, Pseudomonas, rotavirus, Salmonella, Shigella, staphylococci, Toxoplasma gondii, Treponema, or Yersinia was found. ICC identified E. coli and streptococcal antigens in 11 (69%) and 10 (63%) of the 16 cases studied, respectively. Escherichia coli immunoreactivity was located in ulcers, within the lamina propria, and along fissures. Streptococcal immunolabeling occurred within mucosal epithelial cells, in the lamina propria, in ulcers, along fissures, in granulomatous inflammation including multinucleate giant cells, and in lymph nodes. These results suggest that some of the granulomas in CD may result from immunologic processing of bacterial antigens following their penetration through a compromised mucosa. E. coli and streptococcal antigens may contribute to the pathogenesis of CD.