Cell wall-defective variants of pseudomonas-like (group Va) bacteria in Crohn's disease

Several animal transmission studies have indicated that Crohn's disease may be caused by a filterable agent. Filtrates of homogenized tissues were prepared from 8 patients with Crohn's disease, 9 patients with chronic ulcerative colitis, and 20 control patients without inflammatory bowel disease. Conventional bacteriological cultures and hypertonic cultures for cell wall-defective bacterial variants were performed on the filtrates. Bacterial revertants (parent forms) of cell wall-defective variants were obtained from filtrates of various tissues including mesenteric lymph nodes of all patients with Crohn's disease. In no instance were revertants cultured from tissue filtrates of the other patients. The 11 revertants isolated from the 8 patients were identified as Pseudomonas-like bacteria, most closely identifiable with group Va.     

A search for a transmissible agent in Crohn's disease.

Controversy exists as to whether a transmissible agent is responsible for Crohn's disease. Previous reports have suggested that sarcoid-like granulomas can develop in animals following inoculation of homogenates derived from bowel affected by Crohn's disease. This study involved the injection of Crohn's tissue homogenates into experimental animals under a variety of conditions which might be expected to favour the demonstration of such an agent. Homogenates have been inoculated into the ileum of rats, mice, and rabbits and also given inoculated into ileum and footpads of rats which have previously been rendered lymphoedematous by surgical interruption of the draining lymphatics. Bowel homogenates from a total of 17 patients with Crohn's disease have been injected into 91 experimental animals. No macroscopic or microscopic changes indicative of Crohn's disease were detected. Thus study does not support the suggestion that a transmissible agent is present in Crohn's disease.     

Seropositivity in Dutch Crohn's disease patients against primed nude mouse lymph nodes, and the difference with lymphocytotoxic antibodies.

Sera from patients with Crohn's disease have been reported to show positive immunofluorescence with lymph nodes of nude mice primed with a filtrate of intestinal tissue affected with Crohn's disease. An indirect immunofluorescence assay was used to test sera of 63 unrelated patients with Crohn's disease, 21 with ulcerative colitis and 36 control subjects against lymph nodes of athymic nude (nu/nu) mice which had been injected with Crohn's disease and ulcerative colitis intestinal tissue filtrates. Forty nine per cent of Crohn's disease patients, 10% of ulcerative colitis patients and 3% of control sera reacted against lymph nodes of mice injected injected with ulcerative colitis intestinal tissue filtrates, 18% of Crohn's disease sera were with intestinal tissue homogenate from Dutch Crohn's patients. With the lymph nodes of mice injected with ulcerative colitis intestinal tissue filtrates, 18% of Crohn's disease sera were positive, whereas all ulcerative colitis and control sera were negative. Lymph nodes from 18 of the 19 mice injected with Crohn's disease tissue filtrates reacted with Crohn's disease sera, whereas only three of these 19 mice reacted with ulcerative colitis sera. A comparative study, carried out in parallel with Crohn's disease filtrate induced hyperplastic lymph nodes from the Bilthoven colony (W2) and from the New York colony (E671) using sera from 54 Crohn's disease patients from Leiden, showed immunoreactivity with 44 and 57% of the Crohn's disease sera against the two hyperplastic lymph nodes. Thirty six of the 54 Crohn's disease sera (67%) reacted with either or both lymph nodes. Only 11% of the Crohn's disease sera which were examined for immunofluorescence and lymphocytotoxic antibodies had lymphocytotoxic antibodies, whereas 40% and 46% of the same sera showed positive immunofluorescence against E671 and W2, respectively. Absorption studies indicated that lymphocytotoxic antibodies activity and the immunofluorescence against the primed nude mouse lymph node are mediated by different serum antibodies in Crohn's disease. The reproducibility of the nude mouse immunofluorescence test system for a preferential immunoreactivity of Crohn's disease sera against Crohn's disease tissue primed murine lymph nodes has been confirmed by the present study. Further studies are necessary to find out whether crossreactive antigen(s) as recognised by some of the Crohn's disease sera in mice injected with ulcerative colitis tissue filtrate is similar to the antigen(s) detected by Crohn's disease sera in mice injected with Crohn's disease tissue filtrates.     

Cell-Wall Defective Bacteria and Crohn''s Disease: Studies Using Athymic Nude Mice

Indirect immunofluorescence studies were performed to determine whether the antigenic recognition by Crohn's disease (CD) sera of lymphoid tissue from nude (nu/nu) mice injected with CD filtrate is related to cell wall defective pseudomonas-like bacterial revertant forms (CWD-RF). Antisera raised in rabbits against two CWD-RF isolates from CD tissues did not stain lymphomas or hyperplastic lymph nodes produced by CD filtrates, although these tissues demonstrated positive immunofluorescence with CD sera. Pre-absorption of reactive CD sera with CWD-RF did not block this immunofluorescence. Formalinized suspensions of CWD-RF were injected into 36 nu/nu, 12 nu/+, and 31 conventional mice. Thirty other mice were fed suspensions of bacteria. Several nu/nu injected with CWD-RF developed lymphoma (n = 2) and plasma cell hyperplasia (n = 5), none of which reacted with CD sera. Mice fed bacteria did not show intestinal pathology. These studies demonstrate that CWD-RF are not directly related to lymphomagenesis in nu/nu induced by CD filtrates and that different CD serum antibodies are involved in recognition of CWD-RF and lymphoid tissues from nu/nu previously injected with CD filtrates.     

Immunity to cytopathic agents associated with Crohn's disease: a negative study.

Serum and peripheral blood lymphocytes from 10 patients with Crohn's disease and 10 healthy subjects were examined for immunological reactivity against chick embryo cell cultures displaying cytopathic effects after inoculation with 0.2 micro filtrates prepared from Crohn's disease intestinal tissues. Although the assay systems (indirect immunofluorescence, lymphocyte transformation, and cytotoxicity) yielded positive results using well-characterized cytopathic viruses (mumps, measles), neither Crohn's disease nor healthy subjects showed immune reactivity to the chick embryo cell cultures inoculated with Crohn's disease intestinal tissues in any of the assay systems. These experiments provide evidence against the hypothesis that the in vitro cytopathic effect on chick embryo cell cultures produced by Crohn's disease intestinal filtrates are caused by a replicating virus or viruses.     

Neutropenia augments experimentally induced Schistosoma japonicum egg granuloma formation in CBA mice,but not in C57BL/6 mice

The present study was designed to investigate the role of neutrophils during the development of Schistosoma japonicum egg granulomas, in C57BL/6 and CBA mice. Laid eggs were implanted into the liver and monoclonal antibody, RB6-8C5, was used to eliminate neutrophils. After daily antibody treatment between days 9 and 13 of egg implantation, both strains of mice showed a marked decrease in neutrophil infiltration and coagulative hepatocyte necrosis at 2 weeks. At 4 weeks, after antibody administration every other day between days 16 and 26, granuloma formation in C57BL/6 mice was not affected by the treatment, whereas CBA mice exhibited a significant increase of reactions. Neutropenia augmented the Th2 cytokine response (IL-4, IL-13 and IL-5), but not for IFN-gamma at any time point examined and in either strain of mice. Higher levels of IL-4 and IL-13 were noted in CBA mice at early and late stages of granuloma formation, compared to C57BL/6 mice. There was also a striking difference in IL-13 production between the two strains. Our results indicate that neutropenia is associated with a significant augmentation of S. japonicum egg-induced granuloma formation in CBA mice, probably through increase in Th2 cytokines, however, the effects differ between early and late stages and between high and low responders.     

Histological response and antigen transmission in the lymph nodes of athymic nu/nu mice inoculated with Crohn's disease tissue filtrates.

Lymph node histology and antigen transmission in the nu/nu mouse in response to animal inoculation with Crohn's disease tissue filtrates were re-evaluated. We found that a hyperplastic lymph node response in nu/nu mice occurred with Crohn's disease (CD), ulcerative colitis (UC), or other intestinal disease (OID) tissue inoculations. In addition, antigen transmission to lymph nodes as detected by indirect immunofluorescence using CD sera was observed in all inoculation groups. The immunofluorescent reaction also occurred independently of lymph node histology. Thus, we confirm that CD sera recognize an antigen(s) expressed in lymph nodes of athymic mice inoculated with CD tissue filtrates. The antibody (or antibodies) in CD sera was not specific for this 'CD antigen or antigens', however, as tested in the nu/nu mouse system, because the CD sera antibodies also recognised antigens in UC inoculated and OID inoculated animals.     

Search for evidence of a viral aetiology for inflammatory bowel disease

The aetiology of the inflammatory bowel diseases. Crohn's disease, and ulcerative colitis, is still obscure. A viral aetiology has been proposed, based in part on reports that filtrates prepared from tissues of patients with inflammatory bowel disease induce cytopathic effects in tissue culture cells. Our attempts to culture viruses in many cell lines from filtrates prepared from the tissue of 95 patients have been negative, except for one case in which cytomegalovirus was isolated from the tissue of a Crohn's disease patient. Our studies confirm previous reports that intestinal tissue filtrates induce cytopathic effects in inoculated cell cultures, but the effect we observed is non-specific; cytopathic effects were induced in most cell lines tested and with similar frequency irrespective of whether the intestinal filtrates were prepared from Crohn's disease patients, ulcerative colitis patients, or non-inflammatory bowel disease controls. Electron microscopy studies of tissue culture cells exhibiting cytopathic effects have not revealed virus particles. Characterisation of the cytopathic effect inducing factor showed that it was incapable of serial passage in tissue culture, too small to be a conventional virus, resistant to inactivation by ultraviolet light, and heat stable. Our results suggest that the observed cytopathic effect was caused by a non-replicating cytotoxic factor, or factors, released from intestinal tissues of both inflammatory bowel disease and non-inflammatory bowel disease patients.     

The relative risk of inflammatory bowel disease among parents and siblings of Crohn's disease patients

Siblings of patients with Crohn's disease are 17-35 times more likely to develop the disease than members of the general population. Parents of patients with Crohn's disease are 35-70 times more likely to develop Crohn's disease than the general population. The crude risk of siblings or parents of patients with Crohn's disease developing ulcerative colitis may be half that of developing Crohn's disease; their relative risk may be a quarter that of developing Crohn's disease.     

Cold Reacting Antibody in NZB/Bl Mice

NZB/Bl mice produce throughout life cold reacting antibodies (CRA) withspecificity to the I-erythrocyte antigen. These antibodies do not react withautologous erythrocytes since intact mouse erythrocytes do not carry reactiveI-antigen. This antigen on the other hand is widely distributed in NZB/Blmouse tissues. Newborn CF₁ mice injected with cell-free filtrates of NZB/Blmouse spleen produce CRA. The untreated offspring of these animals alsosynthesize this type of antibody suggesting that this immune response is inducedby a self-replicating, ultrafilterable immunogen. CRA in NZB/Bl mice may,therefore, be another example of the induction of an auto-immune response byan infectious agent.

     

PCR detection of Mycobacterium paratuberculosis in Crohn's disease granulomas isolated by laser capture microdissection

BACKGROUND AND AIMS: The uncertainty surrounding the role of Mycobacterium avium subsp paratuberculosis (Map) in Crohn's disease has been compounded by possible contamination from Map present in the lumen microflora. This study used laser capture microdissection (LCM) and polymerase chain reaction (PCR) to detect Map DNA in subepithelial granulomas, isolated from 15 surgically resected, formalin fixed specimens of granulomatous Crohn's disease and from 12 granulomatous disease controls (10 bowel, 2 non-bowel). METHODS: The effect of amplicon size on reliability of PCR from formalin fixed samples was examined by amplifying 435 bp and 133 bp sequences of the human APC gene. After this, nested primers were designed to detect a small fragment (155 bp) of the Map specific IS900 gene in Crohn's granulomas. LCM isolated granulomas from Map culture positive bovine intestine was used as positive control. PCR product specificity was confirmed by direct DNA sequencing. RESULTS: The smaller, but not the larger, fragment of the APC gene amplified reliably in all samples. Amplification of the 155 bp fragment of the IS900 gene detected Map DNA in microdissected Crohn's granulomas in 6 of 15 cases, and in 0 of 12 disease control granulomas. CONCLUSIONS: LCM can be used to detect Map DNA in granulomas in a proportion of patients with Crohn's disease. However, formalin fixation requires that comparatively short DNA fragments of the Map specific IS900 gene be targeted, to permit consistent detection. Detection of Map DNA within granulomas might suggest an infectious aetiology in a subset of patients; alternatively, a transmissible agent may not be involved but mycobacterial DNA may influence pathogenesis by modifying the local cytokine responses.     

Differentiation between intestinal tuberculosis and Crohn's disease in endoscopic biopsy specimens by polymerase chain reaction

OBJECTIVES: It is difficult to differentiate intestinal tuberculosis from Crohn's disease because of similar clinical, pathological, radiological, and endoscopic findings. The purpose of this study was to investigate the value of polymerase chain reaction (PCR) assay in the differentiation intestinal tuberculosis from Crohn's disease, and compare the histopathological features of endoscopic biopsy of the two disorders. METHODS: A total of 39 endoscopic biopsy specimens from patients with intestinal tuberculosis and 30 specimens from patients with Crohn's disease were subjected to pathological analysis retrospectively, Ziehl-Neelsen stain, and PCR assay. RESULTS: Except for granuloma with caseation and confluence, which was the characteristic of intestinal tuberculosis, other pathological features of intestinal tuberculosis and Crohn's disease were very similar or were difficult to find in endoscopic biopsy specimens. The positivity rate by PCR in 39 intestinal tuberculosis specimens was 64.1% (25/39), but was zero by PCR in 30 Crohn's disease specimens. Moreover, in the tissues of intestinal tuberculosis with granulomas similar to those of Crohn's disease, there were 71.4% (10/14) positive by PCR, and there were 61.1% (11/18) positive in intestinal tuberculosis tissues without granulomas. CONCLUSIONS: Biopsy is of limited diagnostic value in the differentiation intestinal tuberculosis from Crohn's disease, and PCR is valuable in the differentiation between intestinal tuberculosis and Crohn's disease.     

Bacterial DNA within granulomas of patients with Crohn's disease--detection by laser capture microdissection and PCR

OBJECTIVES: We previously reported the use of laser capture microdissection (LCM) and PCR to detect the presence of Mycobacterium paratuberculosis DNA in granulomas of patients with Crohn's disease. While this does not imply a cause-effect relationship, it may influence the disease process because bacterial DNA has immunomodulatory effects. The aim of this study was to determine whether DNA from nonmycobacterial commensals, such as Escherichia coli, is also increased in the granulomas of Crohn's disease. METHODS: Archival tissue from 15 surgical cases of Crohn's disease and 10 non-Crohn's granulomatous bowel disease controls were examined. Granulomas were isolated using LCM, and the extracted DNA was examined for presence of E. coli DNA by nested PCR amplification of a 135 base-pair segment of the uidA gene. RESULTS: E. coli DNA was detected in microdissected granulomas in 12/15 Crohn's disease patients and in 1/10 non-Crohn's control granulomas (p < 0.001). Also, E. coli DNA was detected in 8/15 Crohn's full-thickness sections and in 4/10 control full-thickness sections. CONCLUSIONS: E. coli DNA may be detected more frequently in Crohn's granulomas than in other non-Crohn's bowel granulomas. This may indicate a tendency for lumenal bacteria to colonize inflamed tissue, or may be due to increased uptake of bacterial DNA by gut antigen presenting cells. In light of previous detection of M. paratuberculosis DNA in Crohn's granulomas, the nonspecific nature of the type of bacterial DNA present in granulomas is evidence against any one bacterium having a significant causative role in Crohn's disease.     

Th1-polarizing immunization with egg antigens correlates with severe exacerbation of immunopathology and death in schistosome infection

In schistosomiasis mansoni, parasite eggs precipitate an intrahepatic granulomatous and fibrosing inflammatory process, which is mediated by, and dependent on, MHC class II-restricted CD4 T helper (Th) lymphocytes specific for schistosome egg antigens (SEA). In the mouse model of the disease, CBA mice develop large granulomas, whereas in C57BL/6 (BL/6) mice these granulomas are significantly smaller. To further investigate how the prevailing cytokine environment influences the development of the egg-induced immunopathology, we immunized the low-pathology BL/6 mice with SEA in complete Freund's adjuvant (CFA) once before, and once again during, the course of a 7-week infection. This immunization caused a pronounced Th1 shift in the SEA-specific CD4 T cell response, which was detected in the mesenteric lymph nodes (MLNs) and spleens, as well as in the granulomatous lesions themselves. The immunized mice displayed a dramatic enhancement of hepatic egg-induced immunopathology manifested by a marked increase in granuloma size and parenchymal inflammation, leading to early death. Control mice immunized with equivalent amounts of SEA or CFA alone displayed the smaller hepatic lesions in a Th2-dominant environment typically seen in the unimmunized BL/6 mice. Analysis of granuloma and MLN lymphocytes from the SEA/CFA-immunized mice revealed that the proportion of CD4 T cells was unchanged in comparison with the control BL/6 groups and remained significantly lower than that seen in the normally high-pathology CBA strain. These results suggest that the shift toward Th1-type cytokine production by a numerically stable population of CD4 T cells correlates with severe exacerbation of immunopathology in schistosomiasis.     

Immunofluorescence assay using crohn's disease tissue-injected athymic nude mouse lymph nodes in the diagnosis of inflammatory bowel diseases

Sometimes, even after extensive investigative efforts, the diagnosis of inflammatory bowel disease remains in doubt. The accurate diagnosis is important if appropriate therapy is to be instituted. A simple indirect immunofluorescence assay that tests the patient's serum against lymphoid tissues from athymic nude (nu/nu) mice receiving injections of filtrates of Crohn's disease tissue is proposed. Eighty percent of serum samples from patients with active, symptomatic Crohn's disease give positive results of immunofluorescence when tested with these lymphoid tissues. The false-positive rate has been very low (less than 10 percent). Because this assay is fairly sensitive and least invasive, it was used for the clarification of many puzzling cases that were seen at the Albert Einstein College of Medicine over the past three years. Ten of these cases were selected for illustration and discussion and are presented in this report.     

Mechanisms of Schistosoma mansoni egg excretion: Parasitological observations in immunosuppressed mice reconstituted with immune serum

Summary CBA mice which had been deprived of their T cells by a combination of adult thymectomy and injection of rabbit anti-mouse thymocyte serum failed to excrete as many Schistosoma mansoni eggs in their faeces as immunologically intact controls. This failure of parasite egg excretion was not obviously attributable to any marked change in the amount of faecal matter produced, or to a change in the size of the worm burden, or to the number or distribution of eggs in the tissues as a result of T-cell deprivation. S. mansoni eggs freshly isolated from T-cell-deprived mice and injected intravenously into normal animals, induced lung granulomas which were the same size as those induced by injection of eggs from normal donors. The rate of S. mansoni egg excretion was not affected by the density of eggs in the tissues, in as much as there was a linear relationship between the number of tissue-bound eggs and the number of eggs detected in the faeces. Treatment of infected mice with the immunosuppressant hydrocortisone acetate also induced a marked reduction in the rate of egg excretion. Injections of serum derived from chronically infected normal mouse donors increased the rate of egg excretion in both T-cell-deprived and steroid-treated mice, but the degree of reconstitution obtained by daily serum injections was only partial relative to normal egg excretion rates. Treatment of infected normal or deprived serum-treated mice with cobra venom factor to reduce serum complement C3 levels had no effect on the rate of S. mansoni egg excretion.     

Seroreactivity of patients with Crohn's disease with lymph nodes of primed nude mice is independent of the tissue used for priming

Athymic nude mice, injected with a cell-free filtrate of intestinal tissue from patients with Crohn's disease, have been shown to express antigens in their lymph nodes to which sera from patients with Crohn's disease contain antibodies. In the present study, immune reactivity was assessed with a histochemical indirect immunoperoxidase assay on paraplast-embedded tissue sections. The reactive lymph node cells were identified as sinus macrophages. We confirmed earlier findings of immunofluorescence studies that the reaction showed some antibody specificity, the proportion of patients with Crohn's disease who were positive (84%) being higher than of patients with ulcerative colitis (29%). However, reactivity of Crohn's disease sera was found to be antigen nonspecific, as it was equally observed with lymph nodes of mice primed with saline or with homogenates from patients with ulcerative colitis, diverticulitis, or volvulus. Hence, this nude mouse model does not seem appropriate to identify a transmissible etiology of Crohn's disease.     

The Effect of Acquired Immunologic Tolerance on the Development of Leukemia

In these experiments, viable spleen cells from young, healthy AKR micewere injected intravenously into newborn C3H_f/Bi mice. Acquired tolerance tonormal skin and leukemic cell homografts from AKR mice was demonstratedin the C3H animals. This presence of "cellular chimerism" with lymphoidcells from a strain of mice with a high spontaneous incidence of leukemia didnot result in the development of this disease in the host animals. These tolerantC3H_f/Bi mice were also found not to be susceptible as adults to leukemia induction with cell free filtrates from leukemic tissues of AKR mice.

Submitted on November 8, 1961 Accepted on January 12, 1962     

Crohn's disease complicated by granulomatous interstitial nephritis, choroidal neovascularization, and central retinal vein occlusion

Extraintestinal manifestations of Crohn's disease are common. Granulomas may occur in different tissues in Crohn's disease, although kidney granulomas are extremely rare. Although ocular complications of Crohn's disease are infrequent, most ocular manifestations include iritis, uveitis, episcleritis, scleritis, and conjunctivitis. Central retinal vein occlusion has been reported in a few patients with Crohn's disease. The choroidal neovascularization is related to inflammatory disorders such as panuveitis, sarcoidosis. We report a patient with Crohn's disease complicated by granulomatous interstitial nephritis, choroidal neovascularization, and central retinal vein occlusion.     

The granuloma in Crohn's disease.

The number of granulomas in sections of bowel involved by Crohn's disease has been counted and related to length of previous history, treatment with steroids, site of involvement, and the subsequent course of the disease. It was found that a high content of granulomas predicted a good prognosis in the large bowel and anus, but was of no prognostic significance in the small bowel. A large regional variation in granuloma counts was observed from an average of 1 per section in the small bowel to 6 in the colon, 18 in the rectum, and 36 in the anus. Those patients with a long clinical history showed a low granuloma content. The findings are consistent with the view that the granuloma represents an adaptive mechanism for the removal or localisation of the causative agent of Crohn's disease.