Clinical and radiological features suggestive of disseminated tuberculosis in patients with HIV-related abdominal pain

A1054 AGAABSTRACTS • G4313 ANALYSIS OF ANTI-COLONIC AUTOANTIBODIES IN PRIMARY SCLEROSING CHOLANGITIS. J_A_AOdin 1, L Casciola-Rosen 2, A Rosen I. Departments of 1Medicine and 2Dermatology, Johns Hopkins School of Medicine, Baltimore, MD. Patients with primary sclerosing cholangitis (PSC) frequently have concomitant inflammatory bowel disease (IBD), especially ulcerative colitis (UC). Anti-colon and anti-portal tract antibodies as well as perinuclear antineutrophil cytoplasmic antibodies (p-ANCA) have been shown to be prevalent in the sera of patients with PSC suggesting an autoimmune pathogenesis. The identity of the colon target antigen(s) is unclear. Our goal was to further characterize the anti-colon antibodies in sera of patients with PSC and identify any clinical associations such as the presence of IBD. Methods: Sera from twenty-five patients with PSC (biopsy- and ERCPproven) were analyzed by Western blotting of lysates of Caco-2 cells (a human colon epithelial cell line) and HeLa cells (a human cervical epithelial cell line). Antigens recognized by the serum autoantibodies were detected with HRP-labeled goat anti-human IgG followed by enhanced chemiluminescence. Results: 28% of patients with PSC had high titer (up to 5000-fold dilution) IgG autoantibodies targeted against colon antigens migrating at 75 and 57 kDa. 72% of these patients had autoantibodies recognizing both antigens. Surprisingly these autoantibodies were present in patients with or without known IBD. The MW of these antigens differs from any previously reported in the sera of patients with PSC, These antigens did not co-migrate with the common primary biliary cirrhosis associated autoantigens and were not detected in lysates of HeLa cells. Additionally 8% of the sera recognized a 32 kDa antigen in both Caco-2 and HeLa cell lysates possibly representing anti-histone (HI) autoantibodies described previously in UC, though neither patient had known UC. Conclusion: High titer anti-colon IgG autoantibodies directed against novel antigens migrating at 75 and 57 kDa were detected by Western blotting in 28% of patients with PSC. Identification of these antigens may give insight into the pathogenesis of IBD in PSC. • G4314 A NOVEL APPROACH TO IDENTIFY EPITHELIAL DEFENSE MECHANISMS AGAINST NORMAL LUMINAL BACTERIA. H. O~awa. K.Fukushima, I. Sasaki, H.Naito, Y.Funayama, T.Masuko, K.Takahashi, S.Satoh, T.Ueno, A.Hashimoto, S.Matsuno. Tohoku University, Sendai, Japan. Background/Aims: Critical role of normal enteric flora for gut inflammation has been described in IL-2 knockout and HLA-B27 transgenic rodents. Since intestinal epithelial cells (IEC) represent a key barrier against bacteria, the identification of IEC gene products involved in this response may lead to a better understanding of mucosal defense under physiological and pathological conditions such as ulcerative colitis. The aim of this study was to investigate the small bowel and colon IEC gene expression induced by luminal bacteria. Methods: ICR mice were kept under specific pathogen-free (SPF) or sterile (germ free; GF) conditions. GF mice were fed chow contaminated with feces from SPF mice at 3 days, 7 days, 14 days before sacrifice at the age of 8 weeks. IEC were separately isolated from small bowel and colon from SPF, GF, and ex-GF mice (4 in each group). Patterns of IEC mRNA expression before and after bacterial reconstitution were examined by differential display. Cloning, sequencing, and northern blot analysis were performed to confirm the mRNA expression. Results: After bacterial reconstitution, acute self-limited inflammation developed in the colon but not the small bowel of ex-GF, with no evidence of residual chronicity. In 60 displays, we cloned 4 different bands differentially-expressed between small bowel and colon before and after reconstitution. One represented the cryptdin-related sequence 4C (CRS4C) which was strongly expressed only in small bowel IEC after but not before reconstitution. Another represented serum amyloid A1 (SAA1) mRNA which was strongly expressed after reconstitution only in colon IEC. The enhanced SAA1 mRNA expression in colon IEC was also detected in human mucosal inflammation, including ulcerative colitis. Conclusion: The genes detected by this reconstitution model seem to translate the response of IEC against normal bacterial flora. Since mice with normal genetic background are used, this model is ideally suited to study the response of small bowel and colon epithelium against enteric constituents in health and intestinal inflammation. • G4315 RABBIT COLONIC MUCINS INDUCE AUTOANTIBODIES AND MAY CAUSE ULCERATIVE COLITIS-LIKE MUCOSAL DAMAGE. S. Ohara, M. Igarashi*, S. Nakano*, T. Kubota*, F. Ogawa, K. Hotta. Dept, of Biochem. and * Int. Med., Sch. of Med., Kitasato Univ., Sagamihara228, JAPAN. Autoimmunity has been suggested in the etiology of ulcerative colitis. However, auto-antigens have not been identified so far. We have previously reported that the rat colonic mucins induce anti-colonic mucin antibodies in rats, and the mucin-immunized rats suffer from a colitis-like disorder. In the present study, we perform the same kind of experiment using rabbits. Methods: Japanese white rabbits were used. The purification of colonic mucins has been described as follows. Briefly, distal colonic tissue was homogenized in Tris-HC1 buffer (pH 7.2) containing 2 % Triton X-100 and 6

GASTROENTEROLOGYVol. 114, No. 4 M urea. Mucins were purified from the extract using gel-filtration and CsC1 equilibrium centrifugation. The rabbits were sensitized by subcutaneous injections of 1 mg of mucins emulsified with Freund's complete adjuvant. Two and three weeks later, their sensitivity was boosted with 0.5 mg mucins emulsified with Freund's incomplete adjuvant. Control rabbits were injected with only the adjuvant on the same time schedule. One week after the last immunization, sera were collected from the mucin-immunized rabbits and control rabbits. We performed ELISA and Westem blot analysis for detection of anti-mucin antibodies. A pathologic observation was performed using histochemical study. Results: ELISA and Western blot analysis showed that the immunized rabbits had anti-mucin antibodies. Macroscopically, we could not detect mucosal damage, but histochemical study showed infiltration of inflammatory cells and erosion at the colonic mucosa, Conclusion: Our results suggest that colonic mucins are one of the autoantigens of ulcerative colitis. • G4316 THE ROLE OF CD8÷ T CELLS FOR DEVELOPMENT OF COLITIS IN Gcti2-DEFICIENT MICE. L. Ohman*, U. Rudolph, L. Birnbaumer, G. R. Harriman. and E. H6rnauist* *University of G6teborg, Sweden and Baylor College of Medicine, Houston, TX. Mice with targeted deletion of the G protein subunit Gtxi2 develop an inflammatory bowel disease closely resembling ulcerative colitis. Histopathologically the Gcti2-/- mice show acute and chronic mucosal inflammation with ulcerations, crypt abscesses and loss of goblet cells. The inflammation is limited to the colonic mucosa with no skip areas, and the small intestines appear normal. Immunological characterization of the intestinal mucosa has revealed a local increase in memory CD4 ÷ T cells and proinflammatory Thl-type cytokines in Gtxi2-/- mice with colitis. We have previously shown that CD8 ÷ T cells exert a local suppression of mucosal immune responses in normal mice in that CD8-deficient mice exhibit a three-fold stronger gut mucosal immune response following oral immunization, but not systemically following parenteral immunization, as compared to wild type mice. To further elucidate the role of local suppression of mucosal immune responses in development of colitis in this animal model if IBD, Gcti2-/- mice were bred with CD8-/- mice to obtain Gcti2-deficient mice lacking CD8÷ T cells. Transmission of the Gai24- genotype on the CD8-/- background did not show a mendelian distribution: Significantly fewer Gai2-/- mice and correspondingly more Gcti2+/+ mice than expected were found. However, development of colitis was not found to be influenced by the absence of CD8÷ T cells. Thus, Gcti2-/-CD8-/- double knockout mice had to be sacrificed due to severe disease symptoms at a mean age of 15_+4 weeks, and Gai2-/CD8+/- and Geti2-/-CD8+/+ mice were sacrificed at a mean age of 16 ± 7 weeks. Flow cytometric analysis of the CD4 ÷ population in the mesenteric and caudal lymph nodes (draining the small intestine, cecum, plus ascending colon, and distal colon and rectum, respectively) of Gcti2-/-CD8-/- double knockout mice demonstrated an increased frequency of CD4÷ T cells expressing a memory phenotype, i.e. CD44hi,CD62L]°. These CD4÷ T lymphocytes also exhibited an increased expression of the mucosal homing receptors tx4137 and ctEI37, as compared to Gai2+/-CD8-/- mice. The overall frequency of CD4 ÷ T ceils was not increased in the Gai2-/-CD8-/- mice. However, the frequency of CD3+CD4-8- T ceils was increased in both locations. A corresponding decrease in the frequency of CD19 ÷ B lymphocytes was found both in the mesenteric and caudal lymph nodes. Still, the frequency of B lymphocytes expressing the co-stimulatory molecules B7.1 (CD80), and to a lesser extent B7.2 (CD87) was increased in both locations. Analysis of cytokine production revealed a marked increase in proinflammatory Thl-type cytokines in inflamed colons, but not small intestines of Gtxi2-/-CD8-/- mice, as compared to Gt~i2+I-CD8-1- mice. IFN-), and IL-113 levels were increased 11 and 49-fold, respectively. Low levels of IL-4 was detected, but the production was not different between the two strains. No spontaneous production of IL-2, IL-3 or IL-12 was seen. In conclusion, the absence of suppressive CD8÷ T cells in the intestinal mucosa does not aggravate colitis in Gcti2-deficient mice. • G4317 CLINICAL AND RADIOLOGICAL FEATURES SUGGESTIVE OF DISSEMINATED TUBERCULOSIS IN PATIENTS WITH HIVRELATED ABDOMINAL PAIN. EA O'Keefe*, R Wood*, A Van Zyl#. Departments of Medicine* and Radiology#, University of Cape Town, South Africa. Disseminated TB (TB) is the most common cause of abdominal pain in South African AIDS patients. It is important to identify simple diagnostic criteria that differentiate this condition from other causes of HIV-related abdominal pain as it responds well to antituberculous therapy and surgery should be avoided. METHODS: HIV patients presenting to an HIV-referral centre in Cape Town with abdominal pain and a CD4 count <200 were studied prospectively. TB was defined by positive mycobacterial cultures from blood, bone marrow or 2 other sites. The association between the predominant site of pain, fever, weight loss, diarrhoea, hepatomegaly, respiratory symptoms, abnormal chest radiograph (CXR), abdominal ultrasound (US) findings and the presence or absence of TB was assessed.

Immunology, Microbiology, and Inflammatory Disorders AI055

April 1998

RESULTS Forty four patients were studied (25 Black, 10 Mixed race, 9 white) of whom 14 (32%), all non-white, had TB. Other common etiologies were CMV (14%) and cryptosporidiosis (14%). Abdominal pain tended to be generalized more often in TB (p=0.07) but otherwise pain site was not predictive of etiology. Fever, hepatomegaly and respiratory symptoms were associated with TB (p<0.05). Ninety percent of TB patients had abnormal CXRs (pleural effusions, adenopathy, atypical infiltrates, reticulonodular pattern) compared with 37% of others. The presence of adenopathy, abscesses and ascites on US was 67%, 33%, 42% sensitive and 91%, 96%, 91% specific, respectively, for TB. CONCLUSIONS: The presence of fever, hepatomegaly, respiratory symptoms, abnormal CXR or adenopathy, ascites or abscesses on US in advanced HIV patients with abdominal pain is highly suggestive of TB, and antituberculous therapy should be initiated pending TB culture results. • G4318 EFFECT OF IRON ON MUCOSAL T h l CYTOKINF~ IN THE IL-10 KNOCK-OUT MOUSE. B. Oldenburg l, D.M. Rennick 2, G.P van Berge Henegouwen1, B.S. van Asbeck 3 and J.C. KoningsbergerL 1 Dept. of Gastroenterology and 3Medicine, University Hospital Utrecht, The Netherlands and 2DNAX Research Institute, Palo Alto, California. Background. A substantial part of the patients with inflammatory bowel disease (IBD) develop anemia of iron deficiency in the course of the disease and therefore are treated with oral iron supplements. Iron however, may influence inflammatory activity in IBD by participating in a Fenton-type reaction with H202 producing the highly reactive hydroxyl radical. To test the hypothesis that iron exerts pro-inflammatory activity through production of reactive oxygen metabolites, we studied the effect of dietary and locally administered iron on mucosal Thl cytokines in IL 10-/- mice, as a model of IBD. Methods. IL 10-/- and wild type (WT) mice were fed a standard (35 mg/kg FeSO4) or "high iron" (500 mg/kg FeSO4) diet after weaning. After 4 weeks mice were sacrified. Furthermore, a group of adult IL 10-/- mice received "iron-enema's" containing 0.2 ml of 1 mM Ferro(ll)ammonium sulphate. Controls were given PBS-enema's. This was repeated on the third and fifth day after which the mice were sacrified. Colon tissue cultures of all groups were prepared: after dissecting and washing in PBS (+ 5% FCS), colons were resuspended in RPMI-1640, supplemented with 10% FCS, 50 mM 2-ME, penicillin and streptomycin (200/200 U/ml). Supernatants were harvested after 48 hrs incubation and IL-lcc, IL-6, TNF~t and yIFNlevels were assessed (ELISA). Results. All pro-inflammatory cytokines were higher in IL 10-/- mice receiving the high iron diet compared with IL 10-/- mice receiving the standard diet. Elevation of all these cytokines also was noted in "iron enema"treated IL 10-/- mice. Results are summerized in the table (mean-+ SD, *indicates p<0.05; IL 10-/- standard vs high iron diet; IL 10 -/- iron vs PBS enema).

WT standard diet

IL- 1cc pg/ml

IL-6 ng/ml

y-IFN ng/mi

TNF-c~ pg/ml

14 -+ 14

9 -+2

0.7 -+ 0.6

64 -+ 20

WT high iron diet

0

7-+3

0.1 -+0,1

62-+8

ILl0-/- standard diet

38-+14

11-+2

0.2-+0.1

143-+41

IL10-/- high iron diet

107_+177 15-+3"

1.7-+2.4"

247_+189

ILl0-/- PBS enema

50 -+ 59

5+2

1.8 -+ 1.4

118 -+ 35

ILl0-/- iron enema

308 -+ 121" 7 -+3

3.3 -+ 2.3

247 +- 157"

Conclusion. High dietary or locally administered iron leads to elevated proinflammatory cytokine levels in the colon of IL 10-/- mice, which may lead to an increase in colonic inflammation. This research was made possible by a partial grant from ASTRA.

G4319 DETECTION OF LAMINA PROPRIA CELLS PRODUCING IgG AGAINST TROPOMYOSIN ISOFORM 5 IN ULCERATIVE COLITIS. E.K. Onuma, K.M. Das. UMDNJ-Robert Wood Johnson Medical School, New Brunswick, NJ. Colon tissue-bound IgG antibody from ulcerative colitis (UC) patients but not the IgG eluted from colons of patients with Crohn's disease (CD) reacts with a 40kDa protein, p40 (J Clin Invest 76:311-18, 1985). p40 has been purified from the colon to homogeneity, partially sequenced, and the two peptides derived therefrom showed 93-100% identity with the cytoskeletal protein tropomyosin (J Immunol 150:2487-93, 1993). Human tropomyosin isoform 5 (hTM5) is the predominant tropomyosin isoform present in colonic epithelial cells. AIM: To use the ELISPOT assay to detect immunoglobulin-secreting lamina propria cells and to examine if the IgG producing cells in UC are predominately against hTM5. MATERIALS & METHODS: Lamina propria mononuclear cells (LPMC) were isolated from colonoscopy biopsy specimens (6-8 specimens from each patient) from the sigmoid colon at 20-30 cm from the anus. The yield of LPMC ranged from 4 to 10xl05 cells from each patient. Patients included 5 with symptomatic UC, 3 with active colitis due to CD and

3 non-inflammatory bowel disease (non-IBD) controls. The ELISPOT assay was used to detect cells producing total Ig and IgG, as well as IgG against purified hTM5. Background was determined by nonspecific binding against human albumin, and this value was subtracted from the experimental values. IgG producing cells are expressed as % of total Ig producing cells and those binding against hTM5 are expressed as % of IgG producing cells. RESULTS: Of the LPMCs secreting immunoglobulins detected by the ELISPOT assay, the percentage of IgG producing cells is significantly higher in the UC patients (10.26_+ 1.95%) compared to the non-IBD controls (4-+ 1.12%) (p<0.05). There is a slightly higher percentage of IgG producing cells in CD (10.13-+2.95) compared with the non-IBD controls, but this was not statistically significant. In UC, significantly more LPMCs produce IgG against hTM5 (48.56 -+ 8.20%) compared to the CD (4.87 +-2.89%) (p<0.005) and the non-IBD controls (11.13 -+ 5.62%) (p<0.01). There was no significant difference of IgG production against hTM5 in CD and non-IBD controls. CONCLUSIONS: In UC and not in CD and non-IBD controls, the increased number of IgG producing cells in the lamina propria is committed to produce IgG against hTM5. Future studies focusing on the specific antibody against hTM5-peptide(s), relationship to disease state, and differences, if any, in various parts of colon, may provide further understanding on the autoimmune mechanism in UC. G4320 ANTINEUTROPHIL CYTOPLASMIC ANTIBODIES IN ASIAN PATIENTS WITH INFLAMMATORY BOWEL DISEASE SHOW NO CORRELATION WITH PROTEINASE 3, LACTOFERRIN, MYELOPEROXIDASE, ELASTASE, CATHEPSIN G AND LYSOZYME: A SINGAPORE STUDY. Choon Jin Ooi,*Bee Lian Lira, Wei Kuen Cheong, *Ah Ee Ling, Han Seong Ng. Dept of Gastroenterology and *Pathology, Singapore General Hospital.

The pathogenetic importance of antineutrophil cytoplasmic antibodies (ANCA) in IBD is unknown and studies of target antigen localisations have been inconsistent. Objective: In this first study ever in Singapore, we looked at the occurrence of perinuclear ANCA (pANCA) and cytoplasmic ANCA (cANCA) in IBD. In patients positive for ANCA, we attempted to localise target antigens by using Proteinase 3, Lactoferrin, Myeloperoxidase,Elastase, Cathepsin G and Lysozyme. Methods: A prospective study was carried out in the outpatient clinics from July to November 1997. There were 50 patients with IBD and 25 controls from patients with functional bowel disease. Out of the 50 patients with IBD, 10 (20%) had Crohn's disease (CD) and 40 (80%) had ulcerative colitis (UC). All sera from patients and controls were screened for ANCAs by indirect immunofluorescence (IFr) using the Euroimmun 6 biochips IFT system for the determination of autoantibodies against cytoplasm of granulocytes, exocrine pancreas and goblet cells. Sera giving a typical perinuclear staining reaction with the neutrophils fixed with ethanol and cytoplasmic staining reaction with neutrophils fixed with formalin were regarded as pANCA. If sera showed perinuclear staining with ethanol fixed neutrophils but negative with formalin fixed neutrophils, atypical-pANCA (apANCA) were reported. Samples giving reaction with the neutrophiles, irregardless of patterns in the IFT, were tested with the Euroimmun ANCA profile ELISA test kit. The ELISA test kit provides a semi-quantitative assay for human autoantibodies of the IgG class against 6 antigens: Proteinase 3, Lactoferrin, Myeloperoxidase, Elastase, Cathepsin G and Lysozyme. Results: A total of 75 patients were studied; 50 with IBD (median age:39) and 25 controls (15 men, 10 women)(median age: 41). Ten (1 man, 9 women) had CD (median age: 35.5) and 40 (25 men, 15 women) had UC (median age: 39.5). There was no racial predilection among the Chinese, Malays or Indians. In CD, 1 was positive for cANCA, 2 for pANCA and 3 for exocrine/acini. In UC, 4 were positive for pANCA, 15 for apANCA, 1 for cANCA, 7 for goblets cells and 2 for exocrine/acini. In CD and UC the proportion positive for ANCA was 30% and 50% respectively. Among the controls, while none had ANCA, there was 1 each positive for exocrine/acini and goblets cells respectively. Of all those with IBD and positive for ANCA (n: 23) only one patient had anti-myeloperoxidase. No antibodies were detected against the other 5 antigens tested. Conclusion: pANCA and apANCA are the predominant ANCA patterns seen in IBD with an obvious predominance in UC. This pilot Singapore study involving only Asian subjects seem to concur with Western studies in that there is no ANCA association with Proteinase 3, Lactoferrin, Myeloperoxidase, Elastase, Cathepsin G and Lysozyme. The authors acknowledge Euroimmun Lab for providing the IFT and ELISA kits.