April 1995 Immunology, Microbiology, and Inflammatory Disorders A801

• EFFECT OF NITRIC OXIDE SYNTHASE (NOS) INHIBITION ON DEXTRAN SULFATE SODIUM (DSS)-INDUCED COLITIS INRATS AND MICE. ~ , Y. Chela and M. B. Grisham. Dept. of Physiology and Biophysics, LSU Medical Center, Shreveport, LA 71130.

Nitric oxide (NO), a multifarious modulator of immune and inflammatory responses, has been implicated in the pathogenesis of chronic colitis. The objective of this study was to assess the role that NO may play in mediating the pathophysiology of DSS-induced colitis. Methods: Female BALB/C mice (18-22g; n > 5 per group) or male Sprague-Dawley rats (180-220g; nffi8 per group) received two 7-day cycles of 5% DSS (45,000MW) in their drinking water, with an intervening "/-day period of water alone. One mouse and one rat group each received either aminoguanidine (AG; 15#moles/kg/day) or N°nitro-L-arginine methyl ester (L-NAME; 15tLmoles/kg/day) by gavage 2-days prior to DSS treatment and daily thereafter during the 21 day DSS-eyelic administration period. Colonic macroscopic injury and inflammation scores, wet-to-dry ratios and nitric oxide synthase activities were quantified and used as indices of injury and inflammation. Results: Administration of 5% DSS to mice did not alter colonic NOS activity compared to control colonic activity (15,6+2.23 vs 11.0-1-0.96 pmoles/min/mgprotein). Control and 5% DSS eNOS activities were significantly reduced by L-NAME (10.4+1.00 and 14.6+2.07 vs 4 .9+1.01 pmoles/min/mg protein, respectively; p<0 .05) , but not by AG (12.4-l-1.50 pmoles/min/mg protein). Neither NOS inhibitor significantly affected iNOS. DSS administration to mice also significantly reduced colon length (9.94+0.23 vs 6.54+0.21 era; p<0 .05 ) however this alteration was not attenuated by the administration of AG or L-NAME. DSS-treated mice developed edema as reflected by significant increases in colonic wet-to-dry ratios compared to controls (5;30+0.25 vs 4 .0+0.17, respectively; p<0.05) . Neither the DSS induced edema nor the macroscopic injury and inflammation scores were attenuated by AG or L-NAME. Interestingly, DSS administration to rats produced a reduction in eNOS (17.05:3.12 vs 2 .3+0.67 pmoles/min/mg protein) and iNOS (7.35:1.54 vs 4.35:0.87 pmoles/min/mgprotein) activities compared to controls. As in the mouse model of DSS colitis, NOS inhibitors had no protective effect on bowel shortening, increases in colon wet-to-dry ratios or in macroscopic injury and inflammation scores. Conclusions: NOS inhibition does not attenuate the colitis induced by oral administration of DSS to mice or rats. We conclude that NO does not play a major role in mediating the pathophysiology of DSS-induced colitis. (Supported by DK47663)

ANORECTAL SURGERY IN HIV-INFECTED PATIENTS; CLINICAL OUTCOME AND ITS RELATION TO IMMUNE STATUS. ECJ Consten JFM Slors, HJ Noten, J Oosting, SA Danner, JJB van Lanschot, Departments of Surgery, Internal Medicine (AIDS Unit) and Clinical Epidemiology, Academic Medical Centre, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands.

The anorectum is commonly involved in HIV-infected patients. The aim of this study was to determine the spectrum of anorectal disease, its surgical treat- ment, clinical outcome and its relation to immune status. Methods: Medical records of all H/V-infected patients visiting the Academic Medical Centre of the University of Amsterdam between January 1984 and January 1994 were reviewed. Patients with anoreetul pathology requiring surgical treatment were divided into 5 different groups with respect to type of anorectal lesion. Group A consisted of patients with common anorectal disease; group B condylomata acuminata; group C perianal sepsis; group D anorectal ulceration; group E malignancies. Results: During the study period 1117 H/V-positive patients had been admitted to the Academic Medical Centre; 748 of those patients (67%) had been defined as having AIDS according to the CDC case definition. A total of 355 patients (32%) underwent general surgical treatment. Eighty-three patients (7.4%) needed 204 surgical consultations (13% conservative, 87% operative) for 170 anurectal lesions. Fitly-one (61%) of these patients were found to have multiple anoreetal pathology. Operative intervention resulted in adequate wound healing and symptomatic relief in 59% of patients. Adequate wound healing without relief of symptoms was found in 24% of patients. Inadequate wound healing was found in 17% of patients. Type of anorectal disease was related to inadequate wound healing (p < 0.001) and to preoperative CD4+-lympheoyte counts (p < 0.01). Inadequate wound healing and most severely disturbed immune status were encountered in group C, D and E. Within these groups Iow CD4+-lymphocyte counts were a risk factor for inadequate wound healing (p = 0.004). Median postoperative survival was highest (4.7 years) in group A and lowest (0.6 years) in group D and E and was related to type of anorectal disease (p = 0.0001). Conclusion: The incidence of anorectal pathology in HIV-infected patients is high. The spectrum of anorectal disease is complex. Type of anorectal disease is strongly related to immune status, wound healing and postoperative survival. In about 60% of patients surgical intervention results in symptomatic relief and adequate wound healing.

• LOCAL EXCISION AND MUCOSAL ADVANCEMENT FOR ANORECTAL ULCERATION IN HIV-INFECTED PATIENTS. ECJ Consten, JFM Slors, SA Dauner, GJA Offerhaus, JFWM Bartelsman, JJB van Lansehot, Departments of Surgery, Gastroenterology, Pathology and Internal Medicine (AIDS Unit), Academic Medical Centre, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands.

In H/v-infected patients no effective surgical procedure has been described for anorectnl ulceration resistant to antibiotie/antiviral therapy. These patients used to be treated by excision. Due to disappointing therapeutic results, we combIned excision with mucosal advancement in an attempt to resurface the defect. The aim of this retrospective study was to determine the effectiveness of surgical excision of anorceta] ulcers, with or without mucosal advancement, on patients' complaints. Methods: Medical records of H/v-infected patients presenting with anorcctal ulcers between 1984 and 1994 were reviewed. Patients were divided Into two groups with respect to treatment offered. All patients presenting between 1984 and 1990 (group AJ were treated consecutively by excision only; those presenting between 1991 and 1994 (group B) underwent consecutively exclsiun combined with mucosal advancement. Patient characteristics, duration of preoperative ulcer disease, immune status, cultures and histology of enorectal ulcers, and results of the two different types of surgical treatment were identified. Surgical treatment was considered successful. if relief of symptoms was achieved within 4 weeks after the operation. Results: In this study 26 patients suffered from anorectal ulceration; all homosexual man (mean age: 39 years). Group A consisted of 16 patients who underwent ulcer excisions only. Group B consisted of 13 patients who underwent ulcer exeisiuns with mucesal advancement; 3 patients previously had had one or more unsuccessful ulcer excisions without mucosal advancement. Duration of preoperative ulcer disease was on average 5 months In both groups. CD4÷-lymphocyte counts (group A: 110xl06/l; group B: 150x106/1) were comparable for both groups. Anorectal ulceration was related to HSV infection in 30% and to CMV irLfecdon in 29% of the patients. All biopsies were negative for malignancy. Excision of an anorectul ulcer was successful in 7 of the 16 patients in group A. Symptom relief was achieved in 12 of the 13 patients in group B. In group B surgical treatment appeared to be significantly better in comparison with the results in group A (p = 0,02). Conclusion: Operative treatment by excismn in combination with mucosal advancement is significantly more effective than excision only for persistent symptomatic anoreetal ulcers, unresponsive to medical therapy.

FLUORESCENCE MICROSCOPIC SCREENING OF ENTERIC PATHO- GENS IMPORTANT IN THE CHRONIC DIARRHEA OF AIDS: A NEW DIAGNOSTIC SCREENING APPROACH. C.N. Conteas, O.G.W. Bedin, T.M. Sowerby, J. Donovan. Division of Gastroenterology, Southern Calitomia Permanente Medical Group, Los Angeles, CA. Microbiology Reference Laboratory, Cypress, CA.

Aim: Chronic diarrhea m AIDS patients caused by mycobacteda, Crvo- ~ . ~ , Is~Dora belli, ~ sp. andmicrosporidia sp. are often difficult to detect by common laboratory techniques. A need exists for a rapid, simple and direct system by which pathologic samples might be examined for these pathogens. Fluorescence microscopy has recently become a .p~ular methodology for the detection of certain parasites such as mlcrospoddia sp. (Conteas CN et al., Gastro 1994; 106(4 part 2):A668. Van Gool T et al. 1994;46: 94,9. Vfivre Jet aL Folia Parasitologia 1993;40:273-4) and cryptosporidia (Van Gool T et aL J Clin Path 1993;46:694-9). These demonstrated epifluorescence to be a simple, rapid, sensitive and direct method for parasite detection. Recent ob- servations of an epifluorescentproperty of auramine-rhodamine in the presence of a 520-580 nm U.V. filter presents an important possible screening methodology for pathogen detection. Our goal was to develop a rapid screening methodology for stool, jejunal aspirates, mucosal touch preparations, and mucosal biopsies using the phenomenon of pathogen epifluorescence. Methods: Microscopic slides made of stool, jejunal aspirates, mucosal touch preparations and mucosal biopsy specimens (deparaffinized in xylene) from AIDS patients with chronic diarrhea are heated and methanol fixed and then stained with auramine-rhodamine. The stain is applied for 20 mintJtes, water washed, destained in acid alcoho for 20 m nutes, water washed and countemta ned wth potass um

ermanganate for 21 minutes. Air dded specimens are examined for epi- orescence at 250X under a 520'580 nm U.V. filter. The same slide is

treated with a drop of Fungi-Fl~r Stain (Polyscience, Inc. Warrington, PA) and immediately examined trader a 330-380 nm U.V. filter. Results: The auramine-rhodamine stain easily detected the presence of mycobac~ teria, CrvDtosooddium ~ , ~ i ~ and ~ sp. They presented with a characteristic and individually distinct epifiuorescent morphology. Fungi,Fluor Stain produced the specific epifiuorescence of microsporidia species with a characteristic brilliant blue halo effect. This methodology detected pathogens equally well in stool, jejunal aspirates, touch preparations and biopsy tissue specimens. Conclusion: Epifluores- cance microscopic evaluation of_ stool, jejunal aspirates and mucosal biopsy specimens presents a simple, rapid and sensitive technique for the detection of mycobactedum, Cryptosporidium a~p~.um ~ belli,

sp. and micresporidia species. The two staining techniffu-ffS allow for rapid detection of these pathogens are easily performed In a routine clinical laboratory setting, and are readily adaptable to large- scale screening of pathologic specimens.