Inflammatory bowel disease. The case for an infectious etiology.

The infectious hypothesis for IBD is certainly not new. The evidence for an infectious agent is tantalizing but not yet convincing. A failure to demonstrate a specific agent in the inflammatory process could mean that many different organisms are involved, or several organisms that, by themselves, do not cause disease interact. All candidate organisms proposed thus far are compatible with either hypothesis. Nevertheless, interaction between an agent(s) in a host modified by immunologic and genetic factors is still an attractive hypothesis for the cause of these illnesses.

Use of rifabutin in treatment of systemic Mycobacterium paratuberculosis infection in mice.

BALB/c mice were infected intraperitoneally with Mycobacterium paratuberculosis and, after allowing the infection to progress for 30 days, were treated with rifabutin at 0, 12.5, 25, and 50 mg/kg of body weight. Rifabutin was administered in drinking water under conditions of water deprivation, whereby the entire daily dose was delivered within a 1-h period. Animals were killed at biweekly intervals from time zero of treatment to 180 days. Spleens and livers from each animal were examined by quantitative bacteriologic culture and histopathology. Restricted water availability was found to be a viable alternative to daily gavage for single-dose bolus administration. Infection, as assessed by bacterial counts, was reduced only in animals that received 50 mg of rifabutin per kg. In these animals, bacterial counts in the liver and spleen were reduced from 7.2 x 10(5) +/- 4.1 x 10(4) and 6.5 x 10(5) +/- 4.1 x 10(4) to 3.0 x 10(3) +/- 1.8 x 10(2) and 3.1 x 10(3) +/- 2.2 x 10(2), respectively, over the 6-month treatment period. Rifabutin may be an appropriate chemotherapeutic drug for long-term treatment of M. paratuberculosis infection and should be considered in any multidrug regimen.

The use of antimycobacterial agents in Crohn's disease.

Seventy-five years ago, a Scottish surgeon, Dalziel, clearly described Crohn's disease (CD) and suggested that it might be caused by a mycobacteria. However, mycobacteria were not isolated from CD tissue until 1978 and 1984. Since then several investigators, using sophisticated polymerase chain reaction (PCR) techniques, found Mycobacterium paratuberculosis sequences in cultures of Crohn's disease tissue in approximately two thirds of patients. Because of a possible mycobacterial etiology, Rutgeerts et al. (J Clin Gastroenterol 1992;15:24-8) treated resected ileocolonic CD patients who had early evidence of recurrence with ethambutol and rifabutin. Patients were followed by colonoscopy to observe changes in the intestinal lesions, but no improvement was noted. Rutgeerts et al. concluded that antimycobacterial therapy did not affect the course of CD. Failure of these antibiotics does not necessarily negate the mycobacterial theory of CD, however. The study population of Rutgeerts et al. was too small, and the treatment period too short. Furthermore, ethambutol shows little or no activity against the proposed Crohn's organism, Mycobacterium paratuberculosis; use of only one active agent (rifabutin) can lead to drug resistance and failure of antibiotic therapy. We hope that Rutgeerts et al. will continue these important studies, but with a more active combination of antimycobacterial drugs for a longer time.

Implications of antibodies to pyruvylated glucose in healthy populations for mycobacterioses and other infectious diseases.

Members of the Mycobacterium avium-Mycobacterium intracellulare (MAI) complex are typeable because each serovar is characterized by its own specific antigenic glycolipid. By means of an enzyme-linked immunosorbent assay, we studied serum specimens obtained from 148 healthy college students for antibodies to these glycopeptidolipids. Ninety-two (61.5%) of the serum specimens were positive to the specific glycolipid antigen from MAI serovar 8. In a study of a pediatric population, antibodies appeared to develop during adolescence. Individuals with overt mycobacterial disease had a significantly lower incidence (tuberculosis patients, 34.5%; leprosy patients, 25%). We found a lower incidence of positive results in a survey of 96 Japanese serum specimens (29.1%), but the results from a survey of sera obtained from Bombay, India, indicated a large degree of reactivity (55.5%). Antibodies to other MAI serovars (serovars 2, 4, and 11) were not found, except antibodies to MAI serovar 21 were seen in the same individuals with antibodies to serovar 8. The dominant epitope of the MAI serovar 8-specific glycopeptidolipid is a terminal pyruvylated 3-O-methylglucose residue [4,6-(1'-carboxyethylidene)-3-O-methyl-alpha-D-glucopyranosyl] unit, whereas that of the MAI serovar 21 has the same terminal pyruvylated glucose devoid of the 3-methoxy group. Thus the antibodies appear specific for the pyruvylated glucose. It is unclear whether the high prevalence of antibodies to these epitopes reflects a high incidence of subclinical colonization or infection with certain MAI serovars or whether they are acquired through contact with some other related antigen source.

Mycobacterium paratuberculosis infection in a colony of stumptail macaques (Macaca arctoides).

Mycobacterium paratuberculosis infection was documented in a colony of stumptail macaque monkeys (Macaca arctoides), with 29 (76%) of 38 monkeys infected and shedding organisms in feces. Thirteen deaths have occurred during the past five years. Animals without overt clinical disease were shedding as many as 2 X 10(6) colony-forming units of M. paratuberculosis/g of feces. Intestinal tissues from animals dying of this disease contained up to 10(8) colony-forming units of M. paratuberculosis/g of tissue. The clinical and pathological features of paratuberculosis in this species were comparable to those reported for paratuberculosis in ruminants and Mycobacterium avium infections in primates. By enzyme-linked immunosorbent assay, antibodies to M. paratuberculosis were found in 79%-84% of the animals. Antibodies could not be detected in six animals with clinical disease. These findings extend the natural host range of M. paratuberculosis to include nonhuman primates and add support to current suggestions that M. paratuberculosis may be pathogenic for humans.

Experimental disease in infant goats induced by a Mycobacterium isolated from a patient with Crohn's disease. A preliminary report.

Pilot studies were done to assess the pathogenicity of a Mycobacterium which had been recovered from the diseased ileum of a patient with Crohn's disease. In four separate studies, pairs of infant goats served as subjects. One of each pair received an oral inoculum of freshly harvested Mycobacterium species strain Linda suspended in cream. A littermate or stablemate which received only cream served as control. Necropsies were done at three, five, six, and 10 months postinoculation. Each of the four inoculated animals developed segmental granulomatous disease of the ileum or ileum and more proximal segments of small intestine, and regional lymph nodes. The earliest lesion occurred in Peyer's patches of the ileum and consisted of granulomatous clusters of epithelioid cells and giant cells, without caseation, which often occurred in a mantle of lymphocytes between the germinal centers and the muscularis mucosae. Nine of 10 such granulomas were free of acid-fast bacilli. In more advanced lesions, there was confluence of granulomas and ulceration of the mucosal surface. Two of the four inoculated animals also had lymphocytic lymphangitis in affected segments. Although the Mycobacterium Linda was recovered from intestinal segments of all four animals, acid-fast bacteria were not demonstrable in the intestines in two of them. Control animals remained free of lesions and acid-fast bacilli and were negative by bacteriologic culture. The Mycobacterium species strain Linda represents an enteric pathogen capable of inducing granulomas of the distal small intestine of susceptible species. The lesions produced have distinct similarities to those occurring in Crohn's disease.

Spheroplastic phase of mycobacteria isolated from patients with Crohn's disease.

Two strains of an unclassified Mycobacterium species were isolated after 18 and 30 months of incubation of media inoculated with resected intestinal tissues from patients with Crohn's disease. These strains represented the third and fourth isolates of this organism from Crohn's disease patients. Ultrastructural examination of this strain and two previously isolated strains revealed the presence of spheroplasts which eventually transformed into the bacillary form of a previously unrecognized Mycobacterium species. These cell wall-deficient forms did not stain with conventional dyes and failed to grow on hypertonic media. Restriction polymorphism of the ribosomal DNA genes was used to determine the relationship between the cell wall-deficient and bacillary forms. Identical restriction patterns of the ribosomal DNA genes were found between the spheroplasts and Mycobacterium sp. isolates with EcoRI, BamHI, and XhoI restriction endonucleases, thus providing definitive evidence of their origin. Unidentified spheroplasts were isolated from an additional 12 patients with Crohn's disease, of which 7 of 10 seroagglutinated with antiserum prepared against the Mycobacterium sp. Spheroplasts were isolated from 16 of 26 (61%) patients with Crohn's disease but not from tissues of 13 patients with ulcerative colitis or 13 patients with other diseases of the bowel. These findings support the role of mycobacteria as etiologic agents in some cases of Crohn's disease.

A negative search for four infectious agents in inflammatory bowel disease.

Diseased resected portions of intestine from patients with inflammatory bowel disease were examined by chicken embryo inoculation for the presence of chlamydia and histologically for the presence of chlamydia, intracellular campylobacters, Tyzzer's bacilli, and cryptosporidia. Chlamydia were not isolated from any of the seven colon specimens tested and chlamydia, intracellular campylobacters, Tyzzer's bacilli, and cryptosporidia were not demonstrated in 39 sections of colon and ileum from 16 IBD patients.

Effects of penicillamine on serum immunoglobulins and immune complex-reactive material in primary biliary cirrhosis.

Although penicillamine is used in the treatment of primary biliary cirrhosis, its mechanism of action in this disease is unknown. As an immunologic action had been attributed to the drug, we investigated whether penicillamine might alter serum immunoglobulin levels or immune complex-reactive material in patients with primary biliary cirrhosis. Immunoglobulin levels and immune complex reactivity were measured and clinical tests were performed in 53 consecutive patients entering a double-blind randomized trial of 750 mg vs. 250 mg of penicillamine. Measurement of immune complex reactivity was determined by laser nephelometry, 125I-C1q binding, and Raji cell assays. Immune complex reactivity was detected by at least one assay in 75% of patients tested before treatment. Sixty-two percent were positive in the C1q assay, 28% in the Raji cell assay, and 39% by nephelometry. After therapy with either dose, we found no change in immune complex-reactive material by any assay. Concentrations of immunoglobulins G and M fell (p less than 0.05) after 12 mo of therapy. Concentrations of immunoglobulin A decreased (p less than 0.05) only in the high-dose group. Correlation was not consistent between results of immune complex assays and clinical liver tests. Although immunoglobulin levels fell during penicillamine therapy, no decrease in immune complex-reactive material was detected. The effect of penicillamine in primary biliary cirrhosis is not mediated through alteration of immune complex-reactive material.

In vitro antimicrobial susceptibility of a Mycobacterium sp. isolated from patients with Crohn's disease.

The in vitro susceptibility of three strains of an unclassified Mycobacterium sp., isolated from three patients with Crohn's disease, to 23 antimicrobial agents was determined by a modified broth dilution method with 7H9 broth containing oleic acid-albumin-dextrose-catalase, Tween 80, and mycobactin J. All three strains were susceptible to streptomycin, viomycin, rifampin, clofazimine, cefazolin, amikacin, and kanamycin and resistant to p-aminosalicylic acid, cycloserine, 2-thiophenecarboxylic acid hydrazide, trimethoprim, diaminodiphenylsulfone, sulfamethoxazole, sulfadimethoxine, polymyxin B, metronidazole, neomycin, and carbenicillin. Variable results between strains were encountered with ethambutol, ethionamide, capreomycin, amoxicillin, and cephalothin.

Possible role of mycobacteria in inflammatory bowel disease. II. Mycobacterial antibodies in Crohn's disease.

An unclassified Mycobacterium species has been isolated from two patients with Crohn's disease (CD). Antibodies to the unclassified mycobacteria cross-reacted with Mycobacterium paratuberculosis. Because of this cross-reactivity, an enzyme-linked immunosorbent assay (ELISA) was used to examine the sera of inflammatory bowel disease (IBD) patients, both CD (N = 56), and ulcerative colitis (UC) (N = 34), for antibodies to M. paratuberculosis, Mycobacterium kansasii, and Mycobacterium tuberculosis. Controls consisted of healthy, PPD-negative individuals (N = 67), and from PPD-positive patients (N = 41). Eighteen resected CD patients were also examined. CD patients had a statistically significant increase in antibody titer (P = 0.0003) to M. paratuberculosis compared to healthy controls. Although patients with positive PPD had elevated titers to this organism, the positive response of CD patients was not related to PPD responsiveness, area of involvement in the gut, nor to activity of the disease process.

Possible role of mycobacteria in inflammatory bowel disease. I. An unclassified Mycobacterium species isolated from patients with Crohn's disease.

A previously unrecognized Mycobacterium species was isolated from two patients with Crohn's disease. The organism is an acid-fast, mycobactin-dependent Mycobacterium that has characteristics which do not conform to any of the presently recognized species. It belongs to the Runyon group III mycobacteria and is most closely related to Mycobacterium paratuberculosis. Animal inoculation revealed pathogenicity for mice when injected intravenously or intraperitoneally, but not for rats, guinea pigs, rabbits, or chickens. The mice developed hepatic and splenic granulomas which contained numerous acid-fast mycobacteria. A 7-day-old goat which was inoculated orally with 50 mg of the organism developed both humoral and cell-mediated immunologic responses in two to three weeks and granulomatous disease of the distal small intestine, with noncaseating tuberculoid granulomas in five months. Acid-fast bacilli were not demonstrable in sections of the intestine, but a single organism was seen in each of two microgranulomas of the mesenteric lymph node. The Mycobacterium species was reisolated from the lymph node but not from intestine. Our findings raise the possibility that a Mycobacterium plays an etiologic role in at least some cases of Crohn's disease.

Characteristics of an unclassified Mycobacterium species isolated from patients with Crohn's disease.

The characteristics of an unclassified Mycobacterium sp. isolated from three patients with Crohn's disease are presented. The organism is extremely fastidious and mycobactin dependent and may require up to 18 months of incubation for primary isolation. Colony morphology is rough. Characteristics are unlike those of any presently defined species. The isolates produced postive niacin, catalase, and 2-week arylsulfatase reactions and were susceptible to neotetrazolium chloride (1:40,000), streptomycin (2 micrograms/ml), and rifampin (0.25 micrograms/ml). Chromogenicity, nitrate reduction, quantitative catalase, Tween hydrolysis, urease, tellurite reduction, pyrazinamidase, and 3-day arylsulfatase tests were negative, and the isolates were resistant to thiophene-2-carboxylic acid hydrazide (10 micrograms/ml) and isoniazid (10 micrograms/ml). Optimum growth in broth was determined to be in 7H9 medium with Dubos oleic albumin complex, Tween 80, and mycobactin J at 37 degrees C without CO2 or agitation and in low medium depth. This Mycobacterium sp. may be a subspecies or biovariant of Mycobacterium paratuberculosis, or it may represent a new species of Mycobacterium. It is suggested that this Mycobacterium sp. may play an etiological role in some cases of Crohn's disease.

Elevated circulating immune complexes in primary sclerosing cholangitis.

Primary sclerosing cholangitis (PSC) is a syndrome of unknown etiology characterized by an association with inflammatory bowel disease in 50% or more cases. Since altered immunity, including circulating immune complexes, has been implicated in the pathogenesis of inflammatory bowel disease, we postulated that humoral immune mechanisms might also be important in the development of PSC. Therefore, as an initial step in testing this hypothesis, we examined sera of patients with PSC for the presence of circulating immune complexes by two independent methods: C1q binding and Raji cell assays. Twenty-four patients with PSC, 16 of whom had coexisting chronic ulcerative colitis, were prospectively selected by predefined biochemical, histologic, and radiographic criteria. Sixteen patients with inflammatory bowel disease and normal liver tests as well as six patients with extrahepatic biliary obstruction served as disease controls. Sera were positive for circulating immune complexes by at least one method in 80% (16/20) of patients with PSC; 70% (14/20 were positive by the Raji cell assay, 58% (14/24) by the C1q binding assay, and 45% (9/20) by both methods. Levels of circulating immune complexes by each assay were higher in sera from patients with PSC than in sera from healthy controls or patients with inflammatory bowel disease alone (p less than 0.01). There were no differences in the levels of circulating immune complexes or in the frequency of positive tests in PSC patients with or without associated inflammatory bowel disease. In addition, there was no difference between the Raji cell binding of sera from six patients with extrahepatic biliary obstruction and six healthy controls tested concurrently. These data are consistent with the hypothesis that immunologic mechanisms may be important in the pathogenesis of PSC.

Hemochromatosis associated with chronic cimetidine therapy?

We report a patient in whom hemochromatosis with high transferrin saturation developed during long-term cimetidine administration. Prior reports have noted impaired iron absorption to be associated with short-term cimetidine use. The effect of chronic cimetidine administration on iron absorption and metabolism has not been studied. We believe this to be the first, and so far the only report of increased iron absorption with long-term cimetidine use.