Above is an electron micrograph I recently took of one of the organisms found in the tissue space at the site of a lesion in the colon of a patient with inflammatory bowel disease (IBD). Our current work involves analyzing tissue samples of IBD patients to identify the organisms present. Below is a portion of a paper we wrote on our preliminary findings. 

PRESENCE OF NOVEL FORMS IN THE COLON

OF INFLAMMATORY BOWEL DISEASE PATIENTS

ABSTRACT

Objective: A number of theories have been proposed to describe the causative agent in inflammatory bowel disease (IBD). However the etiology of IBD remains unknown. The present study was designed to characterize the appearance and the frequency occurrence of novel forms identified in the colon of a group of IBD patients. Methods: Biopsies from 81 colonoscopy patients were obtained and stained with a variety of stains including H&E and modified methenamine silver stain (GMS) for light microscopy. Stained specimens were analyzed under both normal and epifluorescent illumination. Results: Non-fluorescent GMS-stained forms were identified in 75% (± 11 95% CI) of nonspecific colitis patients: 70% (± 28 95% CI) of Crohn's patients: 100 % of ulcerative colitis patients and none were present in benign disease patients. Fluorescent GMS-stained forms were in 66% (± 12 95% CI) of the patients diagnosed with non-specific colitis: in 50% (± 31 95% CI) of the patients diagnosed with Crohn's disease and in 75% (± 42 95% CI) of the patients diagnosed with ulcerative colitis and no examples of fluorescent GMS-stained forms were found in patients identified with benign disease. The appearance of the novel forms were characterized under light and electron microscopy. Conclusions: Novel forms have been identified in the colon of a high proportion of IBD patients.

Inflammatory bowel disease (IBD) is a group of diseases which includes Crohn's disease, ulcerative colitis, and may include nonspecific colitis. Clinical, epidemiological, and experimental studies have been used to attempt to elucidate the etiology of IBD (1, 2). A number of factors, individually or in combination, have been implicated as causative agents for IBD. These factors include genetic (3), microbial (4), viral (5), immunological (6), nutritional (7), thrombosis (8) and environmental (9) agents as possible causative entities. For example, Mycobacterium paratuberculosis (10) , Pseudomonas maltophilia (11) and viruses (5) have been identified in the gut of IBD patients and hence implicated as etiological agents. A key feature associated with IBD is the activation of macrophages as well as other immune system activity. This involvement of the immune system has resulted in a number of proposed immunological and autoimmune models of IBD pathogenesis (12, 13). An increase in the incidence of IBD has also been reported in several of industrial countries which has lead to the suggestion that environmental (abiotic) agents may be responsible for IBD (14). In spite of the many proposed mechanisms and increasing interest in IBD, none of these studies have been able to demonstrate a clear cause and effect relationship and the etiology of IBD remains unknown.

In the present study, colonoscopy was performed and biopsies were taken from 81 patients with a diagnosis of diarrhea or lower GI bleeding during 1997 and 1998. Biopsy samples were fixed in neutral buffered formalin, embedded in paraffin, sectioned at 4um stained and mounted. A variety of different stains for light microscopy were applied including Periodic Acid Schiff (PAS), Feulgen, hematoxylin-eosin (H&E), acid fast stain, and modified methenamine silver (GMS) stain (Richard Allen Scientific-Chromaview). Samples for light microscopy observation were observed and photographed using an Olympus Vanox and IX70 inverted fluorescence microscopes with normal and 420 nm cube. Images were processed using Metamorph/Metaflour image analysis software (Universal Imaging). Non-fluorescent GMS-stained forms were identified and scored under normal light, fluorescent GMS-stained forms were observed and scored under epifluorescent illumination. Confidence limits for the population proportion were calculated using standard procedures (15).

Based on a review of the specimens stained with H&E, a diagnosis of nonspecific colitis was made in 43 patients; Crohn's disease was diagnosed in 10 patients; ulcerative colitis was diagnosed in 3 patients; and non-inflammatory disease was diagnosed in 5 patients. H&E and acid fast stained specimens did not reveal any novel forms. Specimens were then stained and observed under normal illumination with modified methenamine silver stain. Under these conditions a number polymorphic forms were observed that initially were thought to be a microsporidia-like pathogen except that they were negative for acid fast stain and expressed a different morphology. The non-fluorescent forms were found both individually or in groups in epithelial, lamina propia and stromal layers (see figure 1). These non-fluorescent forms were identified in 46 of the 61 nonspecific colitis patients, 7 of the 10 Crohn's patients, 4 of the 4 ulcerative colitis patients and none of the benign disease patients.

Under epifluorescent illumination with GMS stain, another form, often associated with regions of ulceration, was identified (see figure 1). These fluorescent forms were not colocalized with the GMS-stained forms we observed with normal illumination. The fluorescent forms were identified in 40 of the 61 nonspecific colitis patients, 5 of the 10 Crohn's patients, 3 of the 4 ulcerative colitis patients and again, none of the benign disease patients (see figure 2). Confidence intervals were calculated for the proportion of patients with both the non-fluorescent and fluorescent forms. In all cases the proportion of patients with non-fluorescent and fluorescent forms are greater than would be expected from random chance 95% of the time (see table 1).

Granuloma were present in a number of the IBD patients. In patients with granuloma, the non-fluorescent and fluorescent forms appear to be localized in the peripheral edges of the granuloma. Granuloma were found in 6 of the 43 nonspecific colitis patients, in 8 of the 10 Crohn's Disease patients, in 1 of the 3 ulcerative colitis patients and none were found in the benign disease patients.

The etiology of IBD has been extensively investigated and several models have been proposed with no known causal relationship having been established. In the present study, novel forms were identified from biopsies of the colon and the terminal ileum taken from IBD patients. These patients clinically expressed nonspecific chronic cellular inflammation of the colon in one of three different pathologic forms: nonspecific colitis, Crohn's disease, and ulcerative colitis. Some patients had evidence of either granulomata, active inflammation, ulcers with red halos, evidence of active colitis, or classic-appearing Crohn's disease with associated symptoms of diarrhea, abdominal pain or GI bleeding. In the present study previously unreported forms visible with GMS staining were found in a high percentage of these IBD patients. Work in our laboratory is currently underway to characterize the ultrastructure of these novel forms.

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Frequency of Forms in Patients With Different Diagnosis