Title

Studies into the pathogenesis of Crohn's disease

Date of Completion

January 2009

Keywords

Health Sciences, Pathology

Degree

Ph.D.

Abstract

Crohn's disease (CD) is a chronic inflammatory disease of the gastrointestinal tract. It is characterized by segmental transmural inflammation, mucosal ulceration, fistulas and the development of lymphocytic inflammatory aggregates and noncaseating granulomas. Case studies in the past repeatedly suggested that the fundamental alteration in CD occurs in the regional lymphatics of the intestine. The present study focused on the lymphatics and their role in the pathogenesis of CD. One of the objectives was to establish if the granulomas, or alternatively the inflammatory lymphoid aggregates of CD are, or are not, the product of lymphatic inflammation. Lymphatic proliferation and inflammation were evaluated in early and advanced lesions, and characterized new lymphoid aggregates and granulomas within and surrounding lymphatics. Fifty-one tissue blocks from 25 CD patients and twenty-three tissue blocks from 23 control patients were selected. Tissue sections were immunostained with a lymphatic marker (D2-40), a marker for blood vasculature (FVIII), and markers for T cells (CD3), B cells (CD20) and macrophages (CD68). This study demonstrated lymphangectasia, and lymphocytic perilymphangitis in all CD 25 patients, lymphocyte-obstructed lymphatics in 7, granuloma-obstructed lymphatics in 10 and inflammatory lymphoid follicles in all 25. Free-standing granulomas occurred in 20 patients; and in 3 further patients granulomas were in or attached to blood vascular units (arteries and arterioles). The present study, employing immunohistochemistry, revealed, better than standard microscopy, the association of inflammation, granulomas and tertiary lymphoid follicles or organs (TLOs) with the lymphatic vasculature. Disease in some patients was characterized by lymphocytic perilymphangitis and lymphoid follicular disease and in others by granulomas, some of which totally obstruct lymphatics. These findings have etiologic, therapeutic and prognostic implications. ^ A second objective was to investigate the potential role of human herpesvirus-6 (HHV-6) in Crohn's disease (CD). HHV-6 infections are usually asymptomatic reactivations in the immunocompetent, but often severe in immunocompromised individuals. Although primary HHV-6 infection is mainly associated with roseola infantum, it has also been associated with gastroenteritis, diarrhea, and nausea in children. Evidence of HHV-6 infection was determined by serology, immunohistochemistry (INC), and polymerase chain reaction (PCR). Fifty tissue blocks from 23 CD patients and twenty tissue blocks from 20 control patients were examined. In a separate study, sera from 14 others were evaluated for HHV-6 IgG antibodies. Quantitative real-time PCR (qPCR) was used to assess viral loads in ileal tissues. Serology, IHC, PCR and qPCR indicated the presence of HHV-6 in both CD patients and controls. Serologically, there was no significant difference observed in IgG titers between the groups. IHC on tissues revealed an almost equal distribution of HHV-6 immunoreactivity. HHV-6 DNA was detected in 52% (12/23) of CD and 55% (11/20) of control patients by PCR and in 69.5% (16/23) of CD cases and 65% (13/20) of controls by qPCR. Mean viral load in intestinal tissues was similar in CD and controls (33.4 copies/pg DNA and 57.9 copies/pg DNA, respectively). Finding equal evidence of HHV-6 in patients and controls by multiple methods suggests that this virus is ubiquitous and probably not a cause of Crohn's disease. ^