Crohn's Disease Specimens Research Articles

Potential role of protease-activated receptor-2-stimulated activation of cytosolic phospholipase A2 in intestinal myofibroblast proliferation: Implications for stricture formation in Crohn's disease

Background and aimsMyofibroblast hyperplasia contributes to muscularis mucosae thickening and stricture formation in Crohn's disease (CD). Protease-activated receptor-2 (PAR-2) and cytosolic phospholipase A2 (cPLA2) are known regulators of cell growth, but their significance in intestinal myofibroblast proliferation remain to be elucidated. The principle aims of the present study were to investigate if PAR-2 is expressed in the expanded muscularis mucosa in ileal CD specimens, if inflammatory cytokines may stimulate PAR-2 expression in intestinal myofibroblasts, and if PAR-2 and cPLA2 may regulate intestinal myofibroblast growth. MethodsImmunohistochemistry was used for detection of PAR-2 in ileal CD specimens. Studies on PAR-2 expression, PLA2 activation and cell growth were performed in a human intestinal myofibroblast cell line, CCD-18Co. PAR-2 expression was investigated by RT-PCR and immunocytochemistry. PLA2 activity was analyzed by quantification of released 14C-arachidonic acid (14C-AA). Cell growth was examined by 3H-thymidine incorporation and cell counting. ResultsThe thickened muscularis mucosae of the CD specimens showed strong PAR-2 expression. In cultured myofibroblasts, tumor necrosis factor-α (TNF-α) up-regulated PAR-2 mRNA and protein, and potentiated PAR-2-stimulated 14C-AA release by two known PAR-2 activators, trypsin and SLIGRL-NH2. The release of 14C-AA was dependent on cPLA2. Trypsin stimulated the proliferation of serum-starved cells, and inhibition of cPLA2 reduced normal cell growth and abolished the growth-promoting effect of trypsin. ConclusionsThe results suggest that PAR-2-mediated cPLA2 activation might be of importance in intestinal myofibroblast proliferation. The results also point to the possibility that PAR-2 up-regulation by inflammatory cytokines, like TNF-α, may modulate this effect.

Lymphangiogenesis in Crohn’s disease: an immunohistochemical study using monoclonal antibody D2-40

Crohn's disease (CD) is a chronic inflammatory bowel disorder of unknown etiology. An involvement of the intestinal lymphatic system has been suggested. Recently, monoclonal antibodies have become available to distinguish lymphatic vessels from blood vessels. The aim of the study was to examine the distribution of lymphatic vessels in ileal and colic walls of patients affected by CD and compare it with healthy controls and other inflammatory bowel diseases. Twenty-eight cases of CD, 13 cases of other inflammatory bowel diseases, and 10 normal ileal and colic walls were studied. Immunohistochemical staining was performed using the monoclonal antibody D2-40. Quantification of lymphatic vessels was performed by identifying four fields with high density of lymphatics and then counting the number of lymphatic vessels at high resolution. Lymphatic diameter was also evaluated by using an ocular micrometer. Lymphatic vessels showed the highest density in CD specimens. The median number of lymphatics was significantly higher both in ileal and colic samples of CD than the other inflammatory diseases as well as normal controls. Moreover, in patients with CD, diffuse lymphangiectasia was also observed. The present data suggest that lymphangiogenesis and lymphangiectasia probably play a role in the pathogenesis of CD.

Abnormal microbiota composition in the ileocolonic mucosa of Crohnʼs disease patients as revealed by polymerase chain reaction-denaturing gradient gel electrophoresis

Bacteria might play a role in the pathogenesis of Crohn's disease (CD), and patients harbor a different type and density of gut microbiota compared with normal healthy subjects. Thus, the aim of this study was to compare the microbiota adhered to the mucosa of CD patients with that of healthy subjects. Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) of 16S rRNA gene fragments was used to identify the dominant bacterial species present in fresh biopsy samples obtained from the mucosa of 15 healthy and 19 CD subjects. Two patients suffering from ulcerative colitis and 1 suffering from ischemic colitis also were included. Individuals were clustered in 2 groups according to their molecular fingerprint, which differentiated the majority of CD specimens (88.2%) from the majority of healthy/ulcerative colitis/ischemic colitis specimens (82.3%). In addition, the patient-to-patient variability in microbiota was greater within the CD cluster than in the healthy/ulcerative colitis cluster (P = 0.000). One hundred forty-one sequences were obtained from the PCR-DGGE bands that were grouped into 58 different phylotypes, 8 of which were novel. BLAST analysis revealed that 74.5% of the sequences were similar to those of bacteria that have never been cultivated. In CD samples, prevalence values for Clostridium spp Ruminococcus torques and Escherichia coli were significantly higher, whereas Faecalibacterium was more frequently found in healthy specimens. Opportunistic pathogenic gamma-proteobacteria were found occasionally, only in CD mucosal microbiota. Microbiota attached to the ileocolonic mucosa of CD patients is distinguishable from that of healthy subjects. We postulate that individuals who are predisposed to CD are less able to regulate the microbial makeup of their intestines, which leads to an unstable microbial population.

In vivo colonoscopic optical coherence tomography for transmural inflammation in inflammatory bowel disease

Transmural inflammation, a distinguishing feature of Crohn's disease (CD), cannot be assessed by conventional colonoscopy with mucosal biopsy. Our previous ex vivo study of histology-correlated optical coherence tomography (OCT) imaging on colectomy specimens of CD and ulcerative colitis (UC) showed that disruption of the layered structure of colon wall on OCT is an accurate marker for transmural inflammation of CD. We performed an in vivo colonoscopic OCT in patients with a clinical diagnosis of CD or UC using the previously established, histology-correlated OCT imaging criterion. OCT was performed in 40 patients with CD (309 images) and 30 patients with UC (292 images). Corresponding endoscopic features of mucosal inflammation were documented. Two gastroenterologists blinded to endoscopic and clinical data scored the OCT images independently to assess the feature of disrupted layered structure. Thirty-six CD patients (90.0%) had disrupted layered structure, whereas 5 UC patients (16.7%) had disrupted layered structure (P < .001). Using the clinical diagnosis of CD or UC as the gold standard, the disrupted layered structure on OCT indicative of transmural inflammation had a diagnostic sensitivity and specificity of 90.0% (95% CI: 78.0%, 96.5%) and 83.3% (95% CI: 67.3%, 93.3%) for CD, respectively. The kappa coefficient in the interpretation of OCT images was 0.80 (95% CI: 0.75, 0.86, P < .001). In vivo colonoscopic OCT is feasible and accurate to detect disrupted layered structure of the colon wall indicative of transmural inflammation, providing a valuable tool to distinguish CD from UC.

Ex vivo histology-correlated optical coherence tomography in the detection of transmural inflammation in Crohn’s disease

Distinguishing Crohn's disease (CD) from ulcerative colitis (UC) can be difficult. Transmural inflammation, a key feature of CD, cannot be assessed by conventional colonoscopy with biopsy. Optical coherence tomography (OCT) provides high-resolution, cross-sectional images of the gut wall and might become a new diagnostic tool. The aims of this study were to perform histology-correlated OCT on surgical specimens of CD and UC and to determine its diagnostic accuracy. Colectomy specimens from patients with a preoperative diagnosis of CD (N = 24) or UC (N = 24) were studied with OCT in the operating room. OCT and histopathology were assessed blindly, and diagnostic accuracy of OCT was assessed. Eight preoperatively identified UC patients (33%) with transmural inflammation on postoperative histology were diagnosed with CD, and all 8 had a disrupted layered structure on OCT, a characteristic feature of transmural disease. Sixteen UC patients (67%) had superficial inflammation on histology; of them, 13 (81%) had an intact layered structure on OCT. All 24 preoperative CD patients had transmural inflammation on histology, and 23 (96%) had a disrupted layered structure on OCT. Of 585 histology-OCT image sets from the 48 patients, 152 sets (26%) had transmural inflammation on histology. The sensitivity and specificity for OCT to detect transmural disease were 86% and 91%, respectively. Transmural inflammation, as characterized by disruption of the layered structure of colon wall on OCT, is an accurate marker for the diagnosis of CD. Ex vivo OCT predicted transmural inflammation on postoperative histopathology.

Colonic explant production of IL-1and its receptor antagonist is imbalanced in inflammatory bowel disease (IBD).

IBD is associated with an increased activation of intestinal immune cells, which causes overproduction of proinflammatory cytokines such as IL-1beta. IL-1beta is implicated in mediating the sustained inflammatory response. IL-1 receptor antagonist (IL-1Ra), the naturally occurring inhibitor of IL-1, has been shown to have beneficial effects in experimental models of colitis. In this study we investigated the hypothesis that an imbalance between IL-1 and IL-1Ra exists in IBD by measuring their secretion by explant cultures of colonic biopsies. Freshly homogenized biopsies from involved tissue in IBD patients exhibited significantly lower IL-1Ra/IL-1beta ratios than control and uninvolved IBD mucosal tissue. Using explant cultures, in vitro production of IL-1beta and IL-1Ra increased progressively during the 4-18-h culture periods. IL-1beta secretion was higher in supernatants from involved Crohn's disease (CD) and ulcerative colitis tissue compared with control tissue, and IL-1beta levels increased with severity of inflammation. IL-1Ra secretion was not elevated in involved IBD samples, but significantly higher levels were released when moderate to severely involved tissue samples were compared with noninflammatory controls. Similar to freshly homogenized tissue, explant studies showed that the IL-1Ra/IL-1beta ratios were significantly decreased in involved IBD tissue, but not in uninvolved CD or inflammatory control specimens. These data support the hypothesis of an imbalance between IL-1beta and IL-1Ra in IBD.

Sensitive differential detection of genetically related mycobacterial pathogens in archival material.

A polymerase chain reaction (PCR) assay targeted to the immunogenic protein MPB64 gene was used to detect members of the Mycobacterium tuberculosis complex, and an outward-primed PCR (OPPCR) designed on the IS6110 element allowed differentiation between Mycobacterium bovis and Mycobacterium tuberculosis. Additionally, the amplification of IS1110 and 16S ribosomal RNA sequences combined with a dot blotting assay were able to differentially detect Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium paratuberculosis. The validity of the experimental procedure was tested on reference material and formalin-fixed paraffin-embedded samples from patients with tuberculosis, sarcoidosis, or Crohn disease. We demonstrated mycobacterial DNA in 59 of 75 cases with histologic lesions typical of tuberculosis; we detected M tuberculosis and M paratuberculosis in 6 of 25 sarcoidosis cases and in 7 of 20 Crohn disease specimens, respectively. The proposed diagnostic procedure is directly applicable to archival material and allows differentiation of genetically related mycobacterial pathogens in more detail than other molecular methods. It provides a tool for the diagnostic study of tuberculosis, sarcoidosis, and Crohn disease.

Identification of Mycobacterium avium ss. paratuberculosis in Crohn's tissue

Background: Mycobacterium ss. paratuberculosis (MAP) is known to cause chronic granulomatous enteritis in ruminants and primates (Johne s Disease). Many groups have implicated the possible role of the spheroplastic form of MAP in Crohn's Disease (CD) but the isolation and identification of this bacteria have been difficult due to the uncertain location and rigid growth requirements of this organism. Purpose: To investigate the role of MAP in CD using short-term mycobacterial culture media. Methods: 27 CD tissue specimens (7 surgical resected tissue and 20 biopsies) and 36 controls (3 surgical resected tissue and 33 biopsies) were processed and cultured in both 12B* Bactec bottles and Mycobacterial Growth Indicator Tubes (MGIT) with OADC enrichment, Mycobactin J and PANTA antibiotic mixture for up to one year. Bacterial detection and identification was done through Acid fast staining, mycobactin dependency, PCR analysis using two IS900-derived oligonucleotides and hybridization with an internal probe. Results: Microbial growth was found in both culture media, although only MGIT cultures contained MAP. MAP bacterial isolates were confirmed to be acid fast bacilli, mycobactin dependent and positive for the presence of IS900 and hybridization. Presence of MAP in MGIT cultures were detected in 12 weeks of incubation for resected tissue and 40 weeks for biopsies. See table for results. Conclusion: MAP was much more readily found in CD specimens as compared to control specimens. Additionally, MAP was found more readily in resected tissue and required short-term incubation in contrast to endoscopic biopsies. This may indicate that MAP resides submucosally. Our findings suggest that surgical resected tissue cultured in MGIT media is favorable for the rapid cultivation of MAP versus endoscopically acquired biopsies.

Altered Expression of Laminins in Crohn's Disease Small Intestinal Mucosa

Laminins are a large family of heterotrimeric basement membrane molecules that mediate crucial cell functions such as adhesion, proliferation, migration, and differentiation. Up to now, three distinct laminins have been identified in the normal human small intestinal epithelium. Laminin-1 (alpha1beta1gamma1) and laminin-5 (alpha3beta3gamma2) are mainly expressed at the base of villus cells, whereas laminin-2 (alpha2beta1gamma1) is restricted to the bottom of the crypts. The expression of these molecules has not yet been studied in Crohn's disease (CD), but it could be altered, in light of the important changes occurring in the architecture of the crypt-villus axis under the active state of the disease. To test this hypothesis, the expression of laminin alpha1, alpha2, and alpha3 subunits was analyzed in control, inflamed, and corresponding uninflamed CD small intestinal specimens by indirect immunofluorescence and reverse transcriptase-polymerase chain reaction. Surprisingly, alpha1 and alpha3 remained strongly expressed by all villus cells, whereas alpha2, normally expressed in the bottom of the crypts in control and uninflamed CD specimens, was lacking in inflamed CD specimens. However, this loss of alpha2 expression was associated with a significant up-regulation of both alpha1 and alpha3 expression in the crypts of inflamed CD specimens. A significant up-regulation of the alpha1 subunit was also observed in the crypts of uninflamed CD specimens. At the transcript levels, alpha1 was found significantly higher in inflamed than uninflamed CD specimens. Taken together, these observations identify important alterations in laminin expression in the small intestine with CD and suggest that compositional changes in the epithelial basement membrane may play a role in this disease.

Epithelial permeability to proteins in the noninflamed ileum of Crohn's disease?

Background & Aims: Crohn's disease (CD) is associated with a disturbed intestinal barrier. Permeability studies have focused on inert molecules, but little is known about transepithelial transport of macromolecules with antigenic potential in humans. The aim of this study was to quantify permeation and to characterize passage routes for macromolecules in ileal mucosa in CD. Methods: Noninflamed and inflamed ileal mucosa specimens from patients with CD (n = 12) and ileal specimens from patients with colon cancer (n = 7) were studied regarding transmucosal permeation of ovalbumin, dextran (mol wt, 40,000), and 51Cr-EDTA for 90 minutes in vitro in Ussing chambers. Transepithelial passage routes for fluorescent ovalbumin and dextran 40,000 were investigated by confocal microscopy. Results: Noninflamed ileum from CD patients showed increased permeation of ovalbumin compared with ileum from colon cancer patients ( P < 0.05). Dextran permeation was equal in the three groups, whereas 51Cr-EDTA permeability was increased in inflamed ileum. Ovalbumin passed both transcellularly and paracellularly, but dextran followed a strictly paracellular route. Both markers were subsequently endocytosed by cells of the lamina propria. Conclusions: Noninflamed ileal mucosa from patients with CD shows increased epithelial permeability to ovalbumin, probably by augmented transcytosis. This increase in antigen load to the lamina propria could be an initiating pathogenic event in CD. GASTROENTEROLOGY 1999;117:65-72

In situ Rantes and interferon-gamma gene expression in pediatric small bowel Crohn's disease.

Rantes (regulated upon activation, normal T cell expressed and secreted) is a chemotactic cytokine for memory T lymphocytes, monocytes, and eosinophils. The cytokine interferon-gamma (IFN-gamma) plays a key in the immune response. Their distributions and possible roles in the selective accumulation of inflammatory cells in Crohn's disease (CD) were examined by determining the expression of Rantes and IFN-gamma genes in patients with CD using in situ hybridization (ISH) on frozen and paraffin-embedded tissue sections. Intestinal and mesenteric lymph node samples from 9 children who had undergone ileal resection for CD were examined for the presence of epithelioid-giant cell granulomas (EGCG) and Rantes and IFN-gamma messenger RNA (mRNA). Normal pediatric intestine (n = 5) and lymph nodes (n = 2) served as controls. Many cells in all CD specimens in the epithelial compartment, lamina propria, and the EGCG gave positive signal with the Rantes antisense probe. Labelled cells were identified on paraffin sections as lymphocytes, macrophages, and epithelioid cells. There were Rantes-positive cells in the control intestinal tissues, but many Rantes-positive cells in control lymph nodes that showed follicular hyperplasia. IFN-gamma-positive cells were present in all CD ileal and lymph node specimens, predominantly in close contact with EGCC. No positive signal was obtained with the Rantes and IFN-gamma sense control probes. These findings suggest that Rantes and IFN-gamma contribute to the selective accumulation of macrophages and memory T helper lymphocytes inside the granulomas and inflammatory infiltrates that are characteristic of CD.

Crohn's disease and ulcerative colitis mucosal T cells are stimulated by intestinal epithelial cells: Implications for immunosuppressive therapy

Background . Crohn's disease (CD) and ulcerative colitis (UC) are chronic inflammatory diseases, and their pathogenesis is attributed, in part, to alterations of the mucosal immune system. This study was designed to define the possible contribution of epithelial cells to the activation of lamina propria T lymphocytes (LPTs) in CD and UC. Methods . LPTs isolated from CD, UC, and control surgical specimens were cocultured with freshly isolated allogeneic or autologous epithelial cells or epithelial cell lines. Resulting T-cell proliferation was evaluated by tritiated thymidine incorporation on day 5. Results . When intestinal epithelial cells were used to stimulate mucosal T-cell proliferation, CD and UC LPTs were less responsive than conrol LPTs (p < 0.05 and p < 0.03, respectively). This difference between inflamed and control T cells was consistently observed by using a variety of different intestinal epthelial cell types. Conclusions . CD and UC mucosal T cells are hyporesponsive to activation by intestinal epithelial cells when compared with control LPTs. Elucidating the mechanism underlying the differential activation of CD and UC LPTs may help to better understand the immunopathogenesis of these conditions.

IN SITU RANTES AND INTERFERON-γ GENES EXPRESSION IN PEDIATRIC SMALL BOWEL CROHN'S DISEASE

Aims: Rantes (regulated upon activation, normal T cell expressed and secreted) is a chemotactic cytokine for memory T lymphocytes, monocytes and eosinophils. Interferon-γ (IFN-γ) is a key cytokine responsible for immune response. To visualize their distribution and to understand their possible role in the selective accumulation of inflammatory cells in Crohn's disease (CD), we analysed the expression of Rantes and IFN-γ genes from patients with CD using in situ hybridization (ISH) on frozen and paraffin tissue sections. Méthods: We studied intestinal and mesenteric lymph node samples from 9 children who had undergone ileal resection for CD. The samples were examined for the presence of epithelioid-giant cell granulomas (EGCG). Normal pediatric intestine (n=5) and lymph nodes (n=2) served as controls. Radiolabelled Rantes and IFN-γ mRNA probes were used. Results: Positive signal with the Rantes antisense probe was present within numerous cells in all CD specimens in the epithelial compartment, lamina propria and within EGCG. Labelled cells were identified in paraffin sections as lymphocytes, macrophages, and epithelioid cells. Only scattered Rantes positive cells were present in the intestinal control tissues. Numerous Rantes positive cells were observed in control lymph nodes exhibiting follicular hyperplasia. IFN-γ positive cells were present in all CD specimens in ileal and lymph node specimens, predominantly in close contact with EGCC. No positive signal was obtained with Rantes and IFN-γ sense probes. Conclusions: These findings suggest Rantes and IFN-γ participation in the selective accumulation of macrophages and memory T helper lymphocytes inside granulomas and in the inflammatory infiltrate both of which are characteristics of CD.

Inability of normal human intestinal macrophages to form multinucleated giant cells in response to cytokines.

Multinucleated giant cells are an important feature of the granulomatous reaction in Crohn's disease (CD) but their cellular origin is poorly understood. The aim of this study was to discover if intestinal macrophages are capable of generating multinucleated giant cells in vitro in response to cytokine stimulation. Human intestinal macrophages were isolated from the intestinal mucosa of CD and uninflamed surgical specimens. Isolated macrophages were cultured in chamber slides with and without exposure to a panel of cytokines and cell activators. Cell fusion, multinucleated giant cells formation, and the expression of adhesion molecules were assessed at various time intervals. In contrast with the autologous peripheral monocytes cell fusion was very poor in cultures of control intestinal macrophages and virtually no multinucleated giant cells were seen. Control intestinal macrophages seemed to poorly express the adhesion molecules required for cell to cell adhesion and fusion, namely ICAM-1 and LFA-1. None of these functions was affected by the exposure to cytokines, including interferon gamma. In cultures of macrophages isolated from CD tissues multinucleated giant cell formation spontaneously occurred as early as three days and was not enhanced by the addition of cytokines. CD macrophages seemed to highly express both ICAM-1 and LFA-1. These data show that human intestinal macrophages are unable to form multinucleated giant cells in response to stimuli and support the concept that they are downregulated in a number of functions. The data also suggest that macrophages participating in the ganulomatous reaction in CD are recruited from the circulation.

Differential expression of substance P receptors in patients with Crohn's disease and ulcerative colitis

Background & Aims : Although clinical and pathological differences exist between Crohn's disease (CD) and ulcerative colitis (UC), distinguishing features are often absent, making diagnosis and treatment problematic. This study evaluated the differences in the expression of substance P (SP) receptors in patients with CD or UC. Methods : Tissue samples from patients with inflammatory bowel disease or control patients were obtained at surgery, processed for 125I-SP binding, and analyzed by quantitative autoradiography. Results : Patients with CD showed a massive increase in SP receptors in lymphoid aggregates, small blood vessels, and enteric neurons of the small and large bowel relative to controls. Six of 16 CD specimens had no pathological evidence of CD yet continued to express high concentrations of SP receptors. Pathologically positive patients with UC showed high concentrations of SP receptors on colonic lymphoid aggregates and small blood vessels but not enteric neurons. No increased SP binding was evident in clinically and pathologically quiescent UC colons and normal UC ileostomy samples. Conclusions : The increased expression of SP receptors on the enteric neurons of patients with CD distinguishes CD from UC. The persistent increased SP binding in pathologically normal CD tissue may indicate a subclinical disease state. SP receptor expression may have important diagnostic, etiologic, and therapeutic usefulness in inflammatory bowel disease.

Location of tumour necrosis factor alpha by immunohistochemistry in chronic inflammatory bowel disease.

This study determined the location and tissue density of cells immunoreactive for tumour necrosis factor alpha (TNF alpha) in intestinal specimens from 24 patients with chronic inflammatory bowel disease (15 with Crohn's disease, nine with ulcerative colitis) and 11 controls. There was significantly increased density of TNF alpha immunoreactive cells in the lamina propria of both ulcerative colitis and Crohn's disease specimens, although the distribution of these cells differed in the two conditions. In ulcerative colitis most of the TNF alpha immunoreactivity was seen in the subepithelial macrophages, with comparatively less in the deep lamina propria, while in Crohn's disease immunoreactive cells were distributed evenly throughout the lamina propria. Increased submucosal immunoreactivity was found only in Crohn's disease, in which TNF alpha positive macrophages tended to cluster around arterioles and venules, often infiltrating and disrupting vascular endothelium. It is suggested that this degree of TNF alpha production probably contributes significantly to the pathogenesis of both Crohn's disease and ulcerative colitis, by impairing the integrity of epithelial and endothelial membranes, increasing inflammatory cell recruitment, and by prothrombotic effects on the vascular endothelium.

Deposits of terminal complement complex (TCC) in muscularis mucosae and submucosal vessels in ulcerative colitis and Crohn's disease of the colon.

Extensively washed, ethanol fixed and paraffin embedded colonic specimens from 15 patients with ulcerative colitis (UC) and nine patients with Crohn's disease (CD) of the colon, ileal specimens from six patients with CD of the ileum, and histologically normal control specimens obtained from 10 patients operated for colonic carcinoma, were examined by immunohistochemistry with a monoclonal antibody specific for a neoepitope in the C9 part of the terminal complement complex (TCC). The submucosal blood vessels in inflammatory bowel disease (IBD) showed significantly more TCC positivity than the controls, and vascular TCC deposition was statistically related (p less than 0.001) to degree of inflammation. Five of the six ileal CD specimens contained likewise vascular TCC deposits. In addition, five UC specimens and one colonic CD specimen contained TCC-positive fibrils in the muscularis mucosae or submucosa. There was no significant difference in vascular TCC deposits between UC and CD. The results suggested that terminal complement activation takes place in the intestinal lesions of IBD.

Differentiation Between Crohnʼs Disease and Other Inflammatory Conditions by Electron Microscopy

The authors previously have demonstrated axonal necrosis of autonomic nerves in the surgically resected ilea of patients with Crohn's disease both in grossly normal ileal resection margins and in diseased areas. The present study of ileal stomal biopsies was carried out to obviate the possibility that the observed axonal damage might be related to the prolonged surgical manipulations required for ileal resection. The authors present studies of biopsies of ileal stomas and of small bowel from patients with Crohn's disease and various control disorders, including ulcerative colitis. Stomal biopsies were fixed immediately after they were obtained. Widespread, severe axonal necrosis of autonomic nerves was present in all Crohn's disease specimens, regardless of the patient's clinical status or the gross or routine microscopic evaluation of the same specimen. Controls either had no necrosis or displayed a minor degree of focal necrosis involving single axons. The authors conclude that Crohn's disease is accompanied by a severe and extensive necrosis of gut axons, and that such electron microscopic findings may serve to differentiate Crohn's disease from other inflammatory disorders.

Differentiation Between Crohnʼs Disease and Other Inflammatory Conditions by Electron Microscopy

The authors previously have demonstrated axonal necrosis of autonomic nerves in the surgically resected ilea of patients with Crohn's disease both in grossly normal ileal resection margins and in diseased areas. The present study of ileal stomal biopsies was carried out to obviate the possibility that the observed axonal damage might be related to the prolonged surgical manipulations required for ileal resection. The authors present studies of biopsies of ileal stomas and of small bowel from patients with Crohn's disease and various control disorders, including ulcerative colitis. Stomal biopsies were fixed immediately after they were obtained. Widespread, severe axonal necrosis of autonomic nerves was present in all Crohn's disease specimens, regardless of the patient's clinical status or the gross or routine microscopic evaluation of the same specimen. Controls either had no necrosis or displayed a minor degree of focal necrosis involving single axons. The authors conclude that Crohn's disease is accompanied by a severe and extensive necrosis of gut axons, and that such electron microscopic findings may serve to differentiate Crohn's disease from other inflammatory disorders.

Intestinal fine structure in Crohn's disease. Lysosomal inclusions in epithelial cells and macrophages.

Resected intestines from eight patients with Crohn's disease and three control cases were investigated by transmission electron microscopy. Characteristic changes were observed in the mucosa of all Crohn's disease specimens, most typically an infiltration of numerous macrophages into the propria. These cells displayed large lysosomes with inclusions which were mainly dense, irregularly shaped and composed of aggregated particles and bizarre-shaped, myelin-like figures. Similar inclusions were also found in the lysosomes of the surface epithelial cells and the macrophages in the submucosa. This latter layer otherwise consisted of an oedematous, collagenous connective tissue. The muscularis appeared structurally unaffected. Qualitatively, the findings were almost identical in all patients with Crohn's disease, but varied quantitatively without any clear correlation with the clinical histories. Moreover, in all cases typical alterations were found not only in the macroscopically most clearly affected parts of the intestines, but also in grossly normal regions, close to the margins of resection. The analogy of the fine structural findings with those of granulomas produced by injection of bacteria into experimental animals suggests that a microbial invasion of the intestinal wall may have initiated the disease. It therefore seems reasonable to assume that the lysosomal inclusions we observed represent partly degraded bacteria. More occasionally, virus-like particles were found within the lysosomes of the epithelial cells and the macrophages in the underlying propria. In view of the diffuse spread of the alterations it seems possible that there exists a generalized defect in the barrier function of the intestine in Crohn's disease. This could lead to passage of bacteria and/or other agents into the mucosa, followed by an influx of inflammatory cells from the blood. Storage of non-degradable microbial components in macrophages could then be responsible for the initiation and propagation of a chronic inflammatory process, similar to that of other granulomatous disorders.