Experimental model of rat ileitis resembling Crohn’s disease

Crohn’s disease (CD) is a chronic, idiopathic and relapsing inflammatory disease of the gastrointestinal (GI) tract. Current theories point to an impaired immune response to microbes within the intestinal flora in a genetically susceptible host. Although the lymphatic obstruction represents one of the main histopathological diagnostic criteria, its etiology is unknown. Many animal models that, in some respects, resemble human CD have been developed, but these models do not represent the complexity of human disease. We planned to obtain an animal model of CD by surgical manipulation of the enteric lymphatic vessels in the TNBS model of rat ileitis. Surgical approach: 12 F344 male rats were surgically treated by ligature and cut of ileo-colic lymphatic chain near the Treitz. The lymph nodes near the ileocecal valve were injected with a sclerosing agent (control group). Surgical and chemical approach: F344 were treated as above and, at the same time, intraluminally ileum injected with TNBS, a chemical reagent able to induce severe enteritis. 6 rats were sacrificed after one week (group 1). 8 rats received two more weekly TNBS injections and then were sacrificed (group 2). Control and treated rats were sacrificed at the same time. Animals were weighted and monitored for clinical manifestations of GI inflammation. At sacrifice, macroscopical changes were noted and ileum and colon specimens were harvested and processed for histopathological analysis. Most of the animals showed adhesions of bowel loops and thickening of the mesentery. Groups 1 and 2: TNBS induced more severe macroscopical features of inflammation in the intestinal wall, such as fistolae, dilatation of the ileum and sometimes intestinal obstruction. Microscopically, we observed transmural inflammatory infiltrate, fibrosis of mesentery, granulomas, and in the most severe cases, signs of neoangiogenesis, derangement of the muscle layers, ulcers and loss of villi in the mucosa. Besides the microscopical inflammatory changes, we evidenced an increased number of dilated lymphatic vessels. Group 1: one week after a single injection, most of the animals partially solved and showed less severe microscopical damages. The surgical and chemical approach did not induce in this rat model the severe fibrosis and the stricture of intestinal lumen that usually lead to surgery in human CD. However, the histopathological features found in these rats resemble the severe pathological features observed in human CD

Multimodal imaging in the diagnosis and evaluation of intestinal malrotations in adults: a case report

Midgut malrotation is a congenital anomaly referring to either lack of or incomplete rotation of the fetal intestines around the axis of the superior mesenteric artery during fetal development. It is rare in adulthood and the true incidence is difficult to estimate because most patients are asymptomatic. The diagnosis is usually performed with several radiological and surgical methods. We report a case of a woman who presented with cramp-like abdominal pain localized to the right iliac fossa. The patient underwent abdominal ultrasound, radiological examination without and with contrast, and computed tomography with three-dimensional volume rendering reconstruction. Although small bowel follow-through is often enough to recognize the type of malrotation, using multimodal imaging may offer a better definition of this abnormality with a better definition of the kind of malrotation, by adding additional anatomical information. In our case, the imaging clearly showed malrotation of the small bowel with reverse rotation of the colon. Hence a multimodal imaging strategy proved useful for the diagnosis of intestinal malrotation in an adult afflicted by chronic cramp-like abdominal pain

The gastric wall in systemic sclerosis patients: a morphological study

Organ failure secondary to fibrosis is the main cause of morbidity and death in patients with systemic sclerosis. Gastrointestinal tract dysmotility is a major visceral manifestation, clinically ranging from an asymptomatic form to severe paresis. Although the oesophagus is the most frequently affected part of the gastrointestinal tract, all other segments can be involved. The present study was undertaken to evaluate the histopathological changes of the gastric wall in a series of full­thickness biopsies from systemic sclerosis patients who underwent gastric surgery due to severe gastroesophageal involvement. Gastric biopsies were processed for light microscopy and transmission electron microscopy. The histological and ultrastructural observations revealed a generalized fibrosis affecting all the gastric wall layers. The most severe changes were observed in the muscularis mucosae and muscle layers. Wide areas of marked focal fibrosis with dense collagen bundles and elastic fibre deposition surrounding smooth muscle cells were found. Myofilaments and thickened dense bodies were severely disarranged or absent in most smooth muscle cells. Nerve fibres showed ultrastructural alterations, such as oedematous axoplasm and scarce cytoskeletal elements. Abundant elastic and collagen fibres enveloped nerve fibres, nerve endings and interstitial cells of Cajal, thereby separating them from smooth muscle cells and blood microvessels. This study provides evidence for a prominent fibrosis and severe ultrastructural alterations of smooth muscle cells and nerve fibres as the main histopathological hallmarks in the gastric wall of systemic sclerosis patients

Differential expression of junctional adhesion molecules in different stages of systemic sclerosis: pathogenetic implications

Systemic sclerosis (SSc, scleroderma) is characterized by early perivascular inflammation, microvascular endothelial cell (MVEC) activation/damage and defective angiogenesis. Junctional adhesion molecules (JAMs) regulate leukocyte recruitment to sites of inflammation and ischemia-reperfusion injury, vascular permeability and angiogenesis. In the present study, we investigated the possible role of JAMs in SSc pathogenesis.The expression of JAM-A and JAM-C in skin biopsies from 25 patients with SSc (14 early-stage SSc, 11 late-stage SSc) and 15 healthy subjects was investigated by immunohistochemistry and western blotting. Subcellular localization of JAMs in cultured dermal healthy MVECs (H-MVECs) and early-stage SSc-MVECs was assessed by confocal laser scanning microscopy. Serum levels of soluble JAM-A (sJAM-A) and JAM-C (sJAM-C) from 64 SSc patients (25 early-stage SSc, 39 late-stage SSc) and 32 healthy subjects were examined by ELISA.In control skin, constitutive JAM-A expression was observed in dermal MVECs and fibroblasts. In early-stage SSc skin, JAM-A expression was strongly increased in MVECs, fibroblasts and perivascular infiltrating inflammatory cells. In late-stage SSc, JAM-A expression was decreased compared with healthy controls. JAM-C was weakly expressed in control and latestage SSc skin, while it was strongly expressed in MVECs, fibroblasts and inflammatory cells from early-stage SSc skin. Cell surface expression of JAM-A was higher in early-stage SSc-MVECs and increased in H-MVECs upon stimulation with early-stage SSc sera. JAM-C was cytoplasmic in resting H-MVECs, while it was rapidly recruited to the cell surface upon challenging with early-stage SSc sera. Early-stage SSc-MVECs exhibited increased expression and constitutive localization of JAM-C on the cell surface. Circulating levels of sJAM-A and sJAM-C were significantly increased in earlystage SSc compared with healthy controls and late-stage SSc. In addition, sJAM-A and sJAM-C levels were significantly raised in SSc patients with ‘early/active’ nailfold capillaroscopic pattern compared with those with ‘late’ capillaroscopic pattern. Collectively, the results of our study suggest that JAMs may participate in dermal MVEC activation, inflammatory processes and impaired angiogenesis in different stages of SSc

Stiff skin syndrome: evidence for an inflammation-independent fibrosis?

Objectives. Stiff skin syndrome (SSS) is a rare scleroderma-like syndrome of unknown aetiology. A 16-year-old boy presented with thoracic and abdominal asymmetry, and ‘orange peel' cutaneous lesions, with fibrotic stone-hard indurations at the buttocks, thighs and arms leading to secondary joint contractures of the extremities. Our aim was to analyse the expression of extracellular matrix (ECM) molecules and pro-fibrotic cytokines in the dermis and epidermis of SSS. Methods. The diagnosis of SSS was confirmed by clinical and histopathological examination. Collagen type 1 alpha-2 chain (Col1A2), fibronectin-1, thrombospondin-1, TGF-β, connective tissue growth factor (CTGF), IL-6, -1β, ET-1, Fibroblast growth factor receptor 3 (FGFR-3) and MCP-1 expression was analysed in SSS and age- and sex-matched healthy control skin by real-time PCR. VEGF expression was also studied. Results. Histopathological examination showed flattened dermal papillae, a scarce presence of sub-epidermal microvessels and mild dermal fibrosis, but no inflammatory infiltrates. In the SSS dermis, the expression of IL-1β, -6 and MCP-1 was low, whereas VEGF was intensively expressed. No differences were observed for TGF-β, CTGF and ET-1. In contrast, col1A2, fibronectin-1 and thrombospondin-1 were overexpressed in the SSS dermis. Conclusion. In our SSS patient, an overexpression of ECM proteins was detected, whereas no inflammatory infiltrates or up-regulation of pro-fibrotic cytokines were found. The data suggest that fibrosis in SSS might be independent from inflammatio

Bone marrow-derived mesenchymal stem cells from early diffuse systemic sclerosis stimulate in vitro angiogenesis and foster revascularization of ischemic limbs

Systemic sclerosis (SSc) is a chronic connective tissue disorder characterized by widespread microangiopathy, lack of angiogenesis, and fibrosis. Bone marrow-derived mesenchymal stem cells (MSCs) produce and release high amounts of proangiogenic and antiapoptotic factors that inhibit apoptosis of endothelial cells under hypoxic conditions and promote the formation of capillary-like structures in vitro. In the present study, we characterized MSCs from SSc patients for the expression of factors implicated in MSC recruitment at sites of injury, angiogenesis, and fibrosis. We also analyzed whether SSc-MSCs could modulate dermal microvascular endothelial cell (MVEC) angiogenesis in vitro. MSCs were obtained from 5 patients with early diffuse SSc and 5 healthy donors. MSCs were expanded in culture and their phenotype was investigated by flow cytometry, CFU-F assay, and analysis of osteogenic/adipogenic differentiation. MSCs were stimulated with VEGF, TGFβ, or SDF-1. Transcript and protein levels of SDF-1 and its receptor CXCR4, VEGF, TGFβ1 and receptors TβRI/TβRII were evaluated by real-time RT-PCR, Western blotting, and confocal microscopy. VEGF, SDF-1, and TGFβ1 secretion in culture supernatant was measured by ELISA. MVEC capillary morphogenesis was performed on Matrigel with the addition of MSC-conditioned medium. SSc- and healthy-MSCs showed no significant differences in morphology, proliferation rate, CFU-F colony formation or osteogenic/adipogenic differentiation. In SSc-MSCs, the basal expression of proangiogenic SDF-1/CXCR4 and VEGF was significantly increased compared to healthy-MSCs. SSc-MSCs constitutively released higher levels of SDF-1 and VEGF. SDF-1/CXCR4 were upregulated after VEGF stimulation, and CXCR4 redistributed from cytoplasm to cell surface. VEGF was increased by SDF-1 challenge. VEGF, TGFβ and SDF-1 stimulation upregulated TGFβ1, TβRI, and TβRII in SSc-MSCs. SSc-MSC-conditioned medium showed a greater proangiogenic effect on MVECs than healthy-MSCs. Experiments with blocking antibodies demonstrated that MSC-derived cytokines were responsible for this potent proangiogenic effect. One patient who developed gangrene of the upper and lower limbs was treated with 3 intravenous infusions of expanded autologous MSCs. Areas of necrotic skin were reduced after the first MSC infusion. After the third infusion, angiography showed revascularization of the patient’s extremities. Skin section analysis revealed cell clusters with tube-like structures, and angiogenic factors were strongly expressed. SSc-MSCs constitutively overexpress and release proangiogenic factors, and potentiate dermal MVEC angiogenesis in vitro. In SSc patients with severe peripheral ischemia, intravenous infusion of expanded autologous MSCs may foster the recovery of the vascular network

Histopathological modifications in blood-induced haemophilic artropathy

Haemarthrosis triggers haemophilic arthropathy (HA), involving mainly the larger joints. Mechanisms and pathways of blood-induced joint damage are still not completely clear. The molecular triad OPG/RANK/RANKL tightly controls the bone turnover and any change in the balance between OPG and RANKL leads to bone pathological conditions. The aim of this study was to evaluate the expression of RANK/RANKL/OPG system in synovial tissue of haemophilic patients (pts) with severe HA, compared to osteoarthritic (OA) pts. Synovial biopsies from 20 HA pts and from 16 OA pts, obtained during arthroplasty of the knee, were routinally processed for light microscopy. The severity of HA was evaluated according to i) Ultrasonography (US) score, ii) World Federation of Haemophilia orthopaedic joint scale (WFH) and iii) radiographic Petterson method. Synovial sections were stained with H&E to evaluate pathological changes. The expression of RANK, RANK-L, OPG was examined by immunohistochemistry. Moreover, serum levels of sRANKL and OPG from 67 HA pts and 30 controls were measured by ELISA assay. The mean US, WFH and Pettersson score in HA pts were 11(range 0-21 with cut off>5), 39,5 (range:12-57), and 10,4 points (range: 6-12), respectively. Microscopically, the synovium from HA pts showed a large amount of intra- and extracellular haemosiderinic deposits. The lining layer showed mild proliferation and in the sublining area the vessels showed thickened wall, due to a local and chronic inflammatory stimuli. A lower expression of OPG was found in HA synovium compared to OA. RANK and RANK-L positivity was strongly expressed in the lining and sublining both in HA and OA synovial tissue. Serum levels of sRANKL and OPG resulted lower than in controls. Our preliminary data show that, in HA pts, the synovium highly expressed RANK and RANKL, whereas OPG positivity decreased, suggesting an osteoclastic activation. Tissue expression of OPG correlates with serum level of this protein in HA pts and with severity of HA, as assessed by US score