Lymphatic endothelial progenitor cells and vascular endothelial growth factor-C in spondyloarthritis and Crohn's disease: two overlapping diseases?

OBJECTIVES: The role of the lymphatic system in the connection between spondyloarthritis (SpA) and Crohn's disease (CD) remains yet to be elucidated. The aim of the present study was to investigate the circulating levels of lymphatic endothelial progenitor cells (LEPCs) and vascular endothelial growth factor-C (VEGF-C) and their possible correlation with clinical parameters in SpA, SpA associated with CD (SC), and CD. METHODS: Peripheral blood samples from SpA (n=36), SC (n=20) and CD (n=28) patients and 20 age- and sex-matched healthy controls were collected and used for quantification of circulating LEPCs and VEGF-C. LEPCs were identified by fluorescence-activated cell sorting using FITC-CD34, APC-CD133 and PE-VEGFR-3 antibodies. Serum levels of VEGF-C were measured by enzyme-linked immunosorbent assay. The possible correlations between disease duration (< or >10 years; < or >20 years) and clinical activity (BASDAI for SpA or CDAI for CD) and LEPC counts and VEGF-C levels were analysed. RESULTS: Circulating LEPC levels were significantly increased in SpA (p=0.0006) and SC (p=0.0058) patients compared with controls. In CD patients, LEPC counts negatively correlated with disease duration, with lower levels in longstanding disease (>20 years, p=0.018), but were not different from controls. No significant difference in VEGF-C levels was found in SpA, SC and CD compared with controls. Both LEPC and VEGF-C levels were independent of BASDAI and CDAI. CONCLUSIONS: On the basis of our observations, an active mobilisation of lymphatic endothelial cell precursors was observed only for spondylitis involvement.

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 followthrough 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.

Inactivation of urokinase-type plasminogen activator receptor (uPAR) gene induces dermal and pulmonary fibrosis and peripheral microvasculopathy in mice: a new model of experimental scleroderma?

OBJECTIVE: Urokinase-type plasminogen activator receptor (uPAR) is a key component of the fibrinolytic system involved in extracellular matrix remodelling and angiogenesis. The cleavage/inactivation of uPAR is a crucial step in fibroblast-to-myofibroblast transition and has been implicated in systemic sclerosis (SSc) microvasculopathy. In the present study, we investigated whether uPAR gene inactivation in mice could result in tissue fibrosis and peripheral microvasculopathy resembling human SSc. METHODS: The expression of the native full-length form of uPAR in human skin biopsies was determined by immunohistochemistry. Skin and lung sections from uPAR-deficient (uPAR(-/-)) and wild-type (uPAR(+/+)) mice at 12 and 24 weeks of age were stained with haematoxylin-eosin, Masson's trichrome and Picrosirius red. Dermal thickness and hydroxyproline content in skin and lungs were quantified. Dermal myofibroblast and microvessel counts were determined by immunohistochemistry for α-smooth muscle actin and CD31, respectively. Endothelial cell apoptosis was assessed by TUNEL/CD31 immunofluorescence assay. RESULTS: Full-length uPAR expression was significantly downregulated in SSc dermis, especially in fibroblasts and endothelial cells. Dermal thickness, collagen content and myofibroblast counts were significantly greater in uPAR(-/-) than in uPAR(+/+) mice. In uPAR(-/-) mice, dermal fibrosis was paralleled by endothelial cell apoptosis and severe loss of microvessels. Lungs from uPAR(-/-) mice displayed non-specific interstitial pneumonia-like pathological features, both with inflammation and collagen deposition. Pulmonary pathology worsened significantly from 12 to 24 weeks, as shown by a significant increase in alveolar septal width and collagen content. CONCLUSIONS: uPAR(-/-) mice are a new animal model closely mimicking the histopathological features of SSc. This model warrants future studies.

Telocytes in Crohn's disease.

Crohn's disease (CD) is a relapsing chronic inflammatory disorder that may involve all the gastrointestinal tract with a prevalence of terminal ileum. Intestinal lesions have a characteristic discontinuous and segmental distribution and may affect all layers of the gut wall. Telocytes (TC), a peculiar type of stromal cells, have been recently identified in a variety of tissues and organs, including gastrointestinal tract of humans and mammals. Several roles have been proposed for TC, including mechanical support, spatial relationships with different cell types, intercellular signalling and modulation of intestinal motility. The aim of our study was to investigate the presence and distribution of TC in disease-affected and -unaffected ileal specimens from CD patients compared with controls. TC were identified by CD34/PDGFRα immunohistochemistry. In affected CD specimens TC disappeared, particularly where fibrosis and architectural derangement of the intestinal wall were observed. In the thickened muscularis mucosae and submucosa, few TC entrapped in the fibrotic extracellular matrix were found. A discontinuous network of TC was present around smooth muscle bundles, ganglia and enteric strands in the altered muscularis propria. At the myenteric plexus, the loss of TC network was paralleled by the loss of interstitial cells of Cajal network. In the unaffected CD specimens, TC were preserved in their distribution. Our results suggest that in CD the loss of TC might have important pathophysiological implications contributing to the architectural derangement of the intestinal wall and gut dysmotility. Further functional studies are necessary to better clarify the role of TC loss in CD pathophysiology.

Differential expression of junctional adhesion molecules in different stages of systemic sclerosis.

OBJECTIVE: Systemic sclerosis (SSc) 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. This study was undertaken to investigate the possible role of JAMs in SSc pathogenesis. METHODS: JAM-A and JAM-C expression levels in skin biopsy samples from 25 SSc patients and 15 healthy subjects were investigated by immunohistochemistry and Western blotting. Subcellular localization of JAMs in cultured healthy dermal MVECs and SSc MVECs was assessed by confocal microscopy. Serum levels of soluble JAM-A (sJAM-A) and sJAM-C in 64 SSc patients and 32 healthy subjects were examined by enzyme-linked immunosorbent assay. RESULTS: In control skin, constitutive JAM-A expression was observed in MVECs and fibroblasts. In early-stage SSc skin, JAM-A expression was strongly increased in MVECs, fibroblasts, and perivascular inflammatory cells. In late-stage SSc, JAM-A expression was decreased compared with controls. JAM-C was weakly expressed in control and late-stage SSc skin, while it was strongly expressed in MVECs, fibroblasts, and inflammatory cells in early-stage SSc. Surface expression of JAM-A was higher in early-stage SSc MVECs and increased in healthy MVECs stimulated with early-stage SSc sera. JAM-C was cytoplasmic in resting healthy MVECs, while it was recruited to the cell surface upon challenge with early-stage SSc sera. Early-stage SSc MVECs exhibited constitutive surface JAM-C expression. In SSc, increased levels of sJAM-A and sJAM-C correlated with early disease and measures of vascular damage. CONCLUSION: Our findings indicate that JAMs may participate in MVEC activation, inflammatory processes, and impaired angiogenesis in different stages of SSc.

RANK-RANKL-OPG in hemophilic arthropathy: from clinical and imaging diagnosis to histopathology.

OBJECTIVE: Hemarthrosis triggers hemophilic arthropathy, involving the target joints. The histopathogenesis of blood-induced joint damage remains unclear. The triad of receptor activator of nuclear factor-κB (RANK), RANK ligand (RANKL), and osteoprotegerin (OPG; RANK-RANKL-OPG) controls bone turnover. Our aim was to evaluate RANK-RANKL-OPG expression in the synovium of hemophilic patients with severe arthropathy. METHODS: Synovial biopsies were obtained from 18 patients with hemophilic arthropathy and 16 with osteoarthritis (OA) who were undergoing total knee replacement and synovectomy. The severity of hemophilic arthropathy was evaluated according to ultrasonography score, the World Federation of Hemophilia (WFH) orthopedic joint scale, and the radiographic Pettersson score. RANK-RANKL-OPG expression was examined by immunohistochemistry and Western blotting. Serum levels of soluble RANKL (sRANKL) and OPG from an extended group of 67 patients with hemophilic arthropathy and 30 healthy controls were measured by ELISA. RESULTS: The mean ultrasonography, WFH orthopedic joint scale, and Pettersson scores in patients with hemophilic arthropathy indicated severe arthropathy. A decreased expression of OPG was found in hemophilic arthropathy synovium compared with patients with OA. RANK and RANKL immunopositivity was strong in the lining and sublining layers in hemophilic arthropathy synovial tissue. Western blotting confirmed the immunohistological findings. Serum levels of sRANKL and OPG in patients with hemophilia were lower than in healthy controls. CONCLUSION: In hemophilic arthropathy, the synovium highly expressed RANK and RANKL, whereas OPG immunopositivity decreased, suggesting an osteoclastic activation. Low tissue expression of OPG paralleled the serum levels of this protein and the severity of hemophilic arthropathy assessed by ultrasonography, Pettersson, and WFH orthopedic joint scale scores.

Bone marrow-derived mesenchymal stem cells from early diffuse systemic sclerosis exhibit a paracrine machinery and stimulate angiogenesis in vitro.

OBJECTIVE: To characterise bone marrow-derived mesenchymal stem cells (MSCs) from patients with systemic sclerosis (SSc) for the expression of factors implicated in MSC recruitment at sites of injury, angiogenesis and fibrosis. The study also analysed whether the production/release of bioactive mediators by MSCs were affected by stimulation with cytokines found upregulated in SSc serum and tissues, and whether MSCs could modulate dermal microvascular endothelial cell (MVEC) angiogenesis. METHODS: MSCs obtained from five patients with early severe diffuse SSc (SSc-MSCs) and five healthy donors (H-MSCs) were stimulated with vascular endothelial growth factor (VEGF), transforming growth factor ß (TGFß) or stromal cell-derived factor-1 (SDF-1). Transcript and protein levels of SDF-1 and its receptor CXCR4, VEGF, TGFß(1) and receptors TßRI and TßRII were evaluated by quantitative real-time PCR, western blotting and confocal microscopy. VEGF, SDF-1 and TGFß(1) secretion in culture supernatant was measured by ELISA. MVEC capillary morphogenesis was performed on Matrigel with the addition of MSC-conditioned medium. RESULTS: In SSc-MSCs the basal expression of proangiogenic SDF-1/CXCR4 and VEGF was significantly increased compared with H-MSCs. SSc-MSCs constitutively released higher levels of SDF-1 and VEGF. SDF-1/CXCR4 were upregulated after VEGF stimulation and CXCR4 redistributed from the cytoplasm to the cell surface. VEGF was increased by SDF-1 challenge. VEGF, TGFß and SDF-1 stimulation upregulated TGFß(1), TßRI and TßRII in SSc-MSCs. TßRII redistributed from the cytoplasm to focal adhesion contacts. SSc-MSC-conditioned medium showed a greater proangiogenic effect on MVECs than H-MSCs. Experiments with blocking antibodies showed that MSC-derived cytokines were responsible for this potent proangiogenic effect. CONCLUSION: SSc-MSCs constitutively overexpress and release bioactive mediators/proangiogenic factors and potentiate dermal MVEC angiogenesis.

Evidence for the prevention of enthesitis in HLA-B27/hß(2)m transgenic rats treated with a monoclonal antibody against TNF-α.

Transgenic rats with high expression of HLA-B27 and human ß(2) -microglobulin (B27TR) develop a multisystem inflammatory disease resembling human inflammatory bowel disease (IBD) and spondyloarthropaties (SpA). Tumour necrosis factor α (TNF-α) has a crucial role in sustaining chronic inflammation in the gut and joints. The aim of this work was to evaluate whether TNF-α blockade could prevent or reduce the inflammation of peripheral joints in B27TR. A first group of 9-week-old B27TR received an anti-TNF-α monoclonal antibody (mAb) or an isotypic IgG2a,k up to the age of 18 weeks. An untreated group was monitored up to the age of 18 weeks and then randomly assigned to a 9-week treatment with anti-TNF-α mAb or IgG2a,k. Each rat was monitored for clinical IBD and peripheral joint manifestations. After sacrifice the colon and hind paws were examined for macroscopical and microscopical pathological changes. Early TNF-α blockade prevented, and late treatment improved IBD signs in B27TR. Erythema, oedema, inflammatory infiltrate close to the tendons and enthesis, proliferating chondrocyte-like cells, signs of new endochondral bone ossification and bone erosion were observed in peripheral joints of four out of six IgG2a,k-treated B27TR, both at 18 and 27 weeks. Immunopositivity for phosphorylated Smad1/5/8 indicated that the process of joint remodelling was activated in B27TR. Some entheses showed chondroid nodules. Anti-TNF-α treatment reduced inflammation and preserved the enthesis organization in most animals. Occasional and transient erythema and oedema were still present in three of six of the late anti-TNF-α-treated animals. Smad1/5/8 signalling was not inhibited by late anti-TNF-α treatment. In B27TR, articular involvement follows IBD onset and develops at entheses. Early TNF-α blockade prevents the onset of IBD and consequently the development of enthesitis in peripheral joints in the B27TR model of human SpA.

Progressive loss of lymphatic vessels in skin of patients with systemic sclerosis.

OBJECTIVE: Systemic sclerosis (SSc) is a connective tissue disorder characterized by microvascular and fibrotic changes in the skin and internal organs. The role of blood vessel dysfunction in the pathogenesis of SSc has been extensively investigated, but few studies have addressed the involvement of the lymphatic vascular system. Our aim was to evaluate dermal lymphatic vessels in patients with SSc according to different phases of skin involvement. METHODS: Skin biopsies were obtained from the forearm of 25 SSc patients (10 early/15 late-stage disease) and 13 healthy controls. Skin sections were immunostained for podoplanin (D2-40), which is selectively expressed in lymphatic endothelial cells, and examined by confocal laser scanning microscopy. Lymphatic vessels were counted in the papillary and reticular dermis. Data were analyzed using Student's t test. RESULTS: The number of lymphatic vessels was significantly reduced in the papillary and reticular dermis of SSc patients compared with controls. In early SSc, lymphatic vessel counts were not different from controls in the papillary dermis, and showed a trend toward a reduction in the reticular dermis. In late SSc, a significant reduction in lymphatic vessels compared with controls was found in both the papillary and reticular dermis. The number of lymphatic vessels in the papillary dermis of late SSc was significantly lower than in early SSc. CONCLUSION: In SSc, lymphatic microangiopathy is linked to the progression of skin involvement. The progressive disappearance of lymphatic vessels may have a critical pathogenetic role in the progression of SSc from an early edematous phase to overt fibrosis.

Autologous mesenchymal stem cells foster revascularization of ischemic limbs in systemic sclerosis: a case report.

BACKGROUND: Mesenchymal stem cells can differentiate into endothelial cells and participate in angiogenesis in adults. In experimental models of acute myocardial infarction, mesenchymal stem cells led to the recovery of cardiac function through the formation of a new vascular network. OBJECTIVE: To describe treatment with intravenous infusions of expanded autologous mesenchymal stem cells in 1 patient with critical limb ischemia due to systemic sclerosis. DESIGN: Case report. SETTING: The rheumatology unit at the University of Florence, Florence, Italy. PATIENT: A woman, aged 34 years, with systemic sclerosis who developed acute gangrene of the upper and lower limbs. INTERVENTION: 3 intravenous pulses of expanded autologous mesenchymal stem cells. MEASUREMENTS: Angiography, skin histopathology, and immunohistochemistry. RESULTS: Areas of necrotic skin were reduced after the first mesenchymal stem-cell infusion. After the third infusion, angiography showed revascularization of the patient's extremities. Skin section analysis revealed cell clusters with tubelike structures, and angiogenic factors were strongly expressed. LIMITATION: Causality cannot be established by a single case. CONCLUSION: In patients with systemic sclerosis who have severe peripheral ischemia, intravenous infusion of expanded autologous mesenchymal stem cells may foster the recovery of the vascular network, restore blood flow, and reduce skin necrosis. PRIMARY FUNDING SOURCE: Fondazione Cassa di Risparmio di Pistoia e Pescia (partial funding).

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 full-thickness 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.

The IL1-like cytokine IL33 and its receptor ST2 are abnormally expressed in the affected skin and visceral organs of patients with systemic sclerosis.

BACKGROUND: Early endothelial cell (EC) activation/damage and profibrotic Th2-associated cytokines play a pivotal role in systemic sclerosis (SSc). Interleukin 33 (IL33) is a novel member of the IL1 family that promotes Th2 responses and inflammation through the ST2 receptor. IL33 is also a chromatin-associated transcriptional regulator in ECs. OBJECTIVE: To investigate the role of the IL33/ST2 axis in SSc. METHODS: Skin biopsies were obtained from 30 patients with SSc (15 early/15 late stage) and 10 healthy subjects. Lung, kidney, heart, oesophagus, stomach, placenta biopsies and bronchoalveolar lavage cells from patients with SSc and controls were also analysed. IL33/ST2 expression was investigated by immunohistology, confocal immunofluorescence microscopy, western blotting and RT-PCR. RESULTS: In skin biopsies from control subjects, constitutive nuclear IL33 protein expression was found in dermal ECs and keratinocytes, while ST2 was weakly expressed in ECs and fibroblasts. In skin biopsies from patients with early SSc, IL33 protein was downregulated or absent in ECs and epidermis while IL33 mRNA was normally expressed or even upregulated. Moreover, ECs, perivascular infiltrating mast cells, CD68-positive macrophages, CD3-positive T cells, CD20-positive B cells and activated fibroblasts/myofibroblasts exhibited strong ST2 expression. In skin biopsies from patients with late SSc, IL33 was constitutively found in most ECs while ST2 immunostaining was weaker. In early SSc, the loss of endothelial IL33 protein and the overexpression of ST2 involved all affected organs. Dermal and pulmonary fibroblasts showed IL33 expression in SSc. CONCLUSION: IL33 and ST2 are abnormally expressed in SSc. In early SSc, upon EC activation/damage IL33 may be mobilised from ECs to signal through ST2 in key profibrotic players such as inflammatory/immune cells and fibroblasts/myofibroblasts.

HLA-B27 transgenic rat: an animal model mimicking gut and joint involvement in human spondyloarthritides.

The HLA-B27 gene is strongly associated with the spondyloarthropathies (SpA), a group of chronic inflammatory diseases affecting the bowel, the joints, and the axial skeleton. Yet the basis for this association remains unknown, despite extensive study in the past years. The HLA-B27 transgenic rat spontaneously develops a disease that strikingly resembles human SpA. The purpose of this article is to review the contribution of studies on the HLA-B27 transgenic rat model aimed to elucidate the intimate relationship between gut and joint involvement in SpA.

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-beta, connective tissue growth factor (CTGF), IL-6, -1beta, 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-1beta, -6 and MCP-1 was low, whereas VEGF was intensively expressed. No differences were observed for TGF-beta, 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 inflammation.

TNFalpha blockade prevents the development of inflammatory bowel disease in HLA-B27 transgenic rats.

Abstract Rats transgenic for HLA-B27 and human beta2microglobulin (B27TR) develop a multi-systemic disease resembling inflammatory bowel disease (IBD) and spondyloarthritis. TNFalpha has a crucial role in chronic inflammation. Our objective was to evaluate the effect of anti-TNFalpha treatment on spontaneous IBD in B27TR. Nine-week-old B27TR received monoclonal anti-TNFalpha or an isotypic IgG2a,k up to age of 18 weeks. A second group was monitored up to 18 weeks and then randomly assigned to anti-TNFalpha or IgG2 a,k treatment. Each rat was monitored for clinical IBD manifestations. After sacrifice, the colon was examined for pathological changes. TNFalpha receptors (TNF-R1, TNF-R2), Fas/Fas-L expression and apoptosis were evaluated. IgG2a,k-treated and untreated B27TR presented signs of IBD at 11 weeks, whereas in anti-TNFalpha-treated B27TR no IBD signs were detected. In the late treatment, IBD signs improved after 1 week. Histopathological analysis of IgG2a,k-treated B27TR colon showed inflammatory signs that were widely prevented by early anti-TNFalpha treatment. Late treatment did not significantly reduce inflammation. TNF-R1 was weakly expressed in intestinal epithelial cells of IgG2a,k-treated B27TR, while it was comparable to controls in anti-TNFalpha-treated animals. TNF-R2 immunopositivity was strongly evident in IgG2a,k-treated B27TR, whereas was absent in anti-TNFalpha-treated rats. RT-PCR confirmed these results. IgG2a,k-treated B27TR showed, at 18 weeks, few Fas-positive cells and an increase of Fas-L-positive cells. At 27 weeks, Fas-/Fas-L-positive cell number was significantly low. Anti-TNFalpha treatment increased Fas-L expression, whereas Fas increased only with the early treatment. TNFalpha blockade is effective in preventing inflammation in early phase of IBD, maintaining the homeostatic balance of apoptosis.

Endothelial/lymphocyte activation leads to prominent CD4+ T cell infiltration in the gastric mucosa of patients with systemic sclerosis.

OBJECTIVE: Although gastrointestinal tract dysfunction is a common feature in patients with systemic sclerosis (SSc; scleroderma), few studies have addressed the pathogenetic mechanisms of gastrointestinal tract involvement in SSc. We previously showed that severe fibrosis and increased expression of profibrotic cytokines are important hallmarks in the gastric wall of patients with SSc. The aim of the present study was to investigate whether immune and/or microvascular abnormalities may account for tissue damage in gastric wall specimens obtained from patients with SSc. METHODS: Gastric biopsy samples from 27 patients with SSc and 15 healthy control subjects were analyzed by immunohistochemistry for CD45/leukocyte common antigen, CD3/T cells, CD4/T helper cells, CD8/cytotoxic T cells, CD20/B cells, CD14/monocytes, CD68/macrophages, cell adhesion molecules CD11a/lymphocyte function-associated antigen 1 (LFA-1), CD49d/very late activation antigen 4 (VLA-4), CD54/intercellular adhesion molecule 1 (ICAM-1), CD106/vascular cell adhesion molecule 1 (VCAM-1), CD31/platelet endothelial cell adhesion molecule 1, and vascular endothelial growth factor (VEGF). RESULTS: T cell infiltration was a prominent finding in gastric specimens from patients with SSc. The CD4+/CD8+ T cell ratio was significantly increased in SSc specimens compared with controls. T cells were found in both lymphocyte aggregates and diffuse infiltrates and strongly expressed the activation markers VLA-4, LFA-1, and ICAM-1. Endothelial cells showed corresponding surface activation with strong expression of VCAM-1 and ICAM-1. Mature B cells were frequently observed arranged in aggregates and rarely were seen in a diffuse pattern. Most lymphocyte aggregates lacked monocyte/macrophages. No difference in microvascular density was observed between SSc specimens and controls. Both SSc and control specimens showed weak or no expression of VEGF. CONCLUSION: Our findings provide the first evidence that endothelial/lymphocyte activation leading to prominent CD4+ T cell infiltration may play a key pathogenetic role within the gastric wall of patients with SSc and may represent an important therapeutic target.

Genetic loci linked to type 1 diabetes and multiple sclerosis families in Sardinia.

BACKGROUND: The Mediterranean island of Sardinia has a strikingly high incidence of the autoimmune disorders Type 1 Diabetes (T1D) and Multiple Sclerosis (MS). Furthermore, the two diseases tend to be co-inherited in the same individuals and in the same families. These observations suggest that some unknown autoimmunity variant with relevant effect size could be fairly common in this founder population and could be detected using linkage analysis. METHODS: To search for T1D and MS loci as well as any that predispose to both diseases, we performed a whole genome linkage scan, sequentially genotyping 593 microsatellite marker loci in 954 individuals distributed in 175 Sardinian families. In total, 413 patients were studied; 285 with T1D, 116 with MS and 12 with both disorders. Model-free linkage analysis was performed on the genotyped samples using the Kong and Cox logarithm of odds (LOD) score statistic. RESULTS: In T1D, aside from the HLA locus, we found four regions showing a lod-score > or =1; 1p31.1, 6q26, 10q21.2 and 22q11.22. In MS we found three regions showing a lod-score > or =1; 1q42.2, 18p11.21 and 20p12.3. In the combined T1D-MS scan for shared autoimmunity loci, four regions showed a LOD >1, including 6q26, 10q21.2, 20p12.3 and 22q11.22. When we typed more markers in these intervals we obtained suggestive evidence of linkage in the T1D scan at 10q21.2 (LOD = 2.1), in the MS scan at 1q42.2 (LOD = 2.5) and at 18p11.22 (LOD = 2.6). When all T1D and MS families were analysed jointly we obtained suggestive evidence in two regions: at 10q21.1 (LOD score = 2.3) and at 20p12.3 (LOD score = 2.5). CONCLUSION: This suggestive evidence of linkage with T1D, MS and both diseases indicates critical chromosome intervals to be followed up in downstream association studies.

Severe fibrosis and increased expression of fibrogenic cytokines in the gastric wall of systemic sclerosis patients.

OBJECTIVE: Systemic sclerosis (SSc) is a connective tissue disorder characterized by fibrosis of the skin and internal organs. Although the esophagus is the most frequently affected part of the gastrointestinal tract, all other segments can be involved. The present study was undertaken to evaluate the fibrotic process and the expression of fibrogenic cytokines in the gastric wall of SSc patients with gastroesophageal involvement. METHODS: Full-thickness surgical and endoscopic gastric biopsy samples were obtained from 14 SSc patients and 10 controls. Tissue sections were either stained with Masson's trichrome or by immunohistochemistry and analyzed for the expression of types I, III, and IV collagen, alpha-smooth muscle actin (alpha-SMA), transforming growth factor beta (TGFbeta), connective tissue growth factor (CTGF), and endothelin 1 (ET-1). RESULTS: In the gastric wall of SSc patients, Masson's trichrome staining and immunohistochemistry for types I and III collagen revealed a high amount of collagen in the lamina propria that increased toward the muscularis mucosae. In addition, muscle layers showed features of atrophy, with wide areas of focal fibrosis surrounding smooth muscle cells. Type IV collagen was present around glands and small vessels, suggesting a thickening of the basal lamina. The expression of the fibrogenic cytokines TGFbeta and CTGF, ET-1, and the myofibroblast marker alpha-SMA was stronger in SSc patients than in controls. CONCLUSION: A pronounced deposition of collagen, the presence of myofibroblasts, and increased expression of several profibrotic factors are important hallmarks in the stomach of patients with SSc. The fibrotic involvement of the gastric wall may account for muscle atrophy leading to stomach hypomotility in SSc.

Variations of neuronal nitric oxide synthase in systemic sclerosis skin.

OBJECTIVE: In systemic sclerosis (SSc), derangement of the peripheral nervous system is linked to vascular tone dysfunction. Nitric oxide (NO) produced by neuronal nitric oxide synthase (nNOS, NOS-I) might play a dynamic role in the control of vascular tone. This study was performed to verify, by immunohistochemical and biochemical analyses, the presence and expression of nNOS and protein gene product 9.5 (PGP 9.5) in SSc skin, in different subsets and various phases of the disease. METHODS: Biopsy samples of clinically involved skin from 32 SSc patients (12 with limited cutaneous SSc [lcSSc] and 20 with the diffuse form [dcSSc]) and skin samples from 6 healthy controls were either immunostained with anti-PGP 9.5 and anti-nNOS antibodies or analyzed by semiquantitative reverse transcription-polymerase chain reaction and Western blotting. RESULTS: Immunohistochemical and biochemical data showed a decrease in PGP 9.5 and nNOS innervation and in their messenger RNA (mRNA) levels in lcSSc and dcSSc skin. In the edematous phase of SSc, a light alteration in cutaneous innervation was initiated and slowly progressed into the sclerotic phase, becoming most evident in the atrophic phase. Levels of nNOS mRNA were significantly lower between the edematous phase and the sclerotic phase in both dcSSc and lcSSc skin, which was attributable to the earlier occurrence of more severe pathologic alterations. CONCLUSION: Total cutaneous innervation and nNOS innervation slowly disappear in the skin of SSc patients. Expression of nNOS depends on the severity of tissue damage in SSc, and increased synthesis of NO also contributes to this process. It remains to be determined whether the changes in cutaneous innervation are due to the disease itself or whether these changes contribute to the pathogenesis and evolution of SSc.

Cardiac hypertrophy and microvascular deficit in kinin B2 receptor knockout mice.

Experimental and clinical evidence suggests kinin involvement in adaptive myocardial growth. Kinins are growth-inhibitory to cardiomyocytes. Knockout of kinin B2 receptor (B2R) signaling causes dilated and failing cardiomyopathy in 129/J mice, and a 9-bp deletion polymorphism of human B2R is associated with reduced receptor expression and exaggerated left ventricular growth response to physical stress. We reasoned that genetic background and aging may significantly influence the impact of B2R mutation on cardiac phenotype. The theory was challenged in C57BL/6 mice, a strain that naturally differs from the 129/J strain, carrying 1 instead of 2 renin genes. C57BL/6 B2R knockouts (B2R-KO) showed higher blood pressure and heart rate levels (P<0.05) compared with wild-type controls (WT) at all ages examined. At 12 months, left ventricular contractility and diastolic function were mildly altered (P<0.05) and histological and morphological analyses revealed ventricular hypertrophy and cardiomyocyte enlargement in B2R-KO (P<0.01). Reparative fibrosis was enhanced by 208% and capillary density reduced by 38% (P<0.01). Functional and structural alterations induced by B2R deletion in C57BL/6 mice were less severe than those reported previously in the 129/J strain. We conclude that interaction of B2R signaling with other genetic determinants influences aging-related changes in myocardial structure and function. These findings may help us understand the role of kinins in the development of cardiac failure.