Cellular census of human fibrosis defines functionally distinct stromal cell types and states.

Fibrotic disorders are some of the most devastating and poorly treated conditions in developed nations, yet effective therapeutics are not identified for many of them. A major barrier for the identification of targets and successful clinical translation is a limited understanding of the human fibrotic microenvironment. Here, we construct a stromal cell atlas of human fibrosis at single cell resolution from patients with Dupuytren's disease, a localized fibrotic condition of the hand. A molecular taxonomy of the fibrotic milieu characterises functionally distinct stromal cell types and states, including a subset of immune regulatory ICAM1+ fibroblasts. In developing fibrosis, myofibroblasts exist along an activation continuum of phenotypically distinct populations. We also show that the tetraspanin CD82 regulates cell cycle progression and can be used as a cell surface marker of myofibroblasts. These findings have important implications for targeting core pathogenic drivers of human fibrosis.

Identification of TNFR2 and IL-33 as therapeutic targets in localized fibrosis.

Dissecting the molecular landscape of fibrotic disease, a major unmet need, will inform the development of novel treatment strategies to target disease progression and identify desperately needed therapeutic targets. Here, we provide a detailed single-cell analysis of the immune landscape in Dupuytren's disease, a localized fibrotic condition of the hand, and identify a pathogenic signaling circuit between stromal and immune cells. We demonstrate M2 macrophages and mast cells as key cellular sources of tumor necrosis factor (TNF) that promotes myofibroblast development. TNF acts via the inducible TNFR2 receptor and stimulates interleukin-33 (IL-33) secretion by myofibroblasts. In turn, stromal cell IL-33 acts as a potent stimulus for TNF production from immune cells. Targeting this reciprocal signaling pathway represents a novel therapeutic strategy to inhibit the low-grade inflammation in fibrosis and the mechanism that drives chronicity.

Characterisation of the inflammatory response in Dupuytren's disease.

BACKGROUND: Dupuytren's disease is characterised by fibrotic nodule and cord formation in the palmar aponeurosis. The pathophysiology of the disease is still unknown, although cell stress and subsequent activation of immune mechanisms seems to be crucial. MATERIALS AND METHODS: Surgically obtained tissue and blood samples of 100 Dupuytren patients were processed by immunohistochemistry, flow cytometry, as well as immunoscope analysis. Macroscopically normal aponeurotic tissue served as control. RESULTS: Locally, microvascular alterations and massive infiltration by mononuclear cells (CD3+, CD4 > CD8, CD45RO > CD45RA, S100 protein, CD56, CD68, scarce CD19 and mast cells) forming perivascular clusters were found in DD tissue. Cytokine profiling of fibromatosis tissue-derived T-cells showed a Th1/TH17-weighted immune response. Immunoscope analysis revealed a restricted T-cell receptor α/ß repertoire pointing to an (auto)antigen-driven process. CONCLUSION: The striking accumulation of immune cells, expression of leukocyte adhesion molecules, as well as pro-inflammatory and pro-fibrotic cytokines near markedly narrowed vessels supports the theory that the abnormal proliferation of fibroblasts and production of extracellular matrix proteins in DD seems to be related to immune-mediated microvascular damage. The restricted T-cell receptor repertoire of intra-lesional T-cells points to an antigen-driven process. T-cells seem to play an important role in the development of Dupuytren's disease.

The contribution of HLA molecules to Dupuytren's contracture in a Southeast Brazilian population.

PURPOSE: The aim of the present study was to investigate the HLA phenotype in Dupuytren's contracture (DC) patients in order to verify the correlation of these alleles with risk factors for development of DC in the Brazilian population. METHODS: This was a case-controlled study of 25 DC patients and 443 healthy individuals with no history of HLA-associated diseases. HLA class I and class II typing was performed using the polymerase chain reaction sequence-specific primer method. RESULTS: The HLAB*18 phenotype was observed in 32% of the patients and 10.5% of controls. However, P values did not remain significant after correction. DISCUSSION: Although we observed an increased tendency of DC patients to possess the HLA-B*18 allele, the results were not statistically significant after correction. This allele was higher in patients of Italian and/or Spanish ethnicity, localities with frequencies higher than 18.0% and 14.0% respectively. Further investigation with a larger cohort of DC patients is required to confirm the potential role of HLA in this disease.

Contribuicao das moleculas de antigeno de histocompatibilidade leucocitaria (HLA) para a contratura de Dupuytren em uma populacao do Sudeste do Brasil / The contribution of HLA molecules to Dupuytren's contracture in a Southeast Brazilian population

Objetivo: O objetivo deste estudo foi investigar o fenótipo do HLA em pacientes com contratura de Dupuytren (CD) para verificar a correlação desses alelos com os fatores de risco para o desenvolvimento da CD na população brasileira. Métodos: Este foi um estudo de caso-controle de 25 pacientes com CD e 443 indivíduos saudáveis sem histórico de doenças associadas ao HLA. As tipagens classe I e classe II do HLA foram feitas utilizando o método iniciador de sequências específicas da reação em cadeia da polimerase. Resultados: O fenótipo HLA-B*18 foi observado em 32% dos pacientes e 10,5% do grupo controle. Contudo, os valores de p não permaneceram significativos após correção. Discussão: Apesar de termos observado um aumento na tendência de os pacientes com CD terem o alelo HLA-B*18, os resultados não foram estatisticamente significativos após correção. Esse alelo foi maior em pacientes de etnia italiana e/ou espanhola, locais com frequências superiores a 18% e 14%, respectivamente. São necessárias investigações adicionais com uma coorte maior de pacientes com CD para confirmar o possível papel do HLA nessa doença. .

Purpose: The aim of the present study was to investigate the HLA phenotype in Dupuytren's contracture (DC) patients in order to verify the correlation of these alleles with risk factors for development of DC in the Brazilian population. Methods: This was a case-controlled study of 25 DC patients and 443 healthy individuals with no history of HLA-associated diseases. HLA class I and class II typing was performed using the polymerase chain reaction sequence-specific primer method. Results: The HLAB*18 phenotype was observed in 32% of the patients and 10.5% of controls. However, P values did not remain significant after correction. Discussion: Although we observed an increased tendency of DC patients to possess the HLA-B*18 allele, the results were not statistically significant after correction. This allele was higher in patients of Italian and/or Spanish ethnicity, localities with frequencies higher than 18.0% and 14.0% respectively. Further investigation with a larger cohort of DC patients is required to confirm the potential role of HLA in this disease. .

[Assessment of functional activity of phagocytic system in patients suffered from Dupuytren's contracture with hepatic fibrosis].

Dupuytren's contracture--a pathology that should not be seen as an isolated lesion tendon-aponeurotic structures of the palmar surface of the hand, but as a disease that requires careful research and a comprehensive, differentiated approach to treatment. The aim of the study was to investigate the functional activity of the phagocytic system of Dupuytren's contracture patients with chronic hepatitis and liver fibrosis in liquidators of the CHNPP accident consequences. The resulting study 188 patients aged 45-65 years showed the correlation of the data of the functional activity of phagocytic system with degree of liver fibrosis, thus objectively assess the patient's condition and make the appropriate correction in the diagnostic criteria as well as in medical and rehabilitative programs.

Assessment of potential cross-reactivity of human endogenous matrix metalloproteinases with collagenase Clostridium histolyticum antibodies in human sera obtained from patients with Dupuytren's contracture.

Collagenase Clostridium histolyticum (CCH) contains a fixed ratio of class I (AUX-I) and class II (AUX-II) collagenases and is used as treatment for Dupuytren's contracture. These two Zn-dependent enzymes, produced by the Gram-positive bacterium Clostridium histolyticum, are related functionally to matrix metalloproteinases (MMPs) which, among other functions, degrade the extracellular matrix. Since AUX-I and AUX-II exhibit sequence similarities to human MMPs, we assessed MMP-1 (interstitial collagenase), MMP-2 (gelatinase A), MMP-3 (stromelysin 1), MMP-8 (collagenase 2), and MMP-13 (collagenase 3) for cross-reactivity with anti-AUX-I and anti-AUX-II antibodies in patient serum. Serum samples from 71 subjects enrolled in a long-term clinical study (58 males and 13 females; 63 ± 10 years old [mean ± standard error]) were evaluated for cross-reactivity with the five MMPs using the two validated enzyme-linked immunosorbent assays (ELISAs). Inhibition cutoff points for anti-AUX-I and anti-AUX-II antibodies were based on assay inhibition obtained with a nonspecific protein, bovine gamma globulin, which was tested for each clinical sample. No MMP cross-reactivity was found for any of the 71 clinical antibody-positive sera evaluated. Sequence identity assessments indicated minimal, nonmeaningful alignments of the MMPs and AUX-I/AUX-II. Furthermore, clinical adverse event assessments indicated no safety signals related to MMP inhibition. The bioanalytical results, sequence identity, and clinical assessments consistently did not demonstrate cross-reactivity between CCH antidrug antibodies and endogenous human matrix metalloproteinases. The results presented here suggest that treatment of Dupuytren's contracture patients with CCH does not lead to any clinical adverse events associated with MMP inhibition.

Kaposi's sarcoma after repeated surgical procedures in an immunocompetent patient: the lymphatic hypothesis.

A 63-year-old Swiss patient developed acquired nodules on his right palm after 3 localized surgeries, called 'needle fasciotomy', for Dupuytren's disease. Kaposi's sarcoma (KS) was diagnosed in a biopsy of a nodule. A positive immunolabeling and serology for human herpesvirus 8 has been found, but human immunodeficiency virus and hepatitis C identification remained negative. The nodules were limited to the surgically traumatized area. This first report of a nonimmunocompromised patient developing a KS after repeated surgeries in a unique peculiar localized area with a dense lymphatic network sustains the hypothesis that tissue alterations involving the lymphatic system could play a central role in the occurrence of KS.

Frequência dos alelos HLA de Classe I e classe II em indivíduos com contratura de Dupuytren no Estado de São Paulo / Frequency of HLA Class I and Class II subjects with Dupuytren's contracture in the State of Sao Paulo

A contratura de Dupuytren é uma doença do tecido conectivo, resultante de um desequilíbrio fibroproliferativo, em que se formam nódulos e cordões fibrosos na fáscia palmar. Com a evolução da doença os cordões se retraem longitudinalmente em direção aos dedos causando a contratura digital que determina o aspecto característico da doença, levando ao diagnóstico diferencial com hanseníase. Sua incidência é maior na raça branca de origem européia, atingindo principalmente adultos do sexo masculino com idade superior a 50 anos. Muitos fatores de risco têm sido associados à contratura de Dupuytren, como fumo, álcool, trauma local, epilepsia e Diabetes mellitos, entre outros. Embora de etiologia desconhecida, é bem aceito o papel hereditário desta patologia. Estudos com marcadores genéticos, sugerem a associação do HLA-DR3 e autoanticorpo para o colágeno na contratura de Dupuytren. Recentemente foi demonstrada associação genética significativa entre HLA-DRB1*15 e risco de desenvolver a doença em pacientes do Norte Europeu. Assim, o presente estudo teve por objetivo, identificar a Freqüência dos alelos HLA de classe I e classe II em pacientes com a contratura de Dupuytren, atendidos no Instituto Lauro de Souza Lima. Ao mesmo tempo, investigar a presença de algum alelo que pudesse sugerir associação com fator de risco para o desenvolvimento da doença nessa população. Trata-se de um estudo inédito não havendo até o momento, estudo associando HLA e doença de Dupuytren na população brasileira. Para isso foram estudados 25 pacientes com diagnóstico da contratura de Dupuytren, com média etária de 54,24 anos e 443 controles saudaveis de mesma etnia e regiao geográfica. Foi possível observar a predominância do sexo masculino (68%) em relação ao feminino e a manifestação mais tardia no grupo das mulheres, concordadndo com dados da literatura. Foi também constatado alto índice de atividades manuais de moderada a pesada no grupo estudado e a descendência européia na totalidade...

Imiquimod: a potential weapon against Dupuytren contracture.

Dupuytren disease is a proliferative fibroplasia of the subcutaneous palmar tissue, occurring in the form of nodular and cords. Evidence is certainly accumulating for raised levels in Dupuytren's tissue of growth factors known to stimulate fibroblasts, Interleukin-1, basic fibroblast growth factor, transforming growth factor-beta, prostaglandin-F2, prostaglandin-E2, platelet derived growth factor and connective tissue growth factor have been suggested to have a role. Immune modification of profibrotic cytokines would provide a novel means to treat dupuytren contracture. Imiquimod cream 5% (Aldara) is an immune modifier, that downregulates transforming growth factor-beta and fibroblast growth factor-2 (the two most important cytokine in producing fibrosis). Based on previous mentioned evidence we suggest: imquimod as a potential drug for dupuytren contracture treatment.

Alterations in the extracellular matrix proteoglycan profile in Dupuytren's contracture affect the palmar fascia.

Dupuytren's disease is a palmar fibromatosis associated with changes in fibroblast activity that also affect the metabolism of extracellular matrix components. In contrast to disease connected alterations in collagen and non-collagenous glycoproteins (mainly fibronectin), the metabolism of proteoglycans, being glycosaminoglycan modified glycoproteins, is poorly understood. Thus, the aim of the present study was the characterization of matrix proteoglycans (PGs) derived from normal fascia and Dupuytren's fascia. Extracted and purified PGs (particularly small PGs) were analysed for content, molecular mass, immunoreactivity and glycosaminoglycan chain structure. The matrix of normal fascia mainly contains decorin [small dermatan sulfate (DS) PG] with biglycan (another small DSPG) and large chondroitin sulfate(CS)/DSPG representing minor components. Dupuytren's disease is associated with the remodeling of matrix PG composition. The most prominent alteration is an accumulation of biglycan frequently bearing DS chains with higher molecular masses. Moreover, the amount of large CS/DSPG is increased. In contrast, decorin displays changes affecting mainly DS chain structure reflected in (i) an increase in some chain molecular masses, (ii) an enhanced content of iduronate disaccharide clusters, and (iii) oversulfation of disaccharide repeats. The PG alterations observed in Dupuytren's fascia may influence the matrix properties and contribute to disease progression.

Activation markers of connective tissue in Dupuytren's contracture: relation to postoperative outcome.

We investigated 103 consecutive patients operated on for Dupuytren's contracture (DC) to find out the relation between the expression of activation markers of connective tissue in surgical specimens obtained prospectively and recurrence of disease. The history of the disease and present state of the operated hand were obtained a mean of 4 years (range 2.5-6) after the latest operation. Immunohistochemical staining for anticollagen type IV, integrin alpha5, laminin, smooth muscle beta-actin, procollagen type I, and desmin was evaluated. Almost half of the patients noticed recurrences during the study period, one fifth within six months of operation. No differences in the expression of any of the markers investigated were found, either earlier or later than six months postoperatively, in patients with or without recurrent bending. Furthermore, there were no associations between sex, age at onset, number of operations, heredity, diabetes mellitus, or drugs taken for cardiovascular disease, and the expression of any of the immunohistochemical markers. The individual characteristics that place a person at high risk are not obviously related to ongoing production of connective tissue at the time of operation or to connective tissue activity in its conventionally-used sense.

T-and B-lymphocyte subsets in patients with Dupuytren's disease. Correlations with disease severity.

Previous reports have indicated that inflammatory mechanisms may be involved in the pathogenesis of Dupuytren's disease and it has even been suggested that this condition is a T-cell mediated autoimmune disorder. We investigated peripheral blood lymphocyte subsets from 21 patients with Dupuytren's disease and compared them with ten healthy blood donors. The Dupuytren's patients had an increase in DR+ T-cells compared with healthy controls. Furthermore, patients with both palmar and plantar involvement had a higher percentage of DR+ T-cells than those with only the palm affected. The percentage of circulating CD5+ B-cells was lower in the Dupuytren's patients compared with the control group; this feature was marginally significant for the whole group of Dupuytren's patients but was strongest in the group of patients with both palmar and plantar involvement. These findings support previous suggestions that immunological mechanisms, involving activated T-cells and probably also B-cells, are involved in the pathogenesis of Dupuytren's disease.

[Expression of various monoclonal antibodies in nodules and band stage in Dupuytren's disease]. / Expression von diversen monoklonalen Antikörpern im Knoten- und Strangstadium des Morbus Dupuytren.

In 42 seronegative patients (n = 6 normal fascia, n = 12 nodal stage of Dupuytren's contracture, n = 24 stage IV) we performed immunohistochemical examinations of specimens of four locations of the digitopalmar fascia by means of indirect immunofluorescence tests. We determined the expression of monoclonal antibodies against vimentin, leucocyte common antigen (LCA, CD 45), four macrophage-antigens (27E10, RM3/1, 25F9, CD68), PDGFR-beta subunit (platelet-derived growth factor receptor). In 12 specimens of nodal stage disease macrophage-antigens typical for late inflammatory phase was detected. Other macrophage phenotypes were found to a lesser degree. All of these antigens were negative in normal palmar fascia and only sporadically positive in stage IV disease. PDGFR, which has not been investigated in digitopalmar flexion contracture before, has been expressed in cells of Dupuytren's disease as well as in normal fascia to a minor degree. In nodal stage disease, PDGFR-expression was not generally increased, although the majority of these cases showed intensively dyed clusters. EGFR could neither be detected in nodal or cord-stage of Dupuytren's disease, nor in normal digitopalmar fascia.

Molecular genetic and immunohistochemical analysis of the tumor suppressor genes Rb and p53 in palmar and aggressive fibromatosis.

This pilot project analyzed the tumor suppressor genes p53 and Rb in 13 cases of aggressive fibromatoses and six cases of palmar fibromatoses (Dupuytren contracture). Immunohistochemistry, reverse transcription polymerase chain reaction, polymerase chain reaction followed by single-strand confirmation polymorphism analysis, and Southern blot to detect gene rearrangements were used. No abnormalities were detected in p53. The aggressive fibromatoses demonstrated a lack of Rb immunohistochemical staining and decreased mRNA for Rb. No structural mutation in the coding sequence of the Rb gene was detected. The decreased level of Rb gene expression, despite a normal coding sequence, may indicate increased proliferation and may suggest potential treatment schemes.

[The factors promoting dystrophic pathology of the hand and duodenal peptic ulcer in women]. / O faktorakh, sposobstvuiushchikh distroficheskoi patologii kisti i iazvennoi bolezni dvenadtsatiperstnoi kishki u zhenshchin.

The authors have made an attempt to make exact the reasons of the frequent association of hand dystrophic pathology and duodenal ulceration disease in women. It is revealed that pointed diseases are to be the late symptoms of equally directed alterations in the organism, including neuro-humoral and immune mechanisms of adaptation, occurring in women even in sexual maturing period and being the favourable background for occurrence of various diseases including hand dystrophic pathology and duodenal ulcerative disease.

Prevalence of HLA-DR3 and autoantibodies to connective tissue components in Dupuytren's contracture.

Forty-six patients with Dupuytren's contracture (DC) and 55 control persons were HLA-typed for class I and HLA-DR class II antigens and were investigated for the presence of autoantibodies against elastin (ELAB) and collagen types I-IV (ACA I-IV). Using the chi 2 test, obtained from 2 x 2 contingency tables, a significant association was found between DC and HLA-DR3 and autoantibodies to types I-IV collagen. ELAB and ACA I and III were significantly correlated with HLA-DR3 in the whole group of patients plus controls. In analogy to other diseases with autoimmunologic features the presence of HLA-DR3 therefore seems to indicate a higher risk for the formation of connective tissue autoantibodies. The remodeling processes during the course of fibrosis in DC might be responsible for this autoantibody formation.