[Interest of reTURB for pTa high grade bladder urothelial carcinoma]. / Intérêt de la résection complémentaire (ou « second look ¼) pour les carcinomes urothéliaux de vessie classés pTa haut grade.

INTRODUCTION: Second look TURB (Transurethral Resection of Bladder Tumor) is recommended for high-risk pT1 tumors. It is well acquired for tumors classified pT1 high grade but its interest is still discussed for high-grade pTa tumors in the absence of high level of evidence. We evaluated the impact of second-look resection for the high-grade pTa bladder tumor. METHODS: We performed a retrospective study in 2 centers from 2007 to 2016. We included all urothelial tumors classified pTa high grade. We studied the anatomopathological findings of reTURB and its consequences on survival without recurrence and progression. RESULTS: Eighty-four patients were included. Thirty-five patients (41.7%) had reTURB and residual tumor was found in 42.9% of cases. The anatomopathology of reTURB was in 20% of cases high grade pTa, in 14.3% of cases pTis, and in 8.6% of cases pT1. Forty-three patients had recurrence, 13 reTURB patients (30.2%). In the patients who had a reTURB, 12 had recurrence (34.3%) against 31 without reTURB, (63.3%). After the first TURB, 45 patients (53.6%) had bladder instillation: 38 received BCG (45.2%) and 7 ametycin (8.3%). The main factor decreasing recurrence was BCG adjuvant therapy (HR=0.4 [0.2-0.9], P=0.02). The absence of reRTUV appeared to be a recurrence factor, but the result was not statistically significant (HR=1.4 [0.7-3], P=0.3). CONCLUSION: reTURB confirms that residual tumor is often found. His interest in survival without recurrence remains to be proved by a prospective study with a larger number of patients. LEVEL OF EVIDENCE: 3.

Papillary endothelial hyperplasia (Masson's tumor) in children.

The intravascular papillary endothelial hyperplasia (IPEH/Masson's tumor) is a rare benign tumor of the skin and subcutaneous vessels. We report, in four pediatric cases, clinical presentation, care (diagnostic and surgical) of Masson's tumor in children. Two boys (two years) and two girls (four and six years) showed a pain subcutaneous tumor (one to five centimeters). They were in the transverse abdominal muscle, between two metatarsals, at the front of thigh and in the axilla. Imaging performed (MRI, Doppler ultrasound) evoked either a hematoma, a lymphangioma or hemangioma. The indication for removal was selected from pain and/or parental concern. The diagnosis was histologically. A lesion persisted in residual form (incomplete initial resection), and is currently not scalable for eleven years. DISCUSSION: This tumor is characterized by excessive proliferation and papillary endothelial cells in the vessels, following a thrombotic event. It is found mainly in adults (no specific age), and preferentially localizes in the face and limbs. The clinical differential diagnosis of this tumor is angiosarcoma. The imagery has not allowed in our series to diagnose but still essential to eliminate differential diagnoses. Only surgical excision with histological examination can differentiate. Our study emphasizes the possibility of pediatric cases with two cases of unusual locations (abdominal and axilla). Clinical presentations we met, now lead us to direct our histologist looking for a Masson tumor in any child with a subcutaneous tumor and/or intramuscular pain, sudden onset, and vascular appearance (after excluding an arteriovenous malformation).

Contribution of Raman spectroscopy in nephrology: a candidate technique to detect hydroxyethyl starch of third generation in osmotic renal lesions.

BACKGROUND AND OBJECTIVES: HydroxyEthyl Starch (HES) has been one of the most commonly used colloid volume expanders in intensive care units for over 50 years. The first and second generation HES, with a high molecular weight (≥200 kD) and a high degree of substitution (≥0.5), has been associated with both renal dysfunction and osmotic nephrosis-like lesions in histological studies. Recently, third generation HES (130 kD/<0.5) has also been shown to impair renal function in critically ill adult patients although tubular accumulation of HES has never been proven in the human kidney. Our objective was to demonstrate the potential of Raman micro-imaging to bring out the presence of third generation-HES in the kidney of patients having received the volume expander. DESIGN: Four biopsies presenting osmotic nephrosis-like lesions originated from HES-administrated patients with impaired renal function were compared with HES-negative biopsies (n = 10) by Raman microspectroscopy. RESULTS: The first step was dedicated to the identification of a specific vibration of HES permitting the detection of the cellular and tissue accumulation of the product. This specific vibration at 480 cm(-1) is assigned to a collective mode of the macromolecule; it is located in a spectral region with a limited contribution from biological materials. Based on this finding, HES distribution within tissue sections was investigated using Raman micro-imaging. Determination of HES positive pixels permitted us to clearly distinguish positive cases from HES-free biopsies (proportions of positive pixels from the total number of pixels: 23.48% ± 28 vs. 0.87% ± 1.2; p = 0.004). CONCLUSIONS: This study shows that Raman spectroscopy is a candidate technique to detect HES in kidney tissue samples currently manipulated in nephrology departments. In addition, on the clinical aspect, our approach suggests that renal impairment related to third generation HES administration is associated with osmotic nephrosis-like lesions and HES accumulation in the kidney.

[Endothelin-1 and receptor A: predictive value for biochemical relapse on patients with advanced and metastatic prostate cancer]. / Endothéline-1 et récepteur A: valeur pronostique pour la reprise évolutive biologique dans l'adénocarcinome de prostate localement avancé et métastatique ganglionnaire.

INTRODUCTION: Pathological endothelin axis is known to be involved in prostate cancer progression. Our study evaluates immunohistochemical expression of ET-1 and ET-AR on prostate biopsy specimen and the predictive value for biochemical relapse on patients with advanced and metastatic cancer. We also evaluated the impact of ET-1 and ET-AR expression on local progression and metastatic bone progression for these patients. PATIENTS AND METHOD: From 1992 to June 2009, 44 patients with clinical T3 stage and metastatic lymph nodes were included. PSA levels, Gleason score in biopsy cores, number of invaded lymph nodes, the existence of nodular capsule transgression and hormonal treatment given to the patient, were analyzed. Biopsy cores were submitted to immunohistochemical study of the expression of ET-1 and ET-AR. Semi-quantitative ET-1 and ET-AR staining assessment was always realised by the same pathologist. RESULTS: The average age of the cohort was 65.6 (standard deviation 6.3), median PSA level was 52.8 ng /ml (3-227), median time of follow-up was 70 months (6-144). Biochemical relapse was observed in 62.8%. Statistically significant stronger ET-1 expression was observed in biopsies of patients with a biochemical relapse (p=0.014). Eighty percent of patients with a biochemical relapse had a high level of ET-AR expression, but no statistical significance has been shown (p=0.109). The relative risk for progression under hormonal therapy was 1.9 in case of high level of ET-1 expression and biochemical relapse was confirmed 8 months earlier in average. High level of ET-AR expression on biopsy cores may indicate earlier local progression and metastatic bone progression but there were no statistical proof. CONCLUSION: In our study, the strength of ET-1 expression in prostate cancer biopsy cores is a prognostic factor of biochemical relapse for cT3 stage patients with metastatic lymph nodes. We have not been able to prove that ET-1 is an independent prognostic factor. A high level of ET-AR expression on prostate biopsy cores is not, in our study, a prognosis factor for predicting the biochemical relapse.

T-cell receptor gamma gene rearrangement in cutaneous T-cell lymphoma: comparative study of polymerase chain reaction with denaturing gradient gel electrophoresis and GeneScan analysis.

BACKGROUND: The usefulness of T-cell receptor gene rearrangement (TCR-GR) analyses for differentiating cutaneous T-cell lymphoma (CTCL) from benign inflammatory disorders (BID) has been insufficiently studied to date. OBJECTIVES: To evaluate the diagnostic value of TCR-GR analyses, comparing polymerase chain reaction (PCR) with denaturing gradient gel electrophoresis (DGGE) analysis and BIOMED-2 standardized protocol PCR with GeneScan analysis (BIOMED-2-GS). METHODS: Both types of PCR were performed in 157 patients evaluated for initial features suggestive of CTCL between 1996 and 2007. After clinical and histological review, the final diagnosis was CTCL in 77 cases and BID in 80 cases. RESULTS: DGGE and BIOMED-2-GS had a similar diagnostic value for distinguishing CTCL from BID, with a sensitivity of 74% and 77%, respectively, and a specificity of 86%. The observed concordance between both methods was 90% and the kappa coefficient was 0.79. Positivity rates did not depend on the PCR method but varied according to the type of CTCL (73-75% in mycosis fungoides, 90-100% in Sézary syndrome, 40-60% in lymphomatoid papulosis and 100% in other types). The positivity rate in BID was 14% with both methods. The most frequent BID with a monoclonal pattern were drug-induced cutaneous lymphoid hyperplasia, erythrodermic psoriasis and pityriasis lichenoides chronica. CONCLUSIONS: BIOMED-2-GS analysis of the TCRgamma gene is as sensitive and specific as DGGE for CTCL diagnosis. In addition, BIOMED-2-GS is less time-consuming and gives more information concerning the size and nature of TCR-GR.

[Prostate biopsies endothelin-1 expression: pt3a stage prognostic factor?]. / Expression immunohistochimique de l'endothéline-1 sur biopsies prostatiques : marqueur pronostique d'adénocarcinome localement avancé ?

AIM: The study evaluated the immunohistochemistry expression of endothelin-1 (ET-1) by prostate cancer (PCa) in prostate biopsies as an extracapsular stage (pT3a) prognostic factor. MATERIAL AND METHOD: Sixty-eight radical prostatectomies (RP) were performed for clinically localised PCa (35 pT2 and 33 pT3a according to the 2002 pTNM classification). Age, digital rectal examination, initial PSA, biopsy Gleason score, positive biopsies ratio, specimen Gleason score, biopsy and RP specimen perineural neoplasic invasion, PCa DNA ploidy, PCa Ki-67 DNA image cytometry and biopsy and RP specimen ET-1 immunohistochemistry expression for both group were compared. Semi-quantitative ET-1 staining assessment was realised by the same pathologist. RESULTS: pT3a group initial PSA was higher (p=0.032). No statistically difference was noticed between pT2 and pT3a groups for positive biopsies ratio, biopsy perineural neoplastic invasion and biopsy DNA ploidy determination. Biopsy Gleason score > or =7 was predictive of a pT3a stage (p=0.03). Statistically higher intensity of ET-1 PCa expression was observed in biopsies and specimens in pT3a group than in pT2 group (p<0.001 and p=0.01). In multivariate analysis, biopsy ET-1 PCa expression was an independent risk factor of pT3a stage with specificity 79 %, sensibility 69 %, predictive positive value 77 % and negative positive value 72 %. Combined with initial PSA > or =7, values were respectively 100 %, 76.9 %, 100 % and 57.1 %. CONCLUSION: Endothelin-1 (ET-1) prostate cancer biopsy expression in our study was an independent prognostic factor of extracapsular stage (pT3a). Further studies will assess the relevance of ET-1 expression study in clinically localised PCa for active surveillance, curative treatment or targeted adjuvant therapy management.

Airway epithelial cell migration dynamics. MMP-9 role in cell-extracellular matrix remodeling.

Cell spreading and migration associated with the expression of the 92-kD gelatinase (matrix metalloproteinase 9 or MMP-9) are important mechanisms involved in the repair of the respiratory epithelium. We investigated the location of MMP-9 and its potential role in migrating human bronchial epithelial cells (HBEC). In vivo and in vitro, MMP-9 accumulated in migrating HBEC located at the leading edge of a wound and MMP-9 expression paralleled cell migration speed. MMP-9 accumulated through an actin-dependent pathway in the advancing lamellipodia of migrating cells and was subsequently found active in the extracellular matrix (ECM). Lamellipodia became anchored through primordial contacts established with type IV collagen. MMP-9 became amassed behind collagen IV where there were fewer cell-ECM contacts. Both collagen IV and MMP-9 were involved in cell migration because when cell-collagen IV interaction was blocked, cells spread slightly but did not migrate; and when MMP-9 activation was prevented, cells remained fixed on primordial contacts and did not advance at all. These observations suggest that MMP-9 controls the migration of repairing HBEC by remodeling the provisional ECM implicated in primordial contacts.

Pathomechanisms of cyst formation in pulmonary light chain deposition disease.

Cystic lung light chain deposition disease (CL-LCDD) is a recently described rare disorder characterised by numerous cysts and diffuse monoclonal nonamyloid light chain deposits surrounded by macrophagic giant cells. The mechanisms responsible for cyst development remain unknown. The objectives of the present study were to analyse the major components of the pulmonary extracellular matrix in CL-LCDD and to determine the influence of metalloproteinases (MMPs) by comparison with other cystic lung disorders. A virtually complete degradation of the elastic network was found in CL-LCDD. To a lesser degree, loss of fibrillar and basement membrane collagens was also observed. Macrophagic giant cells expressed MMP-1, MMP-2, MMP-9, MMP-12 and MMP-14 and in situ zymography highlighted a strong gelatinolytic activity. As in CL-LCDD, cystic lesions in Langerhans' cell histiocytosis (LCH) and lymphangioleiomyomatosis (LAM) were characterised by the lack of elastic fibres. Similarly, MMP were expressed in CL-LCDD and LCH but the labelled cells were different. In contrast, few MMPs were detected in LAM. In conclusion, elastolysis is common to cystic lung light chain deposition disease and other cystic lung disorders, suggesting its implication in cyst formation. Moreover, in cystic lung light chain deposition disease, a role of metalloproteinases in elastolysis is strongly indicated by the metalloproteinase expression and activity pattern.

Human papillomavirus 16 E6, L1, L2 and E2 gene variants in cervical lesion progression.

The human papillomavirus (HPV) 16 E6 genome variant 350G has been found to be more prevalent in women with persistent infection and cervical disease progression than the HPV16 E6 prototype 350T. In this study, we examined whether women who progressed to a high-grade lesion, yet were infected with the prototype 350T, showed variants in other HPV genes such as L1, L2 and E2. Although we detected variants within these genes, they could not explain this phenomenon. Indeed they correlated similarly with variant 350G and prototype 350T. These data indicate that polymorphisms in HPV16 E6 rather than in the other analyzed genes play a role in determining the risk for cervical lesion progression and that additional factors are likely to be required as well.

In vivo stimulation of connective tissue accumulation by the tripeptide-copper complex glycyl-L-histidyl-L-lysine-Cu2+ in rat experimental wounds.

The tripeptide-copper complex glycyl-L-histidyl-L-lysine-Cu2+ (GHK-Cu) was first described as a growth factor for differentiated cells. Recent in vitro data showed that it possesses several properties of a potential activator of wound repair. We investigated the effects of GHK-Cu in vivo, using the wound chamber model described previously (Schilling, J.A., W. Joel, and M.T. Shurley, 1959. Surgery [St. Louis]. 46:702-710). Stainless steel wire mesh cylinders were implanted subcutaneously on the back of rats. The animals were divided into groups that received sequential injections into the wound chamber of either saline (control group) or various concentrations of GHK-Cu. At the end of the experiments, rats were killed, wound chambers were collected, and their content was analyzed for dry weight, total proteins, collagen, DNA, elastin, glycosaminoglycans, and specific mRNAs for collagens and TGF beta. In the GHK-Cu-injected wound chambers, a concentration-dependent increase of dry weight, DNA, total protein, collagen, and glycosaminoglycan contents was found. The stimulation of collagen synthesis was twice that of noncollagen proteins. Type I and type III collagen mRNAs were increased but not TGF beta mRNAs. An increase of the relative amount of dermatan sulfate was also found. A control tripeptide, L-glutamyl-L-histidyl-L-proline, had no significant effect. These results demonstrate that GHK-Cu is able to increase extracellular matrix accumulation in wounds in vivo.

Quantitative videomicroscopic analysis of the sociologic behavior of non-invasive and invasive tumor cell lines.

To analyze the spatial distribution of tumor cell lines with different invasive properties, we used time-lapse videomicroscopic recordings associated with software programs we have developed for quantification. We observed that non-invasive tumor cells rapidly formed small clusters which aggregated to form larger clusters, whereas highly invasive tumor cells remained isolated and did not form clusters. An attraction index computed from a cellular automaton model was used to quantify the degree of attraction-repulsion between cells. The results suggest that the cluster formation by noninvasive cells is not related to a global attraction model and that the random (dispersed) distribution of invasive cells is not related to cell repulsion. According to these results, we can conclude that random cell movement combined with the intrinsic properties of cells explains the phenomenon of cluster formation.

[Multiple recurrent papillomas of the breast: case report]. / Papillomes multiples récidivants du sein: à propos d'un cas.

We report the case of a patient presenting multiple recurrent papillomas of the breast. This mild mammary pathology, developed at the expense of the galactophoric ducts, recurred four times, in a nodular way, in spite of in sano surgical resection. The retrospective study of the cell proliferation and the DNA quantification of the cells constitutive of the papillomas, on several samples, underlined evolutive lesions, which could be predictive of a malignant degeneration. This observation allows us to discuss the management of this rare mild mammary pathology, which can sometimes lead to invasive breast carcinoma.

Detection of the plasmin system in human mammary pathology using immunofluorescence.

The presence and localization of the plasmin system components urokinase (UPA), tissue type plasminogen activator (TPA), plasminogen (PG), a neoantigen expressed by the plasmin-alpha 2-antiplasmin complex, and plasmin inhibitors alpha 2-antiplasmin (AP) and alpha 2-macroglobulin (MG) have been tested by immunofluorescence on sections of 11 benign and 40 malignant lesions of the breast in an attempt to apply a morphological approach to the problem of tumor invasion in vivo. In benign lesions, TPA was seen in secretions of mammary glands and MG was seen in edematous zones. In one involuting lactating adenoma, UPA, TPA, PG, PAP, and AP were associated with glandular cells. UPA was detected in 11 carcinomas, TPA in 22, PG in 31, PAP in 12, AP in 23, and MG in all 40. All these components were essentially present in invasive territories, with a cellular labeling for UPA and TPA and a fluorescent staining frequently at the periphery of tumoral foci for PG and PAP. AP was more closely associated with cancer cells than MG, which was present in the stroma. Intraductal proliferations were rarely positive and there was no correlation between the localization of PG and the distribution of a basement membrane glycoprotein laminin. These data argue strongly for the involvement of the plasmin system in the infiltrating process of the stroma. This system seems to play a limited role in the breakdown of basement membrane in breast carcinomas in vivo.

Less HLA-G expression in Plasmodium falciparum-infected third trimester placentas is associated with more natural killer cells.

During pregnancy, maternal immune tolerance of the fetal semi-allogeneic graft is partly the consequence of extravillous trophoblast HLA-G expression and its interaction with natural killer (NK) cells. Plasmodium falciparum malaria is frequently associated with maternal and fetal complications. Local HLA-G expression and the number of NK cells were evaluated immunohistochemically in P. falciparum-infected and uninfected placentas (15 each) collected in a seasonal malaria-hypoendemic area. In control placentas, HLA-G was almost always expressed in extravillous trophoblast whereas, in infected placentas, it was significantly more weakly expressed in extravillous trophoblast but was also detected in intervillous space macrophages. NK cells were evaluated in intervillous and intravillous spaces and in basal plate. NK cells were always more abundant in basal plate than in intervillous and intravillous spaces in infected or control placentas. For each area, more NK cells were seen in infected than control placentas. These data suggest that HLA-G down-regulation and more NK cells in placentas may be among the mechanisms involved in poor birth outcome associated with P. falciparum infection.

Usefulness of HPV testing in the follow-up of untreated cervical low grade lesions.

The aim of the present work was to evaluate the usefulness of high-risk human papillomavirus (HR-HPV) testing for the follow-up of women with untreated low grade cervical squamous cell lesions (LSIL). For that, 412 women with a cytological diagnosis of LSIL at entry were monitored by cytology, HR-HPV testing with the Hybrid Capture II assay (HC-II) and colposcopy. Our primary endpoint was clinical progression defined by the presence of a high grade cervical intraepithelial neoplasia (CIN2 and CIN3) at the biopsy. At baseline, histological control revealed 10 CIN2 and 11 CIN3 only in the cohort of women HR-HPV+. In the follow-up, 4 CIN2 and 8 CIN3 were detected, always in the women initially HR-HPV+. Thus, the recurrence of a HR-HPV+ infection clearly selects a population at high-risk for CIN2-3. The semi-quantitative appreciation of the viral load with HC-II could not be used as a good prognostic factor for the follow-up of women with LSIL. HR-HPV testing reduces the number of cytology and colposcopy examinations in the follow-up of women aged >35 years when HPV testing is initially negative. Thus HR-HPV testing should be reserved for the follow-up of this population of women initially HR-HPV+ and proposed 6 to 12 months after the cytological diagnosis of LSIL.

Comparison of liquid based cytology and histology for the evaluation of HER-2 status using immunostaining and CISH in breast carcinoma.

BACKGROUND: HER-2 amplification is an important prognostic biomarker and treatment determinant in breast carcinoma. AIMS: To correlate immunocytochemical (ICC) expression of HER-2 and gene amplification determined by chromogenic in situ hybridisation (CISH) using liquid based cytology (LBC) with immunohistochemistry (IHC) and CISH using histological samples of the same breast carcinomas. METHODS: Frozen sections and cytobrushings of 103 breast carcinomas were analysed. Four techniques were performed on each tumour: two on LBC samples (ICC, and CISH, both graded as positive, indeterminate, or negative) and two on histological samples (IHC and CISH). Two cell lines (MCF-7, negative; BT 474, positive) were used as controls for cytological analysis. A complementary fluorescence in situ hybridisation technique was carried out in histological samples with low amplification (4-10 dots/nucleus). RESULTS: Interobserver agreement for the four techniques calculated by the kappa coefficient indicated a substantial agreement. Nine cases failed in cytology because of poor cellularity. Among 94 cases, 19 were amplified; 73, 12, and 9 tumours were scored 0 or 1+, 2+, and 3+, respectively by IHC and 75, 13, and 6, respectively, by ICC. CISH found no amplification in 72 tumours. Correlations between the IHC and CISH results in the histological and cytological samples were always significant. CONCLUSIONS: Her-2 status could be determined in LBC samples and correlated well with reference histological methods using in situ hybridisation. ICC was less reliable because of the presence of the cytoplasmic membrane. However, these results should be confirmed by a large multicentre study.

[Gestational trophoblastic diseases: a retrospective study from 1997 to 2003]. / Maladies trophoblastiques gestationnelles: étude rétrospective de 1997 a 2003.

BACKGROUND: Trophoblastic diseases correspond to a very heterogeneous group of rare pathology in young women which fertility should be preserved. PATIENT AND METHODS: We conducted a retrospective study from 1997 to 2003, including all patients with molar pregnancy or trophoblatic tumor in our department of Obstetrics and Gynecology. RESULTS: Fifteen patients were identified with 9 molar pregnancies, 5 trophoblastic tumors and 1 placental tumor of implantation site. The outcome was favorable for 14, and one patient died from her metastatic disease. For 4 patients we asked our university colleague for the optimal approach. DISCUSSION: Management of molar pregnancies is well established. Persistent gestational disease is more rare and problematic with potential metastatic dissemination. Multidisciplinary care is often needed. CONCLUSION: Persistent gestational disease should be managed in a highly specialized centre, as developed in the Lyon University Hospital.

Expression of gelatinase A and its activator MT1-MMP in the inflammatory periprosthetic response to polyethylene.

Wear debris of polyethylene prosthetic components is known to induce a host granulomatous reaction which recruits numerous macrophages and multinucleated giant cells. By releasing cellular mediators of a nonspecific inflammatory reaction, activated phagocytic cells are thought to play a key role in osteolysis leading to aseptic loosening of the prosthesis. Matrix metalloproteinases (MMPs) have been implicated in this destructive process by their ability to degrade extracellular matrix components of bone and adjacent connective tissue. To investigate the roles of gelatinase A, its activator MT1-MMP, and the MMP inhibitors TIMP-1 and TIMP-2 in aseptic loosening of polyethylene prostheses, immunohistochemistry (IHC) and in situ hybridization (ISH) were performed on periprosthetic pseudosynovial interface tissues. Gelatinase A and MT1-MMP were strongly detected immunohistochemically in macrophages and multinucleated giant cells in contact with polyethylene wear debris. In contrast to MT1-MMP, gelatinase A mRNAs were not found in phagocytic cells but in surrounding fibroblasts, thereby suggesting cooperation between macrophages and fibroblasts in this process. While TIMP-1 was expressed essentially in hyperplastic pseudosynoviocytes as assessed by IHC and ISH, TIMP-2, MT1-MMP, and gelatinase A were colocalized in phagocytic cells. These data support the concept of progelatinase A activation involving a trimolecular complex (MT1-MMP-TIMP-2-gelatinase A) mechanism. Thus, this study demonstrated that gelatinase A and its activator might contribute to the aseptic loosening of polyethylene prostheses.

Gamma-interferon inhibits extracellular matrix synthesis and remodeling in collagen lattice cultures of normal and scleroderma skin fibroblasts.

Three-dimensional collagen lattice cultures of fibroblasts mimic the in vivo situation better than monolayer cultures. Here, skin fibroblasts from scleroderma patients and healthy controls were cultivated in collagen lattices, and the effects of recombinant human gamma-interferon (IFN-gamma) on these cultures investigated. IFN-gamma inhibited collagen lattice retraction in a dose-dependent way at concentrations ranging from 10 to 10,000 U/ml. This effect was independent of any alteration to the cell proliferation within the lattices. The inhibition was of the same order of magnitude in normal and pathological fibroblasts. The synthesis of collagen and non-collagen proteins, particularly fibronectin, was increased in scleroderma cultures. It was inhibited in both normal and scleroderma fibroblasts by IFN-gamma, with a maximal effect at the concentration 1000 U/ml, but the inhibition of protein synthesis was far more intense in scleroderma than in normal cells. In situ hybridization, Northern blot and dot blot analyses showed that mRNA coding for pro alpha 1(I) collagen was decreased in IFN-gamma-treated cells, indicating an effect at the pretranslational level. IFN-gamma also inhibited glycosaminoglycan synthesis, but in scleroderma cells only. This study shows that IFN-gamma regulates cell behavior in three-dimensional collagen matrices: (i) it decreases protein and specifically glycosaminoglycan synthesis in scleroderma fibroblasts, (ii) it modulates the interactions between cells and matrix that lead to the retraction of the lattice. Whereas collagen synthesis is largely decreased in lattice cultures like in vivo, it remains increased in the case of scleroderma compared to normal fibroblasts and may be down-regulated by IFN-gamma. Similar conclusions may be drawn for fibronectin and glycosaminoglycans. The inhibitory effect of IFN-gamma on the retraction capacity of fibroblasts and on their ability to synthesize increased amounts of extracellular matrix macromolecules may be of potential interest for therapeutic use of IFN-gamma in scleroderma patients.

Expression and activation of matrix metalloproteinases in wounds: modulation by the tripeptide-copper complex glycyl-L-histidyl-L-lysine-Cu2+.

We investigated the expression and activation of matrix metalloproteinases in a model of experimental wounds in rats, and their modulation by glycyl-L-histidyl-L-lysine-Cu(II), a potent activator of wound repair. Wound chambers were inserted under the skin of Sprague-Dawley rats and received serial injections of either 2 mg glycyl-L-histidyl-L-lysine-Cu(II) or the same volume of saline. The wound fluid and the neosynthetized connective tissue deposited in the chambers were collected and analyzed for matrix metalloproteinase expression and/or activity. Interstitial collagenase increased progressively in the wound fluid throughout the experiment. Glycyl-L-histidyl-L-lysine-Cu(II) treatment did not alter its activity. Matrix metalloproteinase-9 (gelatinase B) and matrix metalloproteinase-2 (gelatinase A) were the two main gelatinolytic activities expressed during the healing process. Pro-matrix metalloproteinase (pro-form of matrix metalloproteinase)-9 was strongly expressed during the early stages of wound healing (day 3). In the wound fluid, it decreased rapidly and disappeared after day 18, whereas in the wound tissue, matrix metalloproteinase-9 expression persisted in the glycyl-L-histidyl-L-lysine-Cu(II) injected chamber until day 22. Pro-matrix metalloproteinase-2 was expressed at low levels at the beginning of the healing process, increased progressively until day 7, then decreased until day 18. Activated matrix metalloproteinase-2 was present in wound fluid and wound tissue. It increased until day 12, then decreased progressively. Glycyl-L-histidyl-L-lysine-Cu(II) injections increased pro-matrix metalloproteinase-2 and activated matrix metalloproteinase-2 during the later stages of healing (days 18 and/or 22). These results demonstrate that various types of matrix metalloproteinases are selectively expressed or activated at the various periods of wound healing. Glycyl-L-histidyl-L-lysine-Cu(II) is able to modulate their expression and might significantly alter wound remodeling.