A competitive enzyme-linked immunosorbent assay for quantification of tetrastatin in body fluids and tumor extracts.

Basement membrane collagens or derived fragments are measured in biological fluids such as blood and urine of patients and appear to be useful for diagnosis, prognostication, or treatment monitoring as proposed for endostatin, a fragment of collagen XVIII, or tumstatin, a fragment of collagen IV. Tetrastatin, the NC1 alpha 4 collagen IV domain, was previously reported to inhibit tumor growth and angiogenesis. The aim of this study was to develop and validate a method to measure tetrastatin concentrations in human fluids. We developed a competitive enzyme-linked immunosorbent assay (ELISA). It allowed measuring tetrastatin levels in human serum, bronchial aspiration and bronchoalveolar lavage fluids, and lung tissue extracts. The tetrastatin level was significantly higher in tumor tissues than in healthy lung tissues. Tetrastatin competitive ELISA could be useful to quantify tetrastatin in tissues and biological fluids for the diagnosis or prognostication of diseases in which basement membrane metabolism may be altered, especially tumor progression.

Elastin peptides regulate HT-1080 fibrosarcoma cell migration and invasion through an Hsp90-dependent mechanism.

BACKGROUND: The elastin-derived peptides (EDPs) exert protumoural activities by potentiating the secretion of matrix metalloproteinases (MMP) and the plasminogen-plasmin activating system. In the present paper, we studied heat-shock protein 90 (Hsp90) involvement in this mechanism. METHODS: HT-1080 fibrosarcoma cell migration and invasion were studied in artificial wound assay and modified Boyden chamber assay, respectively. Heat-shock protein 90 was studied by western blot and immunofluorescence. Matrix metalloproteinase-2 and urokinase plasminogen activator (uPA) were studied by gelatin ± plasminogen zymography and immunofluorescence. Heat-shock protein 90 partners were studied by immunoprecipitation. Messenger RNA expression was studied using real-time PCR. Small interfering RNAs were used to confirm the essential role of Hsp90. RESULTS: We showed that kappa-elastin and VGVAPG elastin hexapeptide stimulated Hsp90, pro-MMP-2 and uPA secretion within 6 h, whereas AGVPGLGVG and GRKRK peptides had no effect. No increase of mRNA level was observed. Heat-shock protein 90-specific inhibitors inhibit EDP-stimulated HT-1080 cell-invasive capacity and restrained EDP-stimulated pro-MMP-2 and uPA secretions. The inhibitory effect was reproduced by using Hsp90-blocking antibody or Hsp90 knockdown by siRNA. Heat-shock protein 90 interacted with and stabilised uPA and pro-MMP-2 in conditioned culture media of HT-1080 fibrosarcoma cells. CONCLUSIONS: Taken together, our results demonstrate that EDPs exert protumoural activities through an Hsp90-dependent mechanism involving pro-MMP-2 and uPA.

Evidence for a protective role of the STAT5 transcription factor against oxidative stress in human leukemic pre-B cells.

STAT5 transcription factors are involved in normal B lymphocyte development and in leukemogenesis. We show that the inhibition of STAT5A expression or activity in the NALM6, 697 and Reh leukemic pre-B cell lines, results in a higher spontaneous apoptosis and an increased FAS-induced cell death. However, the molecular mechanisms underlying the altered pre-B cell survival are unclear. We used a proteomic approach to identify proteins that are differentially regulated in cells expressing (NALM6Δ5A) or not a dominant negative form of STAT5A. Among the 14 proteins identified, six were involved in the control of the oxidative stress like glutathione (GSH) synthetase and DJ-1. Accordingly, we showed increased levels of reactive oxygen species (ROS) in NALM6Δ5A cells and suppression of the increased sensitivity to Fas-mediated apoptosis by the GSH tripeptide. Similar results were observed when NALM6 cells were treated with TAT-STAT5Δ5A fusion proteins or STAT5A shRNA. In addition, the 697 and Reh pre-B cells were found to share number of molecular changes observed in NALM6Δ5A cells including ROS generation, following inhibition of STAT5 expression or function. Our results point out to a hitherto undescribed link between STAT5 and oxidative stress and provide new insights into STAT5 functions and their roles in leukemogenesis.

[Effect of elastin peptides on the production of matrix metalloproteinase 2 by human skin fibroblasts in culture]. / Influence des peptides d'élastine sur la production de la métalloprotéinase matricielle-2 par les fibroblastes du derme humain en culture.

Soluble elastin-derived peptides from alkaline or elastase hydrolysis of insoluble elastin, as well as tropoelastin, increase matrix metalloproteinase-2 (MMP-2) production by human skin fibroblasts in culture as determined by gelatin zymography and ELISA. Such an effect is time and concentration dependent; it can be reproduced by synthetic elastin: VGVAPG, PGAIPG, and laminin: LGTIPG, hexapeptides and inhibited by lactose and is therefore elastin receptor-mediated. The steady state levels of MMP-2 mRNAs are invariant following elastin-fibroblasts interaction. Inhibition of phospholipase C (D-609), ADP-ribosylation factor (brefeldin), protein kinase C (RO-318220) and phospholipase D (1-propanol) totally abolished the elastin-mediated increase of MMP-2 production. It suggested that the post-transcriptional mechanism controlling the elastin-mediated overproduction of MMP-2 involved a cascade leading to phospholipase D activation.

Conformational dependence of collagenase (matrix metalloproteinase-1) up-regulation by elastin peptides in cultured fibroblasts.

We have established that treatment of cultured human skin fibroblasts with tropoelastin or with heterogenic peptides, obtained after organo-alkaline or leukocyte elastase hydrolysis of insoluble elastin, induces a high expression of pro-collagenase-1 (pro-matrix metalloproteinase-1 (pro-MMP-1)). The identical effect was achieved after stimulation with a VGVAPG synthetic peptide, reflecting the elastin-derived domain known to bind to the 67-kDa elastin-binding protein. This clearly indicated involvement of this receptor in the described phenomenon. This notion was further reinforced by the fact that elastin peptides-dependent MMP-1 up-regulation has not been demonstrated in cultures preincubated with 1 mm lactose, which causes shedding of the elastin-binding protein and with pertussis toxin, which blocks the elastin-binding protein-dependent signaling pathway involving G protein, phospholipase C, and protein kinase C. Moreover, we demonstrated that diverse peptides maintaining GXXPG sequences can also induce similar cellular effects as a "principal" VGVAPG ligand of the elastin receptor. Results of our biophysical studies suggest that this peculiar consensus sequence stabilizes a type VIII beta-turn in several similar, but not identical, peptides that maintain a sufficient conformation to be recognized by the elastin receptor. We have also established that GXXPG elastin-derived peptides, in addition to pro-MMP-1, cause up-regulation of pro-matrix metalloproteinase-3 (pro-stromelysin 1). Furthermore, we found that the presence of plasmin in the culture medium activated these MMP proenzymes, leading to a consequent degradation of collagen substrate. Our results may be, therefore, relevant to pathobiology of inflammation, in which elastin-derived peptides bearing the GXXPG conformation (created after leukocyte-dependent proteolysis) bind to the elastin receptor of local fibroblasts and trigger signals leading to expression and activation of MMP-1 and MMP-3, which in turn exacerbate local connective tissue damage.

Development of an ex vivo model of pig kidney perfused with human lymphocytes. Analysis of xenogeneic cellular reactions.

BACKGROUND: Because of the explosive nature and the extremely rapid process of hyperacute rejection (HAR), significant infiltration of the xenograft by immunocompetent cells is not observed, and the role and the mechanism of action of cell-mediated rejection in discordant xenografts are therefore still under discussion. METHOD: We developed an experimental approach using pig kidneys perfused with human peripheral blood lymphocytes (PBL) in which the immunologic barrier of hyperacute rejection was excluded and which mimics the in vivo situation. RESULTS: PBL retention in the kidney was evaluated at 20-minute intervals for 3 hours. Retention increased from 30% to 80% with the time of perfusion and was specific because significantly fewer syngeneic lymphocytes were retained. Phenotype analysis of recovered PBL showed a significant decrease in natural killer (NK) cells. Immunohistochemical studies revealed the presence of NK cells and T lymphocytes in the glomerular and interstitial tubular structures of the kidney. Functional studies showed a progressive cessation of diuresis and augmentation of renal vascular resistance when the kidney was perfused with PBL. Electron microscopy examinations of kidney sections perfused with PBL showed swollen endothelial zones, suggesting alterations to and damage of the endothelium. CONCLUSIONS: This system provides a valuable model for the study of early discordant xenogeneic cellular rejection and demonstrates the predominance of xenograft infiltration by NK cells.

Regulation of matrix metalloproteinase-2 (gelatinase A, MMP-2), membrane-type matrix metalloproteinase-1 (MT1-MMP) and tissue inhibitor of metalloproteinases-2 (TIMP-2) expression by elastin-derived peptides in human HT-1080 fibrosarcoma cell line.

Soluble kappa-elastin peptides were shown to stimulate the expression of MMP-2 (but not MMP-9) by human fibrosarcoma HT-1080 cells, both at the protein and mRNA levels; maximal effect being observed at a concentration of 25 microg/ml of kappa-elastin. The stimulatory effect could be reproduced using Val-Gly-Val-Ala-Pro-Gly (VGVAPG) peptide, an elastin-derived hydrophobic hexapeptide which represented the elastin receptor binding sequence of tropoelastin. Furthermore, treatment of cells with lactose (30 mM), which dissociated 67-kDa elastin binding protein (EBP) from cell surfaces, completely abolished this effect, suggesting that the elastin receptor could mediate such a response. Using a specific monoclonal antibody, 67-kDa EBP was detected in HT-1080 membrane preparations by Western immunoblotting. Following treatment with 25 microg/ml kappa-elastin or 200 microg/ml VGVAPG, increased levels of the active 62-kDa form of MMP-2 were found in HT-1080 cell extracts. Stimulation of MT1-MMP mRNA expression by treatment with elastin-derived peptides (EDPs) was shown by competitive polymerase chain reaction (PCR). A reverse zymography analysis revealed that EDPs also stimulated TIMP-2 (but not TIMP-1) production by HT-1080 cells. Competitive PCR confirmed increased TIMP-2 mRNA expression by such treatment. These results suggest that occupancy of the 67-kDa elastin receptor by elastin-derived peptides enhanced both expression and activation of proMMP-2 and consequently, could promote the invasive/metastatic ability of tumor cells expressing this receptor.

Unusual endocervical polypoid tumor with endocrine cells: an immunohistochemical and ultrastructural analysis.

Light microscopic, immunohistochemical, and ultrastructural features of an unusual polypoid tumor of endocervix are reported. Numerous polypeptide hormone and amine-producing endocrine cells were disclosed. Main conventional characteristics were the architectural growth pattern, with infolding glands giving rise to small secondary glands, the hypermucinous benign-appearing epithelium of endocervical type, and, possibly, the stromal smooth muscle. Ultrastructural analysis showed a highly differentiated tumor. Glandular elements were surrounded by a basal lamina. Mucinous cells, several endocrine cell types, amphicrine cells, nonsecretory ciliated cells, ciliated mucinous cells, and possible reserve cells were observed. This tumor departs appreciably from normal mucosa and common varieties of endocervical polyp, particularly its distinctive endocrine profile. The present case does not correspond to a well-defined type of endocervical neoplasia. It shares morphologic analogies with mucinous tumor of ovary. The malignant potential of this lesion as well as its relationship with minimal deviation adenocarcinoma remain questionable.

Isolation and characterization of syncytiotrophoblast plasma membrane from human placenta.

Human full-term syncytiotrophoblast plasma membranes isolated by mechanical procedures (sieving and ultrasonic disintegration), purified by phase centrifugation, form a single band of 1.052 +/- 0.002 g/ml density in percoll gradient. The purity of the preparation was assessed by electron microscopy, enzyme analysis and beta 2-microglobulin determination.

Endocrine cells in the female genital tract.

Endocrine cells are normal inhabitants of the para-urethral, Bartholin's and endocervical glands and of mesonephric rests. All these cells were characterized as serotonin-storing cells. In the para-urethral and Bartholin's glands, serotonin-containing cells were most often found in the transitional epithelium of excretory ducts. Endocrine cells participated in some pathological conditions. Abundant argentaffin cells were observed among the terminal ductules in chronic bartholinitis and serotonin-storing cells were identified in a peculiar ectocervical epithelium. Numerous serotonin-storing cells were detected in a well-differentiated adenocarcinoma of cervix occurring in a patient with the Peutz-Jeghers syndrome. Argyrophilic cells were present in cases of endometrial carcinomas; a striking feature was the demonstration of gut peptide hormones in an unusual type of endometrial adenocarcinoma. Finally, serotonin-storing cells were a constituent of Brenner tumours. It is suggested that a similar endocrine pattern may be shared by tissues originating from both Müllerian ducts and the urogenital sinus.

Carcinoid tumor occurring in a teratoid malformation of the kidney. An immunohistochemical study.

A case of a carcinoid tumor of the kidney that was intimately related to a mixed dysplastic and teratomatous lesion is reported. This lesion displayed focal transitional, mucinous, and endocrine differentiations. Immunofluorescence studies permit the identification of three immunoreactive products: somatostatin, glucagon, and serotonin. It is suggested that the carcinoid tumor arises from this peculiar lesion, which exhibits cells of endocrine lineage.

[Cirrhosis induced by vitamin A]. / Cirrhose induite par la vitamine A.

The case of a 75-year-old woman who had been treated for 12 years by vitamin A (250,000 UI/day) for psoriasis is presented. During the hospitalisation, hepatic cirrhosis was detected and attributed to hypervitaminosis A based on disease history, clinical hypervitaminosis A features, increased vitamin A blood values and histological patterns, i. e. perisinusoidal fibrosis and spontaneous fluorescence of lipid vacuoles within fat-storing cells. Thus, although rare, vitamin A intoxication can be responsible for cirrhosis.

Endocrine cells in the anal canal.

Endocrine cells are normal inhabitants of the anal canal. While numerous endocrine cells are distributed throughout anal ducts and crypts, few are dispersed in the anal transitional zone. All these cells were characterized as serotonin-storing cells, and this endocrine profile is quite distinctive from that of adjacent mucosae. Rectal epithelium contains serotonin, somatostatin, enteroglucagon, BPP and HPP immunoreactive cells; endocrine cells are lacking in the pectinal folds and perianal skin. It is suggested that this distinctive hormonal profile may be regarded as a specific marker of this anal territory. The same pattern is found in the fetal transitional lining of anal canal. Evidence of serotonin-storing cells in the transitional epithelium of anal glands and crypts and in the ATZ epithelium, reinforces the homology between these linings and urothelium. The presence of a similar fetal epithelium implies that ATZ epithelium in adults is not necessarily metaplastic. All derivatives of the cloaca may therefore share the same endocrine profile.

[Malignant fibrous histiocytoma with granular cells]. / Histiocytome fibreux malin avec cellules granuleuses.

This report describes a malignant tumor of soft tissue which combined granular cells with an histologic pattern of malignant fibrous histiocytoma. Immunohistological and ultrastructural studies were performed. Neo-plastic cells were devoid of S-100 Protein but contained lysozyme. Ultrastructural findings showed granular cells containing numerous lysosomes and non-granular cells exhibiting fibroblastic and histiocytic differentiations. This lesion prompts us to consider either a malignant granular cell tumor or a malignant fibrous histiocytoma.

[Merkel cell tumor]. / Tumeur à cellules de Merkel.

Two cases of skin carcinoma which display endocrine differentiation are reported. In the relevant literature, these neoplasms are considered to be of Merkel cells lineage. These two carcinomas demonstrated salient morphological features. The first tumor contained small aggregates of cells with a deeply indented nucleus. It is postulated that these formations represent foci of Merkel cells maturation. It is suggested that this distinctive focal histological feature may permit recognition of these neoplasms. The second tumor exhibited an admixture of endocrine and epidermoid differentiation. Such observations prompt us to postulate that Merkel cells and keratinocytes originate from the same stem cell.

[Interstitial pneumopathy in subjects treated with amiodarone]. / Pneumopathies interstitielles chez les sujets traités par l'amiodarone.

Five cases of interstitial fibrinogenic pneumopathy in addition to the 17 cases already published have considerably increased the suspicion that amiodarone might be responsible for this type of lung lesion. Moreover, it has been firmly established that 10 (56%) of 18 patients with fibrinogenic interstitial pneumopathy had taken the drug. Amiodarone therefore seems to play an important role in the genesis of iatrogenic pulmonary fibroses. These, however, are rare, even though they probably remain undiagnosed in a substantial number of cases.

Argyrophilic cells in mammary carcinoma.

Breast tumor tissues were treated by the Grimelius procedure and examined for the presence of argyrophilic cells. Carcinomas found to contain argyrophilic cells did not include classic carcinoid tumors; the group was, in fact, heterogeneous, comprising poorly differentiated ductal carcinomas, lobular carcinomas, carcinomas of uncertain origin, and colloid carcinomas. Colloid tumors were the most frequently encountered. The prominence of argyrophilic cells in colloid carcinomas raises the possibility that development into mucin-producing cells is propitious for endocrine differentiation.

Endocrine cells in the prostate gland, urothelium and Brenner tumors. Immunohistological and ultrastructural studies.

Endocrine cells are a normal constituent of the prostate gland, prostatic urethra and urinary bladder mucosa. Positive results using immunohistochemical technics were obtained only with antiserotonin antibodies. In normal tissues, there was a close similarity between the distribution of argyrophilic cells (Grimelius) and serotonin-storing cells. Some striking features were the patchy distribution of endocrine cells, the presence of slender cytoplasmic processes occasionally reaching the luminal surface and the paucity of specialized cells in bladder mucosa. It is unlikely that endocrine cells participate in conventional neoplasms of prostate and bladder. Exceptions are lobular hyperplasia, certain adeno-carcinomas of prostate and inverted papilloma of bladder. An ultrastructural study permitted the distinction of two types of endocrine cells characterized by a different morphology of their granules. Another relevant finding was the presence of serotonin-storing cells in Brenner tumors. The latter observation emphasizes the close similarity between this neoplastic epithelium and urothelium. This implies that endocrine cells may be of mesodermal derivation.