Weight loss surgery improves the metabolic status in an obese rat model but does not affect bladder fibrosis associated with high fat diet feeding.

BACKGROUND: Bladder dysfunction has one of the highest prevalences as a comorbidity of obesity in industrialized countries. The aetiopathogenesis of obesity-associated bladder dysfunction is still obscure, but there is growing evidence that general metabolic changes in obese patients may be in part responsible. As demonstrated recently, high fat diet (HFD) significantly alters the protein expression in the urinary bladder, activates multiple signalling pathways associated with cell survival and inflammation and ultimately provokes bladder fibrosis in an obese rat model. The study aimed to elucidate the role of matrix metalloproteases (MMPs) and their specific tissue inhibitors of metalloproteases (TIMPs) in obesity-related bladder extracellular matrix (ECM) remodelling and the effect of weight loss surgery via sleeve gastrectomy (SG) on phenotype and molecular parameters. METHODS: Twenty-four male Sprague-Dawley rats were used for (i) characterization of the HFD phenotype and (ii) evaluation of alterations following SG. Metabolic status, the degree of bladder fibrosis and tissue expression and activity of MMP2, MMP9, MMP14, TIMP1 and TIMP2 were analysed by immunohistochemistry, enzyme-linked immunosorbent assay and activity assays. Statistical differences were calculated by analysis of variance or independent Student's t-test. A P-value <0.05 was considered statistically significant. RESULTS: In HFD rats, we found significant alterations in lipid metabolism, fat mass, free fatty acid profile, insulin resistance and inflammatory markers. Voided volume was significantly decreased, and bladder showed marked fibrosis. MMPs and TIMPs were differentially regulated depending on animal status (controls, chow diet, HFD, and SG- and sham-operated animals) in both urothelium and detrusor smooth muscle. Although animal weight and most metabolic parameters were positively affected by SG, bladder fibrosis persisted. The limitations of this study were 1 month follow-up and lack of direct measurement of bladder function. CONCLUSIONS: Early diagnosis of the bladder dysfunction associated with obesity is essential to allow targeted early intervention, that is, before manifestation of potentially irreversible ECM fibrotic alterations.

Zymographic analysis and characterization of MMP-2 and -9 forms in human sound dentin.

The role and function of dentin matrix metalloproteinases (MMPs) are not well-understood, but they may play a key role in dentinal caries and the degradation of resin-bonded dentin matrices. To test the null hypothesis that MMP-9 is not found in dentin matrix, we used gelatin zymography to extract and isolate all molecular forms of gelatinolytic MMPs in demineralized mature sound dentin powder obtained from extracted human molars, characterizing and identifying the enzymes by Western blotting. Gelatinolytic MMPs were detected in extracts of demineralized dentin matrix and identified as MMP-2 and MMP-9. Acidic extracts (pH 2.3) yielded 3-8 times more MMP activity than did EDTA (pH 7.4). Their activation may contribute to dentin matrix degradation, which occurs during caries progression and following resin bonding. Inhibition of MMP-2 and -9 proteolytic activity may slow caries progression and increase the durability of resin-dentin bonds.

Matrix metalloproteinase inhibitors as anticancer therapeutics.

Matrix metalloproteinases (MMPs), also designated as matrixins, play a central role in many biological processes and are involved both in physiologic cellular processes and in pathologic situations such as tumor growth, invasion and metastasis. For more than 30 years MMPs have been considered as promising targets for cancer therapy and a number of different synthetic and natural MMP inhibitors have been identified as cytostatic and anti-angiogenic agents and have begun clinical testing in view of their specific implication in malignant tissues. Although preclinical studies were so compelling to encourage several clinical trials, the past years have seen a consistent number of disappointments and limited success. The critical examination of previous studies shed light on new information about the cellular source, substrates and mode of action of MMPs, focusing the attention of future research on the identification of specific MMP targets in tumors at different stage of tumor progression, both in order to improve efficacy and to reduce the side effect profile. In this review we discuss the current view on the feasibility of MMPs as target for therapeutic intervention in cancer, taking into account that the perspective may be of great value for molecular medicine for the twenty-first century, providing intriguing information about the MMPs as mediators in biology and pathology, and as targets for disease therapies.

Flow cytometric evaluation of Vibrio parahaemolyticus adhesion inhibition to human epithelial cells.

BACKGROUND: The present report demonstrates the usefulness of flow cytometry for a quantitative assessment of adhesion inhibition of a Vibrio parahaemolyticus strain to human epithelial cells to acquire more information about the nature of its adhesins. METHODS: The inhibition of the adhesive process to Hep-2 was assayed by adding several monosaccharides to infected cells monolayers. The quantification of the adherent bacteria, labeled with a specific primary antibody plus a secondary fluorescein isothiocyanate-conjugated antibody, was performed by flow cytometry in comparison with light microscopy. The adherence was quantified in terms of the proportion of cells with adherent V. parahaemolyticus and as the mean of adherent bacteria per cell. RESULTS: The adhesion showed a percentage of 98% with a mean fluorescence channel of 331 comparable to those obtained by light microscopy. The addition of monosaccharides resulted in a D-mannose and N-acetyl-galactosamine sensitive adherence. Even if this environmental strain also showed a mannose-sensitive cell-associated hemoagglutination that could mediate V. parahaemolyticus adherence, our results suggest that different sites for an irreversible adherence to host cell are involved. CONCLUSIONS: Flow cytometry in combination with indirect immunofluorescence is an effective tool to investigate the adhesive process of bacteria to epithelial cells because it is more sensitive and reproducible than visual counting of bacteria performed in light microscopy.

Aluminum induces inflammatory and proteolytic alterations in human monocytic cell line.

The increasing exposure to aluminum has been linked with the development of different human pathologies (e.g., breast cancer, myofasciitis, neurodegenerative diseases), probably due to the consistent presence of aluminum salts in widely diffused cosmetic products and vaccines. However, the mechanisms underlying immunologic and proliferative alterations still remain unknown. In the present study we investigated the ability of different aluminum compounds (i.e., aluminum chloride vs Imject® Alum, a mixture of aluminum and magnesium hydroxide) to trigger both inflammatory and proteolytic responses in U-937 human monocytic cell line. We demonstrated, by multiplex immunoassay analyses, that monocytic cells treated with both Imject Alum and aluminum chloride showed different and peculiar expression profiles of 27 inflammatory mediators and 5 matrix metalloproteinases, with respect to untreated control cells. In particular, we found dose-dependent significantly increased levels of pro-inflammatory cytokines, growth factors, and chemoattractant chemokines; whereas among metalloproteinases, only collagenolytic protease showed a significant dose-dependent increase in Imject-treated cells with respect to controls and Al-chloride treated cells. Noteworthy, we found only in Imject Alum-treated cells the significant positive correlations among collagenolytic metalloproteinase and increased expression of pro-inflammatory chemokines, suggesting a possible involvement of aluminum in regulating the acute inflammatory responses. In agreement to emerging evidences, for the first time we demonstrated that the treatment of monocyte cells with aluminum-based adjuvant is able to induce an inflammatory status and a proteolytic cascade activation. In fact, the cell treatment with Imject Alum induced increased levels of several cytokines and proteinases, suggesting these monocyte mediators as possible biomarkers for aluminum-linked diseases. The identification of the biochemical pathways involved in Al-induced cell injury pave the way for improving the knowledge on the potential impact of aluminum in human physio-pathology.

Promotion of tumour metastases and induction of angiogenesis by native HIV-1 Tat protein from BK virus/tat transgenic mice.

OBJECTIVE: To characterize the T53 cell line and its clones derived from an adenocarcinoma of BK virus (BKV)/tat transgenic mice and to establish the role of native Tat in tumorigenicity, induction of metastases and angiogenesis. DESIGN AND METHODS: Tat was quantified by flow cytometry and chloramphenicol acetyltransferase (CAT) assays. Tumorigenicity and metastatic ability of cell lines were assayed in nude mice. Production of proteases was evaluated by a plasmin chromogenic assay and gelatinase zymography. The angiogenic effect was studied in vivo with conditioned medium from tumour cell lines. RESULTS: Tat protein was detected in tumour cell lines in amounts from 600-7000 molecules/cell. Conditioned medium from tumour cell lines was able to transactivate an LTR-CAT in HL3T1 cells, indicating release of extracellular Tat. Tumour cell lines, inoculated into nude mice induced angiogenic tumours with remarkable recruitment of host endothelial cells. Metastases were detected in lymph nodes, lungs, kidneys, and heart. Cell lines produced relevant amounts of proteases. Conditioned medium implanted in mice with matrigel induced an angiogenic response, enhanced by addition of heparin. Preincubation with an anti-Tat antibody abolished the angiogenic effect. CONCLUSIONS: Tat from cells from BKV/tat transgenic mice promotes tumorigenesis and formation of metastases and induces angiogenic activity. Angiogenesis occurs at physiological concentrations of Tat lower than 20 ng/ml. The effects of Tat on induction of metastases and angiogenesis appear to be mediated by activation of proteases.

Prostate-specific antigen synthesis and secretion by human placenta: a physiological kallikrein source during pregnancy.

Prostate-specific antigen (PSA), a kallikrein-like serine protease until recently thought to be prostate specific, has been demonstrated in various nonprostatic tissues and body fluids. PSA has been also found in human endometrium and amniotic fluids, even if the significance of this novel expression is unclear. In this study, we have demonstrated by multiple techniques that human placental tissue, obtained at delivery from normal full-term pregnancies, synthesizes and secretes PSA. RT-PCR showed the presence of PSA messenger ribonucleic acid; biochemical, chromatographic, and immunological studies revealed the expression of both free and complexed PSA forms; immunoelectron microscopy indicated the syncytiotrophoblast as the site of PSA synthesis and secretion. Moreover, in vitro experiments demonstrated that PSA production and secretion are up-regulated by 17beta-estradiol, a pregnancy-related steroid hormone. These results suggest that human placenta is a source of the PSA present in amniotic fluid and maternal serum during pregnancy.

Evaluation of leukocyte stabilisation in TransFix-treated blood samples by flow cytometry and transmission electron microscopy.

In this report, we have evaluated the effects of a TransFix-based stabilisation technique on leukocyte scatter characteristics, immunophenotyping, membrane permeability, absolute cell counting and morphology to extend previously reported flow cytometric data focused on the lymphocyte population. We show that scatter characteristics, immunophenotyping and absolute cell counting are well preserved, particularly in the lymphocyte population. Nevertheless, a general increase in membrane permeability, evaluated by propidium iodide (PI) uptake, was observed in TransFix-treated leukocyte subsets. Ultrastructural observations show selective morphological preservation (up to 10 days of storage) of lymphocytes and, to a lesser extent, of monocytes. In contrast, granulocytes have necrosis-like features, although the plasma membrane seems well preserved. Therefore, electron microscopy observations reflect modifications induced in different cell populations as evidenced by flow cytometry (FC). The data indicate that this short-term stabilisation method is particularly suitable for the analysis of human lymphocytes and it is a good procedure for quality control programmes for inter- and intra-laboratory performance evaluation; good results are obtained with respect to antigen definition and absolute cell counting procedures. Any apoptotic pathways in leukocyte subsets are blocked for at least 10 days.

Conjugated bile acids in breast cyst fluids: relationship to cation-related cyst subpopulations.

Gross cystic breast disease is a benign lesion occurring in 7% of adult women. Apocrine changes of epithelium lining the breast cysts cause a higher risk of developing breast cancer. According to the possible role of bile acids in the pathogenesis of cancer, we analysed breast cyst fluids aspirated from 96 women for distribution of conjugated bile acid concentrations in the two subsets of breast cysts. Bile acid levels were correlated to K+ concentrations (P < 0.0001) and mean value was higher in Na/K < 3 metabolically active apocrine cyst as compared with Na/K > 3 flattened cyst (P < 0.001). Because bile acids could play an important role in the pathogenesis and growth of breast cancer, the significantly higher intracystic concentrations of these carcinogen compounds in apocrine Type I cysts might provide a further biological explanation as to why women with apocrine changes may be at higher breast cancer risk and could be useful for the biochemical knowledge occurring in the different functional stages of the gross breast cysts.

Alpha 1-antichymotrypsin complexes in human breast cyst fluids.

Alpha 1-antichymotrypsin, a serum protease inhibitor, was found in 72 breast cyst fluids aspirated from women affected by gross cystic breast disease. When fractionated by gel chromatography, the presence of protein complexes or aggregates was demonstrated. A different distribution of the alpha 1-antichymotrypsin appeared to be related to the ionic composition of the breast cyst fluid; when compared with metabolically active apocrine cysts, a statistically significant increase of alpha 1 protease inhibitor values in flattened epithelial cysts was revealed (P < 0.001). Two-dimensional immunoelectrophoresis showed in apocrine cysts (Na/K ratio < 3) a characteristic double peak of alpha 1-antichymotrypsin immunoprecipitin curve. The relationship between this alpha 1 protease inhibitor and electrolyte profiles may provide further knowledge about the imbalance between proteases and their inhibitors on functional changes of gross cysts and might be useful in studies on their mechanism of formation and relationship to subsequent breast cancer.

beta-Endorphin expression in gross cystic breast disease.

Opioid peptides have a variety of pathophysiologic actions, playing a novel important role in human breast cancer. The expression of beta-endorphin was studied in 84 human breast cyst fluids from gross cystic breast disease-affected patients. The concentration of beta-endorphin in pooled breast cyst fluids was over four-fold higher than in respective plasma with a significant increase in the mean value of the 'metabolically active' apocrine cysts when compared with flattened cysts (P < 0.001). The higher levels of Type I cyst suggest de novo mammary synthesis of endogenous opioid peptides and could represent an altered expression of biosynthetic activity of apocrine breast cells, providing a possible explanation on functional changes of gross cysts, on the mechanism of their formation and a perspective relationship to breast cancer risk.

Lipids status in human breast cyst fluids.

Benign mammary gross cystic disease is the most common breast lesion; women with apocrine changes of epithelium lining the cysts are at higher risk for developing breast cancer than the normal population. Total cholesterol, high- and low-density lipoproteins fractions, triglycerides and phospholipids, lipase activity and total lipid concentrations were measured in cyst fluids and sera from 89 women affected by gross cystic breast disease. Total cholesterol and high-density lipoprotein content were significantly (P < 0.001) greater in pooled cyst fluids than normal sera. Moreover, data analyses show a significant increase in the mean values of total lipids and lipase activity in metabolically active apocrine cysts, when compared to the flattened cysts (P < 0.001). The lipids feature of apocrine cysts could represent an altered expression of biosynthetic activity of the surrounding apocrine cell surface glycolipid and steroidogenic metabolism and may provide further knowledge about the functional stage changes of gross breast cysts.

Development and characterization of hydrogen peroxide-resistant Chinese hamster ovary cell variants--I. Relationship between catalase activity and the induction/stability of the oxidant-resistant phenotype.

Hydrogen peroxide (H2O2)-resistant sublines of Chinese hamster ovary (CHO) cells were isolated by in vitro exposure to the oxidant (treatment for 1 hr followed by 3 days of growth in peroxide-free medium). Stepwise increase in low level H2O2 concentrations produced variants which were progressively more resistant to the growth inhibitory effect elicited by the oxidant. Removal from H2O2 decreased resistance and the curve describing this process was biphasic in nature. In addition, the rate of loss of the H2O2-resistant phenotype was more rapid for the toxicity elicited by low concentrations of hydrogen peroxide, compared to that produced by high concentrations. Changes in total cell proteins were found to parallel the variations in sensitivity to the oxidant, since the protein content constantly increased during the adaptation process and decreases upon removal from H2O2. Catalase activity did not show large variations in resistant sublines with respect to the parental cell line, and these changes were at least partially related to differences in cell size/amount of total cell proteins of the sublines. In addition, the minor changes observed for catalase activity did not correlate with the degree of resistance to growth inhibition elicited by the oxidant. It may therefore be suggested that the H2O2-resistant phenotype of mammalian cells, initially adapted to low--then gradually increased--concentrations of the oxidant, is the result of a complex phenomenon which only partially involves over-expression of catalase.

Lactate dehydrogenase, isoenzyme patterns and cation levels in human breast gross cyst fluid.

In order to investigate the diagnostic and/or prognostic value of total activity and isoenzymatic patterns of lactate dehydrogenase, 24 human breast gross cystic fluids were studied. A comparison of total lactate dehydrogenase activity to the serum level revealed an increased activity in about 63% of the cases examined; moreover, a significant increase in the slow-moving lactate dehydrogenase 4 and 5 isoenzymes was observed in some cyst fluids. The levels of Na+ and K+ concentrations were also analyzed and two classes of cysts were identified: one presenting Na+ and K+ levels similar to those found in extracellular compartment; the other with high K+ and low Na+ levels, characteristic of an intracellular fluid. This latter pattern could indicate an active metabolism of the epithelial cells lining the cysts. This breast cyst fluid also showed increased levels of lactate dehydrogenase 4 and 5 isoenzymes. The correlation between an increased activity of lactate dehydrogenase 4 and 5 isoenzymes and high K+ and low Na+ levels could be the expression of a high biosynthetic activity and of an anaerobic metabolism in some cysts, suggesting the evolution of the breast gross cyst lesion to malignancy. The importance of these observations is discussed.

Ferritin and transferrin levels in human breast cyst fluids: relationship with intracystic electrolyte concentrations.

The concentrations of sodium and potassium and the content of ferritin and transferrin, proteins considered as potential markers for identifying cells undergoing divisional activity, were measured in fluid from 30 human breast cysts. On the basis of the relative electrolyte concentrations, two main classes of cysts were defined. When the cyst fluids were subdivided according to their Na+/K+ ratio, a significant difference was found between menstruating vs. menopausal patients. The relationship between the two major iron-binding proteins and the Na+/K+ ratio may indicate the expression of a local higher biosynthetic activity in apocrine cysts associated with higher cancer risk.

Circulating immune complexes in human breast cyst fluids: relationship with intracystic immunoglobulin and electrolyte levels.

Circulating immune complexes, the major classes of immunoglobulins and electrolyte concentrations were measured in sixty-two breast cyst fluids aspirated in women affected by gross cystic breast disease. Two main classes of cysts were defined according to the Na/K ratio. Appreciable levels of immunoglobulins were found in almost all samples examined; 66% of breast cyst fluids showed increased levels of immune complexes. A highly significant linear correlation between increased values of immune complexes and immunoglobulin M (p less than 0.001) was found in apocrine cysts, characterized by Na/K ratio less than 3. However, a significant inverse linear correlation was found between positive values of immune complexes and lowered levels of immunoglobulins A (p less than 0.001) and G (p less than 0.001) in epithelial cysts with Na/K ratio greater than 3. These data suggest and confirm that the menstrual cycle can also influence or modulate the metabolic activity of human breast cells as a part of the secretory immune system. The relationship between immune complexes and immunoglobulins and electrolyte profiles may provide further knowledge about the immunological features of breast cyst fluid and suggest the possible alteration of immune-response in cystic breast lesions associated with increased cancer risk.

Hydrogen peroxide cytotoxicity under conditions of normal or reduced catalase activity in H2O2-sensitive and -resistant Chinese hamster ovary (CHO) cell variants.

H2O2-sensitive and -resistant sublines of Chinese Hamster Ovary (CHO) cells were tested for their sensitivity to the growth inhibitory effect elicited by increasing concentrations of the oxidant under conditions of normal or reduced catalase activity. Experimental results have demonstrated that, under conditions of reduced catalase activity, the cytotoxic action of H2O2 was differentially regulated in resistant and sensitive cells. Indeed, the parental cell line and cells resistant to low concentrations of H2O2 (V 250 cells) depended on catalase to a lower extent than did highly resistant cells (V 850 cells). It is interesting to note that V 250 cells had more catalase, on a per million cell basis, than V 850 cells. We conclude that acquired resistance to oxidative stress is not entirely dependent on catalase and that the contribution of catalase depends on the degree of resistance to the oxidant.

Biochemical properties of metalloproteinases from the hemolymph of the mussel Mytilus galloprovincialis Lam.

The expression of matrix metalloproteinases (MMP) with gelatinase activity was found in the whole hemolymph of the marine mussel Mytilus galloprovincialis Lam. Cleavage activity was specific for gelatin; very little activity towards human type-IV collagen, and no activity for cold fish gelatin, casein or bovine serum albumin were detected. EDTA and 1,10-phenanthroline were inhibitory, suggesting that mussel MMPs require divalent cations for their proteolytic activity; in fact, the presence of exogenously added divalent ions significantly protected the MMPs from inhibition. No inhibition was detected with serine or cysteine proteinase inhibitors. The specific vertebrate inhibitors as well as the classical vertebrate activator of MMPs were without effect, whereas sulphydryl reducing agents had a strong inhibitory effect. Mussel MMPs showed an exponential curve of thermal-dependent decay that was not protected by the presence of metal ions. Overall the results indicate both similarities and differences between invertebrate and vertebrate gelatinases, providing information for understanding the biological role of these ancient proteinases.