Vitronectin (Vn) glycosylation patterned by lectin affinity assays-A potent glycoproteomic tool to discriminate plasma Vn from cancer ascites Vn.

Changes in glycosylation have been associated with human cancer, but their complexity poses an analytical challenge. Ovarian cancer is a major cause of death in women because of an often late diagnosis. At least one-third of patients presents ascites fluid at diagnosis, and almost all have ascites at recurrence. Vitronectin (Vn) is a multifunctional glycoprotein that is suggested to be implicated in ovarian cancer metastasis and is found within ascites. The present study evaluated the potential of using lectin affinity for characterizing the glycosylation pattern of Vn. Human Vn was purified from 1 sample of ovarian cancer ascites or a pool of plasma samples. Consistent findings were observed with both dot blot and lectin array assays. Based on a panel of 40 lectins, the lectin array revealed discriminant patterns of lectin binding to Vn glycans. Interestingly, almost all the highlighted interactions were found to be higher with Vn from ascites relative to the plasma counterpart. Also, the lectin array was able to discriminate profiles of lectin interactions (ConA, SNA-I, PHA-E, PHA-L) between Vn samples that were not evident using dot blot, indicating its high sensitivity. The model of ConA binding during thermal unfolding of Vn confirmed the higher accessibility of mannosylated glycans in Vn from ascites as monitored by turbidimetry. Thus, this study demonstrated the usefulness of lectins and the lectin array as a glycoproteomic tool for high throughput and sensitive analysis of glycosylation patterns. Our data provide novel insights concerning Vn glycosylation patterns in clinical specimens, paving the way for further investigations regarding their functional impact and clinical interest.

Ascites-induced shift along epithelial-mesenchymal spectrum in ovarian cancer cells: enhancement of their invasive behavior partly dependant on αv integrins.

At least one-third of patients with epithelial ovarian cancer (OC) present ascites at diagnosis and almost all have ascites at recurrence. The presence of ascites, which acts as a dynamic reservoir of active molecules and cellular components, correlates with the OC peritoneal metastasis and is associated with poor prognosis. Since epithelial-mesenchymal transition (EMT) is involved in different phases of OC progression, we have investigated the effect of the unique ascitic tumor microenvironment on the EMT status and the behavior of OC cells. The exposure of three OC cell lines to ascites leads to changes in cellular morphologies. Within ascites, OC cells harboring an initial intermediate epithelial phenotype are characterized by marked dislocation of epithelial markers (E-cadherin, ZO-1 staining) while OC cells initially harboring an intermediate mesenchymal phenotype strengthen their mesenchymal markers (N-cadherin, vimentin). Ascites differentially triggers a dissemination phenotype related to the initial cell features by either allowing the proliferation and the formation of spheroids and the extension of colonies for cells that present an initial epithelial intermediate phenotype, or favoring the migration of cells with a mesenchymal intermediate phenotype. In an ascitic microenvironment, a redeployment of αv integrins into cells was observed and the ascites-induced accentuation of the two different invasive phenotypes (i.e. spheroids formation or migration) was shown to involve αv integrins. Thus, ascites induces a shift toward an unstable intermediate state of the epithelial-mesenchymal spectrum and confers a more aggressive cell behavior that takes on a different pathway based on the initial epithelial-mesenchymal cell features.

Rapid and soft formulation of folate-functionalized nanoparticles for the targeted delivery of tripentone in ovarian carcinoma.

We report the development of folate-functionalized nanoparticles able to target folate receptors, and to deliver a poorly water soluble cytotoxic agent, a tripentone, in ovarian carcinoma. The stability under incubation of lipid nanoparticles formulated by a low-energy phase inversion temperature method was investigated. Thanks to the presence of Labrasol(®), a macrogolglyceride into the composition of the nanocarriers, the conjugation of different quantities of a folate derivate (folic acid-polyethylene glycol2000-distearylphosphatidylethanolamine) to nanoparticles was possible by a rapid, soft, very simple post-insertion process. As determined by dynamic light scattering, nanoparticles present a monodisperse diameter of about 100 nm, a spherical shape as attested by transmission electron micrographs, a weakly negative surface zeta potential, and are able to encapsulate the tripentone MR22388. The presence of folate receptors on SKOV3 human ovarian cancer cells was identified by fluorescent immunocytochemistry. Cellular uptake studies assessed by flow cytometry indicated that these nanoparticles reached the SKOV3 cells rapidly, and were internalized by a folate-receptor mediated endocytosis pathway. Moreover, nanoparticles allowed the rapid delivery of the antitumor agent tripentone into cells as shown in vitro by real-time cellular activity assay. Such folate-lipid nanoparticles are a potential carrier for targeted delivery of poorly water soluble compounds into ovarian carcinoma.

Fabrication of functional fibronectin patterns by nanosecond excimer laser direct write for tissue engineering applications.

Laser direct write techniques represent a prospective alternative for engineering a new generation of hybrid biomaterials via the creation of patterns consisting of biological proteins onto practically any type of substrate. In this paper we report on the characterization of fibronectin features obtained onto titanium substrates by UV nanosecond laser transfer. Fourier-transform infrared spectroscopy measurements evidenced no modification in the secondary structure of the post-transferred protein. The molecular weight of the transferred protein was identical to the initial fibronectin, no fragment bands being found in the transferred protein's Western blot migration profile. The presence of the cell-binding domain sequence and the mannose groups within the transferred molecules was revealed by anti-fibronectin monoclonal antibody immunolabelling and FITC-Concanavalin-A staining, respectively. The in vitro tests performed with MC3T3-E1 osteoblast-like cells and Swiss-3T3 fibroblasts showed that the cells' morphology and spreading were strongly influenced by the presence of the fibronectin spots.

Psychoneuroendocrine characteristics of common obesity clinical subtypes.

OBJECTIVE: To relate psychological profiles, cerebral asymmetry and the hypothalamus-pituitary-adrenal axis (HPA) reactivity to clinical characteristics of common obesity. METHODS: Sixty consecutive adult female overweight and obese patients attending the outpatient endocrine department were included in this study. Clinical evaluation specifically selected a priori the following indexes: obesity age of onset, parenthood obesity, carbohydrate craving, binge eating with purging, obesity degree (defined by the body mass index (BMI)--weight (kg)/height (m(2))), body fat distribution (defined by the abdominal--thigh ratio (A/T)) and initial weight loss after medical treatment. Psychological evaluation was performed with the Minnesota Multiphasic Personality Inventory (MMPI). In the last 30 patients, the Edinburgh Inventory of Manual Preference (EIMP) and the corticotrophin-releasing hormone (CRH) test were also performed. RESULTS: Clinical characteristics defined a priori were independent variables as evaluated by contingency table analysis. Factorial analysis of variance (ANOVA) revealed a significantly different MMPI profile, according to parental obesity, with post-hoc significantly higher scores on the hypochondriasis (Hs), paranoia (Pa), psychasthenia (Pt) and schizophrenia (Sc) scales in patients with obese parents. Obese patients presented significantly higher dichotomized manual preference indexes in relation to overweight patients. Parental obesity, binge eating behaviour with purging, body fat distribution and the dichotomized manual preference index were independent significant factors for the ACTH response in the CRH test, together explaining 41% of the response variability. Age of onset of obesity and the dichotomized manual preference index were independent and significant factors for the cortisol response, together explaining 37% of its variability. A non-normal distribution was found for the ACTH response: high- and low-responders presented significantly different MMPI profiles, with high-responders presenting higher scores on all clinical scales except masculinity/femininity (Mf). CONCLUSION: Overweight/obese subjects with parental obesity present a distinctive personality profile and a higher ACTH response in the CRH test. Cerebral asymmetry may be a relevant factor for obesity development and is associated with the HPA reactivity. HPA reactivity is a sensitive index integrating clinical, psychological and neural asymmetric factors. International Journal of Obesity (2001) 25, 24-32

Human ovarian adenocarcinoma cells synthesize vitronectin and use It to organize their adhesion.

Extracellular matrix components and integrin receptors are frequently altered in cancer, including ovarian adenocarcinoma. Vitronectin (Vn) is a matrix protein mainly synthesized by liver cells; it is present in normal ovarian surface epithelium and differentiated ovarian adenocarcinoma, but is frequently undetectable in undifferentiated carcinoma (F. Carreiras et al., 1996, Gynecol Oncol 62:260-267). Wondering about the cellular origin of Vn in ovarian carcinoma, we searched for evidence of Vn synthesis by these tumors. We demonstrated that three human ovarian adenocarcinoma cell lines were able to synthesize Vn, as revealed by the presence of Vn mRNA and the protein. The Vn matrix promotes adhesion of ovarian tumor cells through alphav integrins. Moreover, during in vitro growth, Vn is progressively organized into a particular pattern in combination with the recruitment of alphav into focal contacts. Our results suggest that Vn synthesis may participate in ovarian adenocarcinoma cell biology and raise the possibility that altered expression of Vn in some ovarian carcinomas could result from a defect in Vn synthesis.

Migration properties of the human ovarian adenocarcinoma cell line IGROV1: importance of alpha(v)beta3 integrins and vitronectin.

Cell migration of ovarian tumoral cells is essential for cell dissemination and for invasion of the submesothelial extracellular matrix (ECM). We have conducted a study of the migratory properties of an ovarian adenocarcinoma cell line (IGROV1) by using 2 distinct methods for the evaluation of cell migration. We found that in a short-term transfilter migration assay, IGROV1 cells migrated toward vitronectin, fibronectin, type IV collagen and laminin in an integrin-dependent manner. When migration was evaluated in a wound healing assay, the restitution of the wounded area was stimulated solely by added, exogenous vitronectin and was almost totally dependent on alpha(v)beta3 integrin function. Moreover, we demonstrated that alpha(v)beta3 was localized in focal contacts restricted to the leading edge of migrating cells, whereas vitronectin notably localized with actin stress fibers and cortical actin. On the other hand, several kinase inhibitors were found to impede migration of IGROV1 induced by vitronectin. It thus appears that alpha(v)beta3-vitronectin interactions lead to the activation of multiple signaling pathway including activation of protein kinase C, phosphatidyl-inositol-3-phosphate kinase and protein tyrosine kinase. The "alpha(v)beta3-vitronectin system" is therefore essential to the migration of human ovarian carcinoma cells.

Transient hyperandrogenemia and its relation to ovulation.

OBJECTIVE: To study serum androgen levels in relation to ovulation. DESIGN: Prospective, controlled clinical study. SETTING: Outpatient endocrine department of a public central hospital. PATIENT(S): Forty-eight consecutive young, nonobese, hirsute women. INTERVENTION(S): Endocrine evaluation between days 2 and 5 and between days 22 and 25 of a spontaneous menstrual cycle. MAIN OUTCOME MEASURE(S): Levels of FSH, LH, E2, P, androstenedione (A), total testosterone (T), DHEAS, and 17alpha-hydroxyprogesterone (17-OHP). RESULT(S): Hyperandrogenemia occurred equally in a persistent (56%) or a transient form (44%). Transient hyperandrogenemia was more common in the early follicular phase in ovulatory cycles and in the second phase in delayed or anovulatory cycles (63% and 37% versus 10% and 90%, respectively). In delayed or anovulatory cycles, A, total T, and DHEAS increased significantly during the delayed follicular phase. CONCLUSION(S): In delayed or anovulatory cycles, transient hyperandrogenemia occurs late in the follicular phase because of previous ineffective steroidogenesis. In contrast, in ovulatory cycles, transient hyperandrogenemia occurs mainly in the early follicular phase. After ovulation, effective aromatization attenuates the condition. During evaluation and medical treatment of hirsutism, physicians should consider the common occurrence of transient hyperandrogenemia and its relation to ovulation.

Dyslipidaemia in female overweight and obese patients. Relation to anthropometric and endocrine factors.

OBJECTIVE: To characterize serum lipid abnormalities in overweight and obese female patients and to quantify the relative importance of associated factors. METHODS: Cross sectional study at the first visit to the out-patient department. 237 consecutive overweight and obese female patients (age 31 +/- 14 y, body mass index (BMI) 34.2 +/- 6.0 kg/m2) were studied. Evaluation included a questionnaire-based assessment of dietary and physical activity habits, determination of anthropometric indexes (BMI, abdominal/thigh ratio (A/T) and conicity index (CI)) and endocrine evaluation. Statistical analysis by factorial ANOVA and multiple regression. RESULTS: Dyslipidaemia was present in 46% of the patients, with hypercholesterolaemia (35%) being more frequent than hypertriglyceridaemia (10%). Age but not dietary habits or physical activity patterns was significantly related to serum lipid concentrations, independently accounting for 6-10% of their variability. Pharmacological drug use resulted in increased serum triglyceride concentrations, explaining less than 5% of their variability. Serum cholesterol concentrations were not significantly related either to anthropometric or to endocrine indexes. For serum triglyceride concentrations, anthropometric indexes accounted for 6% of their variability and endocrine indexes-postprandial insulin, serum cortisol, testosterone and androstenedione together accounted for 32%. CONCLUSION: In mild to moderate female obesity of the peripheral type, dyslipidaemia is common. However the most common abnormality, hypercholesterolaemia is not significantly related to anthropometric or endocrine indexes, while these together account for more than one third of variability in serum triglyceride concentrations.

Expression and localization of alpha v integrins and their ligand vitronectin in normal ovarian epithelium and in ovarian carcinoma.

In an extension of a previous in vitro study [Carreiras et al., Int. J. Cancer 63, 530-536 (1995)] and in an effort to understand the adhesive interactions mediated by integrins within epithelial ovarian tumors, the presence of the alpha v and beta 3 subunits and that of vitronectin (Vn) in ovarian carcinomas at various stages of differentiation and in normal ovarian epithelium were comparatively investigated. The study was performed on material from 34 patients. By immunofluorescence, cryostat sections were analyzed for their expression of alpha v (34 cases), beta 3 (19 cases), and Vn (29 cases). alpha v was expressed in normal epithelium and in highly differentiated tumors as well as in a majority of moderately and poorly differentiated carcinomas with identical staining pattern. beta 3 subunit and Vn were also expressed in normal cases and highly differentiated carcinomas. However, they were lacking in most of the less differentiated tumors. The analysis of cases which were simultaneously tested for the presence of alpha v, beta 3, and Vn revealed that a large proportion of normal ovarian epithelium and highly differentiated tumors simultaneously expressed alpha v, beta 3, and Vn; in contrast, in all moderately and poorly differentiated carcinomas either beta 3 or Vn was absent. The potential role of the alpha v beta 3/Vn system in ovarian epithelium functions is discussed. It is also speculated that modifications of this system in ovarian carcinomas might contribute to tumor progression.

Implication of the alpha v beta 3 integrin in the adhesion of the ovarian-adenocarcinoma cell line IGROV1.

Accumulating evidence suggests that integrins, which participate in many complex cellular processes, are important for tumor progression and metastasis. In order to understand the role of these cell-surface receptors and of their ligands in the biological behavior of ovarian tumor cells, we have examined the expression of integrins in the human ovarian-adenocarcinoma cell line IGROV1. These cells expressed the alpha v sub-unit and used it to attach on vitronectin (Vn). A monoclonal antibody (MAb) (69-6-5) specific to alpha v blocked the attachment of IGROV1 cells on Vn and fibrinogen (Fg), but not on fibronectin (FN) and other adhesive ligands. Immunoprecipitation of surface biotinylated cells followed by Western blotting showed that the alpha v sub-unit was associated with beta 3, but not with beta 1, beta 5 or beta 6. When cells were cultivated on glass coverslips or on Vn sub-stratum in serum-free medium, immunofluorescence staining with MAb 69-6-5 revealed the presence of alpha v at cell-cell contacts and at focal contacts, supporting its active participation in adhesion as part of a functional heterodimer. Furthermore, immunofluorescence data showed the presence of Vn as a fibrillar network in IGROV1 cells cultivated on FN-coated slides. These results suggest that alpha v beta 3 and its Vn ligand may play a important role in the behavior of ovarian epithelial tumors.

Synthesis and cytotoxic activity against L1210 leukemia of new aminocyclopenta[c]thiophenones.

Synthesis of some new hydroxyaminocyclopenta[c]thiophenones was achieved via halogenation reaction, then formation and finally cleavage of an aziridino ring. The in vitro cytotoxic activity of these compounds was evaluated against L1210 leukemia. The importance of the ketohydroxyethylamino sequence for their activities is discussed.

Diabetic retinopathy in a population of 1,302 insulin dependent diabetics (IDDM) diagnosed before 30 years of age.

The authors analyse the data resulting from the first ophthalmological observation of 1,302 insulin dependent diabetics whose age at diagnosis is less than 30 years and who have been observed regularly by the Portuguese Diabetic Association. The prevalence of retinopathy is 41, 6%; 34.3% is non-proliferative and 7.3% is proliferative. Retinopathy is more frequent in males (P < 0.001). The prevalence of retinopathy increases with the duration of diabetes and it is equal to or greater than 80% in people who have had diabetes for 10 years or more. 'Poor' glucose control, the coexistence of other late complications and arterial hypertension increase the risk of retinopathy (P < 0.001).

[Chronic hypoparathyroidism: review of 5 clinical cases]. / Hipoparatiroidismo crónico: revisão de 5 casos clínicos.

In 5 patients with a long clinical evolution of tetany and/or convulsions and with documented hypocalcaemia and hyperphosphatemia, low or inappropriate values of parathormone were detected. Only two of the patients had a history of subtotal thyroidectomy and all presented with basal ganglia calcification, bilateral subcapsular cataracts and prolonged QTc interval in the ECG. After one month of oral therapy with calcium and calcitriol, the values of calcaemia and phosphatemia were in a near-normal range with the exception of a patient in which that normalization was much slower and only occurred after correction of magnesaemia. In this last patient statistical correlation between QTc interval in the ECG and the calcaemia was statistically significant (P less than 0.001). We conclude that the QTc interval can be a useful and accessible index in acute situations of symptomatic hypocalcaemia.