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Background: Bortezomib is a selective and reversible inhibitor of the 26S proteasome that shows potent antitumor activity in vitro and in vivo against several human cancers of adulthood. No data are available on bortezomib activity against human pediatric neuroblastoma. Methods: Ten neuroblastoma cell lines and suspensions of primary neuroblastoma cells from three patients were tested for sensitivity to bortezomib. Colony formation, cell proliferation, cell cycle progression, and apoptosis were evaluated by a clonogenic assay and by measuring [sup.3]H-thymidine incorporation, bromodeoxyuridine uptake, DNA fragmentation, and phosphatidylserine exposure and propidium iodide staining, respectively. Angiogenesis was assessed by the chick embryo chorioallantoic membrane (CAM) assay. Two mouse xenograft models that mimic the growth and spread of neuroblastoma in humans were used to examine in vivo sensitivity of neuroblastoma to bortezomib. All statistical tests were two-sided. Results: Bortezomib inhibited proliferation and colony formation of neuroblastoma cell lines in a time- and dose-dependent manner. The mean bortezomib concentration that caused 50% inhibition of growth was 6.1 nM (95% confidence interval [CI] = 0.9 to 11.3 nM) at 72 hours. Bortezomib-treated neuroblastoma cells were arrested at [G.sub.2]/M and underwent apoptosis (mean percentage of apoptotic cells in four neuroblastoma cell lines treated with 20 nM bortezomib for 24 hours ranged from 20% to 35%, and caspases were activated by two- to fivefold with respect to untreated cells). Similar results were obtained for primary neuroblastoma cells exposed to bortezomib. Bortezomib inhibited angiogenesis in CAMs stimulated by conditioned medium from neuroblastoma cell lines, by neuroblastoma xenografts, and by primary neuroblastoma biopsy specimens (microvessel area: 2.9 x [10.sup.-2] [mm.sup.2], 95% CI = 1.8 x [10.sup.-2] to 3.8 x [10.sup.-2] [mm.sup.2] in CAMs treated with biopsy specimens alone and 1.3 x [10.sup.-2] [mm.sup.2], 95% CI = 1 x [10.sup.-2] to 1.5 x [10.sup.-2] [mm.sup.2] in CAMs treated with biopsy specimens plus bortezomib, P = .024). In both mouse models, mice treated with bortezomib lived statistically significantly longer than control mice (mean survival time in the pseudometastatic model: 74.2 versus 50.3 days, P

Backround: Because only limited evidence suggests that elastic stockings prevent the post-thrombotic syndrome in patients with symptomatic deep venous thrombosis (DVT), these stockings are not widely used. Objective: To evaluate the efficacy of compression elastic stockings for prevention of the post-thrombotic syndrome in patients with proximal DVT. Design: Randomized, controlled clinical trial. Setting: University hospital. Patients: 180 consecutive patients with a first episode of symptomatic proximal DVT who received conventional anticoagulant treatment. Interventions: Before discharge, patients were randomly assigned to wear or not wear below-knee compression elastic stockings (30 to 40 mm Hg at the ankle) for 2 years. Follow-up was performed for up to 5 years. Measurements: The presence and severity of the post-thrombotic syndrome were scored by using a standardized scale. Results: Post-thrombotic sequelae developed in 44 of 90 controls (severe in 10) and in 23 of 90 patients wearing elastic stockings (severe in 3). All but 1 event developed in the first 2 years. The cumulative incidence of the post-thrombotic syndrome in the control group versus the elastic stockings group was 40.0% (95% Cl, 29.9% to 50.1%) versus 21.1% (Cl, 12.7% to 29.5%) after 6 months, 46.7% (Cl, 36.4% to 57.0%) versus 22.2% (Cl, 13.8% to 30.7%) after 1 year, and 49.1% (Cl, 38.7% to 59.4%) versus 24.5% (Cl, 15.6% to 33.4%) after 2 years. After adjustment for baseline characteristics, the hazard ratio for the post-thrombotic syndrome in the elastic stockings group compared with controls was 0.49 (Cl, 0.29 to 0.84; P = 0.011). Limitations: This study lacked a double-blind design. Conclusions: Post-thrombotic sequelae develop in almost half of patients with proximal DVT. Below-knee compression elastic stockings reduce this rate by approximately 50%.

Background: Expression of the c-myb proto-oncogene in neuroblastoma, the most common extracranial solid tumor of infancy, is linked with cell proliferation and differentiation. Neuroblastoma can be selectively targeted via monoclonal antibodies against the disialoganglioside (G[D.sub.2]) tumor-associated antigen. Liposomes coated with anti-G[D.sub.2] antibodies (targeted liposomes) and entrapping a c-myb antisense oligonucleotide have antitumor activity. Because antisense oligonucleotides containing CpG motifs can stimulate immune responses, we evaluated the effect of CpG-containing c-myb antisense oligonucleotides encapsulated within targeted liposomes. Methods: Antisense (myb-as) and scrambled (myb-scr) control oligonucleotides with CpG motifs were encapsulated within GD2-targeted and non-targeted liposomes. Two murine (nude and SCID-bg) xenograft models of neuroblastoma were established. Mice (groups of 10) were injected intravenously with various oligonucleotide and liposome formulations, and life span, long-term survival, immune cell activation, and cytokine release were measured over time. Results: Tumor-bearing mice injected with targeted liposome-CpG-myb-as or targeted liposome-CpG-myb-scr lived longer than mice in any other group, although long-term survival (i.e., more than 120 days) was obtained only in mice injected with targeted liposome-CpG-myb-as. Splenocytes isolated from mice injected with targeted liposome-CpG-myb-as contained activated macrophages, B cells, and natural killer (NK) cells, but only activated NK cells were associated with antitumor cytotoxic activity. In vivo immune cell activation was accompanied by the time-dependent increases in plasma levels of the cytokines interleukin 12 (IL-12; maximum level reached by 2 hours) and interferon gamma (IFN-[lambda]; maximum level reached by 18 hours) and was dependent on the oligonucleotide CpG motif. Ablation of macrophages or NK cells resulted in a loss of in vivo antitumor activity. Conclusion: Immune cell activation, involving the time-dependent activation of macrophages and NK cells, contributes to the antitumor activity of targeted liposome-CpG-myb-as against neuroblastoma and could improve the effectiveness of antitumor targeted liposomes.

Results from epidemiologic studies are controversial with respect to the relation between total dietary fat consumption and breast cancer risk; there is more general agreement that a high-fat diet is associated with aggressive prostate cancer. Recent epidemiologic investigations and laboratory experimentation with animal models suggest that relatively high intakes of long-chain n - 3 fatty acids, and n - 9 fatty acids present in olive oil, reduce breast cancer risk by mechanisms that may involve modification of the biosynthesis of eicosanoids from n - 6 polyunsaturated fatty acids. Although there is only limited support for the hypothesis that total fat intake affects breast cancer risk, there is experimental evidence that n - 6 fatty acids, again via eicosanoid production, may enhance breast cancer invasion and metastasis; n - 3 fatty acids may exert a suppressive effect. Although studies of prostate cancer are less advanced, the indication is that a high fat intake promotes the emergence of the metastatic phenotype; further research is required to establish the roles of the various classes of fatty acids but it does appear that the long-chain n - 3 fatty acids may also retard prostate cancer progression.

Recent data suggest that treatment with n - 3 fatty acids could enhance the susceptibility of plasma low-density-lipoprotein (LDL) to oxidation. Twelve hypertriacylglycerolemic, hemodialyzed patients were treated with 2.5 g n - 3 fatty acids/d for 2 mo. Treatment was then withdrawn for 2 mo (washout phase). Plasma total cholesterol and LDL cholesterol increased significantly (9% and 28%) and plasma triacylglycerols decreased significantly after the n - 3 phase compared with baseline and washout values. LDL susceptibility to oxidation was tested by oxidation of LDL particles with 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH). No significant changes were observed for the lag phase and the peroxidation rate. The vitamin E content of LDL also did not change significantly. The results thus suggest that a daily dosage of 2.5 g n - 3 fatty acids does not enhance LDL susceptibility to oxidation, while retaining its hypotriacylglycerolemic effect.

Fifteen patients with right ventricular dysplasia were investigated by T1-weighted spin- and gradient-echo pulse sequences, using a protocol that enabled beth a subjective analysis of myocardial signal intensity and a quantitative/qualitative analysis of right and left ventricular function. In 8 patients, 3 investigators independently recognized abnormally hyperintense areas in the anatomic sites usually affected by the disease. In 7 of these patients, these areas showed an overlap with adyskinetic areas imaged by beth magnetic resonance imaging (MRI) and echocardiography. In 1 patient who underwent a cardiac transplant, MRI of the explanted heart showed an excellent correlation between the distribution of the lesions and the in vivo/in vitro features. The data were compared with those from an equivalent sample of patients affected by dilated cardiomyopathy. In the latter patients, no focal hyperintensities were attributed to any anatomic sites in the right ventricle, and no focal a-dyskinetic loci were observed. Furthermore, the 2 groups of patients were significantly different in regard to dimensional and functional quantitative parameters. The results suggest that MRI is useful in integrating echocardiographic data and can be helpful in diagnosing this disease in late stages. (Am J Cardiol 1992;70:1S89-159S)

A diet rich in carbohydrates and soluble fiber and low in saturated fat is prescribed for patients with non-insulin-dependent diabetes mellitus (NIDDM). This diet is designed to improve blood glucose (sugar) control, reduce the amount of low-density-lipoprotein (LDL) cholesterol in the blood, and reduce the risk of developing heart disease. Other studies have reported that diets low in saturated fat and rich in polyunsaturated or monounsaturated fat reduce the risk of heart disease. Therefore, a study was performed to compare two diets, one rich in complex carbohydrates and one rich in monounsaturated fat. The subjects included 19 patients with NIDDM who had good control over their blood glucose levels and were not taking insulin. The study was broken down into three phases. For the first two months, all of the subjects followed diets high in carbohydrate and low in fat (carbo diet). During the second two months of the study, all of the subjects followed diets rich in monounsaturated fat (mono diet). For the next two-month period, the all subjects returned to the carbo diet. Blood glucose and cholesterol levels were measured before and after each diet period. Blood glucose and cholesterol levels were the same at the end of each diet period as they were at the beginning of the study. Also, there were no changes in blood triglyceride (fatty acids) levels or high-density-lipoprotein, the beneficial form of cholesterol, during the three diet periods. It is concluded that both diets are well tolerated and do not alter blood glucose or cholesterol levels. This suggests that patients with NIDDM may be able to consume a wider variety of foods without suffering adverse side effects. (Consumer Summary produced by Reliance Medical Information, Inc.)

Background: Advanced-stage neuroblastoma resists conventional treatment; hence, novel therapeutic approaches are required. We evaluated the use of c-myb antisense oligodeoxynucleotides (asODNs) delivered to cells via targeted immunoliposomes to inhibit c-Myb protein expression and neuroblastoma cell proliferation in vitro. Methods: Phosphorothioate asODNs and control sequences were encapsulated in cationic lipid, and the resulting particles were coated with neutral lipids to produce coated cationic liposomes (CCLs). Monoclonal antibodies directed against the disialoganglioside [GD.sub.2] were covalently coupled to the CCLs. [sup.3]H-labeled liposomes were used to measure cellular binding, and cellular uptake of asODNs was evaluated by dot-blot analysis. Growth inhibition was quantified by counting trypan blue dye-stained cells. Expression of c-Myb protein was examined by western blot analysis. Results: Our methods produced [GD.sub.2]-targeted liposomes that stably entrapped 80%-90% of added c-myb asODNs. These liposomes showed concentration-dependent binding to [GD.sub.2]-positive neuroblastoma cells that could be blocked by soluble anti-[GD.sub.2] monoclonal antibodies. [GD.sub.2]-targeted liposomes increased the uptake of asODNs by neuroblastoma cells by a factor of four-fold to 10-fold over that obtained with free asODNs. Neuroblastoma cell proliferation was inhibited to a greater extent by [GD.sub.2]-targeted liposomes containing c-myb asODNs than by nontargeted liposomes or free asODNs. [GD.sub.2]-targeted liposomes containing c-myb asODNs specifically reduced expression of c-Myb protein by neuroblastoma cells. Enhanced liposome binding and asODN uptake, as well as the antiproliferative effect, were not evident in [GD.sub.2]-negative cells. Conclusions: Encapsulation of asODNs into immunoliposomes appears to enhance their toxicity toward targeted cells while shielding nontargeted cells from antisense effects and may be efficacious for the delivery of drugs with broad therapeutic applications to tumor cells. [J Natl Cancer Inst 2000;92:253-61]

Avaliar o procedimento cirúrgico de retalho homodigital, bem como a função do quirodáctilo, a dor, a sensibilidade, a estética e a satisfação do paciente.

In the present paper, an analysis of the stability of the MoS2compound in 17-4 PH stainless steel matrix during the sintering of powder injection moulded samples is presented. A feedstock containing 10% vol. of the solid lubricant phase MoS2, mixed with 17-4 PH stainless steel powder was prepared and injected. The sintering was carried out at various temperatures ranging from 650°C to 1,300°C. The progress of the dissociation process of MoS2as a function of sintering temperature and the formation of new phases were analyzed via X-ray diffraction. The microstructure of the resulting material was analyzed by SEM/EDS. As expected, for temperatures above 650°C, the results confirm the decomposition of MoS2and formation of others sulphides during the sintering cycle. In addition, there occurs dissolution of molybdenum resulting from MoS2decomposition.

Background:Prophylaxis of venous thromboembolism (VTE) in hospitalized medical patients is largely underused. We sought to assess the value of a simple risk assessment model (RAM) for the identification of patients at risk of VTE. Methods:In a prospective cohort study, 1180 consecutive patients admitted to a department of internal medicine in a 2‐year period were classified as having a high or low risk of VTE according to a predefined RAM. They were followed‐up for up to 90 days to assess the occurrence of symptomatic VTE complications. The primary study outcome was to assess the adjusted hazard ratio (HR) of VTE in high‐risk patients who had adequate in‐hospital thromboprophylaxis in comparison with those who did not, and that of VTE in the latter group in comparison with low‐risk patients. Results:Four hundred and sixty‐nine patients (39.7%) were labelled as having a high risk of thrombosis. VTE developed in four of the 186 (2.2%) who received thromboprophylaxis, and in 31 of the 283 (11.0%) who did not (HR of VTE, 0.13; 95% CI, 0.04–0.40). VTE developed also in two of the 711 (0.3%) low‐risk patients (HR of VTE in high‐risk patients without prophylaxis as compared with low‐risk patients, 32.0; 95% CI, 4.1–251.0). Bleeding occurred in three of the 186 (1.6%) high‐risk patients who had thromboprophylaxis. Conclusions:Our RAM can help discriminate between medical patients at high and low risk of VTE. The adoption of adequate thromboprophylaxis in high‐risk patients during hospitalization leads to longstanding protection against thromboembolic events with a low risk of bleeding.

Novel anti-vasculature strategies that are emerging for the treatment of cancer and for the inhibition of angiogenesis may be a promising new tool for the adjuvant therapy of malignant tumours. Over the last fifteen years, several reports have been published concerning the relationship between tumour progression and angiogenesis in experimental models of neuroblastoma in vitro and in vivo. Moreover, a high vascular index in neuroblastoma correlates with poor prognosis, suggesting dependence of aggressive tumour growth on active angiogenesis. Here, we present an overview of the most recent advances in anti-vasculature therapy of neuroblastoma, and describe some preclinical results as well as future perspectives.

Ectodermal dysplasias EDs as defined by FreireMaia FreireMaia 1971; Hum Hered 21: 309–312; FreireMaia 1977; Acta Genet Med Gemellol 26: 121–131 are congenital disorders characterized by alterations in two or more ectodermal structures, at least one of these involving alterations in hair, teeth, nails, or sweat glands. Suggestions for a new definition and, consequently, for a new classification of EDs have being proposed lately, mainly with the purpose of connecting clinical knowledge with recent discoveries of gene mutations responsible for about 30 of EDs. The aim of this review was to update the clinical classification of EDs with recent molecular 64 genes and 3 chromosome regions and clinical data, mainly of EDs of the A group N  186, in order to contribute information for the evaluation of the ED definition proposed by FreireMaia. Our conclusion is that the combination of both procedures—clinical and molecular—only brings advantages for a deeper knowledge of EDs. First, it allows a rapid diagnosis that may become even more precise whenever DNA exams are available. Secondly, the comprehension of the biological mechanisms that cause EDs is needed for the design of efficient prevention and treatment approaches. © 2009 WileyLiss, Inc.

ABO blood groups greatly influence circulating von Willebrand factor (VWF) levels, and O group subjects have lower VWF values. In this study, we investigated whether ABO groups affect VWF survival by monitoring the post-DDAVP (1-desamino-8-d arginine vasopressin) time courses of VWF antigen (VWF:Ag), VWF collagen binding (VWF:CB), and factor VIII (FVIII) in 47 healthy subjects (28 O and 19 non-O blood groups). The elimination half-life (T1/2el) of VWF was found significantly shorter in O than in non-O subjects (10.0 ± 0.8 hours vs 25.5 ± 5.3 hours, respectively; P < .01), as was the T1/2el of VWF:CB (7.9 ± 0.5 hours vs 20.9 ± 4.5 hours; P < .01). A direct linear correlation was found between basal VWF:Ag and T1/2el, subjects with higher VWF levels having longer-surviving VWF. ABO blood groups appeared to strongly influence VWF clearance, but not its synthesis or release from endothelial cells. The VWF propeptide to VWF:Ag ratio, useful for predicting an increased VWF clearance, was found significantly higher in O than in non-O individuals (1.6 ± 0.1 vs 1.2 ± 0.5, P < .001), with values that correlated inversely with T1/2el (P < .001). Based on these findings, we conclude that the lower VWF values in O group individuals is attributable to a shorter VWF survival and circulating VWF values are strongly influenced by its half-life.

Disseminated neuroectoderma-derived tumors, mainly neuroblastoma in childhood and melanoma in the adulthood, are refractory to most current therapeutic regimens and hence the prognosis remains very poor. Preclinical research studies have indicated several agents that show promising therapeutic potential for these neoplasms. However, there appears to be a limitation to their in vivo applicability, mainly due to unfavorable pharmacokinetic properties that lead to insufficient drug delivery to the tumor or metastatic sites or to high systemic or organ-specific toxicity. In this scenario, the focus is on targeted cancer therapy. Encapsulating anticancer drugs in liposomes enables targeted drug delivery to tumor tissue and prevents damage to the normal surrounding tissue. Indeed, sterically stabilized liposomes have been shown to enhance the selective localization of entrapped drugs to solid tumors, with improvements in therapeutic indices. The identification of tumor-associated antigens and/or genes and the relative ease of manipulating the physicochemical features of liposome hold promise for the development of novel therapeutic strategies that selectively target tumor cells. Combined targeting is still investigated, especially the availability to simultaneously target and kill both the cancer cells and the tumor vasculature. Animal models make it possible to link molecular genetics and biochemistry information to the physiological basis of disease and are important predictive tools that offer a frontline testing system for studying the involvement of specific genes and the efficacy of novel therapeutics approaches. Relevant experimental models of human neuroblastoma and melanoma, which better reflect the tumor behavior in patients, are required to evaluate the effectiveness of the various targeted liposomal formulations and their possible systemic and organ-specific toxicity. The most multifunctional targeted liposomes are herein described, with primary attention on testing their efficacy in clinically relevant animal models for the treatment of neuroblastoma and melanoma.

Background: The mechanisms leading to pregnancy-related hypertensive disorders, and pregnancy-induced hypertension (PIH) and pre-eclampsia (PE) in particular, are still not clear. Diagnostic criteria are clinical because specific markers of the condition are lacking. A role of the fibrinolytic system has been suggested. Objectives: We aimed to evaluate the behavior of tissue-type plasminogen activator (t-PA), plasminogen activator inhibitor type 1 (PAI-1), PAI-2, and the placental hormone inhibin-A in women with a normal pregnancy vs. women with pregnancies complicated by PIH or PE. Methods: Blood samples were drawn between the 25th and 30th gestational week (GW) and between the 31st and 36th GW in order to assay t-PA, PAI-1, PAI-2 and inhibin-A; routine biochemical exams, ultrasonography umbilical artery pulsatility index (PI), placental weight and newborn weight were measured. Results: In pregnancies complicated by hypertensive disorders, PAI-1 levels were higher than in controls and increased significantly after the 25th GW, especially in PE, as did inhibin-A. PAI-2 levels were significantly lower after the 30th GW in patients with PIH and PE. The PAI-1/PAI-2 ratio was significantly higher in PE patients than in controls as of the 25th GW, but only after the 30th GW in patients with PIH. Inhibin-A was significantly correlated with fibrinolytic parameters, and inversely with newborn weight. Receiver–operator characteristic curves for PAI-1 and inhibin-A showed a high sensitivity and specificity for PE. PAI-2 correlated with newborn and placental weight, and inversely with PI of the umbilical artery. Conclusions: Fibrinolytic tests (especially PAI-1) and inhibin-A monitoring during pregnancy may help in the early diagnosis of pregnancy-related hypertensive disorders.

The central problem in cancer chemotherapy is the severe toxic side effects of anticancer drugs on healthy tissues. The use of liposomes as drug delivery vehicles for antitumour therapeutics has great potential to revolutionise the future of cancer therapy. As tumour architecture causes liposomes to preferentially accumulate at the tumour site, their use as drug carriers results in the localization of a greater amount of the loaded drug at the tumour site, thus improving cancer therapy and reducing the harmful non-specific side effects of chemotherapeutics. In addition, targeting of liposomal anticancer drugs to antigens expressed or over-expressed on tumour cells provides a very efficient system for increasing the therapeutic indices of the drugs. Animal models allow detailed examination of molecular and physiological basis of diseases and offer a frontline testing system for studying the involvement of specific genes and the efficacy of novel therapeutic approaches. Until recently, the most resorted experimental model of paediatric Neuroblastoma (NB) tumour is the subcutaneous xenograft in nude mice. However, the main disadvantage of this animal model is that it does not reflect the metastatic potential of NB cells, ultimately responsible for poor patient survival. A more realistic view of the clinical potential of targeted therapies could be obtained if a tumour model were available that better reflects the growth of advanced NB in children (i.e. large adrenal gland tumours and multiple small metastatic lesions). All current data support this concept and recommend that orthotopic implantation of tumour cells in recipient animals is mandatory for studies of tumour progression, angiogenesis, invasion, and metastasis. This review will focus on the description of the most clinically relevant animal models established to test the efficacy of targeted liposomal anti-tumour formulations for the treatment of Neuroblastoma.

Photodynamic therapy (PDT) is based on the use of a photozensitising compound which is accumulated by rapidly proliferating cells. Subsequent irradiation with light wavelengths specifically absorbed by the photosensitiser promotes the generation of reactive short-lived oxygen species which cause an irreversible and selective damage. Endovascular interventions to correct obstructive arterial disease have been developed worldwide with excellent short term results. However, long term patency is still limited by the onset of restenosis, due to subsequent intimal hyperplasia (IH). IH is characterized by proliferation and migration of smooth muscle cells (SMC) and extracellular matrix production. Targeting of SMC by photozensitisers can be efficiently achieved by taking advantage of the receptors for low density lipoproteins (LDL) expressed by such cells. Thus, preference is given to hydrophobic compounds which readily partition in the lipid matrix of LDL. We developed a liposomal formulation of a highly hydrophobic photozensitising agent, Zn(II)- phthalocyanine (ZnPc). The liposome-delivered ZnPc was readily taken up by cultured SMC cells and preferentially localized in the Golgi apparatus. Red light irradiation of incubated SMC induced cell death. Extension of these investigations to an in vivo rabbit model showed that ZnPc mainly accumulated in the media layer, where PDT induces the main damage through cellular depletion due to apoptosis of SMC, changes in the extracellular matrix with generation of a barrier to cellular migration, and acceleration of re-endothelization. Initial clinical applications showed that PDT safely and effectively prevents restenosis after angioplasty up to a 6 month follow-up.

Background:Recently, we reported an association between asymptomatic carotid atherosclerosis and venous thromboembolism (VTE) of unknown origin. We hypothesized that patients with VTE of unknown origin would be at a higher risk of developing symptomatic atherosclerosis than patients with VTE induced by known risk factors. Methods:To examine this hypothesis, we studied 1919 consecutive patients followed prospectively after their first VTE episode. The primary outcome was non‐fatal and fatal symptomatic atherosclerotic disease in patients with VTE of unknown origin as compared to those with secondary VTE. An independent committee assessed all study outcomes, and adjusted hazard ratios (HR) were calculated using the Cox's proportional hazards model. Results:After a median follow‐up of 48 and 51 months, respectively, at least one symptomatic atherosclerotic complication was detected in 160 of the 1063 patients (15.1%) with VTE of unknown origin, and in 73 of the 856 (8.5%) with secondary VTE. After adjusting for age and other risk factors of atherosclerosis, the HR for symptomatic atherosclerotic complications in patients with VTE of unknown origin compared to those with secondary VTE was 1.6 (95% confidence intervals; CI: 1.2–2.0). When the analysis was restricted to patients without previous symptomatic atherosclerosis, the HR became 1.7 (95% CI: 1.1–2.4). Conclusions:Patients with VTE of unknown origin have a 60% higher risk of developing symptomatic atherosclerotic disease than do patients with secondary venous thrombosis.

Background:Functional defects of the protein C pathway, detectable in plasma as activated protein C (APC) resistance, are a prevalent risk factor for venous thrombosis. The factor V (FV) Leiden mutation causes APC resistance by interfering with the APC-mediated inactivation of both FVa and FVIIIa. Co-inheritance of FV Leiden and quantitative FV deficiency on different alleles, a rare condition known as pseudo-homozygous APC resistance, is associated with pronounced APC resistance and 50% reduced FV levels, because of non-expression of the non-Leiden FV allele. Objectives:The role of normal FV in modulating the APC resistance phenotype in carriers of FV Leiden was investigated in patients with pseudo-homozygous APC resistance and in model systems. Patients/methods:Four functional plasma assays probing both components of APC resistance (susceptibility of FVa to APC and cofactor activity of FV in FVIIIa inactivation) were employed to compare seven clinically and genetically characterized FV Leiden pseudo-homozygotes to 30 relatives with different FV genotypes (including 12 FV Leiden heterozygotes and seven carriers of FV deficiency) and to 32 unrelated FV Leiden homozygotes. Results and conclusions:All assays consistently indicated that FV Leiden pseudo-homozygotes are significantly more APC-resistant than heterozygotes and indistinguishable from homozygotes. Thrombin generation measurements in FV-deficient plasma reconstituted with purified normal FV and FV Leiden confirmed these observations and showed that the expression of the normal FV allele is an important modulator of APC resistance in FV Leiden heterozygotes. These findings provide an explanation for the higher thrombotic risk of FV Leiden pseudo-homozygotes when compared with heterozygotes.

Fibrin deposition is central to the acute humoral rejection process occurring in the presence of consumptive coagulopathy when pig organs are transplanted into primates.

In order to assess whether the HR2 haplotype of the factor V gene (HR2) increases the risk of venous thromboembolism (VTE) in carriers of antithrombin (AT), protein C (PC) or S (PS) defects, we performed this determination in 336 subjects, who were family members of 66 symptomatic patients with clotting inhibitors defects. We first assessed the presence of previous VTE, and then followed prospectively subjects without prior VTE. VTE episodes had occurred in 26 individuals: 18 in 139 carriers of clotting inhibitors defects alone (annual incidence, 0.55%), four in 33 carriers of clotting inhibitors defects combined with HR2 (0.52%) and four in 151 non‐carriers (0.1%), resulting in a relative risk (RR) for VTE of 4.9 (95% CI: 1.7–14.4) and 4.62 (95% CI: 1.2–18.4), respectively. After an overall follow‐up of 2557 patient‐years, VTE episodes developed in 12 subjects: nine in 121 carriers of clotting inhibitors defects alone (annual incidence, 0.92%), three in 29 carriers of clotting inhibitors defects combined with HR2 (1.0%) and none in 147 non‐carriers. In family members of patients with AT, PC or PS defects the coinheritance of HR2 haplotype does not seem to increase the thromboembolic risk.

Cell-cycle deregulation is an early event of hepatocarcinogenesis. We evaluated the role of changes in activity of nuclear factor κB (NF-κB) and some related pathways in this alteration, and the interference of N-(4-hydroxyphenyl)retinamide (HPR), a retinoid chemopreventive for various cancer types, with these molecular mechanisms and the evolution of preneoplastic liver to cancer. Male F344 rats, initiated according to the ‘resistant hepatocyte’ model of liver carcinogenesis, received weekly 840 nmol of liposomal HPR (SL-HPR)/100 g body wt or empty liposomes, between 5 and 25 weeks after initiation. Inhibition of DNA synthesis and induction of apoptosis occurred in pre-cancerous lesions, 7–147 days after starting SL-HPR, and a decrease in carcinoma incidence and multiplicity was observed 25 weeks after arresting treatment. An increase in NF-κB expression and binding activity, and under-expression of the inhibitor κB-α (IκB-α) were found in preneoplastic liver and neoplastic nodules, 5 and 25 weeks after initiation, respectively. These lesions also showed low expression of Mat1A and low activity of methionine adenosyltransferase I/III, whose reaction product, S-adenosyl-l-methionine, enhances IκB-α expression. SL-HPR prevented these changes and induced a decrease in expression of iNos, c-myc, cyclin D1 and Vegf-A genes, that were over-expressed in preneoplastic liver and nodules, and a decrease in Bcl-2/Bax, Bcl-2/Bad and Bcl-xL/Bax mRNA ratios with respect to the lesions of control rats. Liposomes alone did not influence the parameters tested. These results indicate that signal transduction pathways controlled by NF-κB, nitric oxide and S-adenosyl-l-methionine are deregulated in pre-cancerous lesions. Recovery from these alterations by SL-HPR is associated with chemoprevention of hepatocarcinogenesis. Overall, these studies elucidate some molecular changes, in early stages of hepatocarcinogenesis, and underline their pathogenetic role. Moreover, they demonstrate a partially new mechanism of HPR chemopreventive effect and indicate the potential clinical relevance of this compound for prevention of hepatocellular carcinoma.

Five monkey recipients of a porcine renal xenograft were studied to determine the relationship between fibrin formation in acute humoral xenograft rejection (AHXR) and procoagulant and anticoagulant factor levels to establish whether changes in coagulation parameters could be used to predict AHXR and determine whether AHXR is associated with overt disseminated intravascular coagulopathy (DIC) in this 
 model.

Presents recipes of several Italian dishes by chef Lorenzo Pagnan. Stuffed capsicum; Red cabbage salad; Polenta stacks; Marinated Mushrooms; Potato gnocchi with sage burnt butter; Involtini di vitello; Budino al cioccolato.

Melanoma is a highly malignant and increasingly common tumour. Since metastatic melanoma remains incurable, new treatment approaches are needed. Previously, we reported that the synthetic retinoid N‐(4‐hydroxyphenyl)retinamide (fenretinide, HPR) induces apoptosis in neuroblastoma cells, sharing a neuroectodermal origin with melanoma cells. Since no data exist thus far on the effects of HPR on human melanoma tumours, our purpose was to investigate the in vitromodulation of cell growth and apoptosis by HPR in melanoma cells. Ten human melanoma cell lines were exposed in vitroto increasing concentrations of HPR. Dose‐dependent growth inhibition and cytotoxicity were observed. According to cytofluorimetric analysis, propidium iodide staining and TUNEL assay, HPR‐treated melanoma cells were shown to undergo apoptosis. However, IC50values ranged from 5 to 28 μM, while IC90values were between 10 and 45 μM. These last concentrations are approximately 10‐fold higher than those achievable in patients given oral HPR. To explore the potential of new delivery strategies, HPR was loaded at high concentrations into immunoliposomes directed to disialoganglioside GD2, a tumour‐specific antigen extensively expressed by neuroectoderma‐derived tumours. Treatment of melanoma cells for a short time (2 hr) with HPR‐containing immunoliposomes followed by culture in drug‐free medium gave rise to apoptosis of target cells, whereas cells treated for 2 hr with equivalent concentrations of the free drug survived. The efficacy of immunoliposomal HPR was strongly dependent on the density of GD2 expression in the different cell lines. Int. J. Cancer 81:262–267, 1999. © 1999 Wiley‐Liss, Inc.

Melanoma is a highly malignant and increasingly common neoplasm. Because metastatic melanoma remains incurable, new treatment approaches are needed. Immunoliposomes have been previously shown to enhance the selective localization of immunoliposome‐entrapped drugs to solid tumors with improvements in the therapeutic index of the drugs. Previously, we reported that the synthetic retinoid fenretinide (HPR) is an inducer of apoptosis in neuroblastoma (NB) cells, sharing the neuroectodermal origin with melanoma cells. HPR is a strong inducer of apoptosis also in melanoma cells, although at doses 10‐fold higher than those achievable clinically. Thus, our purpose was to investigate the in vitropotentiation of its cytotoxic effect on melanoma cells in combination with long‐circulating GD2‐targeted immunoliposomes. GD2 is a disialoganglioside extensively expressed on tumors of neuroectodermal origin, including melanoma. Murine anti‐GD2 antibody (Ab) 14.G2a and its human/mouse chimeric variant ch14.18 have been ligated to sterically stabilized liposomes by covalent coupling of Ab to the polyethylene glycol (PEG) terminus. Ab‐bearing liposomes showed specific, competitive binding to and uptake by various melanoma cell lines compared with liposomes bearing non‐specific isotype‐matched Abs or Ab‐free liposomes. Cytotoxicity was evaluated after 2 hr treatment, followed by extensive washing and 72 hr incubation. This treatment protocol was designed to minimize non‐specific adsorption of liposomes to the cells, while allowing for maximum Ab‐mediated binding. When melanoma cells were incubated with 30 μM HPR entrapped in anti‐GD2 liposomes, a significant reduction in cellular growth was observed compared to free HPR, entrapped HPR in Ab‐free liposomes or empty liposomes. Cytotoxicity was not evident in tumor cell lines of other origins that did not express GD2. Growth of NB cells was also inhibited by immunoliposomes with entrapped HPR. Int. J. Cancer 81:268–274, 1999. © 1999 Wiley‐Liss, Inc.

Patients with neuroblastoma (NB) are often refractory to metabolic radiotherapy with radioiodine-labelled metaiodobenzylguanidine (MIBG), generally due to low, if any, expression of the transporter molecule responsible for MIBG uptake in tumor cells. Delivery of anticancer drugs in sterically stabilized (polyethylene glycol (PEG)-containing) immunoliposomes (SIL) is an emerging tool for the selective delivery of antitumor drugs to cells expressing specific antigens. By taking advantage of receptor-mediated endocytosis of targeted liposomes, the delivery of MIBG to NB cells may be enhanced, bypassing the requirement for a drug-specific membrane transporter. NB cells, as well as some neuroectoderma-derived cell lineages, such as melanoma cells, express frequently disiaioganglioside GD2. This surface disiaioganglioside can, therefore, be utilized as a target to selectively deliver MIBG-loaded SILs to these GD2-expressing cells.We thus explored the feasibility to encapsulate 125I-MIBG into anti-GD2 immunoliposomes and investigated the cellular uptake and metabolism of SIL-MIBG compared to free MIBG in a panel of NB and melanoma cell lines in vitro. We successfully loaded free MIBG into stabilized liposomes and cova-iently coupled them to monoclonal anti-GD2 antibodies.The relative expression of MIBG-transporter and GD2 determined the degree of MIBG uptake. Uptake of SIL-encapsulated MIBG by all cell lines was higher than that of free MIBG, the only exception being the highly transport-competent, GD2-negative cell line SK-N-BE2c. Moreover, successful incorporation of MIBG in melanoma cells, which are inherently non competent in taking up the free drug, could be achieved by SIL-MIBG. Interestingly, the intracellular half-life of SIL-MIBG was significantly more prolonged than thai of free MIBG in all NB cell lines, which reportedly cannot efficiently store free MIBG in subcellular compartments. The retention of SIL-MIBG by NB and melanoma cells was similar to that observed with free MIBG in highly storage-efficient pheochromocytoma (PC) cells.Thus, targeting GD2-positive cells with specific MIBG-loaded immunoliposomes appears a novel strategy for tumor cell killing, regardless of their competence to specifically incorporate the free compound.

Serum levels of thyroxine (T4), triiodothyronine (T3), reverse triiodothyronine (rT3) and TSH were measured in euthyroid subjects after a single dose of 4 mg D-thyroxine (DT4) or of 0.25 mg L-thyroxine (LT4). The same parameters and TSH response to TRH were also evaluated in 7 dyslipidemic patients before and after one month of treatment with 6 mg DT4. T4levels increased about 165% at h 4 after DT4and only 47% after LT4; T3levels remained unchanged until h 10 both after DT4and after LT4; rT3levels increased almost 179% after DT4and only 32% after LT4. TSH levels decreased about 30% after both DT4and LT4. In the long term study similar variations of the same parameters were observed: basal TSH levels decreased and TSH response to TRH was inhibited in all patients but one; T4levels increased 62%, T3levels increased 35%, while rT3levels increased 545%. Our results show that: 1) both acute and long-term treatment with DT4suppress TSH secretion; 2) DT4both in acute and in long-term administration, is preferentially dealogenated in the alaninic ring with production of rDT3, instead of in the phenolic ring with production of DT3. This may contribute to explain its lower metabolic activity.

The platelet aggregate filtration pressure test (PAFP) was used to study the aggregant activity of single doses of 200 mg indobufen, administered either orally or by i.v. injection. This double-blind study using placebo in a crossover design was conducted in twelve patients whose baseline aggregation values were high.The compound showed markedly different antiaggregant activity from placebo at the various observation times after administration.The two administration routes did not produce any significant differences.The PAFP test can be useful in identifying atherosclerotic patients with platelet hyperaggregation, and for clinical pharmacology studies with platelet antiaggregant drugs.

Human neuroblastoma (NB) tumours represent a major therapeutic challenge due to the lack of drugs effective in controlling cell proliferation. We previously reported that the synthetic retinoid Fenretinide (HPR) inhibits NB cell growth through the induction of programmed cell death. More recently, various NB cell lines have been shown to be partially resistant, in vitro, to HPR used at in vivo achievable concentrations (1-3 μmol/L). To significantly increase the dose, half-life, and stability of this promising anticancer agent we studied a system of conventional or long-circulating liposomes.In this study, we showed that HPR can be efficiently and stable encapsulated in conventional (CL-HPR) and stabilized liposomes (SL-HPR). Since the leakage of the drug from the liposomes under the experimental conditions used is negligible, it seems that HPR is entering cells via uptake of intact liposomes. Liposome-entrapped HPR completely arrested the growth of NB cells. The effect was dose- and time-dependent. Indeed, SL-HPR at 30 (imol/ L induced, in the cell lines partially resistant to free HPR, a very rapid (24-48 h) fall in thymidine uptake (> 95 %), whereas at 3 μmol/L it exhibited cytostatic effects.Time lapse photomicroscopy showed that NB cells treated with SL-HPR underwent a death process highly reminiscent of apoptosis, with progressive condensation of the cytoplasm around the nucleus and intense cell shrinkage. The cells then rounded up and detached from the plate. Furthermore, propidium iodide staining of the DNA showed that a high proportion of cells treated with SL-HPR displayed a small and brightly staining nucleus; chromatin appeared aggregated into dense masses at the nuclear periphery, a typical feature of apoptotic cells. These findings were confirmed by electronic microscopy, DNA fragmentation assay, DNA content analysis and by a quantitative assay for evaluating programmed cell death based upon the labeling of DNA breaks with tritiated thymidine. HPLC analysis showed that HPR did not become metabolized after uptake into NB cells cultured in vitro, thus indicating that SL-HPR-induced apoptosis results from the action of HPR, itself, and not from its metabolite(s). In conclusion, our study demonstrates that Fenretinide entrapped in conventional or sterically stabilized liposomes dramatically suppresses NB cell growth by inducing programmed cell death.