Fusion of Tumor-Derived Exosomes with Long-Circulating and pH-Sensitive Liposomes Loaded with Doxorubicin for the Treatment of Breast Cancer.

Doxorubicin (DOX) is a chemotherapeutic agent that has been used in the treatment of breast cancer. However, serious toxic effects have limited its use, mainly cardiotoxicity. To minimize the adverse effects, liposomal preparations containing DOX have been developed. These preparations can reach the target in the tumor region as well as bypass the resistance-related problems. An alternative to increased therapeutic efficacy may be the fusion of liposomes with exosomes released from tumor cells to facilitate membrane and fusion interactions, achieving greater cell uptake. Thus, the purpose of this study was the fusion of exosomes derived from breast tumor cells with long-circulating and pH-sensitive liposomes loading DOX (ExoSpHL-DOX) for the treatment of breast cancer. The mean diameter of ExoSpHL-DOX was 100.8 ± 7.8 nm, the polydispersity index was 0.122 ± 0.004, and the encapsulated DOX content was equal to 83.5 ± 2.5%. The fusion of exosomes with long-circulating and pH-sensitive liposomes was confirmed by Fourier transform infrared spectroscopy, Raman spectroscopy, and nano-flow cytometry. The physicochemical characteristics of ExoSpHL-DOX were maintained for 60 days, at 4 °C. The study of the release of DOX from ExoSpHL-DOX in dilution media with different pH values showed the pH sensitivity characteristic of the nanosystem, since 96.6 ± 0.2% of DOX was released from ExoSpHL-DOX at pH 5.0, while at pH 7.4, the release was 70.1 ± 1.7% in the medium. The cytotoxic study against the breast cancer cell line demonstrated that ExoSpHL-DOX treatment significantly reduced the cancer cell viability.

Mediterranean diet is associated with bone mineral density and muscle mass in postmenopausal women.

PURPOSE: This study aimed to investigate the association between the Mediterranean diet (MD), body composition, and bone mineral density (BMD) in postmenopausal women. METHODS: In this cross-sectional study, 105 apparently healthy postmenopausal women aged between 45 and 65 years were included. BMD, percentage body fat, and appendicular lean mass index (ALMI, appendicular lean mass/height squared) were assessed by dual-energy X-ray absorptiometry. Dietary intake was assessed by a validated food frequency questionnaire. Assessment of MD adherence was based on intake of cereals, vegetables, fruits, meats, dairy products, fish, red wine, and olive oil, and expressed as the Mediterranean diet score (MDS). RESULTS: Women with higher adherence to the MD had higher ALMI (6.6 ± 0.8 kg/m2 vs. 6.3 ± 0.7 kg/m2; p = 0.039) and lumbar spine BMD (1.076 ± 0.149 vs. 0.997 ± 0.143 g/cm2; p = 0.007) compared to those with lower MDS. Linear regression analysis adjusted for previous hormone therapy, previous smoking behavior, and habitual physical activity showed an independent positive contribution of MDS to lumbar spine BMD (mean difference 0.088 g/cm2, 95% confidence interval 0.028-0.147; p = 0.004) and ALMI (mean difference 0.296 kg/m2, 95% confidence interval 0.020-0.591; p = 0.049). CONCLUSION: Bone mineral density at the lumbar spine and ALMI were positively associated with the MDS in a sample of postmenopausal women from a non-Mediterranean region.

Dietary intake of isoflavones is associated with a lower prevalence of subclinical cardiovascular disease in postmenopausal women: cross-sectional study.

BACKGROUND: Menopause has been associated with an increased risk of cardiovascular disease. It has been shown that isoflavones protect vascular endothelial cells against induced oxidative stress injury. Therefore, the present study aimed to investigate the association between the dietary intake of isoflavones and the presence of subclinical cardiovascular disease (CVD) in postmenopausal women. METHODS: Ninety-six postmenopausal women [mean (SD) age 55.2 (4.9) years, body mass index (BMI) 27.2 (4.6) kg m-2 ] completed the study protocol. Habitual physical activity was assessed using a digital pedometer, resting metabolic rate was measured by indirect calorimetry and dietary intake was assessed via a validated food frequency questionnaire. Subclinical CVD was defined as carotid artery intima-media thickness (C-IMT) >0.9 mm and/or the presence of one or more atherosclerotic plaques in any of the studied segments. RESULTS: Mean (SD) C-IMT was 0.74 (0.2) mm, 25% of participants were found to have atherosclerotic plaques and the prevalence of subclinical CVD was 35%. Participants with subclinical CVD were more likely to consume less selenium, magnesium, folate and isoflavones, even after adjusting for total energy intake. A multivariate-adjusted regression model showed that a BMI >27 kg m-2 was associated with 90% higher risk of having ≥1 plaque and/or C-IMT >0.9 mm (P = 0.017). Higher oestradiol levels (P = 0.004) and isoflavone intake (P = 0.021) were independently associated with a lower risk of having subclinical CVD. CONCLUSIONS: In the present study, we observed that a higher isoflavone dietary intake was associated with a lower risk of subclinical CVD in postmenopausal women, independent of BMI and endogenous oestradiol levels.

The effect of dental bleaching on pulpal tissue response in a diabetic animal model: a study of immunoregulatory cytokines.

AIM: To evaluate the influence of tooth bleaching on immunoregulatory cytokines production (IL-6, Tumour necrosis factor (TNF)-α and IL-17) in the pulp tissue of normoglycaemic and diabetic rats. METHODOLOGY: Twenty-eight rats were divided into normoglycaemic and diabetic rats (n = 14). Diabetes mellitus (DM) was induced with a single dose of alloxan diluted in citrate buffer via intramuscular injection. After DM confirmation, all rats were sedated and tooth bleaching was performed using 35% hydrogen peroxide on the right maxillary molars for 30 min. Left molars were used as controls. Bleaching resulted in four hemimaxillae groups: normoglycaemic (N), N-bleached (NBle), diabetic (D) and D-bleached (DBle). After 2 and 30 days, rats were euthanized and hemimaxillae processed for analysis by haematoxylin and eosin and immunohistochemistry. Results within and between animals were submitted to Wilcoxon signed-rank and Mann-Whitney tests (P < 0.05). RESULTS: At 2 days, the NBle group had mild, and the DBle had severe inflammatory infiltration in the pulpal tissue (P < 0.05). TNF-α and IL-6 cytokines were associated with increased immunolabelling in the bleached groups compared to nonbleached (P < 0.05). However, IL-17 had increased immunolabelling in the NBle compared to the N and DBle group (P < 0.05). At 30 days, reactionary dentine was observed in the coronal pulp of all bleached teeth and no inflammation was present (P > 0.05). TNF-α cytokines had increased immunolabelling in the DBle group compared to the D group (P < 0.05). However, for IL-6 and IL-17, no difference was observed in this period (P > 0.05). CONCLUSIONS: Tooth bleaching increased IL-6 and TNF-α in the pulp tissue regardless of diabetes mellitus; however, diabetic rats had higher TNF-α levels for longer periods. Tooth bleaching influenced the increase in IL-17 in the early periods in normoglycaemic rats.

Concentration-dependent effect of bleaching agents on the immunolabelling of interleukin-6, interleukin-17 and CD5-positive cells in the dental pulp.

AIM: To evaluate lymphocyte-like cell activation (CD5-positive cells) and the expression of interleukin (IL)-6 and IL-17 in the pulp after tooth bleaching with two concentrations of hydrogen peroxide (H2 O2 ). METHODOLOGY: The right and left maxillary molars from 40 rats were treated randomly with bleaching gel with 20% H2 O2 (BLUE group, 1 application of 50 min), 35% H2 O2 (MAXX group, three applications of 15 min), or placebo gel (control). After 2 and 30 days, the rats were killed (n = 10), and the jaws were processed for histological and immunohistochemistry analysis of the pulp tissue. The scores of inflammation and immunolabelling (IL-6/IL-17) were submitted to Mann-Whitney and Kruskal-Wallis followed Dunn tests, respectively; anova tests were used for comparisons of number of CD5-positive cells and pulp chamber area values (P < 0.05). RESULTS: At 2 days, 60% of specimens of the BLUE group were associated with moderate inflammation in pulp horns, and in the MAXX group with necrosis (P < 0.05). At 30 days, the pulp was organized, and tertiary dentine was formed. The MAXX group had superior immunolabelling of IL-17 at 2 days differing significantly from other groups (P < 0.05). At 2 days, 90% of the specimens of the BLUE group had moderate immunolabelling of IL-6, and 50% of the MAXX group had severe immunolabelling, both significantly different from the control (P < 0.05). There was no significant difference between the groups at 30 days (P > 0.05). CD5-positive cells were present at 2 and 30 days, particularly in the bleached groups (P < 0.05), without significant difference between time periods (P > 0.05). CONCLUSIONS: IL-6 and IL-17 participated in inflammation in the pulp tissue of rats after tooth bleaching, particularly at 2 days. The immunolabelling was greater with increasing H2 O2 concentration. This process was accompanied by the prolonged activation of CD5-positive cells.

The effect of dental bleaching on pulpal tissue response in a diabetic animal model.

AIM: To evaluate pulpal tissue response after dental bleaching in normal and alloxan-induced diabetic rats. METHODOLOGY: Twenty-eight rats were divided into two groups of normoglycaemic and diabetic rats (n = 14). Diabetes mellitus (DM) was induced with alloxan. After DM confirmation, all rats were anaesthetized and dental bleaching was performed with 35% hydrogen peroxide (H2 O2 ) on the right maxillary molars for 30 min. Left molars were used as controls. Bleaching resulted in four hemimaxillae groups: normoglycaemic (N), N-bleached (NBle), diabetic (D) and D-bleached (DBle). After 2 or 30 days, the animals were euthanized and the hemimaxillae were removed, processed for histopathological analysis and stained with haematoxylin-eosin (HE), Masson's trichrome (MT) and picrosirius red (PSR). Results obtained within animals (normoglycaemic or diabetic rats) were submitted to Wilcoxon or paired t-tests, and between animal (normoglycaemic and diabetic rats), to Mann-Whitney test or t-tests. RESULTS: At 2 days, the NBle group had a mild inflammatory infiltration in the pulpal tissue, whilst the DBle had severe inflammation or necrosis (P < 0.05). At 30 days, no inflammation was present. However, a significant difference in pulp chamber area reduction by reactionary dentine deposition was found between the NBle and DBle groups (P < 0.05). At 2 days, fewer immature collagen fibres and more mature collagen fibres were noted in the NBle, D and DBle groups; this was significantly different when compared to the N group (P < 0.05). At 30 days, significantly fewer immature collagen fibres and more mature collagen fibres were noted in NBle compared with DBle group (P < 0.05). CONCLUSIONS: The inflammatory tissue response in rats' teeth after dental bleaching was greater in diabetic rats. Additionally, the increase in reactionary dentine deposition and mature collagen fibres observed in diabetic rats needs further evaluation to confirm the present results.

Expression of an active recombinant lysine 49 phospholipase A(2) myotoxin as a fusion protein in bacteria.

ACL myotoxin (ACLMT) is a K49 phospholipase A(2)-like protein isolated from the venom of the snake Agkistrodon contortrix laticinctus (broad-banded copperhead) that induces necrosis of skeletal muscle. We have previously cloned and sequenced the cDNA coding for ACLMT from a venom gland cDNA library. In order to perform structure and function studies, we have developed an expression system for production of ACLMT as a fusion protein with maltose binding protein (MBP) from the periplasm of bacteria, using the pMAL-p2 expression vector. The cDNA coding for the mature toxin without the signal peptide was amplified by PCR and subcloned into the pMAL-p2 vector. The new plasmid (pMAL-MT) was used to transform BL21(DE3) E. coli cells. Culture of transformed cells induced with IPTG led to the expression of a 60 kDa fusion protein which strongly reacts with anti-native ACLMT antibodies. The fusion protein was purified from the bacterial periplasm by affinity chromatography in an amylose column and by gel filtration. The purified fusion protein (MBP-rACLMT) was able to induce necrosis of skeletal muscle of mice very similar to that caused by the native myotoxin.

[Prognostic value of pre- and postoperative alpha-fetoprotein in the follow-up of patients with surgically-treated hepatocellular carcinoma]. / Valore prognostico dell'alfa-fetoproteina pre- e postoperatoria nel follow-up dei pazienti operati per epatocarcinoma.

A percentage ranging from 60 to 80% of hepatocarcinomas are associated with increased levels of alphafetoprotein (AFP). In the three years following surgical resection there was a 80% possibility of recidivation. The aims of the present study were: a) to evaluate the significance of preoperative AFP assay as a prognostic index of recidivation; b) to evaluate the importance of repeated assays during the postoperative period in order to ensure an early diagnosis of recidivation. Between 1982 and 1989, 62 patients underwent surgery for hepatocarcinoma. Thirty-one patients who had undergone so-called curative surgery were periodically controlled for a period varying between 6 and 55 months, and were included in the present study. The remaining 32 patients were excluded for the following reasons: palliative surgery, postoperative death, postoperative complications unrelated to tumoral recidivation. In all cases AFP assay was carried out preoperatively, one month after surgery, and then every six months. Recidivation was always confirmed on the basis of tomodensitometric and arteriographic data. Before surgery out of a group of 30 patients, 11 showed normal AFP levels (below 20 mg/ml), while 19 had levels between 49 and 7350 mg/ml. Twenty-three patients (74%) reported one case of recidivation during the period between 6 and 40 months. Among the 11 patients who had showed normal preoperative AFP levels, 5 had a recidivation between 12 and 36 months, and 3 of these showed high AFP levels. In 18 out of the 19 patients (90%) with high preoperative AFP levels recidivation was diagnosed between 4 and 40 months following surgery; 4 of these were not associated with a rise in AFP.(ABSTRACT TRUNCATED AT 250 WORDS)

Cyrtoneurina pararescita (Diptera: Muscidae) (Couri, 1995) como novo hospedeiro de Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae) / Cyrtoneurina pararescita (Diptera: Muscidae) (Couri, 1995) as new host of Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae)

This study reports the occurrence of Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae) as parasitoid of Cyrtoneurina pararescita (Diptera: Muscidae) (Couri, 1995) in bovine feces obtained in Goiânia, Goiás state, Brazil. Bovine manure samples, collected at two weeks intervals, were taken to the laboratory and the pupae were extracted by water flotation. Each pupa was placed in capsules of gelatin until the emergence of dipterous or their parasitoids. The parasitism percentage was of 2.4%. This paper reports the first occurrence of N. vitripennis as parasitoid of C. pararescita worldwide.

[Nasopharyngeal colonization and antimicrobial resistance of Streptococcus pneumoniae isolated from children with acute rhinopharyngitis] / Colonização e resistência antimicrobiana de Streptococcus pneumoniae isolado em nasofaringe de crianças com rinofaringite aguda.

OBJECTIVE: To determine the prevalence and risk factors for nasopharyngeal colonization by, and to evaluate antimicrobial susceptibility of Streptococcus pneumoniae strains in children with acute rhinopharyngitis. METHODS: We collected nasopharyngeal swab specimens from 400 children aged 3 months to 5 years and with clinical status of acute rhinopharyngitis from June 16, 1997 to May 20, 1998 at the outpatient clinics of two hospitals in the city of São Paulo. Nasopharyngeal specimens were collected pernasally using a calcium alginate swab and plated immediately after collection onto trypticose soy agar with 5% sheep blood and garamicin 5 mcg/ml. Penicillin susceptibility was determined by oxacillin 1 mcg disk screening test and the minimal inhibitory concentration by the E-test. RESULTS: Pneumococci were recovered from 139 children, indicating a colonization prevalence of 35%. The risk factors analyzed indicated that the colonization was more prevalent in children attending day-care centers, children with siblings younger than 5 years, and children with recent use of antimicrobial agents. The prevalence of penicillin non-susceptible strains was of 16 % (20 strains). All strains were intermediately resistant (0.1mcg/ ml

The disintegrin-like domain of the snake venom metalloprotease alternagin inhibits alpha2beta1 integrin-mediated cell adhesion.

The alpha2beta1 integrin is a major collagen receptor that plays an essential role in the adhesion of normal and tumor cells to the extracellular matrix. Here we describe the isolation of a novel metalloproteinase/disintegrin, which is a potent inhibitor of the collagen binding to alpha2beta1 integrin. This 55-kDa protein (alternagin) and its disintegrin domain (alternagin-C) were isolated from Bothrops alternatus snake venom. Amino acid sequencing of alternagin-C revealed the disintegrin structure. Alternagin and alternagin-C inhibit collagen I-mediated adhesion of K562-alpha2beta1-transfected cells. The IC50 was 134 and 100 nM for alternagin and alternagin-C, respectively. Neither protein interfered with the adhesion of cells expressing alphaIIbeta3, alpha1beta1, alpha5beta1, alpha4beta1 alphavbeta3, and alpha9beta1 integrins to other ligands such as fibrinogen, fibronectin, and collagen IV. Alternagin and alternagin-C also mediated the adhesion of the K562-alpha2beta1-transfected cells. Our results show that the disintegrin-like domain of alternagin is responsible for its ability to inhibit collagen binding to alpha2beta1 integrin.

Orientacoes atuais da metodologia ergonomica na Franca. / Present orientations of ergonomic methodology in France

Os autores apresentam as orientacoes atuais da metodologia ergonomica na Franca, em particular o metodo de analise orientada do trabalho, desenvolvido por Ombredane, Faverge, Montmollin, Leplat e Wisner e enriquecido pelos pesquisadores do Laboratorio e Fisiologia do Trabalho e Ergonomia do Conservatorio Nacional de Artes e Oficios (CNAM), Paris Franca. Trata-se de um metodo de analise particular a cada tipo de situacao de trabalho. O exemplo que ilustra este artigo e sobre uma intervencao em uma industria automobilistica.onde a analise ergonomica identificou, entre outras, as dificuldades posturais sentidas pelos operarios de idade superior a 40 anos, trabalhando em linha de montagem. As recomendacoes ergonomicas feitas permitiram modificacoes que diminuiram tais problemas.E tambem ressaltada a importancia do conhecimento de uma metodologia ergonomica pelos medicos do trabalho e engenheiros de seguranca que, por sua posicao dentro da empresa, podem ter um importante papel na melhoria das condicoes de trabalho

Comunicacao sobre fluorose. / Fluorinese

Esta comunicacao refere-se a identificacao do risco da fluorose, tendo-se verificado a aquisicao desta por trabalhadores de industrias de fertilizantes em Cubatao. O risco foi constatado com base na analise de fluoretos urinarios. Recomenda-se que os colegas se mantenham atentos ao problema em outras regioes, especialmente em fabricas ou mineracoes, onde possa haver o risco