Search for cytotoxic agents in multiple Laurencia complex seaweed species (Ceramiales, Rhodophyta) harvested from the Atlantic Ocean with emphasis on the Brazilian State of Espírito Santo

The development of new anti-cancer drugs of algal origin represents one of the least explored frontiers in medicinal chemistry. In this regard, the diversity of micro- and macroalgae found in Brazilian coastal waters can be viewed as a largely untapped natural resource. In this report, we describe a comparative study on the cytotoxic properties of extracts obtained from the Laurencia complex: Laurencia aldingensis, L. catarinensis, L. dendroidea, L. intricata, L. translucida, L. sp, and Palisada flagellifera. All of these species were collected in the coastal waters of the State of Espírito Santo, Brazil. Four out of the twelve samples initially investigated were found to show significant levels of toxicity towards a model tumor cell line (human uterine sarcoma, MES-SA). The highest levels of cytotoxicity were typically associated with non-polar (hexane) algal extracts, while the lowest levels of cytotoxicity were found with the corresponding polar (methanol) extracts. In this report, we also describe a biological model currently in development that will not only facilitate the search for new anti-cancer drug candidates of algal origin, but also permit the identification of compounds capable of inducing the destruction of multi-drug resistant tumors with greater efficiency than the pharmaceuticals currently in clinical use.

Dose-response effects of systemic anandamide administration in mice sequentially submitted to the open field and elevated plus-maze tests

The endocannabinoid system is involved in the control of many physiological functions, including the control of emotional states. In rodents, previous exposure to an open field increases the anxiety-like behavior in the elevated plus-maze. Anxiolytic-like effects of pharmacological compounds that increase endocannabinoid levels have been well documented. However, these effects are more evident in animals with high anxiety levels. Several studies have described characteristic inverted U-shaped dose-response effects of drugs that modulate the endocannabinoid levels. However, there are no studies showing the effects of different doses of exogenous anandamide, an endocannabinoid, in animal models of anxiety. Thus, in the present study, we determined the dose-response effects of exogenous anandamide at doses of 0.01, 0.1, and 1.0 mg/kg in C57BL/6 mice (N = 10/group) sequentially submitted to the open field and elevated plus-maze. Anandamide was diluted in 0.9% saline, ethyl alcohol, Emulphor® (18:1:1) and administered ip (0.1 mL/10 g body weight); control animals received the same volume of anandamide vehicle. Anandamide at the dose of 0.1 mg/kg (but not of 0.01 or 1 mg/kg) increased (P < 0.05) the time spent and the distance covered in the central zone of the open field, as well as the exploration of the open arms of the elevated plus-maze. Thus, exogenous anandamide, like pharmacological compounds that increase endocannabinoid levels, promoted a characteristic inverted U-shaped dose-response effect in animal models of anxiety. Furthermore, anandamide (0.1 mg/kg) induced an anxiolytic-like effect in the elevated plus-maze (P < 0.05) after exposing the animals to the open field test.

Efetividade da suplementação diária ou semanal com ferro na prevenção da anemia em lactentes

OBJETIVO: Avaliar a efetividade da suplementação universal profilática com sulfato ferroso, em administração diária ou semanal, na prevenção da anemia em lactentes. MÉTODOS: Ensaio de campo randomizado com crianças de seis a 12 meses de idade, atendidas em unidades básicas de saúde do município do Rio de Janeiro, em 2004-2005. Foram formadas três coortes concorrentes com suplementação universal com sulfato ferroso com grupos: diário (n=150; 12,5mgFe/dia), semanal (n=147; 25mgFe/semana) e controle (n=94). A intervenção durou 24 semanas e foi acompanhada por ações educativas promotoras de adesão. A concentração de hemoglobina sérica foi analisada segundo sua distribuição, média e prevalência de anemia (Hb<110,0g/L) aos 12 meses de idade. A avaliação da efetividade foi realizada segundo intenção de tratar e adesão ao protocolo, utilizando-se análises de regressão múltipla (linear e de Poisson). RESULTADOS: Os grupos mostraram-se homogêneos quanto às variáveis de caracterização. A intervenção foi operacionalizada com sucesso, com elevada adesão ao protocolo em ambos os grupos expostos a ela, sem diferença estatística entre eles. Após ajuste, somente o esquema diário apresentou efeito protetor. Na análise por adesão, o esquema diário apresentou evidente efeito dose-resposta para média de hemoglobina sérica e prevalência de anemia, não sendo observado nenhum efeito protetor do esquema semanal. CONCLUSÕES: Apenas o esquema diário de suplementação universal com sulfato ferroso dos seis aos 12 meses de idade foi efetivo em aumentar a concentração de hemoglobina sérica e em reduzir o risco de anemia

Atividade física durante a gestação e desfechos fetais: um estudo de casos e controles

OBJETIVO: Analisar a relação entre atividade física durante o segundo trimestre de gestação e baixo peso ao nascer, prematuridade e restrição de crescimento intra-uterino. MÉTODOS: Estudo de caso-controle realizado no município de São Paulo, em 2005. Foram estudados 273 recém-nascidos de baixo peso e 546 controles. Dentre os casos foram selecionadas duas sub-amostras: 117 nascimentos pré-termo e 132 com restrição de crescimento intra-uterino (n=132) e seus respectivos controles. As informações foram obtidas mediante entrevistas com as puérperas e transcrição de dados dos prontuários. Foram realizadas análises de regressão logística múltipla condicional e hierarquizada. RESULTADOS: Foi identificado como fator de proteção para baixo peso ao nascer a realização de atividades leves por mais de sete horas diárias (ORaj:0,61; IC 95 por cento :0,39;0,94), para a qual identificou-se relação do tipo dose-resposta (p de tendência=0,026), e tendência similar na análise da restrição de crescimento intra-uterino (ORaj:0,51; IC 95 por cento :0,26;0,97). A realização de atividades domésticas associou-se como fator protetor tanto contra o baixo peso ao nascer quanto à prematuridade (p de tendência=0,013 e 0,035, respectivamente). Foi detectado efeito de proteção contra prematuridade para a caminhada no lazer. CONCLUSÕES: Atividades físicas leves, como caminhadas, durante o segundo trimestre de gestação exercem efeito protetor independente sobre o baixo peso ao nascer, a prematuridade e a restrição de crescimento intrauterino

From Offshore to Onshore: Multiple Origins of Shallow-Water Corals from Deep-Sea Ancestors

Shallow-water tropical reefs and the deep sea represent the two most diverse marine environments. Understanding the origin and diversification of this biodiversity is a major quest in ecology and evolution. The most prominent and well-supported explanation, articulated since the first explorations of the deep sea, holds that benthic marine fauna originated in shallow, onshore environments, and diversified into deeper waters. In contrast, evidence that groups of marine organisms originated in the deep sea is limited, and the possibility that deep-water taxa have contributed to the formation of shallow-water communities remains untested with phylogenetic methods. Here we show that stylasterid corals (Cnidaria: Hydrozoa: Stylasteridae)-the second most diverse group of hard corals-originated and diversified extensively in the deep sea, and subsequently invaded shallow waters. Our phylogenetic results show that deep-water stylasterid corals have invaded the shallow-water tropics three times, with one additional invasion of the shallow-water temperate zone. Our results also show that anti-predatory innovations arose in the deep sea, but were not involved in the shallow-water invasions. These findings are the first robust evidence that an important group of tropical shallow-water marine animals evolved from deep-water ancestors.

A Vaccine Encoding Conserved Promiscuous HIV CD4 Epitopes Induces Broad T Cell Responses in Mice Transgenic to Multiple Common HLA Class II Molecules

Current HIV vaccine approaches are focused on immunogens encoding whole HIV antigenic proteins that mainly elicit cytotoxic CD8+ responses. Mounting evidence points toward a critical role for CD4+ T cells in the control of immunodeficiency virus replication, probably due to cognate help. Vaccine-induced CD4+ T cell responses might, therefore, have a protective effect in HIV replication. In addition, successful vaccines may have to elicit responses to multiple epitopes in a high proportion of vaccinees, to match the highly variable circulating strains of HIV. Using rational vaccine design, we developed a DNA vaccine encoding 18 algorithm-selected conserved, ""promiscuous"" ( multiple HLA-DR-binding) B-subtype HIV CD4 epitopes - previously found to be frequently recognized by HIV-infected patients. We assessed the ability of the vaccine to induce broad T cell responses in the context of multiple HLA class II molecules using different strains of HLA class II-transgenic mice (-DR2, -DR4, -DQ6 and -DQ8). Mice displayed CD4+ and CD8+ T cell responses of significant breadth and magnitude, and 16 out of the 18 encoded epitopes were recognized. By virtue of inducing broad responses against conserved CD4+ T cell epitopes that can be recognized in the context of widely diverse, common HLA class II alleles, this vaccine concept may cope both with HIV genetic variability and increased population coverage. The vaccine may thus be a source of cognate help for HIV-specific CD8+ T cells elicited by conventional immunogens, in a wide proportion of vaccinees.

Effects of high-dose creatine supplementation on kidney and liver responses in sedentary and exercised rats

This study evaluated the effects of high-dose of short-term creatine supplementation (5g.kg(-1). day(-1) to 1 week) and long-term creatine supplementation (1g.kg(-1). day(-1) to 4-8 weeks) on kidney and liver structure and function of sedentary and exercised Wistar rats ( Exercise sessions consisted of swimming at 80% of maximal work load supported during 5 days per week with daily sessions of 60 minutes throughout the duration of the supplementation). Seventy-two animals ( 245 +/- 5g) were divided into four groups (n = 18): control diet Sedentary ( SED), Creatine diet Sedentary (CRE), control diet Exercised (EXE), and Creatine diet Exercised (EXECRE). Histological and blood biochemical studies were performed after one, four, and eight weeks of creatine supplementation and exercise ( n = 6). No differences were found when comparing SED, EXE and EXECRE groups for kidney and liver structure and function at one, four and eight weeks. However, the CRE group showed higher levels of creatinine (1.1 +/- 0.2 vs. 0.4 +/- 0.1 mg.dl(-1); p < 0.05), and urea ( 37 +/- 3 vs. 19 +/- 1 mg. dl(-1); p < 0.05) when compared with all others groups at four and eight weeks. At eight weeks, the CRE group presented increased levels of ALT (41 +/- 7 vs. 23 +/- 7 U.L(-1); p < 0.05), AST (89 +/- 6 vs. 62 +/- 5 U. L(-1); p < 0.05), GGT (8.0 +/- 0.9 vs. 3.9 +/- 1.0 U. L(-1); p < 0.05), and AP (125 +/- 10 vs. 69 +/- 9 U. L(-1); p < 0.05) also when compared with all others groups. Moreover, the CRE group demonstrated some structural alterations indicating renal and hepatic damage at four and eight weeks, respectively. These results suggest that long-term creatine supplementation (up to 4-8 weeks) may adversely affect kidney and liver structure and function of sedentary but not of exercised rats.

A DNA Vaccine Encoding Multiple HIV CD4 Epitopes Elicits Vigorous Polyfunctional, Long-Lived CD4(+) and CD8(+) T Cell Responses

T-cell based vaccines against HIV have the goal of limiting both transmission and disease progression by inducing broad and functionally relevant T cell responses. Moreover, polyfunctional and long-lived specific memory T cells have been associated to vaccine-induced protection. CD4(+) T cells are important for the generation and maintenance of functional CD8(+) cytotoxic T cells. We have recently developed a DNA vaccine encoding 18 conserved multiple HLA-DR-binding HIV-1 CD4 epitopes (HIVBr18), capable of eliciting broad CD4(+) T cell responses in multiple HLA class II transgenic mice. Here, we evaluated the breadth and functional profile of HIVBr18-induced immune responses in BALB/c mice. Immunized mice displayed high-magnitude, broad CD4(+)/CD8(+) T cell responses, and 8/18 vaccine-encoded peptides were recognized. In addition, HIVBr18 immunization was able to induce polyfunctional CD4(+) and CD8(+) T cells that proliferate and produce any two cytokines (IFN gamma/TNF alpha, IFN gamma/IL-2 or TNF alpha/IL-2) simultaneously in response to HIV-1 peptides. For CD4(+) T cells exclusively, we also detected cells that proliferate and produce all three tested cytokines simultaneously (IFN gamma/TNF alpha/IL-2). The vaccine also generated long-lived central and effector memory CD4(+) T cells, a desirable feature for T-cell based vaccines. By virtue of inducing broad, polyfunctional and long-lived T cell responses against conserved CD4(+) T cell epitopes, combined administration of this vaccine concept may provide sustained help for CD8(+) T cells and antibody responses-elicited by other HIV immunogens.

Determination of Threshold Dose of Photodynamic Therapy to Measure Superficial Necrosis

Background Data: Photodynamic therapy (PDT) involves the photoinduction of cytotoxicity using a photosensitizer agent, a light source of the proper wavelength, and the presence of molecular oxygen. A model for tissue response to PDT based on the photodynamic threshold dose (Dth) has been widely used. In this model cells exposed to doses below Dth survive while at doses above the Dth necrosis takes place. Objective: This study evaluated the light Dth values by using two different methods of determination. One model concerns the depth of necrosis and the other the width of superficial necrosis. Materials and Methods: Using normal rat liver we investigated the depth and width of necrosis induced by PDT when a laser with a gaussian intensity profile is used. Different light doses, photosensitizers (Photogem, Photofrin, Photosan, Foscan, Photodithazine, and Radachlorin), and concentrations were employed. Each experiment was performed on five animals and the average and standard deviations were calculated. Results: A simple depth and width of necrosis model analysis allows us to determine the threshold dose by measuring both depth and surface data. Comparison shows that both measurements provide the same value within the degree of experimental error. Conclusion: This work demonstrates that by knowing the extent of the superficial necrotic area of a target tissue irradiated by a gaussian light beam, it is possible to estimate the threshold dose. This technique may find application where the determination of Dth must be done without cutting the tissue.

Expression of eight genes of nuclear factor-kappa B pathway in multiple myeloma using bone marrow aspirates obtained at diagnosis

Purpose: To evaluate the expression of NF-kappa B pathway genes in total bone marrow samples obtained from MM at diagnosis using real-time quantitative PCR and to evaluate its possible correlation with disease clinical features and survival. Material and methods: Expression of eight genes related to NF-kappa B pathway (NFKB1, IKB, RANK, RANKL, OPG, IL6, VCAM1 and ICAM1) were studied in 53 bone marrow samples from newly diagnosed MM patients and in seven normal controls, using the Taqman system. Genes were considered overexpressed when tumor expression level was at least four times higher than that observed in normal samples. Results: The percentages of overexpression of the eight genes were: NFKB1 0%, IKB 22.6%, RANK 15.1%, RANKL 31.3%, OPG 7.5%, IL6 39.6%, VCAM1 10% and ICAM1 26%. We found association between IL6 expression level and International Staging System (ISS) (p = 0.01), meaning that MM patients with high ISS scores have more chance of overexpression of IL6. The mean value of ICAM1 relative expression was also associated with the ISS score (p = 0.02). Regarding OS, cases with IL6 overexpression present worse evolution than cases with IL6 normal expression (p = 0.04). Conclusion: We demonstrated that total bone marrow aspirates can be used as a source of material for gene expression studies in MM. In this context, we confirmed that IL6 overexpression was significantly associated with worse survival and we described that it is associated with high ISS scores. Also, ICAM1 was overexpressed in 26% of cases and its level was associated with ISS scores.

High-Dose Antipsychotic Medication; A Practical Pocket Checklist

There is evidence that a significant number of patients with schizophrenia and other chronic psychotic psychosis are prescribed high-dose antipsychotic drugs despite the fact that clinical guidelines recommend the routine use of a single antipsychotic drug in a standard dose. The prescriptions for high-dose and combined antipsychotic drugs are relatively common in clinical practice. This occurs despite the fact that results of published trials of high-dose antipsychotic drug treatment for schizophrenia provide little evidence to support effectiveness of using high-dose antipsychotic treatment and most importantly such strategy is not recommended. Moreover, there is mounting evidence of higher incidence of side effects and mortality associated with high dose antipsychotic treatment. Therefore we are presenting a practical pocket checklist which is aimed at minimizing predicted and unpredicted side effects during such treatments.

Primary Hyperparathyroidism as the First Clinical Manifestation of Multiple Endocrine Neoplasia Type 2A in a 5-Year-Old Child

Background: Primary hyperparathyroidism occurs in only 10%-30% of patients with multiple endocrine neoplasia type 2A (MEN2A), rarely as the sole clinical manifestation, and is usually diagnosed after the third decade of life. Summary: A5-year-old girl was referred for prophylactic thyroidectomy as she carried the p.C634R RET mutation. She was clinically asymptomatic, with a normally palpable thyroid and with the cervical region free of lymphadenopathy or other nodules. Preoperative tests revealed hypercalcemia associated with elevation of parathyroid hormone (PTH) (calcium = 11.2mg/dL, calcium ion = 1.48mmol/L, phosphorus = 4.0 mg/dL, alkaline phosphatase = 625U/L, parathyroid hormone (PTH) PTH = 998 pg/mL). A thyroid ultrasound was normal and parathyroid scintigraphy with (99m)Tc-Sestamibi revealed an area of radioconcentration in the upper half of the left thyroid lobe suggesting hyperfunctioning parathyroid tissue. She underwent total thyroidectomy and parathyroidectomy and developed hypocalcemia. The anatomopathological examination showed no histopathological changes in the thyroid tissue and an adenoma of the parathyroid gland, confirming the diagnosis of hyperparathyroidism. Conclusions: Primary hyperparathyroidism can be a precocious manifestation of MEN2A. This case report highlights that asymptomatic hypercalcemia should be scrutinized in children related to patients with MEN2A who carry a mutation in the RET proto-oncogene, especially mutations in the codon 634, before the currently recommended age of 8 years.

RNA interference-mediated knockdown of CD49e (alpha 5 integrin chain) in human thymic epithelial cells modulates the expression of multiple genes and decreases thymocyte adhesion

Background: The thymus is a central lymphoid organ, in which bone marrow-derived T cell precursors undergo a complex process of maturation. Developing thymocytes interact with thymic microenvironment in a defined spatial order. A component of thymic microenvironment, the thymic epithelial cells, is crucial for the maturation of T-lymphocytes through cell-cell contact, cell matrix interactions and secretory of cytokines/chemokines. There is evidence that extracellular matrix molecules play a fundamental role in guiding differentiating thymocytes in both cortical and medullary regions of the thymic lobules. The interaction between the integrin alpha 5 beta 1 (CD49e/CD29; VLA-5) and fibronectin is relevant for thymocyte adhesion and migration within the thymic tissue. Our previous results have shown that adhesion of thymocytes to cultured TEC line is enhanced in the presence of fibronectin, and can be blocked with anti-VLA-5 antibody. Results: Herein, we studied the role of CD49e expressed by the human thymic epithelium. For this purpose we knocked down the CD49e by means of RNA interference. This procedure resulted in the modulation of more than 100 genes, some of them coding for other proteins also involved in adhesion of thymocytes; others related to signaling pathways triggered after integrin activation, or even involved in the control of F-actin stress fiber formation. Functionally, we demonstrated that disruption of VLA-5 in human TEC by CD49e-siRNA-induced gene knockdown decreased the ability of TEC to promote thymocyte adhesion. Such a decrease comprised all CD4/CD8-defined thymocyte subsets. Conclusion: Conceptually, our findings unravel the complexity of gene regulation, as regards key genes involved in the heterocellular cell adhesion between developing thymocytes and the major component of the thymic microenvironment, an interaction that is a mandatory event for proper intrathymic T cell differentiation.

Trends in dermatomyositis- and polymyositis-related mortality in the state of Sao Paulo, Brazil, 1985-2007: multiple cause-of-death analysis

Background: Dermatomyositis (DM) and polymyositis (PM) are rare systemic autoimmune rheumatic diseases with high fatality rates. There have been few population-based mortality studies of dermatomyositis and polymyositis in the world, and none have been conducted in Brazil. The objective of the present study was to employ multiple-cause of-death methodology in the analysis of trends in mortality related to dermatomyositis and polymyositis in the state of Sao Paulo, Brazil, between 1985 and 2007. Methods: We analyzed mortality data from the Sao Paulo State Data Analysis System, selecting all death certificates on which DM or PM was listed as a cause of death. The variables sex, age and underlying, associated or total mentions of causes of death were studied using mortality rates, proportions and historical trends. Statistical analysis were performed by chi-square and H Kruskal-Wallis tests, variance analysis and linear regression. A p value less than 0.05 was regarded as significant. Results: Over a 23-year period, there were 318 DM-related deaths and 316 PM-related deaths. Overall, DM/PM was designated as an underlying cause in 55.2% and as an associated cause in 44.8%; among 634 total deaths females accounted for 71.5%. During the study period, age-and gender-adjusted DM mortality rates did not change significantly, although PM as an underlying cause and total mentions of PM trended lower (p < 0.05). The mean ages at death were 47.76 +/- 20.81 years for DM and 54.24 +/- 17.94 years for PM (p = 0.0003). For DM/PM, respectively, as underlying causes, the principal associated causes of death were as follows: pneumonia (in 43.8%/33.5%); respiratory failure (in 34.4%/32.3%); interstitial pulmonary diseases and other pulmonary conditions (in 28.9%/17.6%); and septicemia (in 22.8%/15.9%). For DM/PM, respectively, as associated causes, the following were the principal underlying causes of death: respiratory disorders (in 28.3%/26.0%); circulatory disorders (in 17.4%/20.5%); neoplasms (in 16.7%/13.7%); infectious and parasitic diseases (in 11.6%/9.6%); and gastrointestinal disorders (in 8.0%/4.8%). Of the 318 DM-related deaths, 36 involved neoplasms, compared with 20 of the 316 PM-related deaths (p = 0.03). Conclusions: Our study using multiple cause of deaths found that DM/PM were identified as the underlying cause of death in only 55.2% of the deaths, indicating that both diseases were underestimated in the primary mortality statistics. We observed a predominance of deaths in women and in older individuals, as well as a trend toward stability in the mortality rates. We have confirmed that the risk of death is greater when either disease is accompanied by neoplasm, albeit to lesser degree in individuals with PM. The investigation of the underlying and associated causes of death related to DM/PM broaden the knowledge of the natural history of both diseases and could help integrate mortality data for use in the evaluation of control measures for DM/PM.

Multiple classical limits in relativistic and nonrelativistic quantum mechanics

The existence of a classical limit describing the interacting particles in a second-quantized theory of identical particles with bosonic symmetry is proved. This limit exists in addition to the previously established classical limit with a classical field behavior, showing that the limit h -> 0 of the theory is not unique. An analogous result is valid for a free massive scalar field: two distinct classical limits are proved to exist, describing a system of particles or a classical field. The introduction of local operators in order to represent kinematical properties of interest is shown to break the permutation symmetry under some localizability conditions, allowing the study of individual particle properties.