Effects of 17ß-estradiol on early gonadal development and expression of genes implicated in sexual differentiation of a South American teleost, Astyanax altiparanae.

Gonadal sex differentiation in teleost fish shows greater plasticity as compared to other vertebrates, as it can be influenced by a variety of factors such as exogenous sex steroids. Exogenous estrogens, such as 17ß-estradiol (E2), can induce feminization when administered during early embryonic development. However, the mechanisms underlying the E2-induced feminization are not fully understood, especially in Neotropical species. Therefore, the aim of this study was to evaluate the effects of E2 administration on the phenotypic sex characteristics, histological assessment of the gonads, and the expression of selected genes in Astyanax altiparanae exposed to dietary E2 prior to gonadal differentiation. At 4 days post-hatch (dph), groups of 30-40 undifferentiated larvae were fed with a diet containing varying amounts of E2 for 28 days, and fish were sampled at 90 dph. Previous studies revealed that ovary formation in A. altiparanae occurred at 58 dph, whereas the first sign of testis formation was found at 73 dph. In relation to the control, E2 exposure increased the proportion of phenotypic females in 120% and 148.4% for 4 and 6 mg E2/Kg, respectively. However, histological analysis revealed that treatments did not affect gonadal sex ratio between males and females, but induced intersex (testis-ova) in the group treated with 6 mg E2/Kg food. Treatment with E2 also altered gonadal transcript levels of a selected number of genes implicated in sexual differentiation. Males overexpressed dmrt1, sox9 and amh following E2 treatment as compared to control. Females showed increased mRNA levels of dmrt1 and sox9, which might be related to the down-regulation of cyp19a1a after E2 exposure. In summary, E2 exposure during early gonadal development affected male secondary characteristics without changing the gonadal sex ratio, and altered expression of genes implicated in sexual differentiation.

Vitamin D receptor (VDR) polymorphisms are associated to spontaneous preterm birth and maternal aspects.

Preterm birth (PTB) is featured by less than 37weeks of gestational age or fewer than 259days since the first day from the last menstrual period. Complications of PTB are the major cause of neonatal deaths, several factors are linked to PTB increased risk including immunological and genetics. Vitamin D plays an important role in immune response modulation and its action occurs through the vitamin D receptor (VDR), which recently has been described as overexpressed in human placenta during the pregnancy. Herein we assessed two single nucleotide polymorphisms (SNPs) FokI (rs2228570 A>G) and Cdx-2 (rs11568820 T>C), within VDR, using TaqMan fluorogenic probes, and differential susceptibility to SPTB. We assessed 104 pregnant women with SPTB and 85 women with normal birth in a Northeastern Brazilian population. Statistically significant differences for both SNPs where found when comparing allele and genotype frequencies in both groups: the T allele for rs2228570 and A allele for rs11568820 were significantly more frequent in SPTB group than in normal birth group (p=0.000013 and p=0.00466, respectively). The rs11568820 A/A genotype was associated to clinical/demographic variables such as: premature birth (p=0.007), neonate weight (p=0.039), presence of infection during pregnancy (p=0.011) and premature birth among multiparous (p=0.015). The rs2228570 T/T genotype associated with gestational diabetes mellitus (p=0.044) and chorioamnionitis during pregnancy (p=0.043). In conclusion our findings indicate an association between polymorphisms FokI and Cdx-2 within VDR gene and SPTB, suggesting their involvement in the triggering of these syndromes.

Utilização de plasma rico em plaquetas de coelhos com poucos leucócitos e hemácias para a consolidação de ossos / Use of plasma rich in platelets of rabbits with few leukocytes and erythrocytes to consolidate bones

Objetivou-se utilizar o plasma rico em plaquetas, obtido por técnica capaz de produzir um produto autólogo, com reduzido número de leucócitos e hemácias, e avaliar, por meio de radiografias, a capacidade de cicatrização de uma falha óssea induzida no rádio de coelhos. Foram coletados 10,5mL de sangue por via intracardíaca, o qual foi distribuído em três tubos de hemossedimentação contendo citrato de sódio. Os tubos contendo as amostras foram submetidos a uma centrifugação a 2.000rpm (670,8G) por 20 minutos, e da coluna de sedimentação foram aspirados de cada tubo 1.000µL de plasma para a redução do volume sobrenadante. Aspirou-se o plasma acima do anel de leucócitos e transferiu-se para outro tubo para centrifugação a 2.000rpm por 10 minutos. O conteúdo plaquetário no fundo do tubo foi ressuspendido e homogeneizado a 1.000µL do plasma sobrenadante para formar o PRP líquido. Uma falha de 1,0cm foi realizada no rádio esquerdo de cada coelho. O tempo de consolidação foi observado por meio de exame radiográfico aos 45 e 90 dias de pós-operatório (PO). As imagens foram comparadas entre o grupo controle (G1) e o grupo que recebeu o implante de PRP por via transdérmica (G2). A evolução da cicatrização foi verificada com auxílio do programa Adobe Photoshop CC e em uma escala de cinza que representa o contraste. No G2 foi verificada média de 63% de contraste aos 45 dias de PO e de 65% aos 90 dias de PO. No G2, aos 45 dias de PO, a média encontrada foi de 42,7%, e aos 90 dias de PO, 31,25%, indicando que houve evolução do processo de reparação óssea em comparação ao grupo que não recebeu o implante de PRP. O método de obtenção de plasma rico em plaqueta autólogo de coelhos reduziu o número de leucócitos e hemácias e recuperou o número de plaquetas, sendo este maior ou igual aos valores fisiológicos para a espécie. O PRP obtido foi capaz de acelerar o processo de consolidação óssea em coelhos.

The aim of this study was to use the platelet-rich plasma obtained by a technique capable of producing an autologous product, with a reduced number of white blood cells and red blood cells and assessed by radiographs, the healing ability of a bone defect induced in the radio rabbits. 10.5mL of blood was collected via intracardiac blood sedimentation and distributed into three tubes containing sodium citrate. The tubes containing the samples were subjected to centrifugation at 2,000 rpm (670.8G) for 20 minutes and the sediment column were aspirated from each plasma tube1,000μL to reduce the supernatant volume. The plasma was aspirated from above the leukocytes ring and transferred to another tube for centrifugation at 2,000 rpm for 10 minutes. The platelet content in the tube bottom was resuspended and homogenized to 1,000μL plasma supernatant PRP to form the liquid. A1.0 cm failure was performed on the left radio of each rabbit. The healing time was observed by means of radiographic examination at 45 and 90 days after surgery. The images were compared between the control group (G1) and the group receiving PRP implant transdermally (G2). The healing progress was assessed with the help of Adobe Photoshop program and a gray scale that represents the contrast. G2 had an average of 63% contrast at 45 days PO and 65% at 90 days PO. In G2, at 45 days PO the average was 42.7% and at 90 days PO 31.25% indicating that there was an increase in bone repair process compared to the group that did not receive the PRP implant. The method of obtaining an autologous platelet rich plasma of rabbits reduced the number of leukocytes and erythrocytes and platelets recovered which is greater than or equal to the physiological range for the species. The obtained PRP was able to accelerate the process of bone healing in rabbits.

Evaluation of vascular effect of Photodynamic Therapy in chorioallantoic membrane using different photosensitizers.

Photodynamic Therapy (PDT) is a local treatment that requires a photosensitizing agent, light and molecular oxygen. With appropriate illumination, the photosensitizer is excited and produces singlet oxygen that is highly reactive and cytotoxic. Tumor vascular network is essential for the tumor growth and the understanding of vascular response mechanisms enables an improvement in the PDT protocol for cancer treatment. Compounds of porphyrin (Photogem®) and chlorin (Photodithazine®) were the photosensitizers tested. The incubation times varied from 20 to 80 min and the concentration ranged between 0.1 and 100 µg/cm(2). Different light doses were used between 4.8 and 40 J/cm(2) with irradiance varying between 80 and 100 mW/cm(2). The light dose of 30 J/cm(2) was used in the intravenous photosensitizer application. The membrane images were made from 0 to 300 min after treatment. The vascular response was evaluated by the average vessel area. Different responses was observed depending on the photosensitizer concentration and administration form. Intravenous application has been more efficient to produce vessel constriction and the most pronounced effect was observed for the chlorin.

Juvenile angiofibroma: major and minor complications of preoperative embolization.

INTRODUCTION: Juvenile angiofibromas (JA) are highly vascular, benign tumours for which surgery is the treatment of choice. In most services, embolisation is performed prior to resection. Nevertheless, there are few data on the complications of preoperative embolisation for JA. AIM: To describe major and minor complications of preoperative embolisation in a 32-year experience of patients undergoing surgical resection of JA at a tertiary hospital. METHODS: Retrospective chart review study of 170 patients who underwent surgical resection of JA at a tertiary hospital between September 1976 and July 2008. RESULTS: All patients were male. Age ranged from 9 to 26 years. Ninety-one patients had no complications after embolisation. Overall, 105 complication events occurred of which four major and 101 minor. CONCLUSION: In our series, preoperative embolisation for JA produced no irreversible complications and no aesthetic or functional sequelae. The vast majority of complications were transient and amenable to clinical management.

Antimicrobial action of chelating agents: repercussions on the microorganism development, virulence and pathogenesis.

Infections caused by resistant microorganisms often fail to respond to conventional therapy, resulting in prolonged illness, increased treatment costs and greater risk of death. Consequently, the development of novel antimicrobial drugs is becoming more demanding every day since the existing drugs either have too many side-effects or they tend to lose effectiveness due to the selection of resistant strains. In view of these facts, a number of new strategies to obstruct vital biological processes of a microbial cell have emerged; one of these is focused on the use of metal-chelating agents, which are able to selectively disturb the essential metal metabolism of the microorganism by interfering with metal acquisition and bioavailability for crucial reactions. The chelation activity is able to inhibit the biological role of metal-dependent proteins (e.g., metalloproteases and transcription factors), disturbing the microbial cell homeostasis and culminating in the blockage of microbial nutrition, growth and development, cellular differentiation, adhesion to biotic (e.g., extracellular matrix components, cell and/or tissue) and abiotic (e.g., plastic, silicone and acrylic) structures as well as controlling the in vivo infection progression. Interestingly, chelating agents also potentiate the activity of classical antimicrobial compounds. The differences between the microorganism and host in terms of the behavior displayed in the presence of chelating agents could provide exploitable targets for the development of an effective chemotherapy for these diseases. Consequently, metal chelators represent a novel group of antimicrobial agents with potential therapeutic applications. This review will focus on the anti-fungal and anti-protozoan action of the most common chelating agents, deciphering and discussing their mode of action.

In vivo anti-angiogenic effects further support the promise of the antineoplasic activity of methyl jasmonate.

Molecular plant components have long been aimed at the angiogenesis and anti-angiogenesis pathways, and have been tested as sources for antineoplasic drugs with promising success. The present work deals with the anti-angiogenic effects of Methyl Jasmonate. Jasmonate derivatives were demonstrated to selectively damage the mitochondria of cancer cells. In vitro, 1-10 mM Methyl Jasmonate induced the cell death of the human umbilical vein endothelial cells (HUVEC) and the Murine melanoma cells (B16F10), while micromolar concentrations were ineffective. In vivo, comparable concentrations were toxic and reduced the vessel density of the Chorioallantoic Membrane of the Chicken Embryo (CAM). However, 1-10 microM concentrations produced a complex effect. There was increased capillary budding, but the new vessels were leakier and less organised than corresponding controls. It is suggested that not only direct toxicity, but also the drug effects upon angiogenesis are relevant to the antineoplasic effects of Methyl Jasmonate.

Survey of canine heartworm in the city of Recife, Pernambuco, Brazil.

Six hundred and eleven random-source dogs (338 male, 273 female) one year of age or older, from six sections of the city of Recife, Pernambuco, were examined antemortem for circulating microfilariae Dirofilaria immitis and Dipetalonema reconditum adult heartworm (D. immitis) antigen, and examined postmortem for adult heartworms. The prevalence of heartworm infection was 2.3% (14/611), as determined by necropsy for adult worms, and 1% (6/611) had circulating microfilariae of D. immitis; thus, 57.1% of the heartworm-infected dogs had occult infections. The results of serological testing indicated that 1.3% (8/611) of the dogs were positive for adult heartworm antigen. A total of 42 (6.9%) of the dogs had microfilariae of D. reconditum; 40 of these had only D. reconditum and two additional dogs had microfilariae of both species, D. immitis and D. reconditum.

Conformational stability and antibody response to the 18kDa heat-shock protein formulated into different vehicles.

Protein stability is one of the most important obstacles for successful formulation in the development of new-generation vaccines. Here, the 18kDa heat-shock protein (18kDa-hsp) was chemically modified though conjugation with bovine serum albumin or by esterification with N-hydroxysuccinimide ester of palmitic acid. The biologically active conformation of the protein was preserved after chemical modification. The immune responses to the recombinant 18kDa-hsp from Mycobacterium leprae were studied in different presentations: free, copolymerized with bovine serum albumin in aggregates (18kDa-hsp-BSA), and either surface linked to liposomes or entrapped into liposomes. Measuring the antibody production of immunized genetically selected mice has compared the adjuvant effects of liposomes and proteic copolymer. Among the two liposome preparations, the strongest response was obtained with the surface-exposed antigen-liposomes. The copolymer 18kDa-hsp-BSA conferred a high titer of antibody in injected mice, and persisted 70 d after immunization. This approach should prove very useful for designing more effective vaccines by using 18kDa-hsp as carrier protein.

Kinetic studies on the membrane form of intestinal alkaline phosphatase.

We have purified different membrane and soluble forms of alkaline phosphatase from human placenta and bovine intestine. The enzymes will be used as markers in immunoconjugates and/or as model for membrane enzyme studies. The membrane form of alkaline phosphatase extracted from bovine intestine was purified on Q-Sepharose and on L-histidyldiazobenzyl-phosphonic acid-agarose columns to remove phosphodiesterase activity. The purified enzyme had a molecular mass of 61 kDa, Km of 1208 microM, and Vmax 240 mumol pNP/min when assayed in 1 M diethanolamine, 0.5 mM MgCl2 buffer, pH 9.8, containing 10 to 2250 microM of pNPP at 37 degrees C. In the present investigation we studied the effect of salts and inositol derivatives on this enzyme activity, which was found to depend on 0.5 mM Mg2+, and to be fully inhibited by 1.2 mM Hg2+. Vanadate (0.5 mM) and Zn2+ (0.5 mM) reduced the Km value by 43% and 84%, respectively. Inositol (2 mM) and inositol-2-monophosphate (2 mM) reduced the activity by 23% and 17%. Inositol-1-monophosphate (0.5 mM) and cyclic-inositol-(1:2)-monophosphate (0.5 mM) enhanced their Km value by at least 30% compared to p-nitrophenylphosphate.

Kinetic studies on the membrane form of intestinal alkaline phosphatase

We have purified different membrane and soluble forms of alkaline phosphatase from human placenta and bovine intestine. The enzymes will be used as markers in immunoconjugates and/or as model for membrane enzyme studies. The membrane formof alkaline phosphatase extracted from bovine intestine was purified on Q-Sepharose and on L-histidyldiazobenzylphosphonic acid-agarose columns to remove phosphodiesterase activity. The purified enzyme had a molecular mass of 61 kDa, Km of 1208 µM, and Vmax 240 µmol pNP/min when assayed in 1 M diethanolamine, 0.5 mM MgCl2 buffer, pH 9.8, containing 10 to 2250 µM of pNPP at 37§C. In the present investigation we studied the effect of salts and inositol derivatives on this enzyme activity, which was found to depend on 0.5 mM Mg2+, and to be fully inhibited by 1.2 mM Hg2+. Vanadate (0.5 mM) and Zn2+ (0.5 mM) reduced the Km value by 43 percent and 84 percent, respectively. Inositol (2 mM) and inositol-2-monophosphate (2 mM) reduced the activity by 23 percent and 17 percent. Inositol-1-monophosphate (0.5 mM) and cyclic-inositol-(1:2)-monophosphate (0.5 mM) enhanced their Km value by at least 30 percent compared to p-nitrophenylphosphate

Characterization of different membrane forms of placental alkaline phosphatases.

We have extracted and purified four alkaline phosphatase forms from human term placenta. The enzymes are dependent on Mg2+ for their activity. They can be distinguished by different responses to Zn2+, vanadate and inositol derivatives.

EEG and serum prolactin studies in relation to transcutaneous stimulation of central motor pathways.

Eight adult volunteers had EEG recordings and serial serum prolactin estimations performed both before and after a session of transcutaneous stimulation of the central motor pathways using the technique of Merton and Morton. No significant changes in either the EEG traces or in the serum prolactin values were detected.

Transferrin in maternal and cord blood: the relationship with birthweight.

The relationship between the serum level of maternal transferrin in late pregnancy and the birthweight of the infant was investigated. Mothers with low levels of serum transferrin may give birth to low-birthweight babies, but this was not a general phenomenon. In very-low-birthweight infants, the birthweight correlated with the level of maternal transferrin. It is concluded that the maternal serum transferrin level does not reflect the maturity of the fetus or the birthweight of term neonates.

IgM antibody to filarial antigens in human cord blood: possibility of transplacental infection.

In a hospital in Sri Lanka, antifilarial antibody in maternal and umbilical cord blood was determined by indirect immunofluorescence, enzyme-linked immunosorbent assay and radio-immunoassay. Anti-filarial antibody was detected in 18 of 340 cord blood samples. Specific IgM antibody was detected in 12 cord blood samples. The foetal IgM antibody was probably in response to a transplacental transfer of filarial antigens. The exposure of the foetus to filarial antigens might be beneficial, increasing resistance to infection, or detrimental by inducing at least partial tolerance.