Abnormal methylation at the KvDMR1 imprinting control region in clinically normal children conceived by assisted reproductive technologies

Genomic imprinting alterations have been shown to be associated with assisted reproductive technologies (ARTs) in animals. At present, data obtained in humans are inconclusive; however, some epidemiological studies have demonstrated an increased incidence of imprinting disorders in children conceived by ARTs. In the present study, we focused on the effect of ARTs [IVF and intracytoplasmic sperm injection (ICSI)] on the epigenetic reprogramming of the maternally methylated imprinting control region KvDMR1 in clinically normal children. Qualitative and quantitative methylation at KvDMR1 were assessed by the methylation-specific PCR approach and by the methylation-sensitive enzymatic digestion associated with real-time PCR method, respectively. DNA was obtained from peripheral blood of 12/18 and umbilical cord blood and placenta of 6/18 children conceived by IVF or ICSI. The methylation patterns observed in this group were compared with the patterns observed in 30 clinically normal naturally conceived children (negative controls) and in 3 naturally conceived Beckwith-Wiedemann syndrome patients (positive controls). Hypomethylation at KvDMR1 was observed in 3/18 clinically normal children conceived by ARTs (2 conceived by IVF and 1 by ICSI). A discordant methylation pattern was observed in the three corresponding dizygotic twins. Our findings corroborate the hypothesis of vulnerability of maternal imprinting to ARTs. Furthermore, the discordant methylation at KvDMR1 observed between dizygotic twins could be consequent to one of the following possibilities: (i) a differential vulnerability of maternal imprints among different embryos; or (ii) epimutations that occurred during gametogenesis resulting in the production of oocytes without the correct primary imprint at KvDMR1.

Frequency and prognostic relevance of cancer testis antigen 45 expression in multiple myeloma

Objective. This study aims to analyze the expression of cancer testis antigen 45 (CT45) in normal tissues and in plasma cell disorders and to identify possible associations with clinical data and prognosis in multiple myeloma (MM) patients. Materials and Methods. Expression of CT45 was studied in 20 normal tissues (testis, placenta, skeletal muscle, bladder, lung, spleen, heart, brain and fetal brain, thymus, uterus, stomach, mammary gland, pancreas, prostate, small intestine, kidney, adrenal gland, spinal cord, colon, and one pool of 10 normal bone marrow samples) and bone marrow aspirates from 3 monoclonal gammopathies of undetermined significance, 5 solitary plasmacytomas, 61 newly diagnosed MM patients and MM cell line U266 by reverse transcriptase polymerase chain reaction. Results. CT45 was positive in 3 of 20 (15%) normal tissues tested: lung, brain (both fetal and adult), and spinal cord. Among monoclonal gammopathies, CT45 was positive in 2 of 5 (40%) solitary plasmacytomas bone marrow aspirates, 10 of 61 (16%) MM bone marrow aspirates, and in the U266 MM cell line. Conclusions. We did not find associations between bone marrow histology and CT45 expression. However, we demonstrated for the first time that positive expression of CT45 was associated with poor prognostic (international Staging System) and poor outcomes in MM patients, meaning that CT45-positive cases presented seven times more chance of worse evolution than the negative ones. (C) 2009 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc.

Vascular endothelial growth factor genotypes and haplotypes are associated with pre-eclampsia but not with gestational hypertension

Vascular endothelial growth factor (VEGF) is relevant for normal pregnancy, and abnormalities in VEGF functions are associated with hypertensive disorders of pregnancy. Because there are few studies on how VEGF genetic polymorphisms affect susceptibility to pre-eclampsia (PE), and no studies on how they affect susceptibility to gestational hypertension (GH), we compared VEGF genotype and haplotype distributions in normotensive and hypertensive pregnancies. Genotypes and haplotypes for VEGF polymorphisms (C-2578A, G-1154A and G-634C) were determined in 303 pregnant women (108 healthy pregnant, HP; 101 with GH and 94 with PE). When white and non-white pregnant women were considered together, no significant differences were found in the distributions of VEGF genotypes or haplotypes (P > 0.05) in the three groups. However, with only white subjects, significant differences were found in genotypes distributions for two (C-2578A and G-634C) VEGF polymorphisms (both P < 0.05) between the HP and the PE groups. Importantly, the haplotype including the variants C-2578, G-1154 and C-634, which is associated with higher VEGF gene expression, was less common in the PE group compared with the HP group (4% versus 16%; P = 0.0047). However, we found no significant differences in VEGF haplotypes distributions when the HP and GH groups were compared (P > 0.05). These findings suggest a protective effect for the `C-2578, G-1154 and C-634` haplotype against the development of PE, but no major effects of VEGF gene variants on susceptibility to GH.

Generation of Polyclonal Antibodies Against Recombinant Human Glucocerebrosidase Produced in Escherichia coli

Deficiency of the lysosomal glucocerebrosidase (GCR) enzyme results in Gaucher`s disease, the most common inherited storage disorder. Treatment consists of enzyme replacement therapy by the administration of recombinant GCR produced in Chinese hamster ovary cells. The production of anti-GCR antibodies has already been described with placenta-derived human GCR that requires successive chromatographic procedures. Here, we report a practical and efficient method to obtain anti-GCR polyclonal antibodies against recombinant GCR produced in Escherichia coli and further purified by a single step through nickel affinity chromatography. The purified GCR was used to immunize BALB/c mice and the induction of anti-GCR antibodies was evaluated by enzyme-linked immunosorbent assay. The specificity of the antiserum was also evaluated by western blot analysis against recombinant GCR produced by COS-7 cells or against endogenous GCR of human cell lines. GCR was strongly recognized by the produced antibodies, either as cell-associated or as secreted forms. The detected molecular masses of 59-66 kDa are in accordance to the expected size for glycosylated GCR. The GCR produced in E. coli would facilitate the production of polyclonal (shown here) and monoclonal antibodies and their use in the characterization of new biosimilar recombinant GCRs coming in the near future.

Reproductive toxic effects of Tityus serrulatus scorpion venom in rats

Tityus serrulatus is the most venomous scorpion in Brazil. Little is known about the effect of maternal exposure to the venom on fetal development. We investigated the effect of low to moderate doses of the venom (0.3 or 1.0 mg/kg s.c. on either day 5 or day 10 of gestation) on pregnant rats and on their offspring. For dams, we observed their body weight gain and reproductive parameters. For the offspring, we observed their body weight and weight of internal organs and the number of live and dead fetuses, and we investigated whether the venom caused external, visceral, skeletal or histopathological alterations in the offspring. The offspring were examined on gestational day 21. Injection of the venom on gestational day 5 did not change the reproductive parameters of the dams, their weight or fetuses` weight. Rats that received the high dose of the venom (1.0 mg/kg) on gestational day 10 had heavier placentas and heavier fetuses with heavier lungs. Injections on day 10 of gestation did not alter the reproductive parameters of the dams nor their weight gain at either dose. The venom did not cause malformations of the fetal skeleton or viscera and did not delay fetal development with either dose. In conclusion, subcutaneous administration of 0.3 or 1.0 mg/kg T. serrulatus venom to pregnant Wistar rats at either day 5 or day 10 of gestation did not cause maternal or clear fetal toxicity. Subtle increases in placental weight and fetal body and lung weights observed following treatment with 1.0 mg/kg on day 10 of gestation were not associated with histopathological findings. Whether these observations represent a reaction to treatment and, if so, the underlying mechanisms and their toxicological impact remain to be examined further in future studies. (C) 2008 Elsevier Inc. All rights reserved.

Transplacental Transfer of Iron in the Water Buffalo (Bubalus bubalis): Uteroferrin and Erythrophagocytosis

The objectives of this investigation were to understand transplacental transport of iron by secreted uteroferrin (UF) and haemophagous areas of water buffalo placenta and clarify the role(s) of blood extravasation at the placental-maternal interface. Placentomes and interplacentomal region of 51 placentae at various stages of gestation were fixed, processed for light and transmission electron microscopy, histochemistry and immunohistochemistry. Haemophagous areas were present in placentomes collected between 4 and 10 months of pregnancy. Perl`s reaction for ferric iron was negative in placentomes, but positive in endometrial glands. Positive staining for UF indicated areas in which it was being taken up by phagocytosis and/or fluid phase pinocytosis in areolae of the interplacentomal mesenchyme, with little staining in endometrial stroma. Imunohistochemistry detected UF in trophectoderm of haemophagous regions of placentomes and in other parts of the foetal villous tree, but the strongest immunostaining was in the epithelial cells and lumen of uterine glands. Ultrastructural analyses indicated that erythrophagocytosis was occurring and that erythrocytes were present inside cells of the chorion that also contained endocytic vesicles and caveolae. Results of this study indicate that both the haemophagous areas of placentomes and the areolae at the interface between chorion and endometrial glands are important sites for iron transfer from mother to foetal-placental tissues in buffalo throughout pregnancy.

Cell cycle and apoptosis in normal and cloned bovine near-term placentae

The bovine maternal epithelium is composed of cuboidal cells interspersed with low columnar cells having centrally located nuclei. Bovine trophoblast is composed of two cell types: mononuclear trophoblastic and giant trophoblastic cells that can have two or more nuclei. Number of apoptotic cells and proliferative cells are variable in both cell populations. This study compared tissue growth and apoptosis by flow cytometry in the cell population found at distinct placental regions (central region of placentomes, <= 1-cm microplacentomes and the interplacentomal region) between normal and cloned near-term bovine pregnancies. After a morphological comparison between regions and groups (controls vs. clones), a lesser proportion of diploid to tetraploid cells was observed in the central region of placentomes and in microplacentomes from cloned-derived pregnancies. In addition, cloned animals had a fewer apoptotic cells in the central region of the placentome and in interplacentomal region and a greater proliferative capacity in all regions (cells in G(2)/M) near term as opposed to control animals. These results may reveal the existence of a relationship between such changes in the proportions of uterine and trophoblastic epithelial cells at the end of pregnancy and normal placental function. This could be related to faulty placentation in early pregnancy, placental insufficiency during pregnancy or lack of placental and/or fetal maturation in late pregnancy, which may contribute to someof the abnormalities after in vitro embryo manipulations, such as poor preparation and initiation of parturition, prolonged gestation and lesser post-natal survival in some cloned animals. (C) 2008 Elsevier B.V. All rights reserved.

Placentation in the Amazonian manatee (Trichechus inunguis)

Evidence from several sources supports a close phylogenetic relationship between elephants and sirenians. To explore whether this was reflected in similar placentation, we examined eight delivered placentae from the Amazonian manatee using light microscopy and immunohistochemistry. In addition, the fetal placental circulation was described by scanning electron microscopy of vessel casts. The manatee placenta was zonary and endotheliochorial, like that of the elephant. The interhaemal barrier comprised maternal endothelium, cytotrophoblasts and fetal endothelium. We found columnar trophoblast beneath the chorionic plate and lining lacunae in this region, but there was no trace in the term placenta of haemophagous activity. The gross anatomy of the cord and fetal membranes was consistent with previous descriptions and included a four-chambered allantoic sac, as also found in the elephant and other afrotherians. Connective tissue septae descended from the chorionic plate and carried blood vessels to the labyrinth, where they gave rise to a dense capillary network. This appeared to drain into shorter vessels near the chorionic plate. The maternal vasculature could not be examined in the same detail, but maternal capillaries ran rather straight and roughly parallel to the fetal ones. Overall, there is a close resemblance in placentation between the manatee and the elephant.

The pathogenesis of oral lichen planus

Both antigen-specific and non-specific mechanisms may be involved in the pathogenesis of oral lichen planus (OLP). Antigen-specific mechanisms in OLP include antigen presentation by basal keratinocytes and antigen-specific keratinocyte killing by CD8(+) cytotoxic T-cells. Non-specific mechanisms include mast cell degranulation and matrix metalloproteinase (MMP) activation in OLP lesions. These mechanisms may combine to cause T-cell accumulation in the superficial lamina propria, basement membrane disruption, intra-epithelial T-cell migration, and keratinocyte apoptosis in OLP. OLP chronicity may be due, in part, to deficient antigen-specific TGF-beta1-mediated immunosuppression. The normal oral mucosa may be an immune privileged site (similar to the eye, testis, and placenta), and breakdown of immune privilege could result in OLP and possibly other autoimmune oral mucosal diseases. Recent findings in mucocutaneous graft-versus-host disease, a clinical and histological correlate of lichen planus, suggest the involvement of TNF-alpha, CD40, Fas, MMPs, and mast cell degranulation in disease pathogenesis. Potential roles for oral Langerhans cells and the regional lymphatics in OLP lesion formation and chronicity are discussed. Carcinogenesis in OLP may be regulated by the integrated signal from various tumor inhibitors (TGF-beta1, TNF-alpha, IFN-gamma, IL-12) and promoters (MIF, MMP-9). We present our recent data implicating antigen-specific and non-specific mechanisms in the pathogenesis of OLP and propose a unifying hypothesis suggesting that both may be involved in lesion development. The initial event in OLP lesion formation and the factors that determine OLP susceptibility are unknown.

Placental growth hormone is not suppressed by oral glucose loading in normal human pregnancy

Placental growth hormone (PGH) progressively replaces pituitary growth hormone in the maternal circulation from mid-gestation onwards in human pregnancy. Our previous investigations have shown that placental growth hormone concentrations correlate well with foetal growth. Despite the apparent correlation between PGH and birthweight, the physiology of its secretion during pregnancy has not been well defined. We investigated the response of maternal serum PCH to oral glucose loading in pregnant women (n = 24) who demonstrated normal glucose tolerance at a mean gestation of 29 weeks. Mean (SEM) fasting PGH concentrations were high (36.9 [6.4] ng/ml). No suppression of PGH was noted at one, two or three hours after a 75 g oral glucose load. Similarly, no changes were noted in growth hormone binding protein or in calculated free PGH over the course of the glucose tolerance test. As expected, insulin concentrations rose sixfold and insulin like growth factor binding protein 1 concentrations fell by 20% with glucose loading. Cot-relation analysis showed maternal weight, BMI, fasting serum glucose serum insulin to be significantly correlated with the babies' birthweight. Our results support the proposition that PGH concentrations in maternal serum are not Suppressed by oral glucose loading in non-diabetic mothers.

Identification of residues which regulate activity of the STE20-related kinase hMINK

Activity of the STE20-related kinase hMINK was investigated. hMINK was expressed widely, though not ubiquitously, in human tissues: highest levels being found in haematopoietic tissues but also in brain, placenta, and lung. Mutagenesis revealed that T-191. and Y-193 in the substrate recognition loop of the catalytic domain were critical for kinase activity against exogenous substrates and autophosphorylation. A mutation on T-187 showed reduced enzymatic activity against exogenous substrates but retained autophosphorylationactivity. Phosphorylation was confirmed by the use of a phospho-specific T-187 antibody. hMINK activated the JNK signal transduction pathway and optimal JNK activation occurred when the C-terminus was deleted. In addition, overexpression of the C-terminal domain devoid of kinase activity also resulted in significant activation of the JNK pathway. These data suggest that hMINK requires an activation step that dissociates the C terminal, thereby freeing the catalytic domain to interact with substrates. Models for receptor-mediated activation of hMINK are discussed. (C) 2002 Elsevier Science (USA). All rights reserved.

Institucionalização de estruturas e processos de TI : uma análise das pressões isomórficas que podem influenciar o processo de institucionalização da TI de um órgão do judiciário brasileiro

Orlikowski e Barley (2001) apontam a importância da pesquisa em TI sob a ótica dos Estudos Organizacionais, principalmente quando é utilizada a análise neoinstitucional, porque dessa forma há uma compreensão mais sistêmica sobre como a tecnologia está entrelaçada nas complexas redes interdependentes, sociais, econômicas e políticas. O estudo de caso ocorreu em um órgão do judiciário, onde foram obtidos dados por meio de entrevistas, que foram triangulados com uma pesquisa documental. A dissertação busca elucidar como pressões isomórficas podem influenciar estruturas e processos de TI recomendados pela legislação em vigor, de forma a contribuir para gestores de organizações públicas durante a definição de prioridades para a TI. Para analisar o estágio institucional utilizou-se a classificação proposta por Tolbert e Zucker (1996) e como resultado, apesar das pressões isomórficas coercitivas, obteve-se que poucas estruturas e processos se institucionalizaram totalmente e, aparentemente, só aquelas que traziam com si outros tipos de pressões isomórficas é que parecem ter maior chance de chegar ao último estágio institucional. Foi constatada que a organização, durante o processo de institucionalização, pode incorrer em novas falhas, gerando mitos burocráticos e ritos cerimoniais que por exemplo, podem causar maior descolamento da preocupação por efetividade em seus resultados. Para determinados problemas, apontados durante as entrevistas, não foi possível encontrar influência de nenhuma pressão isomórfica. Tal fato sugere que a teoria neoinstitucional não conseguiu explicar todo o cenário da organização.

Prevalência de Chlamydia trachomatis em casos de partos pretermo atendidos no Hospital Universitário Cassiano Antonio Moraes, Vitória - ES

Introdução: O parto pretermo (PPT) é um dos principais determinantes da morbimortalidade neonatal, acarretando consequências adversas para saúde. As causas são multifatoriais, sendo a infecção intrauterina a razão mais provável para explicar a maioria destes desfechos. Acredita-se que a infecção por Chlamydia trachomatis (CT) também esteja envolvida no PPT e rotura prematura de membranas (ROPREMA). Objetivo: Determinar a prevalência de CT em parturientes e os possíveis fatores de risco relacionados com os casos de partos prematuros atendidos no Hospital Universitário Cassiano Antonio Moraes. Métodos: Estudo de corte transversal, realizado entre parturientes que apresentaram PPT em um Hospital Universitário em Vitória - ES, entre junho de 2012 e agosto de 2013. As participantes responderam a um questionário contendo dados sóciodemográficos, comportamentais e clínicos. Foi coletada uma amostra de urina para rastreio de CT usando reação em cadeia da polimerase. Resultados: A prevalência de PPT durante o período do estudo foi de 26%. Um total de 378 casos de PPT foram registrados, entre eles 323 mulheres foram testadas para o CT; quarenta e cinco (13,9%) tiveram um resultado positivo, sendo que 31,6% possuiam até 24 anos e as mulheres infectadas pela CT eram mais jovens do que as demais (p = 0,022). Um total de 76,2% eram casadas/em união estável, e CT foi mais frequente entre as solteiras (p = 0,018); 16,7% relataram primeira relação sexual com menos de 14 anos de idade. As causas de PPT foram materno-fetais em 40,9%, ROPREMA em 29,7% e trabalho de parto prematuro em 29,4%. Na análise multivariada, ser casada foi um fator de proteção [OR = 12:48 (IC 95%: 0,24-0,97)]. Nenhuma das demais características foram associadas com a infecção por CT. Conclusões: Este estudo evidencia uma alta prevalência de infecção por CT entre parturientes com PPT. Essa alta prevalência reforça a necessidade da definição de estratégias de rastreamento e assistência durante o pré-natal.

Potencialidades dos extrativos do cerne da madeira de teca como preservativo natural e sua influência na cor de madeiras claras

Este estudo teve como objetivo avaliar o potencial preservativo dos extrativos do cerne da madeira de teca (Tectona grandis) e a capacidade dos mesmos na mudança de coloração de madeiras claras. Para tanto, os resíduos gerados no processamento mecânico do cerne da madeira de teca com 20 anos de idade foram coletados e utilizados para realização de extrações. Para avaliar a influência dos extrativos de teca na cor e resistência natural da madeira foi utilizado o alburno da madeira de teca com 10 anos, além da madeira de Pinus sp., em função de ser uma madeira de coloração clara e baixa resistência natural. Foram realizadas extrações em água quente e etanol absoluto. Para determinação da concentração das soluções de tratamento foi realizado um ensaio de toxidez ao fungo Postia placenta. Após definida a concentração, as soluções extraídas foram preparadas para a impregnação. Além disto, foi utilizada uma terceira solução, composta pela combinação das soluções extraídas em água quente e etanol absoluto. Para cada solução testada foi realizado o tratamento pelo método de célula-cheia (Bethell). Para testar a eficiência das soluções preparadas com extrativos de teca, foram realizados leituras colorimétricas e ensaios biológicos com fungos e térmitas xilófagos. A combinação dos extrativos testados promoveu um escurecimento e reduziu a desuniformidade da cor, fazendo com que as madeiras tratadas se aproximassem mais da cor da madeira de cerne do que das amostras sem tratamento das respectivas espécies. A solução de extrativos obtida em etanol absoluto e a combinação dos extrativos obtidos em água quente e etanol absoluto promoveram os melhores resultados na resistência da madeira tratada contra fungos e térmitas xilófagos, alterando significativamente a classe de resistência das respectivas espécies tratadas.

Estudo experimental comparativo entre o butil-2-cianoacrilato, a mistura gelatina-resorcina-formaldeído e sutura na estabilização de enxertos de cartilagem em coelhos

Os enxertos de cartilagem constituem-se em boa opção técnica para aprimoramento das rinoplastias. Procura-se um material para sua fixação que seja de simples manuseio, e que provoque mínima reação tecidual. OBJETIVO: Comparar o uso do butil-2-cianoacrilato, mistura gelatina-resorcina-formaldeído (GRF) e sutura na estabilização de enxertos de cartilagem em coelhos. FORMA DE ESTUDO: Experimental. MATERIAL E MÉTODO: Utilizaram-se 15 coelhos adultos da raça Nova Zelândia machos, de cujas orelhas foram ressecados 6 enxertos de cartilagem, fixados no periósteo da calvária e unidos dois a dois com sutura, GRF e cianoacrilato. Após 2, 6 e 12 semanas, grupos de 5 coelhos foram sacrificados e realizou-se avaliação da distância de deslocamento do enxerto e análise histológica do processo inflamatório tecidual e da adesão/deformidade das cartilagens. RESULTADOS: Houve deslocamento menor nas fixadas com GRF do que com cianoacrilato e sutura. O processo inflamatório foi maior nos animais de 2 semanas, decaindo até restar pequeno grau de fibrose em 12 semanas, de modo semelhante nas 3 fixações. Não houve descolamento nem deformidade em nenhuma dupla de cartilagens fixada com sutura e o maior número de cartilagens descoladas e deformadas se deu com o cianoacrilato. O número de cartilagens deformadas foi diretamente proporcional ao número de descoladas. Os dados foram significativos estatisticamente (p<0,05). CONCLUSÃO: A mistura gelatina-resorcina-formaldeído mostrou ser um método de estabilização de enxertos de cartilagem em coelhos superior ao butil-2-cianoacrilato em todas as fixações, e à sutura na fixação cartilagem-osso.