In vivo evidence of angiogenesis inhibition by ß2-glycoprotein I subfractions in the chorioallantoic membrane of chicken embryos.

The vascular network expansion and functioning are important factors affecting normal intra-uterine fetal development. This study addressed the previously reported antiangiogenic potential of beta-2-glycoprotein I (ß2GPI) in vivo in the chick embryo model of angiogenesis. The effects of two naturally occurring ß2GPI forms on the development of the chorioallantoic membrane (CAM) vessels and the chicken embryo were investigated. ß2GPI monomers and dimers were obtained by fractioned purification and characterized using SDS-PAGE, immunoblot, and ELISA. The egg exposure was performed by injection of small volumes of 2.5 µg/mL solutions of the ß2GPI subfractions. Angiogenesis was evaluated through quantitative measurements of vascular architecture parameters in the captured CAM images, using computational analysis of texture contrasts and computer vision techniques. Quantitative information was assigned to the CAM vasculature modifications. In vivo, the ß2GPI dimer completely halted the formation of CAM vessels and led to embryo death after 48 h of exposure. The ß2GPI monomer allowed the embryo to develop up to the 10th day, despite early changes of CAM vessels. The impaired normal vessel growth proceeded as a self-limited effect. The ß2GPI monomer-exposed eggs showed reduced vascularization on the 6th day of incubation, but embryos were viable on the 10th day of incubation, with ingurgitated CAM vessels implying sequelae of the angiogenesis inhibition. Both subfractions impaired CAM vasculature development. The ß2GPI dimer proved to be largely more harmful than the ß2GPI monomer. ß2GPI modification by cleavage or dimerization may play a role in angiogenesis control in vivo.

In vivo evidence of angiogenesis inhibition by β2-glycoprotein I subfractions in the chorioallantoic membrane of chicken embryos

The vascular network expansion and functioning are important factors affecting normal intra-uterine fetal development. This study addressed the previously reported antiangiogenic potential of beta-2-glycoprotein I (β2GPI) in vivo in the chick embryo model of angiogenesis. The effects of two naturally occurring β2GPI forms on the development of the chorioallantoic membrane (CAM) vessels and the chicken embryo were investigated. β2GPI monomers and dimers were obtained by fractioned purification and characterized using SDS-PAGE, immunoblot, and ELISA. The egg exposure was performed by injection of small volumes of 2.5 µg/mL solutions of the β2GPI subfractions. Angiogenesis was evaluated through quantitative measurements of vascular architecture parameters in the captured CAM images, using computational analysis of texture contrasts and computer vision techniques. Quantitative information was assigned to the CAM vasculature modifications. In vivo, the β2GPI dimer completely halted the formation of CAM vessels and led to embryo death after 48 h of exposure. The β2GPI monomer allowed the embryo to develop up to the 10th day, despite early changes of CAM vessels. The impaired normal vessel growth proceeded as a self-limited effect. The β2GPI monomer-exposed eggs showed reduced vascularization on the 6th day of incubation, but embryos were viable on the 10th day of incubation, with ingurgitated CAM vessels implying sequelae of the angiogenesis inhibition. Both subfractions impaired CAM vasculature development. The β2GPI dimer proved to be largely more harmful than the β2GPI monomer. β2GPI modification by cleavage or dimerization may play a role in angiogenesis control in vivo.

In vivo evidence of angiogenesis inhibition by ß2-glycoprotein I subfractions in the chorioallantoic membrane of chicken embryos

The vascular network expansion and functioning are important factors affecting normal intra-uterine fetal development. This study addressed the previously reported antiangiogenic potential of beta-2-glycoprotein I (β2GPI) in vivo in the chick embryo model of angiogenesis. The effects of two naturally occurring β2GPI forms on the development of the chorioallantoic membrane (CAM) vessels and the chicken embryo were investigated. β2GPI monomers and dimers were obtained by fractioned purification and characterized using SDS-PAGE, immunoblot, and ELISA. The egg exposure was performed by injection of small volumes of 2.5 µg/mL solutions of the β2GPI subfractions. Angiogenesis was evaluated through quantitative measurements of vascular architecture parameters in the captured CAM images, using computational analysis of texture contrasts and computer vision techniques. Quantitative information was assigned to the CAM vasculature modifications. In vivo, the β2GPI dimer completely halted the formation of CAM vessels and led to embryo death after 48 h of exposure. The β2GPI monomer allowed the embryo to develop up to the 10th day, despite early changes of CAM vessels. The impaired normal vessel growth proceeded as a self-limited effect. The β2GPI monomer-exposed eggs showed reduced vascularization on the 6th day of incubation, but embryos were viable on the 10th day of incubation, with ingurgitated CAM vessels implying sequelae of the angiogenesis inhibition. Both subfractions impaired CAM vasculature development. The β2GPI dimer proved to be largely more harmful than the β2GPI monomer. β2GPI modification by cleavage or dimerization may play a role in angiogenesis control in vivo.

Sperm Bundles in the Seminal Vesicle of the Crematogaster victima (Smith) Adult Males (Hymenoptera: Formicidae).

This study establishes the presence of spermatodesm in the seminal vesicles of sexually mature males of Crematogaster victima (Smith). In this species, the spermatozoa are maintained together by an extracellular matrix in which the acrosomal regions are embedded. This characteristic has not yet been observed in any other Aculeata. However, the sperm morphology in this species is similar to that described for other ants. The spermatozoa measure on average 100 µm in length, and the number of sperm per bundle is up to 256. They are composed of a head formed by the acrosome and nucleus; this is followed by the flagellum, which is formed by the centriolar adjunct, an axoneme with a 9 + 9 + 2 microtubule pattern, two mitochondrial derivatives, and two accessory bodies. The acrosome is formed by the acrosomal vesicle and perforatorium. The nucleus is filled with compact chromatin with many areas of thick and non-compacted filaments. Both mitochondrial derivatives have the same shape and diameters. The presence of sperm bundles in sexually mature males differentiates C. victima from other ants; however, the similarities in the sperm ultrastructure support the monophyly of this insect group.

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.

Changes in gene expression profiles of bovine embryos produced in vitro, by natural ovulation, or hormonal superstimulation.

Embryos produced by hormonal superstimulation have been used as an in vivo control in most published research on embryo gene expression. However, it is not known if this is the most appropriate control for gene expression profile studies. We compared the expression of GRB-10, IGF-II, IGF-IIR, MnSOD, GPX-4, catalase, BAX, and interferon-tau genes, in embryos produced in vivo by hormonal superovulation (SOV), by in vitro fertilization (IVF) or in vivo without any hormonal stimulus (NOV). GRB-10 was less expressed in NOV than IVF embryos, whereas no differences were found for the other genes. The genes related to stress response were then grouped and compared; the sum of expression of MnSOD, GPX-4, and catalase genes tended to be greater in IVF than NOV embryos. A correlation analysis was performed; we found a distinct behavior for NOV embryos when compared with SOV and IVF in the expression of GRB-10, IGF-II and IGF-IIR genes. However, the behavior of these genes was similar in SOV and IVF embryos. We conclude that ovarian hormonal stimulation can affect embryos by altering gene expression. Although this conclusion was based on investigation of only a few genes, we suggest that SOV embryos should be used with caution as a control in gene expression studies.

beta 2-glycoprotein I (apolipoprotein H) modulates uptake and endocytosis associated chemiluminescence in rat Kupffer cells.

beta 2-Glycoprotein I (beta 2 GPI) is known to influence macrophage uptake of particles with phosphatidylserine containing surfaces, as apoptotic thymocytes and unilamellar vesicles in vitro. Nevertheless, effects upon macrophage activation induced by this interaction are still unknown. beta 2 GPI influence upon the reactive species production by Kupffer cells was evaluated in order to investigate whether beta 2 GPI modulates the macrophage response to negatively charged surfaces. Chemiluminescence of isolated non-parenchymal rat liver cells was measured after phagocytosis of opsonized zymosan or phorbolymristate acetate (PMA) stimulation, in the presence and absence of large unilamellar vesicles (LUVs) containing 25 mol% phosphatidylserine (PS) or 50 mol% cardiolipin (CL) and complementary molar ratio of phosphatidylcholine (PC). beta 2 GPI decreased by 50% the chemiluminescence response induced by opsonized zymosan, with a 66% reduction of the initial light emission rate. PMA stimulated Kupffer cell chemiluminescence was insensitive to human or rat beta 2 GPI. Albumin (500 micrograms/ml) showed no effect upon chemiluminescence. beta 2 GPI increased PS/PC LUV uptake and degradation by Kupffer cells in a concentration-dependent manner, without leakage of the internal contents of the LUVs, as shown by fluorescence intensity enhancement. LUVs opsonized with antiphospholipid antibodies (aPL) from syphilitic patients increased light emission by Kupffer cells. Addition of beta 2 GPI to the assay reduced chemiluminescence due to opsonization with purified IgG antibodies from systemic lupus erythematosus (SLE or syphilis (Sy) patient sera. A marked net increase in chemiluminescence is observed in the presence of Sy aPL antibodies, whereas a decrease was found when SLE aPL were added to the assay, in the presence or absence of beta 2 GPI. At a concentration of 125 micrograms/ml, beta 2 GPI significantly reduced Kupffer cell Candida albicans phagocytosis index and killing score by 50 and 10%, respectively. The present data strongly suggest that particle uptake in the presence of beta 2 GPI is coupled to an inhibition of reactive species production by liver macrophages during the respiratory burst, supporting the role of beta 2 GPI as a mediator of senescent cell removal.

RC-IAL cell line: sensitivity of rubella virus grow.

OBJECTIVE: The rapid growth of the rubella virus in RC-IAL2 with development of cytopathic effect, in response to rubella virus infection, is described. For purposes of comparison, the rubella virus RA-27/3 strain was titered simultaneously in the RC-IAL, Vero, SIRC and RK13 cell lines. METHODS: Rubella virus RA-27/3 strain are inoculated in the RC-IAL cell line (rabbit Kidney, Institute Adolfo Lutz). Plates containing 1.5x10(5) cells/ml of RC-IAL line were inoculated with 0.1ml s RA-27/3 strain virus containing 1x 10(4)TCID50/0.1ml. A 25% cytopathic effect was observed after 48 hours and 100% after 96 hours. The results obtained were compared to those observed with the SIRC, Vero and RK13 cell lines. Rubella virus was detected by immunohistochemistry. RESULTS: With the results, it was possible to conclude that the RC-IAL cell line is a very good substrate for culturing rubella virus. The cells inoculated with rubella virus were examined by phase contrast microscopy and showed the characteristic rounded, bipolar and multipolar cells. The CPE in RC-IAL was observed in the first 48 hours and the curve of the increased infectivity was practically the same as observed in other cell lines. CONCLUSIONS: These findings are important since this is one the few cell lines described in the literature with a cytopathic effect. So it can be used for antigen preparation and serological testing for the diagnosis of specific rubella antibodies.

Multi-criteria decision making--an approach to setting priorities in health care.

The objective of this paper is to present a multi-criteria decision making (MCDM) approach to support public health decision making that takes into consideration the fuzziness of the decision goals and the behavioural aspect of the decision maker. The approach is used to analyse the process of health technology procurement in a University Hospital in Rio de Janeiro, Brazil. The method, known as TODIM, relies on evaluating alternatives with a set of decision criteria assessed using an ordinal scale. Fuzziness in generating criteria scores and weights or conflicts caused by dealing with different viewpoints of a group of decision makers (DMs) are solved using fuzzy set aggregation rules. The results suggested that MCDM models, incorporating fuzzy set approaches, should form a set of tools for public health decision making analysis, particularly when there are polarized opinions and conflicting objectives from the DM group.

[Comparison of methods to test the "in vitro" cytotoxicity of biocompatible materials]. / Comparação de métodos para testar a citotoxicidade "in vitro" de materiais biocompatíveis.

OBJECTIVE: A comparison of the sensitivity of the agar diffusion method with that of extraction using cell-lines RC-IAL (fibroblastic of rabbit kidney) and HeLa (epithelial carcinoma cells from the cervix uteri of the human uterus), in the in vitro evaluation of materials of medical and hospital. MATERIALS AND METHODS: Fifteen samples chosen at random, from among the already known positives and negatives in our stock, were tested and identified as cotton, form, latex, cellulose and acrylic. Besides the samples mentioned, many SDS (GIbco) concentrations were tested experimentally in RC-IAL and HeLa cell cultures. RESULTS: Of the 50 samples tested, 44(88%) were positive by both methods. However, when the SDS were compared by using the two methods, positive results were noted in the concentrations of from 0.5 to 0.05 microgram/ml in the agar diffusion ans extraction methods. A cytotoxic effect was only noted in the concentrations of up to 0.25 microgram/ml. CONCLUSION: When the SDS was used, differences favorable to the agar diffusion method were observed in the two cell lines, in two concentrations; that is, the sensitivity of this method was quantitatively greater on inspection than that of the extraction method, as well as being the simpler method to use.

Distribuição da microbiota anemófila em ambiente hospitalar (Campina Grande, PB) / Anemophilic microbiota distribuition in a hospital environment (Campina Grande, PB)

No ambiente hospitalar é frequente a presença de microrganismos oportunistas que ao infectar pacientes imunodeprimidos, idosos e crianças causam surtos hospitalares severos. Todavia, os microrganismos selecionados neste ambiente apresentam multi-resistência aos antimicrobianos de uso rotineiro, dificultando a antibioticoterapia. Neste trabalho foi avaliada a distribuição e a diversidade da microbiota anemófila de 15 salas de um hospital da rede privada (Campina Grande, PB), e analisando o padrão de resistência das bactérias isoladas. Os resultados de 4 coletas mostraram uma diversidade de 20 gêneros de fungos e 10 de bactérias (5 grupos de Gram-positivos e 5 de Gram-negativos). O centro cirúrgico, o berçário e a UTI-adulto foram os locais de maior contaminação. Penicillium spp, Cladosporium spp e FNE foram os fungos de maior frequência de isolamento. Dentre as bactérias, Staphylococcus aureus, oxacilina resistente esteve presente na maioria dos ambientes. Netilmicina e amicacina foram os antibióticos com maior sensibilidade e ampicilina com o menor. Recomenda-se intensificar a desinfecção e assepsia do hospital assim como investigar a relação entre as frequências de isolamento de microrganismos patogênicos e oportunistas com as condições climáticas.

Permeation of superoxide anion through the bilayer of vesicles of a synthetic amphiphile.

Large unilamellar vesicles, prepared with dioctadecyldimethylammonium chloride, entrap nitroblue tetrazolium. Addition of solid KO2, or production of superoxide anion by riboflavin photolysis, to nitroblue tetrazolium-containing dioctadecyldimethylammonium vesicles results in the formation of monoformazan above the phase-transition temperature of the bilayer. Below the phase-transition temperature the yield of monoformazan is negligible. These results demonstrate that superoxide anion permeates vesicles above the phase-transition temperature of the bilayer.

[RC-IAL: rabbit kidney continuous cell line--characteristics and substrate for viral replication]. / RC-IAL: linhagem celular contínua de rim de coelho--características esubstrato para replicação de vírus.

A rabbit kidney cell line RC-IAL, isolated in 1976 and at present at 150a passage, has had its characteristics analysed. The cells presented morphology similar to fibroblasts throughout their culture. The cellular growth proportion remained unaltered from its isolation, with a cloning efficiency of around 9%. The line showed growth dependent on anchorage and chromosomic analysis presented the modal number of the species with small variations to about one chromosome, to a total of about 50%. The line's species of origin was confirmed through indirect immunofluorescence reaction and susceptibility to some viruses with cytopathic effect was verified with vaccinia, cowpox, herpes simplex types 1 and 2 and rubella viruses. This cellular substract is free from contaminating agents, thus satisfying the conditions for its use in scientific work, especially that relating to public health.

[Detection of cytotoxicity of biocompatible materials in MRC-5, HeLa, and RC-IAL cell lines]. / Detecção da citotoxicidade de materiais biocompatíveis nas linhagens celulares MRC-5, HeLa e RC-IAL.

The sensitivity of diploid and heteroploid cell lines for detection of cytotoxicity using the agar diffusion method on cell culture, was tested with ascorbic acid solution of different concentrations. A total of 562 samples of 21 various materials were tested. The heteroploid cell line, RC-IAL, showed in relation to the MRC-5 and HeLa cell lines, greater sensitivity because it showed the presence of cytotoxic effect with the lowest concentration used (10 and 25 micrograms/ml) of ascorbic acid and showed greater diameter of cytotoxic halo in 15 samples and equal diameter in 16 of the 43 positive samples (7.6%). Out of 43 positive samples, the MRC-5 line did not show cytotoxicity in 3 sponge samples and 1 of acrylic resin. The PVC (polyvinylchloride) and polyethylene rarely showed positivity, while, the plastic, cotton and acrylic resin demonstrated cytotoxicity in about 5% of samples. We thus suggest the use of the RC-IAL and HeLa cell lines for continuation of this type of analysis at Adolfo Lutz Institute.

DNA alkylation by carbon-centered radicals.

1. Over the last two decades, the prevalent view in chemical carcinogenesis has been that most free radicals do not bind to DNA. Recent studies, however, are demonstrating formation of adducts between DNA and free radicals such as hydroxyl radicals and aromatic cation radicals. 2. Within this context, we discuss the recent work from our group demonstrating DNA alkylation by carbon-centered radicals formed during biotransformation of genotoxic hydrazine derivatives both in vitro and in vivo. 3. The mutagenic potential of the identified methyl radical adduct, C8-methylguanine, is discussed, and other possible biological sources of carbon-centered radicals are presented.

DNA alkylation by carbon-centered radicals

1. Over the last two decades, the prevalent view in chemical carcinogenesis has been that most free radicals do not bind to DNA. Recent studies, however, are demonstrating formation of adducts between DNA and free radicals such as hydroxyl radicals and aromatic cation radicals. 2. Within this context, we discuss the recent work from our group demonstrating DNA alkylation by carbon-centered radicals formed during biotransformation of genotoxic hydrazine derivatives both in vitro and in vivo. 3. The mutagenic potential of the identified methyl radical adduct, C8-methylguanine, is discussed, and other possible biological sources of carbon-centered radicals are presented

Formation of methyl radicals during the catalase-mediated oxidation of formaldehyde hydrazone.

It has been suggested that formaldehyde hydrazone, a condensation product of hydrazine with formaldehyde, plays an important role in hydrazine-promoted DNA methylation in vivo. The present study demonstrated by spin-trapping experiments with 5,5-dimethyl-1-pyrroline-N-oxide and tert-nitrosobutane that catalase-mediated oxidation of formaldehyde hydrazone generates methyl radicals. Both the use of two spin-traps and parallel studies of oxygen consumption were important for excluding possible artefacts of spin-trapping experiments with tert-nitrosobutane. Hydrazine was also oxidized by catalase but only hydroxyl radicals were detected. Metabolic activation of formaldehyde hydrazone to methyl radicals may be of importance in regard to hydrazine-mediated toxicity and carcinogenicity.

Preliminary characterization of a new virus isolated from the spinal fluid of patients with meningitis in São Paulo, Brazil.

A total of 138 patients with the age of 4 months to 57 years were attended in different hospitals of São Paulo State with aseptic meningitis. A probable new agent was isolated from the cerebrospinal fluid of 35 of 53 specimens examined. Replication of the agent with similar characteristics was detected by CPE produced in the MDCK cell line. Virus-like particles measuring about 40 nm in diameter were observed by negative staining electron microscopy. No hemagglutinating activity was detected at pH 7.2 by using either human, guinea pig, chicken and at pH ranged 6.0-7.2 with goose red blood cells. The agent was not pathogenic to newborn or adult mice. Virus infectivity as measured by CPE was sensitive to chloroform and not inhibited by BuDR, suggesting that agent is an enveloped virus with RNA genome.

Serologic studies on the behaviour of influenza virus type A in persons of greater Säo Paulo during 1976, 1978 and 1979

Foram estudados soros de pessoas de diferentes grupos etários coletados em 1976, 1978 e 1979 para verificar a presença de anticorpos inibidores da hemaglutinaçäo contra diversas estirpes de vírus da influenza A dos subtipos H3N2 e H1N1. A ocorrência da infecçäo pelo subtipo H3N2 foram detectados em 1976 e 1978 mas em 1979, a circulaçäo desse subtipo de vírus foi limitada. A prevalência de anticorpo contra A/Säo Paulo/1/78 (H1N1) foi significativamente maior do que para A/USSR/90/77 (H1N1) em 1978. No entanto em 1979, a estirpe predominante foi A/USSR/90/77 (H1N1). As pessoas com idade inferior a 20 anos foram as mais afetadas pelo subtipo H1N1, enquanto que indivíduos com mais de 20 anos já apresentavam anticorpos para esse subtipo em 1976, antes do resurgimento dessa estirpe. A infecçäo pelo vírus H3N2 em 1978 ocorreu em 65,4% de crianças do grupo etário de 0-4 anos; 47,0% de criancas do mesmo grupo tinham anticorpos para vírus H1N1 em 1979. Anticorpos para o vírus da influenza suina foram detectadas em 60% de pessoas com mais de 60 anos de idade