Vaccination with recombinant modified vaccinia virus Ankara prevents the onset of intestinal allergy in mice.

BACKGROUND: Modified vaccinia virus Ankara (MVA)-encoding antigens are considered as safe vaccine candidates for various infectious diseases in humans. Here, we investigated the immune-modulating properties of MVA-encoding ovalbumin (MVA-OVA) on the allergen-specific immune response. METHODS: The immune-modulating properties of MVA-OVA were investigated using GM-CSF-differentiated BMDCs from C57BL/6 mice. OVA expression upon MVA-OVA infection of BMDCs was monitored. Activation and maturation markers on viable MVA-OVA-infected mDCs were analyzed by flow cytometry. Secretion of INF-γ, IL-2, and IL-10 was determined in a co-culture of BMDCs infected with wtMVA or MVA-OVA and OVA-specific OT-I CD8(+) and OT-II CD4(+ ) T cells. BALB/c mice were vaccinated with wtMVA, MVA-OVA, or PBS, sensitized to OVA/alum and challenged with a diet containing chicken egg white. OVA-specific IgE, IgG1, and IgG2a and cytokine secretion from mesenteric lymph node (MLN) cells were analyzed. Body weight, body temperature, food uptake, intestinal inflammation, and health condition of mice were monitored. RESULTS: Infection with wtMVA and MVA-OVA induced comparable activation of mDCs. MVA-OVA-infected BMDCs expressed OVA and induced enhanced IFN-γ and IL-2 secretion from OVA-specific CD8(+ ) T cells in comparison with OVA, wtMVA, or OVA plus wtMVA. Prophylactic vaccination with MVA-OVA significantly repressed OVA-specific IgE, whereas OVA-specific IgG2a was induced. MVA-OVA vaccination suppressed TH 2 cytokine production in MLN cells and prevented the onset of allergic symptoms and inflammation in a mouse model of OVA-induced intestinal allergy. CONCLUSION: Modified vaccinia virus Ankara-ovalbumin (MVA-OVA) vaccination induces a strong OVA-specific TH 1- immune response, likely mediated by the induction of IFN-γ and IgG2a. Finally, MVA-based vaccines need to be evaluated for their therapeutic potential in established allergy models.

Cell entry targeting restricts biodistribution of replication-competent retroviruses to tumour tissue.

Virotherapy is currently being developed for many different types of viruses including replication-competent murine leukaemia virus (MLV) as a novel tool in cancer therapy. However, there is the risk of insertional mutagenesis associated with this virus, making careful preclinical studies necessary before its first application in man. We have previously generated conditionally replication-competent MLV variants that require activation by tumour-associated proteases to become infectious. Here we analysed in a comparative study the spreading of non-targeted and of such tumour-targeted MLV variants to tumour and extratumoural organs in immunodeficient mice. Both virus types were able to efficiently infect tumour cells after systemic administration. The non-targeted virus, however, also infected extratumoural organs like bone marrow, spleen and liver efficiently. In contrast, the targeted viruses revealed in a quantitative analysis of virus spreading an up to 500-fold more selective infection of tumour tissue than the non-targeted virus. The data raise serious doubts about a safe clinical use of non-targeted MLV. Engineering the virus to become activatable by tumour-associated proteases can significantly improve the safety of MLV.

Populational genetic structure of free-living maned wolves (Chrysocyon brachyurus) determined by proteic markers

Os produtos protéicos de 20 locos gênicos foram analisados eletroforeticamente em hemolisados e plasma de 28 lobos-guarás (Chrysocyon brachyurus) de uma área da região Nordeste do Estado de São Paulo, Brasil. A área de estudo foi dividida em 3 subáreas, considerando os rios Mogi-Guaçu e Pardo como barreiras ao fluxo gênico. O grau de polimorfismo e o nível de heterozigosidade (intraloco e média) estimados neste estudo foram semelhantes aos observados por outros autores para lobos-guarás e outras espécies de canídeos de vida livre. As diferentes amostras e a amostra total demonstraram freqüências genotípicas nos locos polimórficos consistentes com o modelo de equilíbrio genético. Os valores da estatística-F evidenciaram ausência de endocruzamento e de estruturação populacional e, como conseqüência, foram encontrados baixos valores de distância genética entre as amostras correspondentes a cada subárea.

NADP-dependent isocitrate dehydrogenase of Astyanax scabripinnis (Pisces, Characidae) from three altitudes at Grande Stream, Campos do Jordão, SP

Foram analisadas propriedades eletrocinéticas, térmicas e cinéticas dos produtos dos locos que codificam o isocitrato desidrogenase NADP-dependente (IDHP; E.C. 1.1.1.42) como Astyanax scabripinnis (Pisces, Characidae) em três diferentes alturas do Ribeirão Grande, Estado de São Paulo, Brasil. Nas populaçäes das três altitudes foram detectados dois locos da IDHP, bidirecionalmente divergentes, um único em músculo esquelético, o IDHP-A*, e um único em fígado, o IDHP-B*, ambos polimórficos. O único alelo variante no loco IDHP-A*, A*128, mostrou maior freqüência na população de 1.920 m (0,494). Dentre os nove alelos variantes detectados no loco IDHP-B* (*37, *57, *69, *79, *85, *114, *119, *124 e *140), os alelos *37 e *79 apareceram somente na população de 1.800 m. Valores de Qui-quadrado revelaram que para o loco IDHP-A* somente a população de 700 m não está em equilíbrio de Hardy-Weinberg, enquanto para o loco IDHP-B* nenhuma população se encontra em equilíbrio. Qui2 da homogeneidade revelou que as populaçäes são significativamente diferentes em suas freqüências fenotípicas para ambos os locos. Valor médio de FST de Wright (0,036 e 0,320, IDHP-A* e IDHP-B*, respectivamente) foi de 0,178 para populaçäes das três altitudes, o que significa que 82% da diversidade gênica total foi detectada entre indivíduos da mesma população. Estabilidades em temperatura ambiente (16§ a 21§C) e valores de Km e Vmax aparentes de extratos de músculo esquelético de cada fenótipo-A sugerem diferentes papéis das isoformas-A na lipogênese aumentada que ocorre nos peixes em baixas temperaturas.

Populational genetic structure of free-living maned wolves (Chrysocyon brachyurus) determined by proteic markers

Electrophoretic analysis of presumptive twenty gene loci products was conducted in hemolisates and plasma samples of twenty-eight maned wolves (Chrysocyon brachyurus) from an area in northeastern São Paulo State, Brazil. The area sampled was divided into three sub-areas, with the Mogi-Guaçu and Pardo rivers regarded as barriers to the gene flow. The polymorphism degree and heterozygosity level (intralocus and average) estimated in this study were similar to those detected by other authors for maned wolves and other species of wild free-living canids. The samples of each sub-area and the total sample exhibited genotype frequencies consistent with the genetic equilibrium model. The values of the F-statistics evidenced absence of inbreeding and population subdivision and, consequently, low genetic distances were found among the samples of each area.

Os produtos protéicos de 20 locos gênicos foram analisados eletroforeticamente em hemolisados e plasma de 28 lobos-guarás (Chrysocyon brachyurus) de uma área da região Nordeste do Estado de São Paulo, Brasil. A área de estudo foi dividida em 3 subáreas, considerando os rios Mogi-Guaçu e Pardo como barreiras ao fluxo gênico. O grau de polimorfismo e o nível de heterozigosidade (intraloco e média) estimados neste estudo foram semelhantes aos observados por outros autores para lobos-guarás e outras espécies de canídeos de vida livre. As diferentes amostras e a amostra total demonstraram freqüências genotípicas nos locos polimórficos consistentes com o modelo de equilíbrio genético. Os valores da estatística-F evidenciaram ausência de endocruzamento e de estruturação populacional e, como conseqüência, foram encontrados baixos valores de distância genética entre as amostras correspondentes a cada subárea.

Isoform expression in the multiple soluble malate dehydrogenase of Hoplias malabaricus (Erythrinidae, Characiformes)

Kinetic properties and thermal stabilities of Hoplias malabaricus liver and skeletal muscle unfractionated malate dehydrogenase (MDH, EC 1.1.1.37) and its isolated isoforms were analyzed to further study the possible sMDH-A* locus duplication evolved from a recent tandem duplication. Both A (A1 and A2) and B isoforms had similar optima pH (7.5-8.0). While Hoplias A isoform could not be characterized as thermostable, B could as thermolabile. A isoforms differed from B isoform in having higher Km values for oxaloacetate. The possibly duplicated A2 isoform showed higher substrate affinity than the A1. Hoplias duplicated A isoforms may influence the direction of carbon flow between glycolisis and gluconeogenesis

Astyanax scabripinnis (Pisces: Characidae) hemoglobins: structure and function

Electrophoretic patterns of hemoglobins, Root effect, Bohr effect in blood and stripped hemoglobin, Hb-O2 affinity GTP modulation of Astyanax scabripinnis (lambari), caught at three different altitudes in Ribeiräo Grande, near Campos do Jordäo (Säo Paulo), are described. All populations showed the same electrophoretic patterns: two cathodal components in starch gel. Normal Bohr effect values were found in these three populations both in blood (phi = -0,11) and stripped hemoglobin (phi = -0,12). Different blood O2 affinities collected in fish of these 3 populations were detected. GTP has a large influence on Hb-O2 binding properties in A. scabripinnis. Stripped hemoglobin shows small Root effect. The addition of triphosphated nucleotides increases this effect. GTP is more effective than ATP on enhancing Root effect. Oxygen availability in water can be the factor responsible for differences found in blood O2 affinity

Functional properties of the three hemoglobin phenotypes of Nelore cattle

Blood and stripped hemoglobin from Nelore cattle individuals were submitted to oxygen equilibrium experiments in both gasometric and spectrophotometric methods. No difference was detected in oxygen affinity and Bohr effect among Hb A, Hb B and Hb AB in experiments with both blood and stripped hemoglobin, in the presence and absence of the chloride ion. However, n values (Hill plots) were higher in Hb B and Hb AB (>2) than in Hb A (<2)

Astyanax scabripinnis (Pisces: Characidae) hemoglobins: structure and function

Electrophoretic patterns of hemoglobins, Root effect, Bohr effect in blood and stripped hemoglobin, Hb-O2 affinity GTP modulation of Astyanax scabripinnis (lambari), caught at three different altitudes in Ribeirão Grande, near Campos do Jordão (São Paulo), are described. All populations showed the same electrophoretic patterns: two cathodal components in starch gel. Normal Bohr effect values were found in these three populations both in blood (phi = -0,11) and stripped hemoglobin (phi = -0,12). Different blood O2 affinities collected in fish of these 3 populations were detected. GTP has a large influence on Hb-O2 binding properties in A. scabripinnis. Stripped hemoglobin shows small Root effect. The addition of triphosphated nucleotides increases this effect. GTP is more effective than ATP on enhancing Root effect. Oxygen availability in water can be the factor responsible for differences found in blood O2 affinity.

Padrão eletroforético de hemoglobina, efeito Root, efeito Bohr em sangue e em Hb stripped e efeito do GTP na afinidade Hb-O2 de Astyanax scabripinnis (lambari) coletados em três altitudes diferentes no Ribeirão Grande, na região de Campos do Jordão (São Paulo), são descritos. Todas as populações mostraram os mesmos padrões eletroforéticos: dois componentes catódicos em gel de amido. Efeito Bohr normal foi encontrado para as três populações, tanto em sangue (fi = -0,11) quanto em Hb stripped (fi = -0,12). Detectamos afinidades diferentes da hemoglobina ao O2 entre as três populações estudadas. GTP possui forte influência nas propriedades de ligação da hemoglobina ao oxigênio em Astyanax scabripinnis. Em hemoglobina stripped, o efeito Root é baixo. A adição de nucleotídeos trifosfatados aumenta esse efeito. GTP é mais efetivo que ATP no aumento desse efeito. A disponibilidade de O2 na água pode ser o fator responsável pelas diferenças encontradas nas afinidades da hemoglobina pelo oxigênio.

Characterization of the humoral immune response and virus replication in cats experimentally infected with feline foamy virus.

Cats were experimentally infected with cell culture-adapted feline foamy virus (FFV, spumaretrovirinae) isolate FUV. FFV was consistently recovered from peripheral blood leukocytes and throat samples of FFV-infected cats starting 2 to 3 weeks postinfection (p. i.), indicative of the establishment of persistent FFV infections. Viral persistence was established, even despite neutralizing antibodies that appeared early after infection. The humoral immune response toward FFV was quantitatively and qualitatively analyzed over time. FFV Gag-specific antibodies were first detected 2 weeks p. i. and increased further; reactivities to the other structural and nonstructural FFV proteins appeared slightly delayed. Reactivities against FFV Pol and Gag proteins were detectable by immunoblotting and radioimmunoprecipitation, whereas the latter techniques had to be employed for the unambiguous detection of FFV Env-, Bet-, and Bel 1-specific antibodies.

Adaptative features of enzymes from family sciaenidae--II. Studies on phosphoglucose isomerase (PGI) of fishes from the south coast of Uruguay.

1. The electrokinetic and thermostability properties of phosphoglucose isomerase (PGI) in three species of temperate fishes (Perciformes, Acanthopterygii)--Cynoscion striatus, Macrodon ancylodon and Micropogonias furnieri--have been analyzed in order to study the adaptative 2. Unlike most diploid fishes the PGI of these species seemed to be encoded by three PGI loci. 3. The subunits encoded by these loci occurred at different levels in the different tissues and organs analyzed. 4. Genetic variants at two loci (Pgi-A and Pgi-B2) were detected in Cynoscion striatus, and at one (Pgi-A) in Micropogonias furnieri. 5. The product of these loci could be separated in three PGI regions based on their electrophoretic distribution and thermostability properties. 6. The more anodal region (including isozymes and allozymes) was more thermolabile than the less anodal one, which is predominant in a single tissue skeletal muscle. 7. Relative activities of PGI isozymes were compared by Klebe's method to determine pattern of divergence of duplicated gene expression in the three species studied.

Hemoglobin, hematology, intraerythrocytic phosphates and whole blood Bohr effect from lotic and lentic Hypostomus regani populations (São Paulo-Brasil).

Hemoglobin electrophoretic pattern, whole blood Bohr effect, intraerythrocytic phosphates and some hematological parameters were determined and compared between lotic and lentic Hypostomus regani specimens. The electrophoretic patterns of specimens from both kinds of environments showed four anodic fractions. The relative concentration of the fraction I (the most anodal) is significantly different between specimens from lotic and lentic ambients. Both lentic and lotic specimens presented a low Bohr effect: phi = -0.012 to lentic habitat and phi = -0.187 to lotic one. ATP/GTP ratio are higher than 1.0 for lotic environment specimens and lower than 1.0 for lentic ones.

Biological aspects of amazonian fishes I. Red blood cell phosphates of schooling fishes (genus Semaprochilodus: Prochilodontidae).

Red blood cell soluble phosphates from Semaprochilodus taeniurus, S. insignis and a possible hybrid jaraqui-açu, from Amazon basin, were studied by column chromatography. The Semaprochilodus school and the jaraqui-açu shows an intermediate percentage of total phosphorus content as GTP but not as ATP. GTP/ATP ratio for jaraqui-açu and S. taeniurus were 1.5 and for S. insignis, 3.0.

Adaptative features of ectothermic enzymes. III--Studies on phosphoglucose isomerase (PGI) from five species of tropical fishes of the superorder Ostariophysi.

1. The electrokinetic and thermostability properties of phosphoglucose isomerase (PGI) in five species of tropical fish (Ostariophysi)-Leporinus friderici, L. silverstrii, Schizodon nasuttus, Hypostomus sp and Pimelodus maculatus--have been studied in order to study the adaptative nature of protein heterogeneity found in ectotherms. 2. Unlike most diploid fishes, the PGI of these species seemed to be encoded by three, four or five loci. 3. The subunits encoded by these loci occurred at different levels in the different tissues and organs analyzed. 4. Genetic variants at the three PGI loci were detected in two species of the family Anostomidae (L. friderici and S. nasuttus). In the family Pimelodidae, P. Maculatus showed a genetic variant as the Pgi-1 locus. 5. The product of these loci could be separated in three PGI regions based on their electrophoretic mobility, tissue distribution and thermostability properties. 6. The more anodal region (including isozymes and allozymes) was more thermolabile then the less anodal one, which is predominant in a single tissue-skeletal muscle.