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.

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.

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

Electrokinetic, thermic, and kinetic properties of products of NADP-dependent isocitrate dehydrogenase (IDHP; EC 1.1.1.42) loci of Astyanax scabripinnis (Pisces, Characidae) collected at three different altitudes (700 m, 1,800 m, and 1,920 m) of Grande Stream at Campos do Jordão, State of São Paulo, Brazil, were analyzed. Two IDHP bidirectionally divergent loci, a single skeletal muscle, the IDHP-A*, and a single liver IDHP-B*, both polymorphic, were detected in the three different altitude populations. The variant allele *128 at the IDHP-A* locus, had its highest frequency detected in the 1,920 m population (0.494). Among the nine variant alleles detected at the IDHP-B* locus (*37, *57, *69, *79, *85, *114, *119, *124, and *140), the *37 and 79 were detected only in 1,800 m population. Chi-square values showed that only the 700 m population is not in Hardy-Weinberg equilibrium for the IDHP-A* locus, while for the IDHP-B* locus, no population is. Homogeneity Chi2 test indicated that the populations are significantly different in their A and B phenotype frequencies. Wright's FST mean value (0.036 and 0.32, IDHP-A* and IDHP-B*, respectively) was 0.178 for the three altitude populations which means that 82% of total genetic diversity was found among individuals of each one of the populations. Stability at environmental temperatures (16 degrees to 21 degrees C), and apparent Km and Vmax values of each A-phenotype skeletal muscle crude extract suggest different roles of A-isoforms during the increased lipogenesis that occurs in fish at low temperatures.

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.

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.

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.

The bet gene of feline foamy virus is required for virus replication.

Foamy viruses (FV) are complex retroviruses with additional bel genes located between env and the 3' long-terminal repeat. The functions of the bel 2 and bet genes are unknown and both are dispensable for replication of the prototypic human foamy virus in cell cultures. We examined the function(s) of bel 2 and bet of the distantly related feline foamy virus (FFV) in the proviral context. Mutagenesis was used to alter the Bel 2 and Bet or to abrogate their expression. The Bel 2/Bet mutants showed a 1000-fold reduced viral titer in feline kidney cells; in human 293T cells, viral titer was only about 10-fold reduced compared to wild-type FFV. In both cell types, the Bel 2/Bet mutations resulted in a reduced release of FFV particles. The results indicate that FFV Bet is required for efficient virus replication. The functions of the Bel 2 and Bet proteins are discussed.

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.

Measurement of activities of daily living in hospitalized elderly: a comparison of self-report and performance-based methods.

OBJECTIVE: To determine the accuracy of self-reports of physical functioning by hospitalized elderly. DESIGN: Comparison of two measures. PATIENTS AND SETTING: Two-hundred forty-seven medical inpatients (mean age 78.7 years) hospitalized at St. Marys Hospital Medical Center, Madison, WI. MAIN OUTCOME MEASURES: Measures of five activities of daily living by self-report and by performance. RESULTS: The rate of agreement between self-report and performance ADL measures was the lowest in the areas of bathing and dressing where the agreement was 63% and 64%, respectively. When patients reported needing no help in these two tasks, they were measured lower 32% of the time for dressing and 42% for bathing. When patients reported needing help in an activity the agreement rate between patient and occupational therapist varied widely, from only 42% for toileting to 78% for bathing. The two factors which were statistically associated with poor agreement between the two ADL measurements were cognitive impairment (P less than 0.001) and a decline from the pre-hospital level of ADL functioning which had occurred during hospitalization (P less than 0.001). CONCLUSIONS: These data suggest that there may be significant differences between patient assessments and performance-based measurements of ADL functioning in hospitalized elderly at time of discharge. These differences may have implications for the collection of functional measurements for discharge planning or for geriatric research in the hospital environment.

Adaptative features of enzymes from family sciaenidae (Perciformes)--I. Studies on soluble malate dehydrogenase (s-MDH) and creatinine kinase (CK) of fishes from the south coast of Uruguay.

1. Electrophoretic analysis of the soluble malate dehydrogenase (s-MDH) and creatine kinase (CK) isozyme patterns of three species of temperate fishes (Scianidae, Perciformes) indicates at least two loci for s-MDH, Mdh-A and Mdh-B, and four CK, Ck-A, Ck-B, Ck-C and Ck-D. 2. The subunits encoded by these loci occurred at different levels in different tissues and organs analyzed. 3. Through electrophoretic analysis the products of these loci showed different behaviour to changes in temperature. 4. Relative activities of s-MDH and CK isozymes were compared by Klebe's (1975) method to determine pattern of divergence of duplicated gene expression in the three studied species.

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.

Studies on the influence of nitrite on methemoglobin formation in Amazonian fishes.

Twenty one species of fishes, collected from the Rio Solimões and a tributary lake in the Amazon Basin near Manaus, showed a wide range of methemoglobin formation 1 hr after a dose of 30 mg/kg of sodium nitrite i.p. Methemoglobin formation in two experimental fishes, Brycon cf. melanopterum and Semaprochilodus insignis, maintained in tanks in our INPA laboratory, was studied in detail. Both fishes survived a dose of 10 mg/kg of nitrite i.p. but usually died within 3 hr of a dose of 30 mg/kg with levels of blood methemoglobin in excess of 80%. Methemoglobin produced in vitro by addition of nitrite to fresh blood was slowly reduced back to hemoglobin over a period of several hours at room temperature. Hemoglobin in hemolysates was auto-oxidized to methemoglobin at pH 6.1 and below but not at 6.9 and above.

Adaptative features of enzymes from family Sciaenidae--III. Studies on lactate dehydrogenase (LDH) of fishes from the south coast of Uruguay.

1. Electrophoretic analyses of lactate dehydrogenase isozyme patterns of three species of temperate fish belonging to family Sciaenidae (order Perciformes) indicates that at least three LDH loci--Ldh-A, Ldh-B and Ldh-C are active. 2. The subunits encoded by these loci occurred at different levels in the different tissues and organs analyzed. 3. The products of these loci show different response to changes in temperature. The results were obtained by electrophoretic analyses. 4. Thermostability of skeletal muscle, heart and eye lactate dehydrogenase incubated at 60 degrees C for different periods of time were examined. 5. Relative activities of LDH isozymes were compared by Klebe's method to determine pattern of divergence of duplicated gene expression in the three species studied.