Semliki forest virus vectors engineered to express higher IL-12 levels induce efficient elimination of murine colon adenocarcinomas

To evaluate the use of alphavirus vectors for tumor treatment we have constructed and compared two Semliki Forest virus (SFV) vectors expressing different levels of IL-12. SFV-IL-12 expresses both IL-12 subunits from a single subgenomic promoter, while in SFV-enhIL-12 each IL-12 subunit is expressed from an independent subgenomic promoter fused to the SFV capsid translation enhancer. This latter strategy provided an eightfold increase of IL-12 expression. We chose the poorly immunogenic MC38 colon adenocarcinoma model to evaluate the therapeutic potential of SFV vectors. A single intratumoral injection of 10(8) viral particles of SFV-IL-12 or SFV-enh-IL-12 induced>or=80% complete tumor regressions with long-term tumor-free survival. However, lower doses of SFV-enhIL-12 were more efficient than SFV-IL-12 in inducing antitumoral responses, indicating a positive correlation between the IL-12 expression level and the therapeutic effect. Moreover, repeated intratumoral injections of suboptimal doses of SFV-enhIL-12 increased the antitumoral response. In all cases SFV vectors were more efficient at eliminating tumors than a first-generation adenovirus vector expressing IL-12. In addition, the antitumoral effect of SFV vectors was only moderately affected by preimmunization of animals with high doses of SFV vectors. This antitumoral effect was produced, at least partially, by a potent CTL-mediated immune response

The relationship between endometrial cytology during estrous cycle and cutoff points for the diagnosis of subclinical endometritis in grazing dairy cows

The objectives of this study were to assess the effect of the stage of estrous cycle on the percentage of endometrial polymorphonuclear cells (PMN) obtained by cytobrush to determine cutoff values for the diagnosis of subclinical endometritis under pastoral conditions, to measure the prevalence of subclinical endometritis 21 to 62d in milk (DIM), and to evaluate the effect of subclinical endometritis on reproductive performance in grazing dairy cows. The first experiment was conducted on a commercial dairy farm in Buenos Aires province (Argentina), where 17 postpartum cyclic dairy cows without clinical endometritis were selected and synchronized by Ovsynch protocol. Endometrial cytology (cytobrush technique) and blood (tail vessels) samples were obtained on d0, 4, 11, and 18 of the estrous cycle (corresponding to estrus, metestrus, diestrus, and proestrus, respectively) and used for measuring percentage of PMN and P4 concentration, respectively. The percentage of PMN was determined 3times by blinded count by 2 operators. Data were analyzed with PROC MIXED, PROC GENMOD, and PROC FREQ from SAS 9.1. The percentage of PMN did not vary with the stage of the estrous cycle. In addition, PMN counts were below any of the reported thresholds in this study (4%) for most of the cows. Therefore, the risk for false positive test results as a consequence of physiological changes in the counts of PMN during estrous cycle is low. The second experiment was conducted on 4 commercial dairy farms in Buenos Aires province (Argentina), where lactating Holstein dairy cows (n = 418) 21 to 62 DIM without clinical endometritis were studied. Samples of endometrial cytology were collected with the cytobrush technique. Data were analyzed with receiver operator characteristic curves with Sigmaplot 10.0, and with PROC GLIMMIX, PROC PHREG, and PROC LIFETEST from SAS 9.1. Cutoff values for the diagnosis of subclinical endometritis in grazing dairy cows are 8% PMN for 21 to 33 DIM, 6% PMN for 34 to 47 DIM, 4% PMN for 48 to 62 DIM, and overall 5% PMN for 21 to 62 DIM; the prevalence of subclinical endometritis 21 to 62 DIM was 17%. Finally, subclinical endometritis diagnosed at 21 to 62 DIM decreases the hazard for pregnancy (hazard ratio = 0.668; 95% confidence interval = 0.492-0.909) and increases the calving to conception interval by d30 compared with normal cows (median 95% confidence interval = 133 vs. 93, respectively).Facultad de Ciencias Veterinaria

Foliar lead uptake by lettuce exposed to atmospheric fallouts

Metal uptake by plants occurs by soil−root transfer but also by direct transfer of contaminants from the atmosphere to the shoots. This second pathway may be particularly important in kitchen gardens near industrial plants. The mechanisms of foliar uptake of lead by lettuce (Lactuca sativa) exposed to the atmospheric fallouts of a lead-recycling plant were studied. After 43 days of exposure, the thoroughly washed leaves contained 335 ± 50 mg Pb kg−1 (dry weight). Micro-X-ray fluorescence mappings evidenced Pb-rich spots of a few hundreds of micrometers in diameter located in necrotic zones. These spots were more abundant at the base of the central nervure. Environmental scanning electron microscopy coupled with energy dispersive X-ray microanalysis showed that smaller particles (a few micrometers in diameter) were also present in other regions of the leaves, often located beneath the leaf surface. In addition, submicrometric particles were observed inside stomatal openings. Raman microspectrometry analyses of the leaves identified smelter-originated Pb minerals but also secondary phases likely resulting from the weathering of original particles. On the basis of these observations, several pathways for foliar lead uptake are discussed. A better understanding of these mechanisms may be of interest for risk assessment of population exposure to atmospheric metal contamination

Determining thresholds for the diagnosis of subclinical endometritis in Argentina

La endometritis subclínica (ES) afecta negativamente a la reproducción de las vacas de tambo al aumentar el intervalo entre partos. Para su diagnóstico, se debe tomar una muestra citológica del útero y determinar el porcentaje de neutrófilos (PPMN) que se encuentra superficialmente en el endometrio. Distintos PPMN han sido utilizados como puntos de corte de diagnóstico en vacas estabuladas. No es posible extrapolar datos de explotaciones intensivas a las condiciones argentinas de producción, tampoco el uso de valores de corte arbitrarios, debido a que se pueden llegar a interpretar en forma errónea los resultados arribando a diagnósticos incorrectos. Por lo tanto, el objetivo de este trabajo fue determinar la prevalencia de la ES diagnosticada por la técnica de CB en Argentina y buscar los niveles de corte que se correspondan con una disminución de la eficiencia reproductiva. Se muestrearon y evaluaron 487 vacas con flujo vaginal normal entre 21 y 62 días postparto (dpp). Los datos fueron analizados por SAS y SigmaPlot. Las curvas ROC (acrónimo de Característica Operativa del Receptor) obtenidas arrojaron valores de corte de 8, 6, 4 y 5 PPMN para los intervalos 21-33, 34-47, 48-62 y 21-62 dpp respectivamente. Las vacas con ES tuvieron menor tasa de concepción al primer servicio (16,2%) y tuvieron 29 días más de abiertas comparados con vacas sin ES.Subclinical endometritis (SE) adversely affects the reproduction of dairy cows by increasing the number of days open. For diagnosis of subclinical endometritis, it is necessary to take a cytological sample of the uterus and determine the percent of neutrophils (PPMN) present at the endometrial surface. Different percentages have been used as diagnostic cutoffs in dairy cows. It is not possible to extrapolate data of intensive productions to Argentine pastoral conditions, or to use arbitrary cutoff values. This could result in erroneous interpretation of results and misdiagnosis of subclinical endometritis. The aim of this study was to validate cytobrush technique in our country and find cutoff values which correspond to a real decline in reproductive efficiency. For this work 487 cows with normal vaginal discharge between 21 and 62 days postpartum (dpp) were sampled and subclinical endometritis was diagnosed. Data were analyzed by SAS and Sigma Plot. ROC (Receiver Operating Characteristic) curves generated cutoff values of 8, 6, 4 and 5 PPMN for the intervals 21-33, 34-47, 48-62 and 21-62 dpp, respectively. Cows with subclinical endometritis had a 16.2% reduction in first conception rate and had 29 more open days than cows without subclinical endometritis.Fil: Madoz, Laura Vanina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Teriogenología. Cátedra de Reproducción Animal; ArgentinaFil: Plöntzke, J.. Freie Universitat Berlin (freie Universitat Berlin); AlemaniaFil: Albarracín, D.. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Cátedra de Patología Especial; ArgentinaFil: Drillich, M.. Universidad de Medicina Veterinaria; AustriaFil: de la Sota, Rodolfo Luzbel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Teriogenología. Cátedra de Reproducción Animal; Argentin

Increased efficacy and safety in the treatment of experimental liver cancer with a novel adenovirus-alphavirus hybrid vector

An improved viral vector for cancer gene therapy should be capable of infecting tumors with high efficiency, inducing specific and high-level expression of transgene in the tumor and selectively destroying tumor cells. In the design of such a vector to treat hepatocellular carcinoma, we took advantage of (a) the high infectivity of adenoviruses for hepatic cells, (b) the high level of protein expression and proapoptotic properties that characterize Semliki Forest virus (SFV) replicon, and (c) tumor selectivity provided by alpha-fetoprotein (AFP) promoter. We constructed a hybrid viral vector composed of a helper-dependent adenovirus containing an SFV replicon under the transcriptional control of AFP promoter and a transgene driven by SFV subgenomic promoter. Hybrid vectors containing murine interleukin-12 (mIL-12) genes or reporter gene LacZ showed very specific and high-level expression of transgenes in AFP-expressing hepatocellular carcinoma cells, both in vitro and in an in vivo hepatocellular carcinoma animal model. Infected hepatocellular carcinoma cells were selectively eliminated due to the induction of apoptosis by SFV replication. In a rat orthotopic liver tumor model, treatment of established tumors with a hybrid vector carrying mIL-12 gene resulted in strong antitumoral activity without accompanying toxicity. This new type of hybrid vectors may provide a potent and safe tool for cancer gene therapy

Espectrofotometría infrarroja transformada de Fourier para identificar bacterias uterinas patógenas en vacas lecheras

El objetivo del trabajo fue determinar el grado de acuerdo entre dos métodos de identificación bacteriana, marcha bacteriológica convencional y espectroscopía infrarroja transformada de Fourier (TF-IR), así como determinar si la base de datos construida a partir de bacterias aisladas de úteros de vaca lecheras de Europa podía ser utilizada para identificar bacterias uterinas de vacas lecheras de Argentina. Las muestras bacteriológicas fueron tomadas a vacas post parto (n=64) con distinto grado de descarga vaginal (DV). Inicialmente se efectuó diagnostico bacteriológico por marcha bacteriológica convencional (n=27) y posteriormente por TF-IR. El grado de acuerdo entre ambos métodos de identificación bacteriana fue determinado por el coeficiente kappa y el efecto de la DV sobre el aislamiento de Escherichia coli y Trueperella pyogenes fue analizado por regresión logística. El grado de acuerdo entre ambos métodos tuvo un coeficiente kappa de 0,73 (p<0,001). La probabilidad de aislamiento bacteriano (E. coli y T. pyogenes) tendió a incrementarse junto con el grado de DV (p=0,06), siendo 1,88 veces más alta la probabilidad de encontrar un resultado positivo a E. coli o T. pyogenes en vacas con DV fétida que en vacas con DV normal. En conclusión, TF-IR reveló un buen grado de acuerdo con la marcha  acteriológica convencional. Por otro lado, la base de datos producida a partir de bacterias uterinas de vacas lecheras de Europa pudo ser utilizada para la identificación de bacterias uterinas de vacas lecheras de Argentina

Treatment of chronic viral hepatitis in woodchucks by prolonged intrahepatic expression of interleukin-12

Chronic hepatitis B is a major cause of liver-related death worldwide. Interleukin-12 (IL-12) induction accompanies viral clearance in chronic hepatitis B virus infection. Here, we tested the therapeutic potential of IL-12 gene therapy in woodchucks chronically infected with woodchuck hepatitis virus (WHV), an infection that closely resembles chronic hepatitis B. The woodchucks were treated by intrahepatic injection of a helper-dependent adenoviral vector encoding IL-12 under the control of a liver-specific RU486-responsive promoter. All woodchucks with viral loads below 10(10) viral genomes (vg)/ml showed a marked and sustained reduction of viremia that was accompanied by a reduction in hepatic WHV DNA, a loss of e antigen and surface antigen, and improved liver histology. In contrast, none of the woodchucks with higher viremia levels responded to therapy. The antiviral effect was associated with the induction of T-cell immunity against viral antigens and a reduction of hepatic expression of Foxp3 in the responsive animals. Studies were performed in vitro to elucidate the resistance to therapy in highly viremic woodchucks. These studies showed that lymphocytes from healthy woodchucks or from animals with low viremia levels produced gamma interferon (IFN-gamma) upon IL-12 stimulation, while lymphocytes from woodchucks with high viremia failed to upregulate IFN-gamma in response to IL-12. In conclusion, IL-12-based gene therapy is an efficient approach to treat chronic hepadnavirus infection in woodchucks with viral loads below 10(10) vg/ml. Interestingly, this therapy is able to break immunological tolerance to viral antigens in chronic WHV carriers

The relationship between endometrial cytology during estrous cycle and cutoff points for the diagnosis of subclinical endometritis in grazing dairy cows

The objectives of this study were to assess the effect of the stage of estrous cycle on the percentage of endometrial polymorphonuclear cells (PMN) obtained by cytobrush to determine cutoff values for the diagnosis of subclinical endometritis under pastoral conditions, to measure the prevalence of subclinical endometritis 21 to 62d in milk (DIM), and to evaluate the effect of subclinical endometritis on reproductive performance in grazing dairy cows. The first experiment was conducted on a commercial dairy farm in Buenos Aires province (Argentina), where 17 postpartum cyclic dairy cows without clinical endometritis were selected and synchronized by Ovsynch protocol. Endometrial cytology (cytobrush technique) and blood (tail vessels) samples were obtained on d0, 4, 11, and 18 of the estrous cycle (corresponding to estrus, metestrus, diestrus, and proestrus, respectively) and used for measuring percentage of PMN and P4 concentration, respectively. The percentage of PMN was determined 3times by blinded count by 2 operators. Data were analyzed with PROC MIXED, PROC GENMOD, and PROC FREQ from SAS 9.1. The percentage of PMN did not vary with the stage of the estrous cycle. In addition, PMN counts were below any of the reported thresholds in this study (4%) for most of the cows. Therefore, the risk for false positive test results as a consequence of physiological changes in the counts of PMN during estrous cycle is low. The second experiment was conducted on 4 commercial dairy farms in Buenos Aires province (Argentina), where lactating Holstein dairy cows (n = 418) 21 to 62 DIM without clinical endometritis were studied. Samples of endometrial cytology were collected with the cytobrush technique. Data were analyzed with receiver operator characteristic curves with Sigmaplot 10.0, and with PROC GLIMMIX, PROC PHREG, and PROC LIFETEST from SAS 9.1. Cutoff values for the diagnosis of subclinical endometritis in grazing dairy cows are 8% PMN for 21 to 33 DIM, 6% PMN for 34 to 47 DIM, 4% PMN for 48 to 62 DIM, and overall 5% PMN for 21 to 62 DIM; the prevalence of subclinical endometritis 21 to 62 DIM was 17%. Finally, subclinical endometritis diagnosed at 21 to 62 DIM decreases the hazard for pregnancy (hazard ratio = 0.668; 95% confidence interval = 0.492-0.909) and increases the calving to conception interval by d30 compared with normal cows (median 95% confidence interval = 133 vs. 93, respectively).Facultad de Ciencias Veterinaria