An unexpected inhibition of antiviral signaling by virus-encoded tumor suppressor p53 in pancreatic cancer cells

AbstractVirus-encoded tumor suppressor p53 transgene expression has been successfully used in vesicular stomatitis virus (VSV) and other oncolytic viruses (OVs) to enhance their anticancer activities. However, p53 is also known to inhibit virus replication via enhanced type I interferon (IFN) antiviral responses. To examine whether p53 transgenes enhance antiviral signaling in human pancreatic ductal adenocarcinoma (PDAC) cells, we engineered novel VSV recombinants encoding human p53 or the previously described chimeric p53-CC, which contains the coiled-coil (CC) domain from breakpoint cluster region (BCR) protein and evades the dominant-negative activities of endogenously expressed mutant p53. Contrary to an expected enhancement of antiviral signaling by p53, our global analysis of gene expression in PDAC cells showed that both p53 and p53-CC dramatically inhibited type I IFN responses. Our data suggest that this occurs through p53-mediated inhibition of the NF-κB pathway. Importantly, VSV-encoded p53 or p53-CC did not inhibit antiviral signaling in non-malignant human pancreatic ductal cells, which retained their resistance to all tested VSV recombinants. To the best of our knowledge, this is the first report of p53-mediated inhibition of antiviral signaling, and it suggests that OV-encoded p53 can simultaneously produce anticancer activities while assisting, rather than inhibiting, virus replication in cancer cells

Mycobacterium bovis in Swine: Spoligotyping of Isolates from Argentina

A total of 143 Mycobacterium bovis isolates of pigs, from the most productive swine area in Argentina, were typed by spoligotyping. Twenty-two different spoligotypes were identified, and 133 (93%) isolates were grouped into 12 clusters. One of them, designed SB0140, was the most frequent because it held 83 (58%) isolates. This spoligotype also grouped 362 (43%) out of 841 isolates from previously typed cattle and, thus, constitutes the most frequent in our country. In addition, 135 (94%) isolates revealed spoligotypes identical to those of cattle, showing an epidemiological link. On the other hand, there were seven novel spoligotypes, six of which were also unique since they had only one isolate each. This study aimed to identify the spoligotypes of M. bovis isolated from pigs to contribute to a better understanding of the distribution of bovine tuberculosis in the main productive area of Argentina

Detection of Mycobacterium bovis-Infected Dairy Herds Using PCR in Bultk Tank Milk Samples

Bovine tuberculosis (bTB) is a chronic and zoonotic disease due to Mycobacterium bovis. The tuberculosis eradication campaign carried out in Argentina has considerably improved the health situation of the herds. Here we evaluated a strategy to detect M. bovis-infected herds by Touch-Down IS6110 polymerase chain reaction (PCR) in bulk tank raw milk from dairy farms. We evaluated 177 samples from herds with the official tuberculosis free certificate (TFC) and 80 from herds without the certificate, non-tuberculosis-free certificate (NTFC), from 10 departments of Santa Fe province, Argentina. To avoid the effect of Taq polymerase inhibitors, a dilution of DNA template was performed. Positive PCR results were obtained in 102 (40%) of the samples, whereas negative ones were obtained in 155 (60%) of the samples. Importantly, 44% of NTFC and 38% of TFC samples were positive. All samples were subjected to culture in Löwenstein Jensen and Stonebrink media with no positive isolation. The negative predictive value (NPV) of PCR in the TFC group was 95%, while the positive predictive value (PPV) of PCR in the NTFC group was 51%. Based on these results, this work proposes a method that should be applied regularly to detect M. bovis--infected dairy herds, complementary to the official test of tuberculin, or purifed protein derivative (PPD), to control dairy herds, especially those free of tuberculosis.Fil: Zumárraga, Martín José. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Soutullo, Adriana Rosa. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Garcia, Maria Ines. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional del Litoral; ArgentinaFil: Marini, Rocío. Universidad Nacional del Litoral; ArgentinaFil: Abdala, Alejandro Ariel. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Santa Fe. Estación Experimental Agropecuaria Rafaela; ArgentinaFil: Tarabla, Héctor. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Santa Fe. Estación Experimental Agropecuaria Rafaela; ArgentinaFil: Echaide, Susana. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Santa Fe. Estación Experimental Agropecuaria Rafaela; ArgentinaFil: López, Marcela. Dirección Nacional de Instituto de Investigación.Administración Nacional de Laboratorios e Institutos de Salud "Dr. Carlos G. Malbrán"; ArgentinaFil: Zerbini, Elsa Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Dirección Nacional de Instituto de Investigación.Administración Nacional de Laboratorios e Institutos de Salud "Dr. Carlos G. Malbrán"; ArgentinaFil: Canal, Ana. Universidad Nacional del Litoral; ArgentinaFil: Cataldi, Ángel Adrián. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Interlaboratory test: Isolation of Mycobacterium bovis from granulomatous lesions in bovine

Mycobacterium bovis es el agente etiológico de la tuberculosis bovina. La confirmación diagnóstica en el laboratorio se realiza a través del aislamiento bacteriológico. Los ensayos interlaboratorio permiten evaluar el desempeno˜ de cada laboratorio participante comparándolo con otros de capacidades similares. El objetivo del presente estudio fue determinar la eficiencia en el aislamiento de M. bovis. Este estudio contó con la participación de 4 laboratorios, los cuales procesaron a ciego 25 muestras de tejidos con lesiones, seleccionadas previamente por aislamiento. El laboratorio A obtuvo la mayor proporción de aislamientos (68%), seguido del C (60%) y luego del B y D (ambos con el 52%). Las mayores concordancias se observaron entre los pares de laboratorios B-C y B-D (68%). Las diferencias pudieron deberse a factores propios de los procedimientos en cada laboratorio. Este tipo de análisis interlaboratorio permite evidenciar posibles errores en los procedimientos bacteriológicos e identificar puntos críticos del proceso para detectar M. bovis de manera eficiente.Mycobacterium bovis is the causative agent of bovine tuberculosis. The diagnostic laboratory confirmation is made through bacterial isolation. The aim of interlaboratory tests is to assess the performance of each participant in comparison with other of similar capacities. The test objective was to determine the efficiency of isolation of M. bovis. Four laboratories were part of the test and processed 25 blind tissue samples from granulomatous lesions and with previous M. bovis isolation. The laboratory that had the highest proportion of isolates was A (68%), followed by C (60%) and then B and D (both with 52%). The greatest concordance was observed between B-D and B-C laboratories (68%). The differences could be due to specific factors in each laboratory procedures. This type of interlaboratory tests highlights errors in the bacteriology and identifies critical points in the process to detect M. bovis accurately.Fil: Garbaccio, Sergio Gabriel. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Patobiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Barandiaran, Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias; ArgentinaFil: Fernandez, Analía. No especifíca;Fil: Macias, Analía. Universidad Nacional de Río Cuarto. Facultad de Agronomía y Veterinaria; ArgentinaFil: Magnano, Gabriel. Universidad Nacional de Río Cuarto. Facultad de Agronomía y Veterinaria; ArgentinaFil: Martinez Vivot, Marcela. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias; ArgentinaFil: Peyrú, Maite. No especifíca;Fil: Cataldi, Ángel Adrián. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Differential Effects of DNA Double-Strand Break Repair Pathways on Single-Strand and Self-Complementary Adeno-Associated Virus Vector Genomes▿

The linear DNA genomes of recombinant adeno-associated virus (rAAV) gene delivery vectors are acted upon by multiple DNA repair and recombination pathways upon release into the host nucleus, resulting in circularization, concatemer formation, or chromosomal integration. We have compared the fates of single-strand rAAV (ssAAV) and self-complementary AAV (scAAV) genomes in cell lines deficient in each of three signaling factors, ATM, ATR, and DNA-PKCS, orchestrating major DNA double-strand break (DSB) repair pathways. In cells deficient in ATM, transduction as scored by green fluorescent protein (GFP) expression is increased relative to that in wild-type (wt) cells by 2.6-fold for ssAAV and 6.6-fold for scAAV vectors, arguing against a mechanism related to second-strand synthesis. The augmented transduction is not reflected in Southern blots of nuclear vector DNA, suggesting that interactions with ATM lead to silencing in normal cells. The additional functional genomes in ATM−/− cells remain linear, and the number of circularized genomes is not affected by the mutation, consistent with compartmentalization of genomes into different DNA repair pathways. A similar effect is observed in ATR-deficient cells but is specific for ssAAV vector. Conversely, a large decrease in transduction is observed in cells deficient in DNA-PKCS, which is involved in DSB repair by nonhomologous end joining rather than homologous recombination. The mutations also have differential effects on chromosomal integration of ssAAV versus scAAV vector genomes. Integration of ssAAV was specifically reduced in ATM−/− cells, while scAAV integration was more profoundly inhibited in DNA-PKCS−/− cells. Taken together, the results suggest that productive rAAV genome circularization is mediated primarily by nonhomologous end joining

Bovine tuberculosis in domestic pigs: Genotyping and distribution of isolates in Argentina

Bovine tuberculosis is caused by Mycobacterium bovis and affects primarily cattle, among many other mammal species. In this study, 250 isolates of M. bovis collected from pigs slaughtered in Argentina were typed by spoligotyping. Over half of the isolates (66%) grouped into two spoligotypes. Moreover, SB0140 was the most frequent spoligotype detected in the three performed samplings. In addition, 195 isolates were typed through variable number of tandem repeats (VNTR) by selecting 7 loci (MIRU 16–26–31 and ETR A–B–C–D). The relationship among the patterns was performed using a goeBURST algorithm and the main clonal complexes grouped 110 isolates (56%). Although pigs shared genotypes with cattle (n = 21), some patterns were detected only in pigs (n = 14). These findings suggest the pig as a source of M. bovis infection to cattle.Instituto de BiotecnologíaFil: Barandiaran, Soledad. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias; ArgentinaFil: Martinez Vivot, Marcela. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias; ArgentinaFil: Pérez, Andrés M. University of Minnesota. College of Veterinary Medicine; Estados UnidosFil: Cataldi, Angel Adrian. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Zumarraga, Martin Jose. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentin

Bovine tuberculosis in domestic pigs: Genotyping and distribution of isolates in Argentina

Bovine tuberculosis is caused by Mycobacterium bovis and affects primarily cattle, among many other mammal species. In this study, 250 isolates of M. bovis collected from pigs slaughtered in Argentina were typed by spoligotyping. Over half of the isolates (66%) grouped into two spoligotypes. Moreover, SB0140 was the most frequent spoligotype detected in the three performed samplings. In addition, 195 isolates were typed through variable number of tandem repeats (VNTR) by selecting 7 loci (MIRU 16–26–31 and ETR A–B–C–D). The relationship among the patterns was performed using a goeBURST algorithm and the main clonal complexes grouped 110 isolates (56%). Although pigs shared genotypes with cattle (n = 21), some patterns were detected only in pigs (n = 14). These findings suggest the pig as a source of M. bovis infection to cattle.Fil: Barandiaran, Soledad. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Martinez Vivot, Marcela. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Perez, Andres Maximiliano. University of Minnesota; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Cataldi, Ángel Adrián. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Zumárraga, Martín José. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Tuberculosis in swine co-infected with Mycobacterium avium subsp. hominissuis and Mycobacterium bovis in a cluster from Argentina

In Argentina little is known about the epidemiology of tuberculosis (TB) infection in swine. We characterized the epidemiological dynamics of Mycobacterium avium complex (MAC) infection in a swine population of Argentina using molecular tools and spatial analysis techniques. Isolates (n = 196) obtained from TB-like lesions (n = 200) were characterized by polymerase chain reaction. The isolates were positive to either M. bovis (IS6110) (n = 160) or M. avium (IS1245) (n = 16) while the remaining 20 (10·2%) isolates were positive to both M. bovis and M. avium. The detection of both bacteria together suggests co-infection at the animal level. In addition, MAC-positive isolates (n = 36) were classified as M. avium subsp. avium (MAA) (n = 30) and M. avium subsp. hominissuis (MAH) (n = 6), which resulted in five genotypes when they were typed using mycobacterial interspersed repetitive unit, variable number of tandem repeats (MIRU-VNTR). One significant (P = 0·017) spatial clustering of genotypes was detected, in which the proportion of MAH isolates was larger than expected under the null hypothesis of even distribution of genotypes. These results show that in Argentina the proportion of TB cases in pigs caused by M. avium is larger than that reported in earlier studies. The proportion of M. bovis–MAC co-infections was also higher than in previous reports. These results provide valuable information on the epidemiology of MAC infection in swine in Argentina.Instituto de BiotecnologíaFil: Barandiaran, Soledad. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias; ArgentinaFil: Pérez, Andrés M. University of Minnesota. College of Veterinary Medicine; Estados UnidosFil: Gioffre, Andrea. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Martinez Vivot, Marcela. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias; ArgentinaFil: Cataldi, Angel Adrian. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Zumarraga, Martin Jose. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentin