Las células dendríticas plasmacitoides evocan la respuesta efectora de los linfocitos T citotóxicos específicos para Salmonella / The plasmacytoid dendritic cells evoke Salmonella-specific CTL effector response

Resumen Introducción. La función inmunológica de las células dendríticas plasmacitoides durante las infecciones bacterianas, como la de Salmonella spp., es poco conocida. En ese contexto, se analizó su función efectora para presentar antígenos de Salmonella Typhimurium ante linfocitos T citotóxicos. Objetivo. Analizar la respuesta de los linfocitos T citotóxicos específicos para Salmonella evocada por las células dendríticas plasmacitoides. Materiales y métodos. Se usaron células dendríticas plasmacitoides marcadas con éster de succinimidil-carboxifluoresceína, pulsadas con el epítopo de Salmonella OmpC73 Kb- restringido o infectadas con S. Typhimurium como blanco en ensayos de citotoxicidad. Resultados. La lisis específica tuvo significación estadística usando células dendríticas plasmacitoides positivas pulsadas con OmpC73 en todas las relaciones de células efectoras y blanco (E:B) (p≤0,05); en cuanto a las células dendríticas plasmacitoides positivas para S. Typhimurium, solo se observó significación estadística en la relación de 1:100 (p≤0,05) usando las células efectoras OmpC73. Conclusión. Las células dendríticas plasmacitoides pueden evocar la respuesta de los linfocitos T citotóxicos durante la infección con S. Typhimurium.

Abstract Introduction: The immunological role of plasmacytoid dendritic cells (pDC) in bacterial infections such as Salmonella has been poorly documented. Therefore, we analyzed the effector function of these cells by presenting cytotoxic T lymphocytes (CTL) with Salmonella Typhimurium antigens. Objective: To analyze the Salmonella-specific CTL response evoked by pDCs. Materials and methods: We used plasmacytoid dendritic cells stained with carboxyfluorescein succinimidyl ester (CFSE) and pulsed with OmpC73, Salmonella Kb- restricted epitopes or S. Typhimurium as targets for cytotoxicity assays. Results: Specific lysis was shown to be statistically significant in pDC + OmpC73 for all effector:target ratios (p≤0.05). For pDC + S. Typhimurium, statistical significance was only observed at a 1:100 ratio (p≤0.05) using OmpC73. Conclusion: Plasmacytoid dendritic cells evoke CTL response during S. Typhimurium infection.

The role of rpoS, hmp, and ssrAB in Salmonella enterica Gallinarum and evaluation of a triple-deletion mutant as a live vaccine candidate in Lohmann layer chickens

Salmonella enterica Gallinarum (SG) causes fowl typhoid (FT), a septicemic disease in avian species. We constructed deletion mutants lacking the stress sigma factor RpoS, the nitric oxide (NO)-detoxifying flavohemoglobin Hmp, and the SsrA/SsrB regulator to confirm the functions of these factors in SG. All gene products were fully functional in wild-type (WT) SG whereas mutants harboring single mutations or a combination of rpoS, hmp, and ssrAB mutations showed hypersusceptibility to H2O2, loss of NO metabolism, and absence of Salmonella pathogenicity island (SPI)-2 expression, respectively. A triple-deletion mutant, SGDelta3 (SGDeltarpoSDeltahmpDeltassrAB), was evaluated for attenuated virulence and protection efficacy in two-week-old Lohmann layer chickens. The SGDelta3 mutant did not cause any mortality after inoculation with either 1 x 10(6) or 1 x 10(8) colony-forming units (CFUs) of bacteria. Significantly lower numbers of salmonellae were recovered from the liver and spleen of chickens inoculated with the SGDelta3 mutant compared to chickens inoculated with WT SG. Vaccination with the SGDelta3 mutant conferred complete protection against challenge with virulent SG on the chickens comparable to the group vaccinated with a conventional vaccine strain, SG9R. Overall, these results indicate that SGDelta3 could be a promising candidate for a live Salmonella vaccine against FT.

Características definidoras da Resposta Disfuncional ao Desmame Ventilatório como indicadores de acurácia do desmame ventilatório / Defining characteristics of the Dysfunctional Ventilatory Weaning Response as indicators of accuracy of ventilatory weaning / Características definitorias de la Respuesta Disfuncional al Destete Ventilatorio como indicadores de precisión del destete ventilatorio

Objetivou-se avaliar as características definidoras do diagnóstico Resposta Disfuncional do Desmame Ventilatório, como indicadores de acurácia das tentativas de desmame. Estudo observacional de 38 eventos de tentativa de desmame ventilatório em pacientes adultos internados em terapia intensiva. Para as características definidoras foram calculadas: sensibilidade, especificidade, valores preditivos positivos e negativos, acurácia ou sensibilidade, razão de verossimilhança e razão de chances diagnóstica. Também foram consideradas as medianas do número de características definidoras nos eventos de sucesso e insucesso. Foram consideradas acuradas: agitação, deterioração nos gases sanguíneos arteriais em relação aos parâmetros basais, uso moderado da musculatura acessória da respiração, aumento da frequência respiratória em relação aos parâmetros basais e frequência respiratória aumentada de forma significativa em relação aos parâmetros basais. Houve diferença estatística nas medianas do número de características definidoras observadas. Conclui-se que a característica definidora e o número delas influenciariam o sucesso da decisão sobre o desmame.

The study aimed to analyze the defining characteristics of the Dysfunctional Ventilatory Weaning Response as an indicator of the accuracy of ventilatory weaning. Observational study of 38 events of ventilatory weaning in adult patients admitted to intensive care. For the defining characteristics, it was calculated: sensitivity, specificity, positive and negative predictive values, accuracy or efficiency, likelihood ratio positive and negative, and diagnostic odds ratio. It was also considered the median number of defining characteristics in the event of success and failure. It was considered accurate: agitation, deterioration in arterial blood gases from baseline parameters, moderate use of accessory muscles of respiration, increased respiratory rate from baseline parameters and respiratory rate increases significantly with respect to baseline parameters. There was statistical difference in the median number of defining characteristics observed. It was concluded that the defining characteristic and the number of them would influence the success of the weaning decision.

El estudio tuvo como objetivo evaluar las características que definen el diagnóstico de Respuesta Disfuncional al Destete Ventilatorio como indicador de la exactitud del destete ventilatorio. Estudio observacional de 38 eventos de destete ventilatorio en pacientes adultos ingresados en cuidados intensivos. Para las características definitorias se calcularon: sensibilidad, especificidad, valores predictivos positivos y negativos, precisión o sensibilidad, cocientes de probabilidad y odds ratio diagnóstica. Fueran consideradas las medianas del número de características definitorias en casos de éxito o de fracaso. Se consideraron precisas: agitación, deterioro de los parámetros de gases en sangre arterial desde la línea de base, uso moderado de los músculos accesorios de la respiración, aumento de la frecuencia respiratoria a partir de parámetros de línea de base y frecuencia respiratoria aumentada significativamente en comparación con los parámetros de línea de base. Hubo diferencia estadísticamente significativa en la mediana del número de características definitorias observadas. Se concluye que la característica definitoria y el número de ellas influyen en el éxito de la decisión sobre el destete ventilatorio.

Evaluation of vaccine candidate potential of ΔaroA, ΔhtrA and ΔaroAΔhtrA mutants of Salmonella enterica subspecies enterica serovar Abortusequi in guinea pigs.

Salmonella enterica subspecies enterica serovar Abortusequi (S. Abortusequi), a host adapted Salmonella causes abortions, still births and foal mortality in equids. Though known since more than 100 years, it is still a problem in many of the developing countries including India. There is dearth of really good vaccine affording immunity lasting at least for one full gestation. In search of a potential vaccine candidate, three defined deletion mutants (ΔaroA, ΔhtrA and ΔaroAΔhtrA) of S. Abortusequi were tested in guinea pig model for attenuation, safety, immunogenicity, humoral immune response, protective efficacy and persistence in host. The ΔhtrA and ΔaroAΔhtrA mutants were found to be safe on oral inoculation in doses as high as 4.2×109 cfu/animal. Also through subcutaneous inoculation ΔaroAΔhtrA mutant did not induce any abortion in pregnant guinea pigs. All the three mutants did not induce any illness or death in 1-2 week-old baby guinea pigs except ΔhtrA mutant which caused mortality on intraperitoneal inoculation. Inoculation with mutants protected against challenge and increased breeding efficiency of guinea pigs. After >4.5 months of mutant inoculation, guinea pigs were protected against abortifacient dose of wild type S. Abortusequi and mother guinea pigs also conferred resistance to their babies to the similar challenge. Early humoral immune response of S. Abortusequi mutants was characteristic. Faecal excretion of ΔaroA and htrA mutants was detected up to 45 days of inoculation in guinea pigs while ΔaroAΔhtrA mutant could not be detected after 21 days of inoculation. The results indicated that the double deletion mutant (ΔaroAΔhtrA) was the most effective and safe candidate for vaccination against S. Abortusequi through mucosal route of inoculation.

Protective efficacy of maternal antibodies induced by Salmonella toxoid (vaccine).

An attempt was made to evaluate the protective efficacy of maternal antibodies in chicks against salmonellosis. Layer chicks ageing 21 days were individually vaccinated with 100 microg of Salmonella enterica subspecies enterica serovar Weltevreden (BM 1643) toxoid adjuvanted with vitamin E subcutaneously. After 90 days of the primary vaccination the birds were given booster dose of the vaccine. The saline extract of the yolk of eggs laid by the vaccinated birds yielded agglutination and ELISA titres ranging from 43.2 +/- 5.33 to 75.2 +/- 6.26 and 4.987 x 10(3) +/- 0.54 to 5.89x103 +/- 0.56, respectively. Sera of chicks hatched from eggs laid by the vaccinated layers were also subjected to agglutination and ELISA. Agglutination and ELISA titres on the 5th day--post hatching (dph) were 21.6 +/- 1.75 and 4.025 x 10(2) +/- 0.59, while on the 10th dph titers were 13.6 +/- 1.65 and 1.21 x 10(2) +/- 0.60, respectively. It was also observed that only one out of 6 chicks died when challenged with 2 x 10(9) CFU of S. serovar Gallirarum at the age of 7 days showing 83.33% protection. Thus it can be concluded that passive immunity confided by Salmonella enterica subspecies enterica serovar Weltevreden (BM 1643) toxoid can protect chicks against salmonellosis during their early days of life.

Salmonella. typhi OMPs induced immunomodulation in peritoneal macrophages.

The immunomodulatory properties of outer membrane proteins (OMPs) from S. typhi Ty2 were studied in mouse model at 72 hr and 20 days post-infection. Inspite of reduction in the number of macrophages and their protein content observed in the immunized group vis-à-vis infected group, OMPs activated macrophages showed significant upregulation of NO. At 20 days post infection, the level remained almost the same suggesting the prolonged cytotoxic and cytostatic activity due to the long lasting effects of OMPs activated macrophages. Higher activity of SOD in these aged cells pointed out towards the protective efficacy of OMPs to keep the macrophages themselves away from the noxious effects of O2-. Lower level of acid phosphatase in the macrophages from immunized mice group indicated the involvement of oxygen dependent rather than oxygen independent killing process. The enhanced uptake of organisms and their killing could be related to the production of oxygen and nitrogen radicals in the OMPs immunized group.

Immunologic reactivity of a lipopolysaccharide-protein complex of type A Pasteurella multocida in mice

The immunologic reactivity of a lipopolysaccharide (LPS)-protein complex isolated from a potassium thiocyanate extract of a Pasteurella multocida (capsular type A and somatic type 3) strain was evaluated in mice. The LPS-protein complex provided 100% protection in mice against a challenge with the homologous strain. However, when the complex was fractionated into LPS and protein moieties by phenol-water treatment, both components lacked immunogenicity. The complex and extracted components were mitogenic for mouse B lymphocytes with the protein moiety the most active. Although immune serum against the LPS-protein complex protected mice against challenge thereby indicating a role for humoral immunity, the LPS-protein complex of P. multocida was also found to induce cell-mediated immunity. This cell-mediated immunity was demonstrated in mice immunized with the complex by: (1). mitogenic responses of T lymphocytes, (2). induction of delayed type hypersensitivity reaction in the hind footpads, and (3). enhanced resistance to challenge infection with Salmonella enteritidis.