Sumario: I. IntroduccIon: LAs funcIones deL regImen dIscIpLInArIo en eL ambIto penItencIArIo como... more Sumario: I. IntroduccIon: LAs funcIones deL regImen dIscIpLInArIo en eL ambIto penItencIArIo como fundAmento deL mIsmo. II. LA normAtIvA vIgente en LA mAterIA y Los prIncIpIos ApLIcAbLes AL ejercIcIo de LA potestAd dIscIpLInArIA penItencIArIA. 1. La situacion normativa. 2. A vueltas con las relaciones de sujecion especial como paradigma que justifica la regulacion del ambito normativo disciplinario ?de verdad estamos frente a la aplicacion de ius puniendi del estado? 3. Los principios que informan el ejercicio de la potestad disciplinaria penitenciaria: especial referencia al principio de Legalidad. III. LA preservAcIon de LA segurIdAd y eL orden en Los estAbLecImIentos penItencIArIos: InfrAccIones, sAncIones y medIdAs coercItIvAs. 1. Las infracciones previstas en la normativa reglamentaria. 2. Las sanciones correspondientes a las infracciones: especial referencia a la correlacion entre infraccion y sancion. 3. Los medios coercitivos autorizados en el regimen disciplinario. Iv. eL p...
Infection of bovines withMycobacterium boviscauses important financial hardship in many countries... more Infection of bovines withMycobacterium boviscauses important financial hardship in many countries presenting also a risk for humans.M. bovisis known to be adapted to survive and thrive within the intramacrophage environment. In spite of its relevance, at present the information about macrophage expression patterns is scarce, particularly regarding the bovine host. In this study, transcriptomic analysis was used to detect genes differentially expressed in macrophages derived from peripheral blood mononuclear cells at early stages of infection with two Argentinean strains ofM. bovis, a virulent and an attenuated strains. The results showed that the number of differentially expressed genes in the cells infected with the virulent strain (5) was significantly lower than those in the cells infected with the attenuated strain (172). Several genes were more strongly expressed in infected macrophages. Among them, we detected encoding transcription factors, anthrax toxin receptor, cell divisi...
A Mycobacterium bovis strain deleted in mce2A and mce2B genes (M. bovis Δmce2) was tested as an e... more A Mycobacterium bovis strain deleted in mce2A and mce2B genes (M. bovis Δmce2) was tested as an experimental vaccine in cattle challenged with a virulent M. bovis strain. Three-and-a-half-month old calves (n = 5 to 6 per group) were vaccinated and challenged with a virulent strain of M. bovis by the intratracheal route 9 weeks after vaccination. A non-vaccinated group and a group vaccinated with BCG were included as controls. Blood samples were collected to measure IFN-γ by an interferon-gamma release assay (IGRA), cytometry and cytokine responses of bovine purified protein derivative (PPD) restimulated peripheral blood mononuclear cells (PBMCs). The IGRA test showed IFN-γ values similar to pre-vaccination except for the animals vaccinated with M. bovis Δmce2, where a significant increase was observed at 30 days post-vaccination. The expression of IL-2R on CD4(+) cells in response to PPD from the animals vaccinated with Δmce2 increased at 15 days post-vaccination compared to cells from non-vaccinated group. Vaccination of cattle with M. bovis Δmce2 induced the highest (P < 0.05) expression of IFN-γ and IL-17 mRNA upon PPD stimulation of PBMCs compared to vaccination with BCG or that for the non-vaccinated group. There was a weak positive correlation between the production of these proinflammatory cytokines post-vaccination and reduced pathology scores post-challenge. The animals were euthanized and necropsied 100 days after challenge. The group vaccinated with M. bovis Δmce2 displayed a significantly lower histopathological score for lesions in lungs and pulmonary lymph nodes than for the other groups (P < 0.05). A marked positive reaction to tuberculin intradermal test was observed post-vaccination in animals vaccinated with M. bovis Δmce2 compared to those vaccinated with BCG or the non-vaccinated group. In contrast, after challenge, non-vaccinated animals had greater skin test responses than the vaccinated animals. In summary, M. bovis Δmce2 is a promising vaccine candidate to control M. bovis pathogenesis in cattle.
AMycobacterium bovisknockout inp27-p55operon was tested as an antituberculosis experimental vacci... more AMycobacterium bovisknockout inp27-p55operon was tested as an antituberculosis experimental vaccine in animal models. The mutant MbΔp27-p55 was significantly more attenuated in nude mice than its parental strain but more virulent than BCG Pasteur. Challenge experiments in mice and guinea pigs usingM. bovisorM. tuberculosisstrains showed similar protection conferred by MbΔp27-p55 mutant than BCG in terms of pathology and bacterial loads in spleen but lower protection than BCG in lungs. When tested in cattle, MbΔp27-p55 did not induce IL-2 expression and induced a very low production of IFNγ, suggesting that the lack of P27/P55 reduces the capacity ofM. bovisof triggering an adequate Th1 response.
Mycobacterium bovis strain 04-303 was isolated from a wild boar living in a free-ranging field in... more Mycobacterium bovis strain 04-303 was isolated from a wild boar living in a free-ranging field in Argentina. This work reports the draft genome sequence of this highly virulent strain and the genomic comparison of its major virulence-related genes with those of M. bovis strain AF2122/97 and Mycobacterium tuberculosis strain H37Rv.
In this study, a Mycobacterium bovis knockout strain in phoP-phoR and mce2 operons was tested as ... more In this study, a Mycobacterium bovis knockout strain in phoP-phoR and mce2 operons was tested as an antituberculosis experimental vaccine in animal models. The double mutant strain was significantly more attenuated than the wild type strain in inmunocompetent and inmunodeficient mice. Vaccination with the double mutant protected mice against challenge with a virulent M. bovis strain.
Molecular epidemiology has revealed thatMycobacterium tuberculosis(Mtb), formerly regarded as hig... more Molecular epidemiology has revealed thatMycobacterium tuberculosis(Mtb), formerly regarded as highly conserved species, displays a considerable degree of genetic variability that can influence the outcome of the disease as well as the innate and adaptive immune response. Recent studies have demonstrated thatMtbfamilies found worldwide today differ in pathology, transmissibility, virulence, and development of immune response. By proteomic approaches seven proteins that were differentially expressed between a local clinical isolate from Latin-American-Mediterranean (LAM) and from Haarlem (H) lineages were identified. In order to analyze the immunogenic ability, recombinant Rv2241, Rv0009, Rv0407, and Rv2624c proteins were produced for testing specific antibody responses. We found that these proteins induced humoral immune responses in patients with drug-sensitive and drug-resistant tuberculosis with substantial cross-reactivity among the four proteins. Moreover, such reactivity was al...
The identification of bovine tuberculosis (bTB) biomarkers in specific stages of the disease will... more The identification of bovine tuberculosis (bTB) biomarkers in specific stages of the disease will contribute to a better understanding of the immunopathology associated with tuberculosis and to improve the disease diagnosis and prognosis. The aim of this study was to understand the changing profile of the immune responses during the course of infection and to identify biomarkers associated with pathology. Here we describe the immune response developed in experimentally infected cattle with field Mycobacterium bovis strains. Blood samples were taken from each animal at different time points after M. bovis intratracheal infection and lymphocyte subset activation and cytokine mRNA expression were determined from peripheral blood mononuclear cells in response to purified protein derivative (PPDB). We found that CD4 and CD8 activation during the early stages of infection, together with IL-17 gene expression, were positively associated with pathology. The results of this study provide evidences of the role of IL-17 in the immunopathology of tuberculosis and support the use of IL-17 as a potential biomarker with predictive value of prognosis in bTB.
The mce2 operon is one of the four mce operons present in Mycobacterium tuberculosis that encode ... more The mce2 operon is one of the four mce operons present in Mycobacterium tuberculosis that encode exported proteins with a probable role in the virulence mechanisms of this bacterium. In the present study we demonstrated that Rv0586, which encodes a putative GntR-like regulator, is part of the mce2 operon. By using a promoter-lacZ fusion approach and bioinformatics tools, we found that Rv0586 represses the expression of Mce2 proteins and of a putative endonuclease IV, encoded by end (Rv0670) gene. For this reason, we have re-named the repressor protein Mce2R. By gel-shift experiments Mce2R binding was determined to be located within the mce2 promoter region. In addition, two FadR-like operator motifs were identified within the promoter regions of both the mce2 operon and the end gene. These motifs overlap putative -10 and -35 promoter boxes. M. tuberculosis carrying mce2 and end promoter-lacZ fusions were used to infect J774 macrophage-like cells. Expression of beta-galactosidase was induced after phagocytocis, suggesting that some cellular factor could be a key component of the molecular switch regulation expression of the mce2 operon. In conclusion, these results add novel evidence of the complex regulation of mce operon expression.
The mce operons constitute four homologous regions in the Mycobacterium tuberculosis genome, each... more The mce operons constitute four homologous regions in the Mycobacterium tuberculosis genome, each of which has 8–13 ORFs. Although the function of the Mce protein family has not been clearly established, its members are believed to be membrane lipid transporters. Based on functional experiments, we found that the regulator of the mce3 locus, Mce3R, negatively regulates the expression of the Rv1933c–Rv1935c and Rv1936–Rv1941 transcriptional units. These operons are adjacent to one another and divergently transcribed. The predicted functions of most of these genes are related to either lipid metabolism or redox reactions. Bioinformatic analysis of the 5′ UTR sequences of the differentially expressed genes allowed us to define a putative Mce3R motif. Importantly, the Mce3R motif was present six and three times in the mce3R–yrbE3A and Rv1935c–Rv1936 intergenic regions, respectively. Two occurrences of this motif mapped within the two regions of the mce3 operon that were protected by Mce...
Sumario: I. IntroduccIon: LAs funcIones deL regImen dIscIpLInArIo en eL ambIto penItencIArIo como... more Sumario: I. IntroduccIon: LAs funcIones deL regImen dIscIpLInArIo en eL ambIto penItencIArIo como fundAmento deL mIsmo. II. LA normAtIvA vIgente en LA mAterIA y Los prIncIpIos ApLIcAbLes AL ejercIcIo de LA potestAd dIscIpLInArIA penItencIArIA. 1. La situacion normativa. 2. A vueltas con las relaciones de sujecion especial como paradigma que justifica la regulacion del ambito normativo disciplinario ?de verdad estamos frente a la aplicacion de ius puniendi del estado? 3. Los principios que informan el ejercicio de la potestad disciplinaria penitenciaria: especial referencia al principio de Legalidad. III. LA preservAcIon de LA segurIdAd y eL orden en Los estAbLecImIentos penItencIArIos: InfrAccIones, sAncIones y medIdAs coercItIvAs. 1. Las infracciones previstas en la normativa reglamentaria. 2. Las sanciones correspondientes a las infracciones: especial referencia a la correlacion entre infraccion y sancion. 3. Los medios coercitivos autorizados en el regimen disciplinario. Iv. eL p...
Infection of bovines withMycobacterium boviscauses important financial hardship in many countries... more Infection of bovines withMycobacterium boviscauses important financial hardship in many countries presenting also a risk for humans.M. bovisis known to be adapted to survive and thrive within the intramacrophage environment. In spite of its relevance, at present the information about macrophage expression patterns is scarce, particularly regarding the bovine host. In this study, transcriptomic analysis was used to detect genes differentially expressed in macrophages derived from peripheral blood mononuclear cells at early stages of infection with two Argentinean strains ofM. bovis, a virulent and an attenuated strains. The results showed that the number of differentially expressed genes in the cells infected with the virulent strain (5) was significantly lower than those in the cells infected with the attenuated strain (172). Several genes were more strongly expressed in infected macrophages. Among them, we detected encoding transcription factors, anthrax toxin receptor, cell divisi...
A Mycobacterium bovis strain deleted in mce2A and mce2B genes (M. bovis Δmce2) was tested as an e... more A Mycobacterium bovis strain deleted in mce2A and mce2B genes (M. bovis Δmce2) was tested as an experimental vaccine in cattle challenged with a virulent M. bovis strain. Three-and-a-half-month old calves (n = 5 to 6 per group) were vaccinated and challenged with a virulent strain of M. bovis by the intratracheal route 9 weeks after vaccination. A non-vaccinated group and a group vaccinated with BCG were included as controls. Blood samples were collected to measure IFN-γ by an interferon-gamma release assay (IGRA), cytometry and cytokine responses of bovine purified protein derivative (PPD) restimulated peripheral blood mononuclear cells (PBMCs). The IGRA test showed IFN-γ values similar to pre-vaccination except for the animals vaccinated with M. bovis Δmce2, where a significant increase was observed at 30 days post-vaccination. The expression of IL-2R on CD4(+) cells in response to PPD from the animals vaccinated with Δmce2 increased at 15 days post-vaccination compared to cells from non-vaccinated group. Vaccination of cattle with M. bovis Δmce2 induced the highest (P < 0.05) expression of IFN-γ and IL-17 mRNA upon PPD stimulation of PBMCs compared to vaccination with BCG or that for the non-vaccinated group. There was a weak positive correlation between the production of these proinflammatory cytokines post-vaccination and reduced pathology scores post-challenge. The animals were euthanized and necropsied 100 days after challenge. The group vaccinated with M. bovis Δmce2 displayed a significantly lower histopathological score for lesions in lungs and pulmonary lymph nodes than for the other groups (P < 0.05). A marked positive reaction to tuberculin intradermal test was observed post-vaccination in animals vaccinated with M. bovis Δmce2 compared to those vaccinated with BCG or the non-vaccinated group. In contrast, after challenge, non-vaccinated animals had greater skin test responses than the vaccinated animals. In summary, M. bovis Δmce2 is a promising vaccine candidate to control M. bovis pathogenesis in cattle.
AMycobacterium bovisknockout inp27-p55operon was tested as an antituberculosis experimental vacci... more AMycobacterium bovisknockout inp27-p55operon was tested as an antituberculosis experimental vaccine in animal models. The mutant MbΔp27-p55 was significantly more attenuated in nude mice than its parental strain but more virulent than BCG Pasteur. Challenge experiments in mice and guinea pigs usingM. bovisorM. tuberculosisstrains showed similar protection conferred by MbΔp27-p55 mutant than BCG in terms of pathology and bacterial loads in spleen but lower protection than BCG in lungs. When tested in cattle, MbΔp27-p55 did not induce IL-2 expression and induced a very low production of IFNγ, suggesting that the lack of P27/P55 reduces the capacity ofM. bovisof triggering an adequate Th1 response.
Mycobacterium bovis strain 04-303 was isolated from a wild boar living in a free-ranging field in... more Mycobacterium bovis strain 04-303 was isolated from a wild boar living in a free-ranging field in Argentina. This work reports the draft genome sequence of this highly virulent strain and the genomic comparison of its major virulence-related genes with those of M. bovis strain AF2122/97 and Mycobacterium tuberculosis strain H37Rv.
In this study, a Mycobacterium bovis knockout strain in phoP-phoR and mce2 operons was tested as ... more In this study, a Mycobacterium bovis knockout strain in phoP-phoR and mce2 operons was tested as an antituberculosis experimental vaccine in animal models. The double mutant strain was significantly more attenuated than the wild type strain in inmunocompetent and inmunodeficient mice. Vaccination with the double mutant protected mice against challenge with a virulent M. bovis strain.
Molecular epidemiology has revealed thatMycobacterium tuberculosis(Mtb), formerly regarded as hig... more Molecular epidemiology has revealed thatMycobacterium tuberculosis(Mtb), formerly regarded as highly conserved species, displays a considerable degree of genetic variability that can influence the outcome of the disease as well as the innate and adaptive immune response. Recent studies have demonstrated thatMtbfamilies found worldwide today differ in pathology, transmissibility, virulence, and development of immune response. By proteomic approaches seven proteins that were differentially expressed between a local clinical isolate from Latin-American-Mediterranean (LAM) and from Haarlem (H) lineages were identified. In order to analyze the immunogenic ability, recombinant Rv2241, Rv0009, Rv0407, and Rv2624c proteins were produced for testing specific antibody responses. We found that these proteins induced humoral immune responses in patients with drug-sensitive and drug-resistant tuberculosis with substantial cross-reactivity among the four proteins. Moreover, such reactivity was al...
The identification of bovine tuberculosis (bTB) biomarkers in specific stages of the disease will... more The identification of bovine tuberculosis (bTB) biomarkers in specific stages of the disease will contribute to a better understanding of the immunopathology associated with tuberculosis and to improve the disease diagnosis and prognosis. The aim of this study was to understand the changing profile of the immune responses during the course of infection and to identify biomarkers associated with pathology. Here we describe the immune response developed in experimentally infected cattle with field Mycobacterium bovis strains. Blood samples were taken from each animal at different time points after M. bovis intratracheal infection and lymphocyte subset activation and cytokine mRNA expression were determined from peripheral blood mononuclear cells in response to purified protein derivative (PPDB). We found that CD4 and CD8 activation during the early stages of infection, together with IL-17 gene expression, were positively associated with pathology. The results of this study provide evidences of the role of IL-17 in the immunopathology of tuberculosis and support the use of IL-17 as a potential biomarker with predictive value of prognosis in bTB.
The mce2 operon is one of the four mce operons present in Mycobacterium tuberculosis that encode ... more The mce2 operon is one of the four mce operons present in Mycobacterium tuberculosis that encode exported proteins with a probable role in the virulence mechanisms of this bacterium. In the present study we demonstrated that Rv0586, which encodes a putative GntR-like regulator, is part of the mce2 operon. By using a promoter-lacZ fusion approach and bioinformatics tools, we found that Rv0586 represses the expression of Mce2 proteins and of a putative endonuclease IV, encoded by end (Rv0670) gene. For this reason, we have re-named the repressor protein Mce2R. By gel-shift experiments Mce2R binding was determined to be located within the mce2 promoter region. In addition, two FadR-like operator motifs were identified within the promoter regions of both the mce2 operon and the end gene. These motifs overlap putative -10 and -35 promoter boxes. M. tuberculosis carrying mce2 and end promoter-lacZ fusions were used to infect J774 macrophage-like cells. Expression of beta-galactosidase was induced after phagocytocis, suggesting that some cellular factor could be a key component of the molecular switch regulation expression of the mce2 operon. In conclusion, these results add novel evidence of the complex regulation of mce operon expression.
The mce operons constitute four homologous regions in the Mycobacterium tuberculosis genome, each... more The mce operons constitute four homologous regions in the Mycobacterium tuberculosis genome, each of which has 8–13 ORFs. Although the function of the Mce protein family has not been clearly established, its members are believed to be membrane lipid transporters. Based on functional experiments, we found that the regulator of the mce3 locus, Mce3R, negatively regulates the expression of the Rv1933c–Rv1935c and Rv1936–Rv1941 transcriptional units. These operons are adjacent to one another and divergently transcribed. The predicted functions of most of these genes are related to either lipid metabolism or redox reactions. Bioinformatic analysis of the 5′ UTR sequences of the differentially expressed genes allowed us to define a putative Mce3R motif. Importantly, the Mce3R motif was present six and three times in the mce3R–yrbE3A and Rv1935c–Rv1936 intergenic regions, respectively. Two occurrences of this motif mapped within the two regions of the mce3 operon that were protected by Mce...
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