Complications During Inter-Hospital Transfer of Patients with Acute Ischemic Stroke for Endovascular Therapy.

Purpose: Few data are available on complications occurring during inter-hospital transfer from a primary stroke center (PSC) to a comprehensive stroke center (CSC) for endovascular treatment (EVT) after large vessel occlusion (LVO). Therefore, we prospectively studied data from consecutive patients transferred from our PSC to the next CSC during 4 years to determine the incidence and risk factors of complications during transfer. Methods: This observational, single-center study included consecutive patients transferred from January 1, 2015 to December 31, 2018. During inter-hospital transfer, all medical incidents were systematically recorded. A new complete clinical examination was performed on arrival at the CSC. Results: Among the 253 patients transferred to the CSC during the study period, 68 (26.9%) had one or more complications. In 11 patients (4.3%) these were life-threatening and required emergency intervention by a physician. Baseline characteristics were not different between patients with and without complications, except for the LVO location. Specifically, basilar artery (BA) occlusion was strongly associated with complications during the transport (p < 0.0005). Conclusion: Complications occurred in 26.9% of patients during transfer. Only BA occlusion could predict complication during transfer. Future studies should identify variables to help stratifying patients at high and low risk of complications during transportation.

Which Patients Require Physician-Led Inter-Hospital Transport in View of Endovascular Therapy?

INTRODUCTION: The current guidelines advocate the implementation of stroke networks to organize endovascular treatment (ET) for patients with acute ischemic stroke due to large vessel occlusion (LVO) after transfer from a Primary Stroke Centre (PSC) to a Comprehensive Stroke Centre (CSC). In France and in many other countries around the world, these transfers are carried out by a physician-led mobile medical team. However, with the recent broadening of ET indications, their availability is becoming more and more critical. Here, we retrospectively analysed data of patients transferred from a PSC to a CSC for potential ET to identify predictive factors of major complications (MC) at departure and during transport that absolutely require the presence of a physician during interhospital transfer. METHODS: This observational, single-centre study included patients with evidence of intracranial LVO transferred for ET from Perpignan to a 156 km-distant CSC between January 1, 2015 and -December 31, 2018. We compared 2 groups: MC group (patients who required emergency intervention by the medical team due to life-threatening complications, including need of mechanical ventilation at departure) and non-MC group (all other patients who experienced no or only minor complications that could be managed by the emergency paramedics alone). RESULTS: Among the 253 patients who were transferred to the CSC, 185 (73.1%) had no complication, 57 (22.6%) minor complications, and 11 (4.3%) had MC. In multivariate analysis, MC was associated with basilar artery (BA) occlusion (p < 0.0001), initial National Institute of Health Stroke Scale (NIHSS) score >22 (p < 0.005), and history of atrial fibrillation (p < 0.04). Among the 168 patients treated with intravenous thrombolysis (IVT), only 1 patient (0.6%) had MC due to an IVT-related adverse event during transfer. CONCLUSIONS: Physician-led inter-hospital transports are warranted for patients with BA occlusion, initial NIHSS score >22, or history of atrial fibrillation. For the other patients, transfer without a physician may be considered, even if treated with IVT.

Draft Genome Sequence of the Biowarfare Simulant Bacillus atrophaeus Strain 930029.

We report here the draft genome sequence of Bacillus atrophaeus strain 930029. Strain 930029 shows evidence of drift, based on a comparison to the corresponding source strain publicly available today.

Normalization of test and evaluation of biothreat detection systems: overcoming microbial air content fluctuations by using a standardized reagent bacterial mixture.

Test and evaluation of engineered biothreat agent detection systems ("biodetectors") are a challenging task for government agencies and industries involved in biosecurity and biodefense programs. In addition to user friendly features, biodetectors need to perform both highly sensitive and specific detection, and must not produce excessive false alerts. In fact, the atmosphere displays a number of variables such as airborne bacterial content that can interfere with the detection process, thus impeding comparative tests when carried out at different times or places. To overcome these bacterial air content fluctuations, a standardized reagent bacterial mixture (SRBM), consisting in a collection of selected cultivable environmental species that are prevalent in temperate climate bioaerosols, was designed to generate a stable, reproducible, and easy to use surrogate of bioaerosol sample. The rationale, design, and production process are reported. The results showed that 8.59; CI 95%: 8.46-8.72 log cfu distributed into vials underwent a 0.95; CI 95%: 0.65-1.26 log viability decay after dehydration and subsequent reconstitution, thus advantageously mimicking a natural bioaerosol sample which is typically composed of cultivable and uncultivable particles. Dehydrated SRBM was stable for more than 12months at 4°C and allowed the reconstitution of a dead/live cells aqueous suspension that is stable for 96h at +4°C, according to plate counts. Specific detection of a simulating biothreat agent (e.g. Bacillus atrophaeus) by immuno-magnetic or PCR assays did not display any significant loss of sensitivity, false negative or positive results in the presence of SRBM. This work provides guidance on testing and evaluating detection devices, and may contribute to the establishment of suitable standards and normalized procedures.

Morbidity and mortality rates for childhood cancer in Argentina: 2006-2008.

INTRODUCTION: Childhood cancer is a serious public health problem in any country given the large number of years of life lost in an early manner. OBJECTIVE: To describe morbidity and mortality rates for cancer in Argentinean children and adolescents younger than 15 years old in the 2006-2008 three year period. Method. Specific mortality rates and incidence rates per million inhabitants were analyzed in children and adolescents younger than 15 years old by type of tumor and gender. Vital statistics data were used based on the databases provided by the Statistics and Health Information Department of the Ministry of Health of Argentina and the population of 0-14 year old children estimated by the National Statistics and Censuses Institute of Argentina (Instituto Nacional de Estadísticas y Censos, INDEC). In relation to morbidity data, the information published by the Argentine Hospital Oncopediatric Registry (Registro Oncopediátrico Hospitalario Argentino, ROHA) was used. RESULTS: Mortality rate from malignancies was 43.8 per million (3.5% of total deaths in this age group) and the incidence rate was 123.7 per million. Leukemia had the highest specific mortality rate (14.9 per million), followed by tumors in the central nervous system (12.7 per million). The highest incidence rates were also registered for these tumors (45.2 and 15.5 per million, respectively). CONCLUSIONS: Mortality from malignancies accounted for 3.5% of deaths in children and adolescents younger than 15 years old in Argentina. Leukemia and tumors in the central nervous system had the highest specific mortality and incidence rates.

Morbimortalidad por cáncer infantil en la República Argentina: 2006-2008 / Morbidity and mortality rates for childhood cancer in Argentina: 2006-2008

Introducción. El cáncer en los niños es un problema importante de salud pública en un país por el número elevado de años de vida perdidos prematuramente. Objetivo. Describir la morbimortalidad por cáncer en los argentinos menores de 15 años de edad en el trienio 2006-2008. Método. Se analizaron tasas de mortalidad específcas y tasas de incidencia expresadas por millón de habitantes menores de 15 años de edad, según el tipo de tumor y por sexo. Se utilizaron datos de estadísticas vitales a partir de bases de datos provistas por la Dirección de Estadísticas e Información de Salud del Ministerio de Salud y la población de niños de entre 0 y 14 años estimada por el Instituto Nacional de Estadísticas y Censos (INDEC). En relación con los datos de morbilidad, se utilizó la información publicada por el Registro Oncopediátrico Hospitalario Argentino (ROHA). Resultados. La tasa de mortalidad por neoplasias fue de 43,8 por millón (3,5% de las muertes totales en el grupo etario) y la tasa de incidencia, de 123,7 por millón. La leucemia presentó la mayor tasa de mortalidad específca (14,9 por millón), seguida de los tumores del sistema nervioso central (12,7 por millón). Estos tumores también registraron las mayores tasas de incidencia (45,2 y 15,5 por millón respectivamente). Conclusiones. La mortalidad por tumores representó el 3,5% de las muertes en menores de 15 años de la Argentina. La leucemia y los tumores del sistema nervioso central presentaron las mayores tasas de mortalidad específca y de incidencia.(AU)

Introduction. Childhood cancer is a serious public health problem in any country given the large number of years of life lost in an early manner. Objective. To describe morbidity and mortality rates for cancer in Argentinean children and adolescents younger than 15 years old in the 2006-2008 three year period. Method. Specifc mortality rates and incidence rates per million inhabitants were analyzed in children and adolescents younger than 15 years old by type of tumor and gender. Vital statistics data were used based on the databases provided by the Statistics and Health Information Department of the Ministry of Health of Argentina and the population of 0-14 year old children estimated by the National Statistics and Censuses Institute of Argentina (Instituto Nacional de Estadísticas y Censos, INDEC). In relation to morbidity data, the information published by the Argentine Hospital Oncopediatric Registry (Registro Oncopediátrico Hospitalario Argentino, ROHA) was used. Results. Mortality rate from malignancies was 43.8 per million (3.5% of total deaths in this age group) and the incidence rate was 123.7 per million. Leukemia had the highest specifc mortality rate (14.9 per million), followed by tumors in the central nervous system (12.7 per million). The highest incidence rates were also registered for these tumors (45.2 and 15.5 per million, respectively). Conclusions. Mortality from malignancies accounted for 3.5% of deaths in children and adolescents younger than 15 years old in Argentina. Leukemia and tumors in the central nervous system had the highest specifc mortality and incidence rates.(AU)

Morbimortalidad por cáncer infantil en la República Argentina: 2006-2008 / Morbidity and mortality rates for childhood cancer in Argentina: 2006-2008

Introducción. El cáncer en los niños es un problema importante de salud pública en un país por el número elevado de años de vida perdidos prematuramente. Objetivo. Describir la morbimortalidad por cáncer en los argentinos menores de 15 años de edad en el trienio 2006-2008. Método. Se analizaron tasas de mortalidad específcas y tasas de incidencia expresadas por millón de habitantes menores de 15 años de edad, según el tipo de tumor y por sexo. Se utilizaron datos de estadísticas vitales a partir de bases de datos provistas por la Dirección de Estadísticas e Información de Salud del Ministerio de Salud y la población de niños de entre 0 y 14 años estimada por el Instituto Nacional de Estadísticas y Censos (INDEC). En relación con los datos de morbilidad, se utilizó la información publicada por el Registro Oncopediátrico Hospitalario Argentino (ROHA). Resultados. La tasa de mortalidad por neoplasias fue de 43,8 por millón (3,5% de las muertes totales en el grupo etario) y la tasa de incidencia, de 123,7 por millón. La leucemia presentó la mayor tasa de mortalidad específca (14,9 por millón), seguida de los tumores del sistema nervioso central (12,7 por millón). Estos tumores también registraron las mayores tasas de incidencia (45,2 y 15,5 por millón respectivamente). Conclusiones. La mortalidad por tumores representó el 3,5% de las muertes en menores de 15 años de la Argentina. La leucemia y los tumores del sistema nervioso central presentaron las mayores tasas de mortalidad específca y de incidencia.

Introduction. Childhood cancer is a serious public health problem in any country given the large number of years of life lost in an early manner. Objective. To describe morbidity and mortality rates for cancer in Argentinean children and adolescents younger than 15 years old in the 2006-2008 three year period. Method. Specifc mortality rates and incidence rates per million inhabitants were analyzed in children and adolescents younger than 15 years old by type of tumor and gender. Vital statistics data were used based on the databases provided by the Statistics and Health Information Department of the Ministry of Health of Argentina and the population of 0-14 year old children estimated by the National Statistics and Censuses Institute of Argentina (Instituto Nacional de Estadísticas y Censos, INDEC). In relation to morbidity data, the information published by the Argentine Hospital Oncopediatric Registry (Registro Oncopediátrico Hospitalario Argentino, ROHA) was used. Results. Mortality rate from malignancies was 43.8 per million (3.5% of total deaths in this age group) and the incidence rate was 123.7 per million. Leukemia had the highest specifc mortality rate (14.9 per million), followed by tumors in the central nervous system (12.7 per million). The highest incidence rates were also registered for these tumors (45.2 and 15.5 per million, respectively). Conclusions. Mortality from malignancies accounted for 3.5% of deaths in children and adolescents younger than 15 years old in Argentina. Leukemia and tumors in the central nervous system had the highest specifc mortality and incidence rates.

Morbidity and mortality rates for childhood cancer in Argentina: 2006-2008. / Morbidity and mortality rates for childhood cancer in Argentina: 2006-2008.

INTRODUCTION: Childhood cancer is a serious public health problem in any country given the large number of years of life lost in an early manner. OBJECTIVE: To describe morbidity and mortality rates for cancer in Argentinean children and adolescents younger than 15 years old in the 2006-2008 three year period. Method. Specific mortality rates and incidence rates per million inhabitants were analyzed in children and adolescents younger than 15 years old by type of tumor and gender. Vital statistics data were used based on the databases provided by the Statistics and Health Information Department of the Ministry of Health of Argentina and the population of 0-14 year old children estimated by the National Statistics and Censuses Institute of Argentina (Instituto Nacional de Estadísticas y Censos, INDEC). In relation to morbidity data, the information published by the Argentine Hospital Oncopediatric Registry (Registro Oncopediátrico Hospitalario Argentino, ROHA) was used. RESULTS: Mortality rate from malignancies was 43.8 per million (3.5

of total deaths in this age group) and the incidence rate was 123.7 per million. Leukemia had the highest specific mortality rate (14.9 per million), followed by tumors in the central nervous system (12.7 per million). The highest incidence rates were also registered for these tumors (45.2 and 15.5 per million, respectively). CONCLUSIONS: Mortality from malignancies accounted for 3.5

of deaths in children and adolescents younger than 15 years old in Argentina. Leukemia and tumors in the central nervous system had the highest specific mortality and incidence rates.

Roles of chaperone/usher pathways of Yersinia pestis in a murine model of plague and adhesion to host cells.

Yersinia pestis and many other Gram-negative pathogenic bacteria use the chaperone/usher (CU) pathway to assemble virulence-associated surface fibers termed pili or fimbriae. Y. pestis has two well-characterized CU pathways: the caf genes coding for the F1 capsule and the psa genes coding for the pH 6 antigen. The Y. pestis genome contains additional CU pathways that are capable of assembling pilus fibers, but the roles of these pathways in the pathogenesis of plague are not understood. We constructed deletion mutations in the usher genes for six of the additional Y. pestis CU pathways. The wild-type (WT) and usher deletion strains were compared in the murine bubonic (subcutaneous) and pneumonic (intranasal) plague infection models. Y. pestis strains containing deletions in CU pathways y0348-0352, y1858-1862, and y1869-1873 were attenuated for virulence compared to the WT strain by the intranasal, but not subcutaneous, routes of infection, suggesting specific roles for these pathways during pneumonic plague. We examined binding of the Y. pestis WT and usher deletion strains to A549 human lung epithelial cells, HEp-2 human cervical epithelial cells, and primary human and murine macrophages. Y. pestis CU pathways y0348-0352 and y1858-1862 were found to contribute to adhesion to all host cells tested, whereas pathway y1869-1873 was specific for binding to macrophages. The correlation between the virulence attenuation and host cell binding phenotypes of the usher deletion mutants identifies three of the additional CU pathways of Y. pestis as mediating interactions with host cells that are important for the pathogenesis of plague.

Structural insights into RipC, a putative citrate lyase ß subunit from a Yersinia pestis virulence operon.

Yersinia pestis remains a threat, with outbreaks of plague occurring in rural areas and its emergence as a weapon of bioterrorism; thus, an improved understanding of its various pathogenicity pathways is warranted. The rip (required for intracellular proliferation) virulence operon is required for Y. pestis survival in interferon-γ-treated macrophages and has been implicated in lowering macrophage-produced nitric oxide levels. RipC, one of three gene products from the rip operon, is annotated as a citrate lyase ß subunit. Furthermore, the Y. pestis genome lacks genes that encode citrate lyase α and γ subunits, suggesting a unique functional role of RipC in the Y. pestis rip-mediated survival pathway. Here, the 2.45 Å resolution crystal structure of RipC revealed a homotrimer in which each monomer consists of a (ß/α)(8) TIM-barrel fold. Furthermore, the trimeric state was confirmed in solution by size-exclusion chromatography. Through sequence and structure comparisons with homologous proteins, it is proposed that RipC is a putative CoA- or CoA-derivative binding protein.

Evolución de la mortalidad por tumores en las provincias Argentinas 1991-2007 / Cancer mortality in Argentine provinces, 1991- 2007

Objetivo: Analizar la mortalidad por tumores en lasprovincias argentinas y algunos factores que podrían favorecersu incidencia entre 1991 y 2007, habida cuenta de que elcáncer constituye la segunda causa de muerte en el paísy de la heterogeneidad que presenta su distribución en elterritorio. Datos y métodos: Se utiliza información producidapor diversos organismos oficiales sobre la que se realiza unanálisis estadístico descriptivo e inferencial. Resultados: Sedestaca la correlación existente entre la tasa de mortalidadpor tumores y el consumo de alcohol y tabaco, y, en menormedida, con el porcentaje de superficie sembrada; así comotambién la correlación negativa entre la misma y el nivelde pobreza que coincidiría con un retraso en el proceso detransición epidemiológica. Conclusiones: De estos resultadosse desprende la necesidad de profundizar las investigacionessobre los factores que influyen en las defunciones a causade tumores.(AU)

Purpose: To analyze cancer mortality in Argentine andsome factors associated with it during the period 1991-200. Data and methods: Data from government agencies. Descriptive and inferential statistics.Results: Correlations between cancer mortality rate and tobacco and alcohol consumption,poverty level and percentage of sown area are observed. Conclusions: More research aboutfactors associated with cancer mortality is needed.(AU)

Evolución de la mortalidad por tumores en las provincias Argentinas 1991-2007 / Cancer mortality in Argentine provinces, 1991- 2007

Objetivo: Analizar la mortalidad por tumores en lasprovincias argentinas y algunos factores que podrían favorecersu incidencia entre 1991 y 2007, habida cuenta de que elcáncer constituye la segunda causa de muerte en el paísy de la heterogeneidad que presenta su distribución en elterritorio. Datos y métodos: Se utiliza información producidapor diversos organismos oficiales sobre la que se realiza unanálisis estadístico descriptivo e inferencial. Resultados: Sedestaca la correlación existente entre la tasa de mortalidadpor tumores y el consumo de alcohol y tabaco, y, en menormedida, con el porcentaje de superficie sembrada; así comotambién la correlación negativa entre la misma y el nivelde pobreza que coincidiría con un retraso en el proceso detransición epidemiológica. Conclusiones: De estos resultadosse desprende la necesidad de profundizar las investigacionessobre los factores que influyen en las defunciones a causade tumores.

Purpose: To analyze cancer mortality in Argentine andsome factors associated with it during the period 1991-200. Data and methods: Data from government agencies. Descriptive and inferential statistics.Results: Correlations between cancer mortality rate and tobacco and alcohol consumption,poverty level and percentage of sown area are observed. Conclusions: More research aboutfactors associated with cancer mortality is needed.

Biochemical, structural and molecular dynamics analyses of the potential virulence factor RipA from Yersinia pestis.

Human diseases are attributed in part to the ability of pathogens to evade the eukaryotic immune systems. A subset of these pathogens has developed mechanisms to survive in human macrophages. Yersinia pestis, the causative agent of the bubonic plague, is a predominately extracellular pathogen with the ability to survive and replicate intracellularly. A previous study has shown that a novel rip (required for intracellular proliferation) operon (ripA, ripB and ripC) is essential for replication and survival of Y. pestis in postactivated macrophages, by playing a role in lowering macrophage-produced nitric oxide (NO) levels. A bioinformatics analysis indicates that the rip operon is conserved among a distally related subset of macrophage-residing pathogens, including Burkholderia and Salmonella species, and suggests that this previously uncharacterized pathway is also required for intracellular survival of these pathogens. The focus of this study is ripA, which encodes for a protein highly homologous to 4-hydroxybutyrate-CoA transferase; however, biochemical analysis suggests that RipA functions as a butyryl-CoA transferase. The 1.9 Å X-ray crystal structure reveals that RipA belongs to the class of Family I CoA transferases and exhibits a unique tetrameric state. Molecular dynamics simulations are consistent with RipA tetramer formation and suggest a possible gating mechanism for CoA binding mediated by Val227. Together, our structural characterization and molecular dynamic simulations offer insights into acyl-CoA specificity within the active site binding pocket, and support biochemical results that RipA is a butyryl-CoA transferase. We hypothesize that the end product of the rip operon is butyrate, a known anti-inflammatory, which has been shown to lower NO levels in macrophages. Thus, the results of this molecular study of Y. pestis RipA provide a structural platform for rational inhibitor design, which may lead to a greater understanding of the role of RipA in this unique virulence pathway.

Characterization of the Mycobacterium avium subsp. paratuberculosis laminin-binding/histone-like protein (Lbp/Hlp) which reacts with sera from patients with Crohn's disease.

Mycobacterium avium subsp. paratuberculosis (Map) causes a chronic enteric disease in ruminants, called paratuberculosis or Johne's disease. The current model proposes that after ingestion by the host, Map crosses the intestinal barrier via internalization by the M cells. Experimental observations suggest, however, that Map may also transcytose the intestinal wall via the enterocytes, but the mechanisms involved in this process remain poorly understood. Cytoadherence assays performed on epithelial cells with Map revealed that the addition of laminin to the cell culture increases adhesion. A Map protein was isolated by heparin-Sepharose chromatography and identified as a laminin-binding protein like. The gene encoding this protein named Lbp/Hlp was identified in the Map genome sequence at locus MAP3024 (annotated Hup B). The deduced Map Lbp/Hlp amino acid sequence reveals 80% identity with that reported for other mycobacteria. The C-terminal domain involved in adhesion is mainly composed of arginine and lysine residues modified by methylation. In vitro tests demonstrated that recombinant Lbp/Hlp binds laminin, heparin, collagen and epithelial cells. Interestingly, we found that this adhesin corresponds to the antigen described as the target of pANCA and serum antibodies of patients with Crohn's disease.

Yersinia pestis can reside in autophagosomes and avoid xenophagy in murine macrophages by preventing vacuole acidification.

Yersinia pestis survives and replicates in phagosomes of murine macrophages. Previous studies demonstrated that Y. pestis-containing vacuoles (YCVs) acquire markers of late endosomes or lysosomes in naïve macrophages and that this bacterium can survive in macrophages activated with the cytokine gamma interferon. An autophagic process known as xenophagy, which destroys pathogens in acidic autophagolysosomes, can occur in naïve macrophages and is upregulated in activated macrophages. Studies were undertaken here to investigate the mechanism of Y. pestis survival in phagosomes of naïve and activated macrophages and to determine if the pathogen avoids or co-opts autophagy. Colocalization of the YCV with markers of autophagosomes or acidic lysosomes and the pH of the YCV were determined by microscopic imaging of infected macrophages. Some YCVs contained double membranes characteristic of autophagosomes, as determined by electron microscopy. Fluorescence microscopy showed that approximately 40% of YCVs colocalized with green fluorescent protein (GFP)-LC3, a marker of autophagic membranes, and that YCVs failed to acidify below pH 7 in naïve macrophages. Replication of Y. pestis in naïve macrophages caused accumulation of LC3-II, as determined by immunoblotting. While activation of infected macrophages increased LC3-II accumulation, it decreased the percentage of GFP-LC3-positive YCVs (approximately 30%). A viable count assay showed that Y. pestis survived equally well in macrophages proficient for autophagy and macrophages rendered deficient for this process by Cre-mediated deletion of ATG5, revealing that this pathogen does not require autophagy for intracellular replication. We conclude that although YCVs can acquire an autophagic membrane and accumulate LC3-II, the pathogen avoids xenophagy by preventing vacuole acidification.

Replication of Yersinia pestis in interferon gamma-activated macrophages requires ripA, a gene encoded in the pigmentation locus.

Yersinia pestis is a facultative intracellular bacterial pathogen that can replicate in macrophages. Little is known about the mechanism by which Y. pestis replicates in macrophages, and macrophage defense mechanisms important for limiting intracellular survival of Y. pestis have not been characterized. In this work, we investigated the ability of Y. pestis to replicate in primary murine macrophages that were activated with IFN-gamma. Y. pestis was able to replicate in macrophages that were activated with IFN-gamma after infection (postactivated). A region of chromosomal DNA known as the pigmentation (pgm) locus was required for replication in postactivated macrophages, and this replication was associated with reduced nitric oxide (NO) levels but not with reduced inducible NO synthase (iNOS) expression. Y. pestis delta pgm replicated in iNOS-/- macrophages that were postactivated with IFN-gamma, suggesting that killing of delta pgm Y. pestis is NO-dependent. A specific genetic locus within pgm, which shares similarity to a pathogenicity island in Salmonella, was shown to be required for replication of Y. pestis and restriction of NO levels in postactivated macrophages. These data demonstrate that intracellular Y. pestis can evade killing by macrophages that are exposed to IFN-gamma and identify a potential virulence gene encoded in the pgm locus that is required for this activity.

Turning Yersinia pathogenesis outside in: subversion of macrophage function by intracellular yersiniae.

Three bacterial species within the genus Yersinia are causative agents of human disease. Yersinia pestis is transmitted by fleas or in aerosols, infects regional lymph nodes or lungs, and causes the highly lethal disease known as plague. Yersinia enterocolitica and Yersinia pseudotuberculosis are enteric pathogens most commonly associated with self-limiting infections of the mesenteric lymph nodes. Although Y. pestis and the enteropathogenic Yersinia species utilize different modes of transmission and cause different diseases, they rely on a common set of "core" virulence determinants to successfully infect a mammalian host. These virulence factors are encoded on the bacterial chromosome and on an approximately 70-kb plasmid. Once established in lymphoid tissue, all three Yersinia species replicate as aggregates of extracellular bacteria within necrotic lesions or abscesses. At this stage of the infectious process, the bacteria resist phagocytosis by neutrophils, which are able to destroy the bacteria if they are internalized. A type III secretion system encoded on the 70-kb plasmid functions to export multiple proteins (the Yops and LcrV) that are delivered to the extracellular milieu, the plasma membrane, or the cytosol of a host target cell. The Yops and LcrV act in concert to inhibit phagocytosis and downregulate inflammation. Although it is clear that the bulk of bacterial multiplication occurs in an extracellular phase, there is also evidence that all three pathogenic Yersinia survive and multiply in macrophages. Survival and replication of Yersinia in macrophages may occur throughout the infection, but is likely to be of greatest importance at early stages of colonization. That macrophages can serve as permissive sites for bacterial replication in vivo is supported by in vitro experiments, which demonstrate that Y. pestis, Y. peudotuberculosis, and Y. enterocolitica share the ability to survive and multiply in macrophage phagosomes. There is also evidence that the bacteria can subvert the functions of macrophages from within, by inhibiting phagosome acidification (Y. pseudotuberculosis) and the production of nitric oxide (Y. pestis and Y. pseudotuberculosis). Although considerable attention has been focused on how Yersinia subverts the functions of phagocytes from the outside, the study of how these bacteria subvert macrophage functions from the inside will lead to a better overall understanding of Yersinia pathogenesis.

The response regulator PhoP of Yersinia pseudotuberculosis is important for replication in macrophages and for virulence.

Yersinia pestis and Yersinia pseudotuberculosis are closely related facultative intracellular pathogens. The response regulator PhoP was previously shown to be important for Y. pestis survival in macrophages and for virulence in a murine bubonic plague infection assay. Here the importance of PhoP for Y. pseudotuberculosis pathogenesis was investigated. Y. pseudotuberculosis phoP mutants were unable to replicate in low-Mg(2+) medium or in macrophages. phoP(+) Y. pseudotuberculosis strains initiated replication in macrophages after a lag period of approximately 5 h, as shown by fluorescence microscopy and viable count assays. Y. pseudotuberculosis phoP mutants died at a low rate in macrophages; there was no decrease in viability over the first 5 h of infection, and there was a 10-fold decrease in viability between 5 and 24 h of infection. Trafficking of phagosomes containing phoP(+) or phoP mutant Y. pseudotuberculosis was studied by using immunofluorescence microscopy and cathepsin D as a marker for lysosomes. Phagosomes containing phoP mutant Y. pseudotuberculosis acquired cathepsin D at a higher rate than phagosomes containing phoP(+) bacteria. However, the increased rate of marker acquisition for phagosomes containing mutant bacteria was only evident approximately 5 h after infection, suggesting that phoP mutants are able to retard phagosome maturation during the lag phase of intracellular growth. The results obtained with a Y. pestis phoP mutant were similar to those described above, except that the rates of intracellular killing and trafficking to cathepsin D-positive vacuoles were significantly higher. A Y. pseudotuberculosis phoP mutant was 100-fold less virulent than the wild-type strain in a murine intestinal infection model, suggesting that survival and replication in macrophages are important for Y. pseudotuberculosis pathogenesis.

A process for controlling intracellular bacterial infections induced by membrane injury.

Strategies for inhibiting phagolysosome fusion are essential for the intracellular survival and replication of many pathogens. We found that the lysosomal synaptotagmin Syt VII is required for a mechanism that promotes phagolysosomal fusion and limits the intracellular growth of pathogenic bacteria. Syt VII was required for a form of Ca2+-dependent phagolysosome fusion that is analogous to Ca2+-regulated exocytosis of lysosomes, which can be triggered by membrane injury. Bacterial type III secretion systems, which permeabilize membranes and cause Ca2+ influx in mammalian cells, promote lysosomal exocytosis and inhibit intracellular survival in Syt VII +/+ but not -/- cells. Thus, the lysosomal repair response can also protect cells against pathogens that trigger membrane permeabilization.

The ability to replicate in macrophages is conserved between Yersinia pestis and Yersinia pseudotuberculosis.

Yersinia pestis, the agent of plague, has arisen from a less virulent pathogen, Yersinia pseudotuberculosis, by a rapid evolutionary process. Although Y. pestis displays a large number of virulence phenotypes, it is not yet clear which of these phenotypes descended from Y. pseudotuberculosis and which were acquired independently. Y. pestis is known to replicate in macrophages, but there is no consensus in the literature on whether Y. pseudotuberculosis shares this property. We investigated whether the ability to replicate in macrophages is common to Y. pestis and Y. pseudotuberculosis or is a unique phenotype of Y. pestis. We also examined whether a chromosomal type III secretion system (TTSS) found in Y. pestis is present in Y. pseudotuberculosis and whether this system is important for replication of Yersinia in macrophages. A number of Y. pestis and Y. pseudotuberculosis strains of different biovars and serogroups, respectively, were tested for the ability to replicate in primary murine macrophages. Two Y. pestis strains (EV766 and KIM10(+)) and three Y. pseudotuberculosis strains (IP2790c, IP2515c, and IP2666c) were able to replicate in macrophages with similar efficiencies. Only one of six strains tested, the Y. pseudotuberculosis YPIII(p(-)) strain, was defective for intracellular replication. Thus, the ability to replicate in macrophages is conserved in Y. pestis and Y. pseudotuberculosis. Our results also indicate that a homologous TTSS is present on the chromosomes of Y. pestis and Y. pseudotuberculosis and that this secretion system is not required for replication of these bacteria in macrophages.