Características y factores asociados a la mortalidad en pacientes con COVID-19 traqueostomizados: estudio de cohorte retrospectivo en un hospital de Tacna, Perú / Characteristics and factors associated with mortality in tracheostomized patients with COVID-19: a retrospective cohort study in a hospital in Tacna, Peru

RESUMEN Objetivo. Describir las principales características demográficas, clínicas, laboratoriales y terapéuticas e identificar si están asociados con la mortalidad en pacientes traqueostomizados. Material y métodos. Estudio de cohorte retrospectiva en pacientes adultos con diagnóstico de COVID-19, ingresados a UCI (Unidad de Cuidados Intensivos) y que requirieron traqueostomía. Se extrajeron datos demográficos, clínicos, laboratoriales y de tratamiento de las historias clínicas de pacientes que ingresaron al Hospital III Daniel Alcides Carrión de Tacna. Para el análisis de supervivencia se empleó el modelo de riesgos proporcionales de Cox y se calcularon los cocientes de riesgo instantáneos (HR) con sus intervalos de confianza al 95% (IC95%). Resultados. Se evaluaron 73 pacientes, el 72,6% eran hombres, las comorbilidades más comunes fueron obesidad (68,5%), diabetes mellitus tipo 2 (35,6%) e hipertensión arterial (34,2%). El 37% de los participantes fallecieron durante la estancia en UCI. La mediana de tiempo desde la intubación hasta la traqueostomía y la duración de esta fue 17 (RIC: 15−21) y 21 (RIC: 3−39) días, respectivamente. El análisis multivariado mostró que los factores asociados a mortalidad, fueron presentar un valor de procalcitonina > 0,50 ng/dL en el momento de la traqueostomía (HRa: 2,40 IC95%: 1,03−5,59) y el nivel de PaO2/FiO2 menor o igual a 150 mmHg, (HRa: 4,44 IC95%: 1,56−12,60). Conclusiones. Los factores asociados a mortalidad al momento de realizar la traqueostomía fueron presentar un valor de procalcitonina > 0,50 ng/dL y un cociente PaO2/FiO2 menor o igual a 150 mmHg.

ABSTRACT Objective: We aimed to describe the main demographic, clinical, laboratory and therapeutic characteristics and to identify whether they are associated with mortality in tracheostomized patients. Material and methods. Retrospective cohort study in adult patients diagnosed with COVID-19, admitted to ICU (Intensive Care Unit) and requiring tracheostomy. Demographic, clinical, laboratory and treatment data were obtained from the medical records of patients admitted to Hospital III Daniel Alcides Carrión in Tacna. The Cox proportional hazards model was used for survival analysis and hazard ratios (HR) with their 95% confidence intervals (95%CI) were calculated. Results. We evaluated 73 patients, 72.6% were men, the most common comorbidities were obesity (68.5%), type 2 diabetes mellitus (35.6%), and arterial hypertension (34.2%). Thirty-seven percent of the participants died during their stay at the ICU. The median time from intubation to tracheostomy and the duration of tracheostomy was 17 (RIC: 15-21) and 21 (RIC: 3-39) days, respectively. Multivariate analysis showed that the factors associated with mortality were procalcitonin > 0.50 ng/dL at the time of tracheostomy (HRa: 2.40 95%CI: 1.03-5.59) and a PaO2/FiO2 ratio less than or equal to 150 mmHg (HRa: 4.44 95%CI: 1.56-12.60). Conclusions. The factors associated with mortality at the time of tracheostomy were procalcitonin > 0.50 ng/dL and a PaO2/FiO2 ratio less than or equal to 150 mmHg.

Utilidad diagnóstica de los tiempos de positividad de hemocultivos para distinguir verdaderas bacteriemias de contaminantes en base a un sistema automatizado / Diagnostic performance of the time to positivity of blood cultures to distinguish true bacteremia from contaminants based on an automated system

RESUMEN Objetivo. Determinar la utilidad diagnóstica de los tiempos de positividad de hemocultivos para distinguir verdaderas bacteriemias de contaminantes en el sistema automatizado «BACT/ALERT®¼. Materiales y métodos. Se realizó un estudio transversal de tipo pruebas diagnósticas, a partir de una base de datos de muestras de hemocultivos procesadas entre enero del 2016 y agosto del 2021. Se incluyeron todas las muestras de hemocultivos de pacientes con sospecha de bacteriemia, las muestras de hemocultivos fueron ingresadas al sistema «BACT/ALERT®¼ para diferenciar verdaderas bacteriemias de contaminantes. Resultados. Se analizó un total de 33 951 frascos de hemocultivos y se obtuvieron 3875 frascos positivos. El 75,2% (n=2913) del total de hemocultivos positivos fueron verdaderas bacteriemias y 24,8% (n=962) fueron contaminantes. La mediana de tiempo de positividad en los hemocultivos con verdaderas bacteriemias fue significativamente menor (16,3 horas; RIC: 11,2 - 24,9) que la mediana de tiempo de positividad de hemocultivos con contaminantes (22,5 horas; RIC: 18,4 - 31,8; p<0,001). El tiempo de positividad demostró capacidad discriminante para diferenciar verdaderas bacteriemias de contaminantes, con un AUC-ROC de 0,73 (IC95%: 0,71 - 0,75), con 85% y 63% de sensibilidad y especificidad respectivamente para el diagnóstico de contaminantes cuando el tiempo de positividad supera las 16,5 horas. La administración de antibióticos previos a la toma retrasó el tiempo de positividad, en cambio, haber presentado fiebre antes de la toma de muestra acortó el tiempo de positividad. Conclusiones. Nuestros resultados muestran un buen desempeño de los tiempos de positividad de hemocultivos para diferenciar verdaderas bacteriemias de contaminantes utilizando el sistema «BACT/ALERT®¼ cuando el tiempo de positividad fue superior a 16,5 horas.

ABSTRACT Objective. To determine the diagnostic performance of blood culture positivity times for distinguishing true bacteremia from contaminants in the automated "BACT/ALERT®" system. Materials and methods. A cross-sectional, diagnostic test-type study was conducted from a database of blood culture samples processed between January 2016 and August 2021. All blood culture samples from patients with suspected bacteremia were included; blood culture samples were entered into the "BACT/ALERT®" system to differentiate true bacteremia from contaminants. Results. We obtained 33,951 blood cultures samples, of which 3875 were positive. Of the total number of positive blood cultures, 75.2% (n=2913) were true bacteremia and 24.8% (n=962) were contaminants. The median time to positivity in blood cultures with true bacteremia was significantly shorter (16.3 hours; IQR: 11.2 - 24.9) than the median time to positivity of blood cultures with contaminants (22.5 hours; IQR: 18.4 - 31.8; p<0.001). The positivity time showed the capacity to differentiate true bacteremia from contaminants, with an AUC-ROC of 0.73 (95%CI: 0.71 - 0.75), with 85% and 63% sensitivity and specificity respectively for the diagnosis of contaminants when the positivity time exceeds 16.5 hours. The use of antibiotics prior to sampling delayed the time to positivity, while having fever before sampling shortened the time to positivity. Conclusions. Our results show good diagnostic performance of blood culture positivity times to differentiate true bacteremia from contaminants using the "BACT/ALERT®" system when the positivity time was longer than 16.5 hours.

Characteristics and factors associated with mortality in tracheostomized patients with COVID-19: a retrospective cohort study in a hospital in Tacna, Peru. / Características y factores asociados a la mortalidad en pacientes con COVID-19 traqueostomizados: estudio de cohorte retrospectivo en un hospital de Tacna, Perú.

OBJECTIVE: We aimed to describe the main demographic, clinical, laboratory and therapeutic characteristics and to identify whether they are associated with mortality in tracheostomized patients. MATERIAL AND METHODS.: Retrospective cohort study in adult patients diagnosed with COVID-19, admitted to ICU (Intensive Care Unit) and requiring tracheostomy. Demographic, clinical, laboratory and treatment data were obtained from the medical records of patients admitted to Hospital III Daniel Alcides Carrión in Tacna. The Cox proportional hazards model was used for survival analysis and hazard ratios (HR) with their 95% confidence intervals (95%CI) were calculated. RESULTS.: We evaluated 73 patients, 72.6% were men, the most common comorbidities were obesity (68.5%), type 2 diabetes mellitus (35.6%), and arterial hypertension (34.2%). Thirty-seven percent of the participants died during their stay at the ICU. The median time from intubation to tracheostomy and the duration of tracheostomy was 17 (RIC: 15-21) and 21 (RIC: 3-39) days, respectively. Multivariate analysis showed that the factors associated with mortality were procalcitonin > 0.50 ng/dL at the time of tracheostomy (HRa: 2.40 95%CI: 1.03-5.59) and a PaO2/FiO2 ratio less than or equal to 150 mmHg (HRa: 4.44 95%CI: 1.56-12.60). CONCLUSIONS.: The factors associated with mortality at the time of tracheostomy were procalcitonin > 0.50 ng/dL and a PaO2/FiO2 ratio less than or equal to 150 mmHg.

OBJETIVO.: Describir las principales características demográficas, clínicas, laboratoriales y terapéuticas e identificar si están asociados con la mortalidad en pacientes traqueostomizados. MATERIAL Y MÉTODOS.: Estudio de cohorte retrospectiva en pacientes adultos con diagnóstico de COVID-19, ingresados a UCI (Unidad de Cuidados Intensivos) y que requirieron traqueostomía. Se extrajeron datos demográficos, clínicos, laboratoriales y de tratamiento de las historias clínicas de pacientes que ingresaron al Hospital III Daniel Alcides Carrión de Tacna. Para el análisis de supervivencia se empleó el modelo de riesgos proporcionales de Cox y se calcularon los cocientes de riesgo instantáneos (HR) con sus intervalos de confianza al 95% (IC95%). RESULTADOS.: Se evaluaron 73 pacientes, el 72,6% eran hombres, las comorbilidades más comunes fueron obesidad (68,5%), diabetes mellitus tipo 2 (35,6%) e hipertensión arterial (34,2%). El 37% de los participantes fallecieron durante la estancia en UCI. La mediana de tiempo desde la intubación hasta la traqueostomía y la duración de esta fue 17 (RIC: 15−21) y 21 (RIC: 3−39) días, respectivamente. El análisis multivariado mostró que los factores asociados a mortalidad, fueron presentar un valor de procalcitonina > 0,50 ng/dL en el momento de la traqueostomía (HRa: 2,40 IC95%: 1,03−5,59) y el nivel de PaO2/FiO2 menor o igual a 150 mmHg, (HRa: 4,44 IC95%: 1,56−12,60). CONCLUSIONES.: Los factores asociados a mortalidad al momento de realizar la traqueostomía fueron presentar un valor de procalcitonina > 0,50 ng/dL y un cociente PaO2/FiO2 menor o igual a 150 mmHg.

Proximity interactome analysis of Lassa polymerase reveals eRF3a/GSPT1 as a druggable target for host-directed antivirals.

Completion of the Lassa virus (LASV) life cycle critically depends on the activities of the virally encoded, RNA-dependent RNA polymerase in replication and transcription of the viral RNA genome in the cytoplasm of infected cells. The contribution of cellular proteins to these processes remains unclear. Here, we applied proximity proteomics to define the interactome of LASV polymerase in cells under conditions that recreate LASV RNA synthesis. We engineered a LASV polymerase-biotin ligase (TurboID) fusion protein that retained polymerase activity and successfully biotinylated the proximal proteome, which allowed the identification of 42 high-confidence LASV polymerase interactors. We subsequently performed a small interfering RNA (siRNA) screen to identify those interactors that have functional roles in authentic LASV infection. As proof of principle, we characterized eukaryotic peptide chain release factor subunit 3a (eRF3a/GSPT1), which we found to be a proviral factor that physically associates with LASV polymerase. Targeted degradation of GSPT1 by a small-molecule drug candidate, CC-90009, resulted in strong inhibition of LASV infection in cultured cells. Our work demonstrates the feasibility of using proximity proteomics to illuminate and characterize yet-to-be-defined host-pathogen interactome, which can reveal new biology and uncover novel targets for the development of antivirals against highly pathogenic RNA viruses.

Metformin inhibition of mitochondrial ATP and DNA synthesis abrogates NLRP3 inflammasome activation and pulmonary inflammation.

Acute respiratory distress syndrome (ARDS), an inflammatory condition with high mortality rates, is common in severe COVID-19, whose risk is reduced by metformin rather than other anti-diabetic medications. Detecting of inflammasome assembly in post-mortem COVID-19 lungs, we asked whether and how metformin inhibits inflammasome activation while exerting its anti-inflammatory effect. We show that metformin inhibited NLRP3 inflammasome activation and interleukin (IL)-1ß production in cultured and alveolar macrophages along with inflammasome-independent IL-6 secretion, thus attenuating lipopolysaccharide (LPS)- and SARS-CoV-2-induced ARDS. By targeting electron transport chain complex 1 and independently of AMP-activated protein kinase (AMPK) or NF-κB, metformin blocked LPS-induced and ATP-dependent mitochondrial (mt) DNA synthesis and generation of oxidized mtDNA, an NLRP3 ligand. Myeloid-specific ablation of LPS-induced cytidine monophosphate kinase 2 (CMPK2), which is rate limiting for mtDNA synthesis, reduced ARDS severity without a direct effect on IL-6. Thus, inhibition of ATP and mtDNA synthesis is sufficient for ARDS amelioration.

Identification of Common CD8+ T Cell Epitopes from Lassa Fever Survivors in Nigeria and Sierra Leone.

Early and robust T cell responses have been associated with survival from Lassa fever (LF), but the Lassa virus-specific memory responses have not been well characterized. Regions within the virus surface glycoprotein (GPC) and nucleoprotein (NP) are the main targets of the Lassa virus-specific T cell responses, but, to date, only a few T cell epitopes within these proteins have been identified. We identified GPC and NP regions containing T cell epitopes and HLA haplotypes from LF survivors and used predictive HLA-binding algorithms to identify putative epitopes, which were then experimentally tested using autologous survivor samples. We identified 12 CD8-positive (CD8+) T cell epitopes, including epitopes common to both Nigerian and Sierra Leonean survivors. These data should be useful for the identification of dominant Lassa virus-specific T cell responses in Lassa fever survivors and vaccinated individuals as well as for designing vaccines that elicit cell-mediated immunity.IMPORTANCE The high morbidity and mortality associated with clinical cases of Lassa fever, together with the lack of licensed vaccines and limited and partially effective interventions, make Lassa virus (LASV) an important health concern in its regions of endemicity in West Africa. Previous infection with LASV protects from disease after subsequent exposure, providing a framework for designing vaccines to elicit similar protective immunity. Multiple major lineages of LASV circulate in West Africa, and therefore, ideal vaccine candidates should elicit immunity to all lineages. We therefore sought to identify common T cell epitopes between Lassa fever survivors from Sierra Leone and Nigeria, where distinct lineages circulate. We identified three such epitopes derived from highly conserved regions within LASV proteins. In this process, we also identified nine other T cell epitopes. These data should help in the design of an effective pan-LASV vaccine.

Differences in Tissue and Species Tropism of Reptarenavirus Species Studied by Vesicular Stomatitis Virus Pseudotypes.

Reptarenaviruses cause Boid Inclusion Body Disease (BIBD), and co-infections by several reptarenaviruses are common in affected snakes. Reptarenaviruses have only been found in captive snakes, and their reservoir hosts remain unknown. In affected animals, reptarenaviruses appear to replicate in most cell types, but their complete host range, as well as tissue and cell tropism are unknown. As with other enveloped viruses, the glycoproteins (GPs) present on the virion's surface mediate reptarenavirus cell entry, and therefore, the GPs play a critical role in the virus cell and tissue tropism. Herein, we employed single cycle replication, GP deficient, recombinant vesicular stomatitis virus (VSV) expressing the enhanced green fluorescent protein (scrVSV∆G-eGFP) pseudotyped with different reptarenavirus GPs to study the virus cell tropism. We found that scrVSV∆G-eGFPs pseudotyped with reptarenavirus GPs readily entered mammalian cell lines, and some mammalian cell lines exhibited higher, compared to snake cell lines, susceptibility to reptarenavirus GP-mediated infection. Mammarenavirus GPs used as controls also mediated efficient entry into several snake cell lines. Our results confirm an important role of the virus surface GP in reptarenavirus cell tropism and that mamma-and reptarenaviruses exhibit high cross-species transmission potential.

Variables psicosociales predictoras de la no adherencia a los antirretrovirales en personas con VIH-SIDA / Predictive Psychosocial Variables of Non-Adherence to Antiretrovirals in People with HIV-AIDS

Resumen Objetivo: El presente estudio tuvo como objetivo evaluar los niveles de adherencia al tratamiento antirretroviral, así como determinar los factores psicológicos predictores de la no adherencia a dicho tratamiento en pacientes con VIH-SIDA. Método: Diseño descriptivo, transversal y correlacional. La muestra estuvo conformada por 25 personas diagnosticadas con VIH-SIDA que asistieron a los servicios integrales de una asociación civil en la ciudad de Guadalajara, México, y que se encontraban bajo tratamiento antirretroviral. Se utilizó el Cuestionario de adherencia al tratamiento para el VIH-SIDA [CAT-VIH] y el Cuestionario de Factores Relacionados con la Adherencia al Tratamiento para el VIH/SIDA (CFR-AT VIH). Se realizaron análisis descriptivos y no paramétricos para la comparación de las variables de estudio, así como sus respectivas correlaciones. Resultados: Solo 60% de los evaluados presentan una adecuada adherencia al tratamiento con antirretrovirales; además se encontraron diferencias significativas entre adherentes y no adherentes en las variables de autoeficacia (Z=-3.196; p=.001); estrés (Z=-2.033; p=.042); y depresión (Z=-2.409; p=.016). Se obtuvieron correlaciones positivas de la adherencia con autoeficacia (r=.750; p=.000) y apoyo social (r=.462; p=.020) y correlaciones negativas con estrés (r=-.543; p=.005) y depresión (r=-.544; p=.005). Conclusiones: A mayores estados de estrés y depresión, menor serán los niveles de adherencia al tratamiento con antirretrovirales; por lo cual estas variables deben ser tomadas en cuenta para el diseño de estrategias de mejora de la adherencia en personas con VIH-SIDA.

Abstract Objective: Objective: The main aim of this study was to evaluate the levels of adherence to antiretrovirals treatment as well as to determine the psychological factors that predict the non-adherence to antiretrovirals in HIV patients who attend a civil association in Guadalajara, México. Method: It was a descriptive, transversal and correlational design. The sample consisted of 25 patients diagnosed with HIV who attended integral services of a Civil Association. The Adherence to Treatment for HIV/AIDS Questionnaire [CAT-HIV] and the Questionnaire of Factors Related to Adherence to Treatment for HIV/AIDS (CFR-AT HIV) were used. Descriptive and nonparametric analyzes were carried out to compare the study variables, as well as their respective correlations, all through the statistical program SPSS v.20. Results: Only 60% of those evaluated showed adequate adherence to antiretrovirals; in addition, significance differences between adherents and non-adherents in the self-efficacy variable (Z = -3.196, p = .001); stress (Z = -2.033, p = .042); and depression (Z = -2.409, p = .016). Positive correlations of adherence were obtained with self-efficacy (r = .750, p = .000) and social support (r = .462, p = .020) and negative correlations with stress (r = - .543; p = .005) and depression (r = -. 544; p = .005). Conclusions: The higher levels of stress and depression, the lower the levels of adherence to antiretroviral treatment; therefore, these variables must be considered in the design of strategies to improve adherence in people with HIV.

Dimethyl Fumarate Disrupts Human Innate Immune Signaling by Targeting the IRAK4-MyD88 Complex.

Dimethyl fumarate (DMF) is a prescribed treatment for multiple sclerosis and has also been used to treat psoriasis. The electrophilicity of DMF suggests that its immunosuppressive activity is related to the covalent modification of cysteine residues in the human proteome. Nonetheless, our understanding of the proteins modified by DMF in human immune cells and the functional consequences of these reactions remains incomplete. In this study, we report that DMF inhibits human plasmacytoid dendritic cell function through a mechanism of action that is independent of the major electrophile sensor NRF2. Using chemical proteomics, we instead identify cysteine 13 of the innate immune kinase IRAK4 as a principal cellular target of DMF. We show that DMF blocks IRAK4-MyD88 interactions and IRAK4-mediated cytokine production in a cysteine 13-dependent manner. Our studies thus identify a proteomic hotspot for DMF action that constitutes a druggable protein-protein interface crucial for initiating innate immune responses.

PLD3 and PLD4 are single-stranded acid exonucleases that regulate endosomal nucleic-acid sensing.

The sensing of microbial genetic material by leukocytes often elicits beneficial pro-inflammatory cytokines, but dysregulated responses can cause severe pathogenesis. Genome-wide association studies have linked the gene encoding phospholipase D3 (PLD3) to Alzheimer's disease and have linked PLD4 to rheumatoid arthritis and systemic sclerosis. PLD3 and PLD4 are endolysosomal proteins whose functions are obscure. Here, PLD4-deficient mice were found to have an inflammatory disease, marked by elevated levels of interferon-γ (IFN-γ) and splenomegaly. These phenotypes were traced to altered responsiveness of PLD4-deficient dendritic cells to ligands of the single-stranded DNA sensor TLR9. Macrophages from PLD3-deficient mice also had exaggerated TLR9 responses. Although PLD4 and PLD3 were presumed to be phospholipases, we found that they are 5' exonucleases, probably identical to spleen phosphodiesterase, that break down TLR9 ligands. Mice deficient in both PLD3 and PLD4 developed lethal liver inflammation in early life, which indicates that both enzymes are needed to regulate inflammatory cytokine responses via the degradation of nucleic acids.

DDX3 suppresses type I interferons and favors viral replication during Arenavirus infection.

Several arenaviruses cause hemorrhagic fever (HF) diseases that are associated with high morbidity and mortality in humans. Accordingly, HF arenaviruses have been listed as top-priority emerging diseases for which countermeasures are urgently needed. Because arenavirus nucleoprotein (NP) plays critical roles in both virus multiplication and immune-evasion, we used an unbiased proteomic approach to identify NP-interacting proteins in human cells. DDX3, a DEAD-box ATP-dependent-RNA-helicase, interacted with NP in both NP-transfected and virus-infected cells. Importantly, DDX3 deficiency compromised the propagation of both Old and New World arenaviruses, including the HF arenaviruses Lassa and Junin viruses. The DDX3 role in promoting arenavirus multiplication associated with both a previously un-recognized DDX3 inhibitory role in type I interferon production in arenavirus infected cells and a positive DDX3 effect on arenavirus RNA synthesis that was dependent on its ATPase and Helicase activities. Our results uncover novel mechanisms used by arenaviruses to exploit the host machinery and subvert immunity, singling out DDX3 as a potential host target for developing new therapies against highly pathogenic arenaviruses.

[Abdominal unplanned reoperations in the Service of General Surgery, University Hospital of Puebla]. / Reintervenciones quirúrgicas abdominales no planeadas en el Servicio de Cirugía General del Hospital Universitario de Puebla.

INTRODUCTION: The reoperation is considered as the access to the abdominal cavity before complete healing of the surgical wound from a previous operation within the first 60 days after the first procedure. It occurs in 0.5 to 15% of patients undergoing abdominal surgery and generates significant increase in morbidity and mortality in patients undergoing abdominal surgery. OBJECTIVES: Identify the number of unplanned abdominal surgical reoperations and identify the causes of these unplanned reoperations were performed in our department. METHODOLOGY: This is a retrospective study conducted at the University Hospital of Puebla in the period between April 2009 to February 2012, a total of 1,709 abdominal surgeries performed by the Service of General Surgery were included. RESULTS: Ninety-seven cases of reoperation of which 50 cases were not planned surgery cases were identified; 72% (36 cases) from emergency operations, and 28% of elective surgery. CONCLUSIONS: The incidence found in our study is low compared to similar studies. Prospective studies and focus on risk factors and causes of unplanned reoperations are required, in order to know them in detail and, consequently, reduce its incidence and morbidity and mortality they add.

Type I and Type II Interferon Coordinately Regulate Suppressive Dendritic Cell Fate and Function during Viral Persistence.

Persistent viral infections are simultaneously associated with chronic inflammation and highly potent immunosuppressive programs mediated by IL-10 and PDL1 that attenuate antiviral T cell responses. Inhibiting these suppressive signals enhances T cell function to control persistent infection; yet, the underlying signals and mechanisms that program immunosuppressive cell fates and functions are not well understood. Herein, we use lymphocytic choriomeningitis virus infection (LCMV) to demonstrate that the induction and functional programming of immunosuppressive dendritic cells (DCs) during viral persistence are separable mechanisms programmed by factors primarily considered pro-inflammatory. IFNγ first induces the de novo development of naive monocytes into DCs with immunosuppressive potential. Type I interferon (IFN-I) then directly targets these newly generated DCs to program their potent T cell immunosuppressive functions while simultaneously inhibiting conventional DCs with T cell stimulating capacity. These mechanisms of monocyte conversion are constant throughout persistent infection, establishing a system to continuously interpret and shape the immunologic environment. MyD88 signaling was required for the differentiation of suppressive DCs, whereas inhibition of stimulatory DCs was dependent on MAVS signaling, demonstrating a bifurcation in the pathogen recognition pathways that promote distinct elements of IFN-I mediated immunosuppression. Further, a similar suppressive DC origin and differentiation was also observed in Mycobacterium tuberculosis infection, HIV infection and cancer. Ultimately, targeting the underlying mechanisms that induce immunosuppression could simultaneously prevent multiple suppressive signals to further restore T cell function and control persistent infections.

Arenavirus Genome Rearrangement for the Development of Live Attenuated Vaccines.

UNLABELLED: Several members of the Arenaviridae family cause hemorrhagic fever disease in humans and pose serious public health problems in their geographic regions of endemicity as well as a credible biodefense threat. To date, there have been no FDA-approved arenavirus vaccines, and current antiarenaviral therapy is limited to an off-label use of ribavirin that is only partially effective. Arenaviruses are enveloped viruses with a bisegmented negative-stranded RNA genome. Each genome segment uses an ambisense coding strategy to direct the synthesis of two viral polypeptides in opposite orientations, separated by a noncoding intergenic region. Here we have used minigenome-based approaches to evaluate expression levels of reporter genes from the nucleoprotein (NP) and glycoprotein precursor (GPC) loci within the S segment of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV). We found that reporter genes are expressed to higher levels from the NP than from the GPC locus. Differences in reporter gene expression levels from the NP and GPC loci were confirmed with recombinant trisegmented LCM viruses. We then used reverse genetics to rescue a recombinant LCMV (rLCMV) containing a translocated viral S segment (rLCMV/TransS), where the viral NP and GPC open reading frames replaced one another. The rLCMV/TransS showed slower growth kinetics in cultured cells and was highly attenuated in vivo in a mouse model of lethal LCMV infection, but immunization with rLCMV/TransS conferred complete protection against a lethal challenge with wild-type LCMV. Attenuation of rLCMV/TransS was associated with reduced NP expression levels. These results open a new avenue for the development of arenavirus live attenuated vaccines based on rearrangement of their viral genome. IMPORTANCE: Several arenaviruses cause severe hemorrhagic fever in humans and also pose a credible bioterrorism threat. Currently, no FDA-licensed vaccines are available to combat arenavirus infections and antiarenaviral therapy is limited to the off-label use of ribavirin, which is only partially effective and associated with side effects. Here we describe, for the first time, the generation of a recombinant LCMV where the viral protein products encoded by the S RNA segment (NP and GPC) were swapped to generate rLCMV/TransS. rLCMV/TransS exhibited reduced viral multiplication in cultured cells and was highly attenuated in vivo while conferring protection, upon a single immunization dose, against a lethal challenge with wild-type LCMV. Our studies provide a proof of concept for the rational development of safe and protective live attenuated vaccine candidates based on genome reorganization for the treatment of pathogenic arenavirus infections in humans.

Lymphocytic Choriomeningitis Virus Differentially Affects the Virus-Induced Type I Interferon Response and Mitochondrial Apoptosis Mediated by RIG-I/MAVS.

UNLABELLED: Arenaviruses are important emerging human pathogens maintained by noncytolytic persistent infection in their rodent reservoir hosts. Despite high levels of viral replication, persistently infected carrier hosts show only mildly elevated levels of type I interferon (IFN-I). Accordingly, the arenavirus nucleoprotein (NP) has been identified as a potent IFN-I antagonist capable of blocking activation of interferon regulatory factor 3 (IRF3) via the retinoic acid inducible gene (RIG)-I/mitochondrial antiviral signaling (MAVS) pathway. Another important mechanism of host innate antiviral defense is represented by virus-induced mitochondrial apoptosis via RIG-I/MAVS and IRF3. In the present study, we investigated the ability of the prototypic Old World arenavirus lymphocytic choriomeningitis virus (LCMV) to interfere with RIG-I/MAVS-dependent apoptosis. We found that LCMV does not induce apoptosis at any time during infection. While LCMV efficiently blocked induction of IFN-I via RIG-I/MAVS in response to superinfection with cytopathic RNA viruses, virus-induced mitochondrial apoptosis remained fully active in LCMV-infected cells. Notably, in LCMV-infected cells, RIG-I was dispensable for virus-induced apoptosis via MAVS. Our study reveals that LCMV infection efficiently suppresses induction of IFN-I but does not interfere with the cell's ability to undergo virus-induced mitochondrial apoptosis as a strategy of innate antiviral defense. The RIG-I independence of mitochondrial apoptosis in LCMV-infected cells provides the first evidence that arenaviruses can reshape apoptotic signaling according to their needs. IMPORTANCE: Arenaviruses are important emerging human pathogens that are maintained in their rodent hosts by persistent infection. Persistent virus is able to subvert the cellular interferon response, a powerful branch of the innate antiviral defense. Here, we investigated the ability of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) to interfere with the induction of programmed cell death, or apoptosis, in response to superinfection with cytopathic RNA viruses. Upon viral challenge, persistent LCMV efficiently blocked induction of interferons, whereas virus-induced apoptosis remained fully active in LCMV-infected cells. Our studies reveal that the persistent virus is able to reshape innate apoptotic signaling in order to prevent interferon production while maintaining programmed cell death as a strategy for innate defense. The differential effect of persistent virus on the interferon response versus its effect on apoptosis appears as a subtle strategy to guarantee sufficiently high viral loads for efficient transmission while maintaining apoptosis as a mechanism of defense.

Suppression of Fcγ-receptor-mediated antibody effector function during persistent viral infection.

Understanding how viruses subvert host immunity and persist is essential for developing strategies to eliminate infection. T cell exhaustion during chronic viral infection is well described, but effects on antibody-mediated effector activity are unclear. Herein, we show that increased amounts of immune complexes generated in mice persistently infected with lymphocytic choriomeningitis virus (LCMV) suppressed multiple Fcγ-receptor (FcγR) functions. The high amounts of immune complexes suppressed antibody-mediated cell depletion, therapeutic antibody-killing of LCMV infected cells and human CD20-expressing tumors, as well as reduced immune complex-mediated cross-presentation to T cells. Suppression of FcγR activity was not due to inhibitory FcγRs or high concentrations of free antibody, and proper FcγR functions were restored when persistently infected mice specifically lacked immune complexes. Thus, we identify a mechanism of immunosuppression during viral persistence with implications for understanding effective antibody activity aimed at pathogen control.

Updated Frequency of EGFR and KRAS Mutations in NonSmall-Cell Lung Cancer in Latin America: The Latin-American Consortium for the Investigation of Lung Cancer (CLICaP).

INTRODUCTION: Previously, we reported the frequency of epidermal growth factor receptor (EGFR) and KRAS mutations in nonsmall-cell lung cancer (NSCLC) patients in Latin America. The EGFR mutation frequency was found between Asian (40%) and Caucasian (15%) populations. Here, we report the updated distribution of NSCLC mutations. METHODS: A total of 5738 samples from NSCLC patients from Argentina (1713), Mexico (1417), Colombia (1939), Peru (393), Panama (174), and Costa Rica (102) were genotyped for EGFR and KRAS. RESULTS: The median patient age was 62.2 ± 12.3 years; 53.5% were women, 46.7% had a history of smoking, and 95.2% had adenocarcinoma histology. The frequency of EGFR mutations was 26.0% (95% confidence interval [CI], 24.9-27.1; Argentina, 14.4% [12.8-15.6]; México, 34.3% [31.9-36.7]; Colombia, 24.7% [22.8-26.6]; Peru, 51.1% [46.2-55.9]; Panamá, 27.3 [20.7-33.9]; and Costa Rica, 31.4% [22.4-40.4]). The frequency of KRAS mutations was 14.0% (9.1-18.9). In patients with adenocarcinoma, EGFR mutations were independently associated with gender (30.7% females vs. 18.4% males; p < 0.001), nonsmoker status (27.4% vs. 17.1%, p < 0.001), ethnicity (mestizo/indigenous, 35.3% vs. Caucasian, 13.7%, p < 0.001), and the absence of KRAS mutation (38.1% vs. 4.7%; p < 0.001). The overall response rate to EGFR tyrosine kinase inhibitors was 60.6% (95% CI, 52-69), with a median progression-free survival and overall survival of 15.9 (95% CI, 12.420.6) and 32 months (95% CI, 26.5-37.6), respectively. CONCLUSION: Our findings support the genetic heterogeneity of NSCLC in Latin America, confirming that the frequency of EGFR mutations is intermediate between that observed in the Asian and Caucasian populations.

Selective inhibitor of endosomal trafficking pathways exploited by multiple toxins and viruses.

Pathogenic microorganisms and toxins have evolved a variety of mechanisms to gain access to the host-cell cytosol and thereby exert virulent effects upon the host. One common mechanism of cellular entry requires trafficking to an acidified endosome, which promotes translocation across the host membrane. To identify small-molecule inhibitors that block this process, a library of 30,000 small molecules was screened for inhibitors of anthrax lethal toxin. Here we report that 4-bromobenzaldehyde N-(2,6-dimethylphenyl)semicarbazone, the most active compound identified in the screen, inhibits intoxication by lethal toxin and blocks the entry of multiple other acid-dependent bacterial toxins and viruses into mammalian cells. This compound, which we named EGA, also delays lysosomal targeting and degradation of the EGF receptor, indicating that it targets host-membrane trafficking. In contrast, EGA does not block endosomal recycling of transferrin, retrograde trafficking of ricin, phagolysosomal trafficking, or phagosome permeabilization by Franciscella tularensis. Furthermore, EGA does not neutralize acidic organelles, demonstrating that its mechanism of action is distinct from pH-raising agents such as ammonium chloride and bafilomycin A1. EGA is a powerful tool for the study of membrane trafficking and represents a class of host-targeted compounds for therapeutic development to treat infectious disease.