RESUMEN
ABSTRACT: Increased epithelial permeability in sepsis is mediated via disruptions in tight junctions, which are closely associated with the perijunctional actin-myosin ring. Genetic deletion of myosin light chain kinase (MLCK) reverses sepsis-induced intestinal hyperpermeability and improves survival in a murine model of intra-abdominal sepsis. In an attempt to determine the generalizability of these findings, this study measured the impact of MLCK deletion on survival and potential associated mechanisms following pneumonia-induced sepsis. MLCK -/- and wild-type mice underwent intratracheal injection of Pseudomonas aeruginosa . Unexpectedly, survival was significantly worse in MLCK -/- mice than wild-type mice. This was associated with increased permeability to Evans blue dye in bronchoalveolar lavage fluid but not in tissue homogenate, suggesting increased alveolar epithelial leak. In addition, bacterial burden was increased in bronchoalveolar lavage fluid. Cytokine array using whole-lung homogenate demonstrated increases in multiple proinflammatory and anti-inflammatory cytokines in knockout mice. These local pulmonary changes were associated with systemic inflammation with increased serum levels of IL-6 and IL-10 and a marked increase in bacteremia in MLCK -/- mice. Increased numbers of both bulk and memory CD4 + T cells were identified in the spleens of knockout mice, with increased early and late activation. These results demonstrate that genetic deletion of MLCK unexpectedly increases mortality in pulmonary sepsis, associated with worsened alveolar epithelial leak and both local and systemic inflammation. This suggests that caution is required in targeting MLCK for therapeutic gain in sepsis.
Asunto(s)
Pulmón , Quinasa de Cadena Ligera de Miosina , Neumonía , Sepsis , Animales , Ratones , Citocinas , Inflamación , Mucosa Intestinal , Pulmón/metabolismo , Pulmón/patología , Ratones Noqueados , Quinasa de Cadena Ligera de Miosina/genética , Permeabilidad , Neumonía/complicaciones , Sepsis/patología , Uniones Estrechas/fisiologíaRESUMEN
RIPK3 (receptor-interacting protein kinase 3) activity triggers cell death via necroptosis, whereas scaffold function supports protein binding and cytokine production. To determine if RIPK3 kinase or scaffold domains mediate pathology during Pseudomonas aeruginosa infection, control mice and those with deletion or mutation of RIPK3 and associated signaling partners were subjected to Pseudomonas pneumonia and followed for survival or killed for biologic assays. Murine immune cells were studied in vitro for Pseudomonas-induced cytokine production and cell death, and RIPK3 binding interactions were blocked with the viral inhibitor M45. Human tissue effects were assayed by infecting airway epithelial cells with Pseudomonas and measuring cytokine production after siRNA inhibition of RIPK3. Deletion of RIPK3 reduced inflammation and decreased animal mortality after Pseudomonas pneumonia. RIPK3 kinase inactivation did neither. In cell culture, RIPK3 was dispensable for cell killing by Pseudomonas and instead drove cytokine production that required the RIPK3 scaffold domain but not kinase activity. Blocking the RIP homotypic interaction motif (RHIM) with M45 reduced the inflammatory response to infection in vitro. Similarly, siRNA knockdown of RIPK3 decreased infection-triggered inflammation in human airway epithelial cells. Thus, the RIPK3 scaffold drives deleterious pulmonary inflammation and mortality in a relevant clinical model of Pseudomonas pneumonia. This process is distinct from kinase-mediated necroptosis, requiring only the RIPK3 RHIM. Inhibition of RHIM signaling is a potential strategy to reduce lung inflammation during infection.
Asunto(s)
Neumonía , Pseudomonas aeruginosa , Animales , Humanos , Ratones , Pseudomonas aeruginosa/metabolismo , Apoptosis , Inflamación/metabolismo , ARN Interferente Pequeño , Citocinas/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genéticaRESUMEN
The purpose of this study was to assess if rates of firearm trauma within Atlanta geospatially clustered with HIV prevalence and new HIV diagnosis rates. We retrospectively reviewed our Atlanta trauma center's registry for patients sustaining a ballistic firearm trauma from 2014 through 2018. Using the patient's zip code of home residence, we determined the rate of firearm trauma for that zip code. We obtained publicly available rates for HIV that corresponded with these select zip codes to perform a geospatial cluster analysis. The cohort was comprised of 1495 patients and represented 35 zip codes in Atlanta. The mean rate of firearm trauma for the 35 zip codes was 171.1 (±296.4) per 100,000 people. Compared to all Atlanta, the 35 zip codes' mean HIV prevalence (1863.9 vs 924.1, p < .0001) and new HIV diagnosis rate (396.9 vs 199.7, p < .0001) were significantly higher. Rates of firearm trauma and HIV prevalence demonstrated significant geospatial clustering (ß 0.38, 95% CI 0.22-0.53, p < .0001) as did rates of firearm trauma and new HIV diagnoses (ß 0.36, 95% CI 0.18-0.54, p = 0.0002). Our findings provide granular geographic data that could guide targeted HIV screening efforts in communities where our firearm-injured patients live.
Asunto(s)
Infecciones por VIH , Humanos , Estudios Retrospectivos , Infecciones por VIH/diagnóstico , Infecciones por VIH/epidemiología , Análisis por Conglomerados , Sistema de RegistrosRESUMEN
Animal-based research is essential to the study of sepsis pathophysiology, diagnostics, and therapeutics. However, animal models of sepsis are often associated with high mortality because of the difficulty in predicting imminent death based on premortem assessment of the animals. The use of validated visual scoring would allow researchers to systematically identify humane endpoints but visual approaches require high interobserver agreement for accurate results. The objective of this study was to establish a scoring system for mice undergoing cecal ligation and puncture (CLP)-induced sepsis based on 3 visual parameters: respiratory status, activity and response to stimulus (ASR), and eye appearance, with scores ranging from 0 to 3. In the first study, we evaluated interobserver agreement. Veterinary and investigative staff assessed 283 mice with CLP and had substantial to near-perfect agreement for all 3 parameters as evaluated using weighted Cohen κ statistic. The second study assessed the ability of the scoring system and temperature to predict death. The scoring system and subcutaneous transpond- ers were used to monitor C57BL/6J mice (n = 80, male and female) until death or for 7 days after CLP. Results showed that the scoring system discriminates between surviving (n = 26) and nonsurviving (n = 54) septic mice. The scoring system was accurate in predicting death, with an AUC of 0.8997. The sensitivity and specificity of the ASR parameter were 96% and 92%, respectively, and for the eye parameter were 94% and 73%. A sum of the ASR and eye scores that was 5 or more was also predictive of death. Temperature was a quantitative predictor, with sensitivity and specificity of 93% and 92%, respectively. This scoring system refines the CLP model by allowing identification of humane endpoints and avoidance of spontaneous death.
Asunto(s)
Punciones , Sepsis , Humanos , Ratones , Masculino , Femenino , Animales , Ratones Endogámicos C57BL , Punciones/efectos adversos , Ligadura/efectos adversos , Ciego/cirugía , Modelos Animales de EnfermedadRESUMEN
Expression of the tight junction-associated protein junctional adhesion molecule-A (JAM-A) is increased in sepsis, although the significance of this is unknown. Here, we show that septic JAM-A -/- mice have increased gut permeability, yet paradoxically have decreased bacteremia and systemic TNF and IL-1ß expression. Survival is improved in JAM-A-/- mice. However, intestine-specific JAM-A-/- deletion does not alter mortality, suggesting that the mortality benefit conferred in mice lacking JAM-A is independent of the intestine. Septic JAM-A-/- mice have increased numbers of splenic CD44hiCD4+ T cells, decreased frequency of TNF+CD4+ cells, and elevated frequency of IL-2+CD4+ cells. Septic JAM-A-/- mice have increased numbers of B cells in mesenteric lymph nodes with elevated serum IgA and intraepithelial lymphocyte IgA production. JAM-A-/- × RAG-/- mice have improved survival compared with RAG-/- mice and identical mortality as WT mice. Gut neutrophil infiltration and neutrophil phagocytosis are increased in JAM-A-/- mice, while septic JAM-A-/- mice depleted of neutrophils lose their survival advantage. Therefore, increased bacterial clearance via neutrophils and an altered systemic inflammatory response with increased opsonizing IgA produced through the adaptive immune system results in improved survival in septic JAM-A-/- mice. JAM-A may be a therapeutic target in sepsis via immune mechanisms not related to its role in permeability.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Molécula A de Adhesión de Unión , Receptores de Superficie Celular/metabolismo , Sepsis , Animales , Moléculas de Adhesión Celular/genética , Modelos Animales de Enfermedad , Inmunoglobulina A , Ratones , Ratones Endogámicos C57BL , Fagocitosis , Receptores de Superficie Celular/genética , Sepsis/genéticaRESUMEN
BACKGROUND: Gallbladder cancer (GBC) is the most common biliary malignancy frequently metastatic at diagnosis with poor prognosis. While surgery remains the standard for early-stage GBC, the role of surgery in patients with metastatic gastrointestinal cancers is expanding due to improvements in systemic therapies. We sought to evaluate the survival of patients with stage IV GBC undergoing surgery in an era of improved multi-agent systemic therapy. METHODS: A retrospective review of the National Cancer Database was performed. Patients with stage IV GBC who underwent systemic therapy were included. Patients who received radiation therapy, palliative therapy or had missing survival data were excluded. Univariable and multivariable analysis was performed. RESULTS: 4,145 patients were identified between 2004 and 2016. Mean age was 69. Surgery combined with systemic therapy predicted improved median survival compared with chemotherapy alone (11.1mo versus 6.8mo, HR 0.65, p < 0.001). Additionally, receipt of treatment after 2011 predicted improved survival (HR 0.86, p < 0.001). Patients treated with multi-agent chemotherapy in combination with surgery were associated with the greatest hazard ratio benefit (0.40, p < 0.001) versus single agent therapy alone. CONCLUSION: Patients with stage IV gallbladder cancer treated with a combination of surgery and chemotherapy are associated with an improved overall survival compared to chemotherapy alone. Patients receiving care during the more recent era demonstrated improved survival. These results support a role for surgery in selected patients with stage IV gallbladder cancer receiving chemotherapy.
Asunto(s)
Neoplasias de la Vesícula Biliar , Humanos , Anciano , Neoplasias de la Vesícula Biliar/patología , Estadificación de Neoplasias , Modelos de Riesgos Proporcionales , Estudios RetrospectivosRESUMEN
Sepsis induces significant immune dysregulation characterized by lymphocyte apoptosis and alterations in the cytokine milieu. Because cancer patients exhibit a 10-fold greater risk of developing sepsis compared with the general population, we aimed to understand how pre-existing malignancy alters sepsis-induced immune dysregulation. To address this question, we assessed the impact of tumor-specific CD8+ T cells on the immune response in a mouse model of cecal ligation and puncture (CLP)-induced sepsis. Tumor-bearing animals containing Thy1.1+ tumor-specific CD8+ T cells were subjected to CLP, and groups of animals received anti-Thy1.1 mAb to deplete tumor-specific CD8+ T cells or isotype control. Results indicated that depleting tumor-specific T cells significantly improved mortality from sepsis. The presence of tumor-specific CD8+ T cells resulted in increased expression of the 2B4 coinhibitory receptor and increased apoptosis of endogenous CD8+ T cells. Moreover, tumor-specific T cells were not reduced in number in the tumors during sepsis but did exhibit impaired IFN-γ production in the tumor, tumor draining lymph node, and spleen 24 h after CLP. Our research provides novel insight into the mechanisms by which pre-existing malignancy contributes to increased mortality during sepsis.