An analysis of risk factors for carbapenem-resistant Enterobacteriaceae infection.

OBJECTIVES: This study aims to explore the high-risk factors of carbapenem-resistant Enterobacteriaceae (CRE) infection of hospitalised patients in high-risk departments. METHODS: This study is a multicentre, retrospective study. CRE screening positive patients from 1 January 2016 to 31 December 2018 of high-risk departments in five tertiary first-class teaching hospitals in Beijing collect the patients' CRE test specimen information, CRE infection information and outcomes. The patients were divided into a colonisation group and an infection group for comparative analysis. A logistic regression model was established to explore the risk factors of CRE infection. Subgroup analysis was conducted according to invasive procedures and the type of the infection. RESULTS: In total, 344 patients were included in this study, including 85 (24.71%) colonisation and 259 (75.29%) infection; 36.09% CRE colonisation converted to infection, and the mean conversion time from colonisation to infection was 6.5 (4.0, 18.8) days. Renal disease, granulocytosis, invasive procedures and the time from hospital stay to positive CRE were the risk factors for CRE infection. The subgroup analysis showed that the rate of CRE infection in the invasive group was higher than in the non-invasive group (P < 0.001), and the rate of exacerbation or death in the invasive group was also higher than in the non-invasive group (P = 0.019). The average length of ICU and hospitalisation in the healthcare-associated infection group were significantly higher than those in the community infection group, but there was no difference in the proportion of final exacerbation or death between them (P = 0.727). CONCLUSION: Kidney disease, granulocytosis, invasive procedures and CRE detection time are the risk factors for CRE infection. Carrying out CRE screening in patients as early as possible and taking effective intervention measures in time to avoid adverse consequences is all important.

Clinical features and risk factors of severely and critically ill patients with COVID-19.

BACKGROUND: As of June 1, 2020, over 370000 coronavirus disease 2019 (COVID-19) deaths have been reported to the World Health Organization. However, the risk factors for patients with moderate-to-severe or severe-to-critical COVID-19 remain unclear. AIM: To explore the characteristics and predictive markers of severely and critically ill patients with COVID-19. METHODS: A retrospective study was conducted at the B11 Zhongfaxincheng campus and E1-3 Guanggu campus of Tongji Hospital affiliated with Huazhong University of Science and Technology in Wuhan. Patients with COVID-19 admitted from 1st February 2020 to 8th March 2020 were enrolled and categorized into 3 groups: The moderate group, severe group and critically ill group. Epidemiological data, demographic data, clinical symptoms and outcomes, complications, laboratory tests and radiographic examinations were collected retrospectively from the hospital information system and then compared between groups. RESULTS: A total of 126 patients were enrolled. There were 59 in the moderate group, 49 in the severe group, and 18 in the critically ill group. Multivariate logistic regression analysis showed that age [odd ratio (OR) = 1.055, 95% (confidence interval) CI: 1.099-1.104], elevated neutrophil-to-lymphocyte ratios (OR = 4.019, 95%CI: 1.045-15.467) and elevated high-sensitivity cardiac troponin I (OR = 10.126, 95%CI: 1.088 -94.247) were high-risk factors. CONCLUSION: The following indicators can help clinicians identify patients with severe COVID-19 at an early stage: age, an elevated neutrophil-to-lymphocyte ratio and high sensitivity cardiac troponin I.

Nutritional practice in critically ill COVID-19 patients: A multicenter ambidirectional cohort study in Wuhan and Jingzhou.

BACKGROUND AND OBJECTIVES: The novel coronavirus disease (COVID-19) epidemic is spreading all over the world. With the number of cases increasing rapidly, the epidemiological data on the nutritional practice is scarce. In this study, we aim to describe the clinical characteristics and nutritional practice in a cohort of critically ill COVID-19 patients. METHODS AND STUDY DESIGN: This is a multicenter, ambidirectional cohort study conducted at 11 hospitals in Hubei Province, China. All eligible critical COVID-19 patients in the study hospital intensive care units at 00:00, March 6th, 2020, were included. Data collection was performed via written case report forms. RESULTS: A total of 44 patients were identified and enrolled, of whom eight died during the 28-day outcome follow- up period. The median interval between hospital admission and the study day was 24 (interquartile range, 13- 26) days and 52.2% (23 of 44) of patients were on invasive mechanical ventilation. The median nutrition risk in critically ill (mNUTRIC) score was 3 (interquartile range, 2-5) on the study day. During the enrolment day, 68.2% (30 of 44) of patients received enteral nutrition (EN), while 6.8% (3 of 44) received parenteral nutrition (PN) alone. Nausea and aspiration were uncommon, with a prevalence of 11.4% (5 of 44) and 6.8% (3 of 44), respectively. As for energy delivery, 69.7% (23 of 33) of patients receiving EN and/or PN were achieving their prescribed targets. CONCLUSIONS: The study showed that EN was frequently applied in critical COVID-19 patients. Energy delivery may be suboptimal in this study requiring more attention.

Use of recombinant activated factor VII for the treatment of perioperative bleeding in noncardiac surgery patients without hemophilia: A systematic review and meta-analysis of randomized controlled trials.

PURPOSE: To evaluate the efficacy and safety of perioperative use of recombinant activated factor VII (rFVIIa) in noncardiac patients. MATERIALS AND METHODS: We searched electronic databases for randomized controlled trials (RCTs) that involved the use of rFVIIa through December 13, 2019 in noncardiac patients without hemophilia. Two investigators extracted the related data and assessed the quality of the included trials. RESULTS: Eleven RCTs examining 993 perioperative patients were ultimately included. The use of rFVIIa did not decrease all-cause mortality (RR:0.90; 95% CI:0.50,1.64; I2 = 0.0%; P = 0.738), shorten the length of ICU (SMD:-0.15; 95% CI:-0.47,0.17; I2 = 0.0%; P = 0.346) or hospital (SMD:0.42; 95% CI:-0.05,0.89; I2 = 0.0%; P = 0.078) stay, or increase incidence of the thromboembolic events (RR:1.30; 95% CI:0.70,2.41; I2 = 0.0%; P = 0.403) among perioperative patients. However, individual RCT analyses showed that the use of rFVIIa could reduce the volume of blood loss (including prostatic cancer, severe acute pancreatitis (SAP), and spinal disease) and the transfusion of RBCs (including prostatic cancer, SAP, and spinal disease) and FFP (SAP) in a subset of perioperative patients. Publication bias was not present. CONCLUSIONS: For perioperative hemorrhagic patients, rFVIIa-based hemostatic therapy showed no effect on mortality, ICU or hospital LOS, or the rate of thromboembolic events, although it appears to decrease blood loss and reduce the need for blood product transfusion in a subset of patients.

Lactylated Histone H3K18 as a Potential Biomarker for the Diagnosis and Predicting the Severity of Septic Shock.

Objective: To date, there are no studies regarding the lactylation profile and its role in critically ill patients. Thus, we aimed to examine expression of histone H3 lysine 18 (H3K18) lactylation and its role in patients with septic shock. Methods: Thirteen healthy volunteers and 35 critically ill patients from the Department of Surgical Intensive Care Medicine, Beijing Hospital were enrolled in our study. Baseline information and clinical outcomes were obtained prospectively. Lactylation levels of all proteins and H3K18 from peripheral blood mononuclear (PBMC) were determined by western blotting and serum levels of inflammatory cytokines by flow cytometry. Arginase-1 (Arg1) and Krüppel-like factor-4 (Klf4) mRNA expression was evaluated by quantitative real-time PCR (qRT-PCR). Results: Lactylation was found to be an all-protein post-translational modification and was detected in PBMCs from both healthy volunteers and critically ill patients, with a significantly higher relative density in shock patients (t=2.172, P=0.045). H3K18la was expressed in all subjects, including healthy volunteers, with the highest level in septic shock patients (compared with non-septic shock patients, critically ill without shock patients and healthy volunteers P=0.033, 0.000 and 0.000, respectively). Furthermore, H3K18la protein expression correlated positively with APACHE II scores, SOFA scores on day 1, ICU stay, mechanical ventilation time and serum lactate (ρ=0.42, 0.63, 0.39, 0.51 and 0.48, respectively, ρ=0.012, 0.000, 0.019, 0.003 and 0.003, respectively). When we matched patients with septic shock and with non-septic shock according to severity, we found higher H3K18la levels in the former group (t=-2.208, P =0.040). Moreover, H3K18la exhibited a close correlation with procalcitonin levels (ρ=0.71, P=0.010). Patients with high H3K18la expression showed higher IL-2, IL-5, IL-6, IL-8, IL-10, IL-17, IFN-α levels (ρ=0.33, 0.37, 0.62, 0.55, 0.65, 0.49 and 0.374 respectively, P=0.024, 0.011, 0.000, 0.000, 0.000 and 0.000 respectively). H3K18la expression also displayed a positive correlation with the level of Arg1 mRNA (ρ=0.561, P=0.005). Conclusions: Lactylation is an all-protein post-translational modification occurring in both healthy subjects and critically ill patients. H3K18la may reflect the severity of critical illness and the presence of infection. H3K18la might mediate inflammatory cytokine expression and Arg1 overexpression and stimulate the anti-inflammatory function of macrophages in sepsis.

The dynamic changes of serum IgM and IgG against SARS-CoV-2 in patients with COVID-19.

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a worldwide pandemic since it emerged in December 2019. Previous studies have reported rapid antibody response to SARS-CoV-2 in the first 2 to 3 weeks after symptom onset. Here, we retrospectively described the dynamic changes of serum immunoglobulin M (IgM) and IgG specifically against SARS-CoV-2 in later weeks (mainly 4-10 weeks) in 97 hospitalized patients with COVID-19. We observed that serum IgM and IgG, especially in patients with moderate-to-high levels, declined significantly between week 4 to 10 after illness onset. Notably, IgG levels in high percentage of patients (77.5%, 31 of 40) rapidly declined by half, from 212.5 (range, 163.7-420.3) to 96.3 (range, 75.0-133.4) AU/mL, within 1 to 2 weeks in the second month and then sustained at around 100 AU/mL until discharge from hospital. Significant reduction of IgM was also observed as SARS-CoV-2 nucleic acid turned negative (P = .002). In the recovery stage, serum IgG declined significantly (early vs late recovery stage, n = 16, P = .003) with a median reduction of 50.0% (range, 3.7%-77.0%). Our results suggested that the decline of IgM may be an indicator of virus clearance and recovered patients may have a robust immunity against reinfection within at least 3 months after illness onset. Yet, the rapid reduction of IgG by half rises serious concerns on the robustness and sustainability of the humoral immune response in the period after discharge, which is crucial for immunity strategy and developing a vaccine.

Use of Low-Dose Recombinant Factor â ¦a for Uncontrolled Perioperative Bleeding.

BACKGROUND: Recombinant activated factor VIIa (rFVIIa) is a prohemostatic agent initially approved for use in hemophilia patients and has also been used for a diverse range of off-label indications in the context of massive uncontrolled blood loss; however, no convincing evidence exists regarding the optimal dose of rFVIIa to treat uncontrolled bleeding in surgical patients. AIM: To evaluate the effects and safety of a very low dose of rFⅦa in patients with uncontrolled perioperative bleeding in the surgical intensive care unit (ICU). METHODS: 55 patients from Beijing Hospital, who received rFⅦa between July 2004 and November 2018 for uncontrolled perioperative bleeding were included. The controls were matched for age, sex, severity, and operation type. The baseline demographics, survival, changes in bleeding and transfusion, coagulation parameters and complications were analyzed. RESULTS: A low dose of rFⅦa (2.0∼3.6 mg, with a median dose of 39.02 µg/kg) appears to be effective in controlling massive hemorrhage (with an effective rate of 74.55%), and can reduce volume of red blood cell transfusion, improve coagulation status, while has a relatively low risk of thromboembolic complications (3.6%). CONCLUSION: In patients with uncontrolled perioperative bleeding, a low dose of rFⅦa could be used when traditional methods are ineffective.

Cardiac damage in patients with the severe type of coronavirus disease 2019 (COVID-19).

BACKGROUND: Coronavirus disease 2019 (COVID-19) has become a global pandemic. Studies showed COVID-19 affected not only the lung but also other organs. In this study, we aimed to explore the cardiac damage in patients with COVID-19. METHODS: We collected data of 100 patients diagnosed as severe type of COVID-19 from February 8 to April 10, 2020, including demographics, illness history, physical examination, laboratory test, and treatment. In-hospital mortality were observed. Cardiac damage was defined as plasma hypersensitive troponin I (hsTnI) over 34.2 pg/ml and/or N-terminal-pro brain natriuretic peptide (NTproBNP) above 450 pg/ml at the age < 50, above 900 pg/ml at the age < 75, or above 1800 pg/ml at the age ≥ 75. RESULTS: The median age of the patients was 62.0 years old. 69 (69.0%) had comorbidities, mainly presenting hypertension, diabetes, and cardiovascular disease. Fever (69 [69.0%]), cough (63 [63.0%]), chest distress (13 [13.0%]), and fatigue (12 [12.0%]) were the common initial symptoms. Cardiac damage occurred in 25 patients. In the subgroups, hsTnI was significantly higher in elder patients (≥ 60 years) than in the young (median [IQR], 5.2 [2.2-12.8] vs. 1.9 [1.9-6.2], p = 0.018) and was higher in men than in women (4.2 [1.9-12.8] vs. 2.9 [1.9-7.4], p = 0.018). The prevalence of increased NTproBNP was significantly higher in men than in women (32.1% vs. 9.1%, p = 0.006), but was similar between the elder and young patients (20.0% vs. 25.0%, p = 0.554). After multivariable analysis, male and hypertension were the risk factors of cardiac damage. The mortality was 4.0%. CONCLUSIONS: Cardiac damage exists in patients with the severe type of COVID-19, especially in male patients with hypertension. Clinicians should pay more attention to cardiac damage.

Predictors of 1-year mortality in patients on prolonged mechanical ventilation after surgery in intensive care unit: a multicenter, retrospective cohort study.

OBJECTIVES: The requirement of prolonged mechanical ventilation (PMV) is associated with increased medical care demand and expenses, high early and long-term mortality, and worse life quality. However, no study has assessed the prognostic factors associated with 1-year mortality among PMV patients, not less than 21 days after surgery. This study analyzed the predictors of 1-year mortality in patients requiring PMV in intensive care units (ICUs) after surgery. METHODS: In this multicenter, respective cohort study, 124 patients who required PMV after surgery in the ICUs of five tertiary hospitals in Beijing between January 2007 and June 2016 were enrolled. The primary outcome was the duration of survival within 1 year. Predictors of 1-year mortality were identified with a multivariable Cox proportional hazard model. The predictive effect of the ProVent score was also validated. RESULTS: Of the 124 patients enrolled, the cumulative 1-year mortality was 74.2% (92/124). From the multivariable Cox proportional hazard analysis, cancer diagnosis (hazard ratio [HR] 2.14, 95% confidence interval [CI] 1.37-3.35; P < 0.01), no tracheostomy (HR 2.01, 95% CI 1.22-3.30; P < 0.01), enteral nutrition intolerance (HR 1.88, 95% CI 1.19-2.97; P = 0.01), blood platelet count ≤150 × 109/L (HR 1.77, 95% CI 1.14-2.75; P = 0.01), requirement of vasopressors (HR 1.78, 95% CI 1.13-2.80; P = 0.02), and renal replacement therapy (HR 1.71, 95% CI 1.01-2.91; P = 0.047) on the 21st day of mechanical ventilation (MV) were associated with shortened 1-year survival. CONCLUSIONS: For patients who required PMV after surgery, cancer diagnosis, no tracheostomy, enteral nutrition intolerance, blood platelet count ≤150 × 109/L, vasopressor requirement, and renal replacement therapy on the 21st day of MV were associated with shortened 1-year survival. The prognosis in PMV patients in ICUs can facilitate the decision-making process of physicians and patients' family members on treatment schedule.

Inhibition of AIM2 inflammasome-mediated pyroptosis by Andrographolide contributes to amelioration of radiation-induced lung inflammation and fibrosis.

Radiation-induced lung injury (RILI) is one of the most common and fatal complications of thoracic radiotherapy, whereas no effective interventions are available. Andrographolide, an active component extracted from Andrographis paniculate, is prescribed as a treatment for upper respiratory tract infection. Here we report the potential radioprotective effect and mechanism of Andrographolide on RILI. C57BL/6 mice were exposed to 18 Gy of whole thorax irradiation, followed by intraperitoneal injection of Andrographolide every other day for 4 weeks. Andrographolide significantly ameliorated radiation-induced lung tissue damage, inflammatory cell infiltration, and pro-inflammatory cytokine release in the early phase and progressive fibrosis in the late phase. Moreover, Andrographolide markedly hampered radiation-induced activation of the AIM2 inflammasome and pyroptosis in vivo. Furthermore, bone marrow-derived macrophages (BMDMs) were exposed to 8 Gy of X-ray radiation in vitro and Andrographolide significantly inhibited AIM2 inflammasome mediated-pyroptosis in BMDMs. Mechanistically, Andrographolide effectively prevented AIM2 from translocating into the nucleus to sense DNA damage induced by radiation or chemotherapeutic agents in BMDMs. Taken together, Andrographolide ameliorates RILI by suppressing AIM2 inflammasome mediated-pyroptosis in macrophage, identifying Andrographolide as a novel potential protective agent for RILI.

Cholecystokinin type B receptor-mediated inhibition of A-type K+ channels enhances sensory neuronal excitability through the phosphatidylinositol 3-kinase and c-Src-dependent JNK pathway.

BACKGROUND: Cholecystokinin (CCK) is implicated in the regulation of nociceptive sensitivity of primary afferent neurons. Nevertheless, the underlying cellular and molecular mechanisms remain unknown. METHODS: Using patch clamp recording, western blot analysis, immunofluorescent labelling, enzyme-linked immunosorbent assays, adenovirus-mediated shRNA knockdown and animal behaviour tests, we studied the effects of CCK-8 on the sensory neuronal excitability and peripheral pain sensitivity mediated by A-type K+ channels. RESULTS: CCK-8 reversibly and concentration-dependently decreased A-type K+ channel (IA) in small-sized dorsal root ganglion (DRG) neurons through the activation of CCK type B receptor (CCK-BR), while the sustained delayed rectifier K+ current was unaffected. The intracellular subunit of CCK-BR coimmunoprecipitated with Gαo. Blocking G-protein signaling with pertussis toxin or by the intracellular application of anti-Gß antibody reversed the inhibitory effects of CCK-8. Antagonism of phosphatidylinositol 3-kinase (PI3K) but not of its common downstream target Akts abolished the CCK-BR-mediated IA response. CCK-8 application significantly activated JNK mitogen-activated protein kinase. Antagonism of either JNK or c-Src prevented the CCK-BR-mediated IA decrease, whereas c-Src inhibition attenuated the CCK-8-induced p-JNK activation. Application of CCK-8 enhanced the action potential firing rate of DRG neurons and elicited mechanical and thermal pain hypersensitivity in mice. These effects were mediated by CCK-BR and were occluded by IA blockade. CONCLUSION: Our findings indicate that CCK-8 attenuated IA through CCK-BR that is coupled to the Gßγ-dependent PI3K and c-Src-mediated JNK pathways, thereby enhancing the sensory neuronal excitability in DRG neurons and peripheral pain sensitivity in mice.

Aortic intimal intussusception during acute type B aortic dissection endovascular repair.

Aortic intimal intussusception (AoII) is rare, especially during the endovascular repair of acute uncomplicated type B aortic dissection. Here we present a case of 47-year-old man who suffered AoII during the endovascular repair of type B aortic dissection. An abdominal aortic stent was inserted to recanalize the aorta, but failed. He was immediately transferred to our department from the local hospital. Computed tomography angiography confirmed the AoII and showed thrombus in the abdominal aortic stent. Hybrid operation was performed. Final angiography showed patency of the aorta. His postoperative period was uneventful and was discharged on the postoperative 8th day. No complications happened during the 6th month follow-up.

Histone Deacetylase Inhibition Attenuates Cardiomyocyte Hypoxia-Reoxygenation Injury.

BACKGROUND: Cardiac reperfusion injury can have devastating consequences. Histone deacetylase (HDAC) inhibitors are potent cytoprotective agents, but their role in the prevention of cardiac injury remains ill-defined. OBJECTIVE: We sought to determine the therapeutic potential of HDAC inhibitors in an in vitro model of cardiomyocyte hypoxia-reoxygenation (H/R). METHOD: H9c2 cardiomyocytes were subjected to H/R and treated with various classspecific and pan-HDAC inhibitors in equal concentrations (5µM). Biological activity of inhibitors was determined, as a proxy for concentration adequacy, by Western blot for acetylated histone H3 and α-tubulin. Cell viability and cytotoxicity were measured by methyl thiazolyl tetrazolium and lactate dehydrogenase assays, respectively. Mechanistic studies were performed to better define the effects of the most effective agent, Tubastatin-A (Tub-A), on the phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) pathway effectors, and on the degree of autophagy. RESULTS: All inhibitors acetylated well-known target proteins (histone H3 and α-tubulin), suggesting that concentrations were adequate to induce a biological effect. Improved cell viability and decreased cell cytotoxicity were noted in cardiomyocytes exposed to Tub-A, whereas the cytoprotective effects of other HDAC inhibitors were inconsistent. Pro-survival mediators in the PI3K/mTOR pathway were up-regulated and the degree of autophagy was significantly attenuated in cells that were treated with Tub-A. CONCLUSION: HDAC inhibitors improve cell viability in a model of cardiomyocyte H/R, with Class IIb inhibition (Tub-A) demonstrating superior cellular-level potency and effectiveness. This effect is, at least in part, related to an increased expression of prosurvival mediators and a decreased degree of autophagy.

Tubastatin A prevents hemorrhage-induced endothelial barrier dysfunction.

BACKGROUND: Microvascular hyperpermeability resulting from endothelial barrier dysfunction (EBD) is associated with worse clinical outcomes in trauma-induced hemorrhagic shock. We have previously shown that treatment with Tubastatin A (TubA), a histone deacetylase 6 inhibitor, improves outcomes in animal models of shock. In this study, we investigate whether TubA treatment may prevent trauma-related EBD. METHODS: Wistar-Kyoto rats subjected to 40% hemorrhage were treated with TubA or vehicle control. Acute lung injury (ALI) was assessed histologically from tissues harvested 6 hours posthemorrhage. In vitro, human umbilical vein endothelial cells (HUVECs) were cultured in EGM BulletKit medium. Medium was exchanged for glucose-free Dulbecco's Modified Eagle Medium (0.5% fetal bovine serum) with or without TubA, and cells were placed in an anoxic chamber (5% CO2, 95% N2, 20-48 hours). Expression of acetylated tubulin and hypoxia-inducible factor 1α was measured by Western blot. Soluble Intercellular Adhesion Molecule-1 concentration within the medium, a marker of endothelial integrity, was determined using enzyme-linked immunosorbent assay. Monolayers were assessed for permeability via transwell assays using fluorescein isothiocyanate-labeled albumin. RESULTS: Rats treated with TubA had significantly reduced ALI relative to vehicle control. In vitro, TubA significantly attenuated anoxia-induced hyperpermeability, hypoxia-inducible factor 1α expression, and glycocalyx shedding. CONCLUSIONS: Our findings demonstrate that TubA prevents hemorrhage-induced ALI in rats. Additionally, we have shown that TubA prevents anoxia-induced EBD in vitro. Taken together, these results suggest that TubA could attenuate microvascular hyperpermeability related to hemorrhagic shock.

Inhibition of histone deacetylase 6 restores intestinal tight junction in hemorrhagic shock.

BACKGROUND: We recently discovered that Tubastatin-A, a histone deacetylase (HDAC6) inhibitor, can improve survival in a rodent model of hemorrhagic shock (HS), but mechanisms remain poorly defined. In this study, we investigated whether Tubastatin-A could protect intestinal tight junction (TJ) in HS. METHODS: In an in-vivo study with Wistar-Kyoto rats, the rats underwent HS (40% blood loss) followed by Tubastatin-A (70 mg/kg) treatment, without fluid resuscitation. The experimental groups were (1) sham (no hemorrhage, no treatment), (2) control (hemorrhage, without treatment), and (3) treatment (hemorrhage with Tubastatin-A administration). Six hours after hemorrhage, ileum was harvested. Whole cell lysate were analyzed for acetylated α-tubulin (Ac-tubulin), total tubulin, acetylated histone 3 at lysine 9 (Ac-H3K9), ß-actin, claudin-3 and zonula occludens 1 (ZO-1) proteins by Western blot. Histological effects of Tubastatin-A on small bowel were examined. In an in-vitro study, human intestinal epithelial cells (Caco-2) were divided into three groups: (1) sham (normoxia), (2) control (anoxia, no treatment), and (3) treatment (anoxia, treatment with Tubastatin-A). After 12 hours in an anoxia chamber, the cells were examined for Ac-tubulin and Ac-H3K9, cellular viability, cytotoxicity, claudin-3 and ZO-1 protein expression, and transwell permeability study. RESULTS: Tubastatin-A treatment significantly attenuated HS-induced decreases of Ac-tubulin, Ac-H3K9, ZO-1 and claudin-3 proteins in small bowel in-vivo (p < 0.05). In cultured Caco-2 cells, anoxia significantly decreased cellular viability (p < 0.001) and increased cytotoxicity (p < 0.001) compared to the sham group, while Tubastatin-A treatment offered significant protection (p < 0.0001). Moreover, expression of claudin-3 was markedly decreased in vitro compared to the sham group, whereas this was significantly attenuated by Tubastatin-A (p < 0.05). Finally, anoxia markedly increased the permeability of Caco-2 monolayer cells (p < 0.05), while Tubastatin-A significantly attenuated the alteration (p < 0.05). CONCLUSION: Inhibition of HDAC6 can induce Ac-tubulin and Ac-H3K9, promote cellular viability, and prevent the loss of intestinal tight junction proteins during HS and anoxia.

Inhibition of peptidylarginine deiminase attenuates inflammation and improves survival in a rat model of hemorrhagic shock.

BACKGROUND: We have recently shown that inhibition of peptidylarginine deiminase (PAD) improves survival in a rodent model of lethal cecal ligation and puncture. The roles of PAD inhibitors in hemorrhagic shock (HS), however, are largely unknown. The goal of this study was to investigate the effects of YW3-56, a novel PAD inhibitor, on survival after severe HS. METHODS: Mouse macrophages were exposed to hypoxic conditions followed by reoxygenation in the presence or absence of YW3-56. Enzyme-linked immunosorbent assay (ELISA) was performed to measure levels of secreted tumor necrosis factor α and interleukin-6 in the culture medium. Cell viability was determined by methyl thiazolyl tetrazolium assay. In the survival experiment, anesthetized male Wistar-Kyoto rats (n = 10/group) were subjected to 55% blood loss, and treated with or without YW3-56 (10 mg/kg, intraperitoneally). Survival was monitored for 12 h. In the nonsurvival experiment, morphologic changes of the lungs were examined. Levels of circulating cytokine-induced neutrophil chemoattractant 1 (CINC-1) and myeloperoxidase (MPO) in the lungs were measured by ELISA. Expression of lung intercellular adhesion molecules-1 (ICAM-1) was also determined by Western blotting. RESULTS: Hypoxia/reoxygenation (H/R) insult induced tumor necrosis factor α and interleukin-6 secretion from macrophages, which was significantly attenuated by YW3-56 treatment. YW3-56 treatment also increased cell viability when macrophages were exposed to H/R up to 6/15 h and improved survival rate from 20% to 60% in lethal HS rat model. Compared to the sham groups, pulmonary MPO activity and ICAM-1 expression in the HS group were significantly increased, and acute lung injury was associated with a higher degree of CINC-1 levels in serum. Intraperitoneal delivery of YW3-56 significantly reduced pulmonary MPO and ICAM-1 expression and attenuated acute lung injury. CONCLUSIONS: Our results demonstrate for the first time that administration of YW3-56, a novel PAD inhibitor, can improve survival in a rat model of HS and in a cell culture model of H/R. The survival advantage is associated with an attenuation of local and systemic pro-inflammatory cytokines and the protection against acute lung injury after hemorrhage. Thus, PAD inhibition may represent a novel and promising therapeutic strategy for severe HS.

Selective inhibition of histone deacetylase 6 promotes survival in a rat model of hemorrhagic shock.

BACKGROUND: Hemorrhage is the leading cause of preventable trauma-related deaths. We have previously shown that treatment with Tubastatin A (Tub A), a histone deacetylase 6 (HDAC6) inhibitor, can improve survival in a rodent model of septic shock. The aims of the present study were to determine whether selective inhibition of HDAC6 can promote survival in a model of hemorrhagic shock (HS). METHODS: In Experiment I (survival study), Wistar-Kyoto rats were subjected to HS (55% volume blood loss), followed by intraperitoneal injection of either Tub A (70 mg/kg) dissolved in dimethyl sulfoxide (DMSO) or DMSO only (vehicle group) (n = 8 per group). Survival was monitored for 24 hours. In Experiment II (physiologic study), rats were subjected to a sublethal HS (40% blood loss), followed by the same treatment with Tub A (treatment group) or DMSO only (vehicle group, n = 5 per group). All animals were sacrificed 6 hours after hemorrhage, and the heart and liver tissues were harvested. Sham animals were not subjected to hemorrhage and treatment (sham group, n = 5 per group). Cardiac mitochondria were isolated to study the pyruvate dehydrogenase (PDH, an essential enzyme for adenosine triphosphate production) activity. Liver tissue lysates were analyzed for markers of apoptosis (cytochrome c, cleaved caspase 3) and inflammation (high-mobility group box 1) by Western blotting. RESULTS: Severe HS (55% blood loss) was associated with 75% mortality, which was significantly improved by Tub A treatment (37.5% mortality in 24 hours, p = 0.048). Tub A also significantly enhanced the cardiac PDH activity compared with the vehicle group, while suppressing the hepatic high-mobility group box 1 expression, cytochrome c release, and caspase 3 activation. CONCLUSION: Our study has demonstrated for the first time that selective inhibition of HDAC6 can improve survival in a rodent model of HS. The potential mechanisms include enhanced PDH activity, decreased inflammatory drive, and attenuated cellular apoptosis.

Addition of low-dose valproic acid to saline resuscitation provides neuroprotection and improves long-term outcomes in a large animal model of combined traumatic brain injury and hemorrhagic shock.

BACKGROUND: Combined traumatic brain injury (TBI) and hemorrhagic shock (HS) is highly lethal. In a nonsurvival model of TBI + HS, addition of high-dose valproic acid (VPA) (300 mg/kg) to hetastarch reduced brain lesion size and associated swelling 6 hours after injury; whether this would have translated into better neurologic outcomes remains unknown. It is also unclear whether lower doses of VPA would be neuroprotective. We hypothesized that addition of low-dose VPA to normal saline (NS) resuscitation would result in improved long-term neurologic recovery and decreased brain lesion size. METHODS: TBI was created in anesthetized swine (40-43 kg) by controlled cortical impact, and volume-controlled hemorrhage (40% volume) was induced concurrently. After 2 hours of shock, animals were randomized (n = 5 per group) to NS (3× shed blood) or NS + VPA (150 mg/kg). Six hours after resuscitation, packed red blood cells were transfused, and animals were recovered. Peripheral blood mononuclear cells were analyzed for acetylated histone-H3 at lysine-9. A Neurological Severity Score (NSS) was assessed daily for 30 days. Brain magnetic resonance imaging was performed on Days 3 and 10. Cognitive performance was assessed by training animals to retrieve food from color-coded boxes. RESULTS: There was a significant increase in histone acetylation in the NS + VPA-treated animals compared with NS treatment. The NS + VPA group demonstrated significantly decreased neurologic impairment and faster speed of recovery as well as smaller brain lesion size compared with the NS group. Although the final cognitive function scores were similar between the groups, the VPA-treated animals reached the goal significantly faster than the NS controls. CONCLUSION: In this long-term survival model of TBI + HS, addition of low-dose VPA to saline resuscitation resulted in attenuated neurologic impairment, faster neurologic recovery, smaller brain lesion size, and a quicker normalization of cognitive functions.

Nesfatin-1 Suppresses Cardiac L-type Ca²âº Channels Through Melanocortin Type 4 Receptor and the Novel Protein Kinase C Theta Isoform Pathway.

BACKGROUND/AIMS: Nesfatin-1 (NF-1), an anorexic nucleobindin-2 (NUCB2)-derived hypothalamic peptide, acts as a peripheral cardiac modulator and it can induce negative inotropic effects. However, the mechanisms underlying these effects in cardiomyocytes remain unclear. METHODS: Using patch clamp, protein kinase assays, and western blot analysis, we studied the effect of NF-1 on L-type Ca2+ currents (ICa,L) and to explore the regulatory mechanisms of this effect in adult ventricular myocytes. RESULTS: NF-1 reversibly decreased ICa,L in a dose-dependent manner. This effect was mediated by melanocortin 4 receptor (MC4-R) and was associated with a hyperpolarizing shift in the voltage-dependence of inactivation. Dialysis of cells with GDP-ß-S or anti-Gß antibody as well as pertussis toxin pretreatment abolished the inhibitory effects of NF-1 on ICa,L. Protein kinase C (PKC) antagonists abolished NF-1-induced responses, whereas inhibition of PKA activity or intracellular application of the fast Ca2+-chelator BAPTA elicited no such effects. Application of NF-1 increased membrane abundance of PKC theta isoform (PKCθ), and PKCθ inhibition abolished the decrease in ICa,L induced by NF-1. CONCLUSION: These data suggest that NF-1 suppresses L-type Ca2+ channels via the MC4-R that couples sequentially to the ßγ subunits of Gi/o-protein and the novel PKCθ isoform in adult ventricular myocytes.