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BACKGROUND: Genetic locus were identified associated with acute respiratory distress syndrome (ARDS). Our goal was to explore the associations between genetic variants and ARDS outcome, as well as subphenotypes. METHODS: This was a single-center, prospective observational trial enrolling adult ARDS patients. After baseline data were collected, blood samples were drawn to perform whole exome sequencing, single nucleotide polymorphism (SNP)/insertion-deletion to explore the quantitative and functional associations between genetic variants and ICU outcome, clinical subphenotypes. Then the lung injury burden (LIB), which was defined as the ratio of nonsynonymous SNP number per megabase of DNA, was used to evaluate its value in predicting ARDS outcome. RESULTS: A total of 105 ARDS patients were enrolled in the study, including 70 survivors and 35 nonsurvivors. Based on the analysis of a total of 65,542 nonsynonymous SNP, LIB in survivors was significantly higher than nonsurvivors [1,892 (1,848-1,942)/MB versus 1,864 (1,829-1,910)/MB, P=0.018], while GO analysis showed that 60 functions were correlated with ARDS outcome, KEGG enrichment analysis showed that SNP/InDels were enriched in 13 pathways. Several new SNPs were found potentially associated with ARDS outcome. Analysis of LIB was used to determine its outcome predicting ability, the area under the ROC curve of which was only 0.6103, and increase to 0.712 when combined with APACHE II score. CONCLUSIONS: Genetic variants are associated with ARDS outcome and subphenotypes; however, their prognostic value still need to be verified by larger trials. TRIAL REGISTRATION: Clinicaltrials.gov NCT02644798. Registered 20 April 2015.
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A straightforward one-pot, multicomponent approach was developed to synthesize di- and tri-substituted N-sulfonyl formamidines from sulfonyl chlorides, NaN3, ethyl propiolate, and primary/secondary amines under mild conditions without catalysts or additives. Structural analysis of the di-substituted sulfonyl formamidines indicated formation of the E-syn/anti isomeric form. Tri-substituted analogues only formed E-isomers.
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BACKGROUND: Acinetobacter baumannii (A. baumannii) has become one of the most important opportunistic pathogens inducing nosocomial pneumonia and increasing mortality in critically ill patients recently. The interaction between A. baumannii infection and immune response can influence the prognosis of A. baumannii related pneumonia. The target of the present study was to investigate the role of immunodeficiency in A. baumannii induced pneumonia. METHODS: Male BALB/c mice were randomly divided into the normal immunity control (NIC) group, normal immunity infection (NIA) group, immune compromised control (CIC) group, and immune compromised infection (CIA) group (nâ=â15 for each group). Intraperitoneal injection of cyclophosphamide and intranasal instillation of A. baumannii solution were used to induce compromised immunity and murine pneumonia, respectively. The mice were sacrificed at 6 and 24 h later and the specimens were collected for further tests. Seven-day mortality of mice was also assessed. RESULTS: After A. baumannii stimulation, the recruitment of neutrophils in mice with normal immunity increased sharply (Pâ=â0.030 at 6 h), while there was no significant raise of neutrophil counts in mice with compromised immune condition (Pâ=â0.092 at 6 h, Pâ=â0.772 at 24 h). The Th cell polarization presented with pulmonary interleukin (IL)-4 and interferon (IFN)-γ level in response to the A. baumannii in CIA group were significantly depressed in comparison with in NIA group (IFN-γ: Pâ=â0.003 at 6 h; Pâ=â0.001 at 24 h; IL-4: Pâ<â0.001 at 6 h; Pâ<â0.001 at 24 h). The pulmonary conventional dendritic cell accumulation was even found to be inhibited after A. baumannii infection in immunocompromised mice (Pâ=â0.033). Correspondingly, A. baumannii associated pneumonia in mice with compromised immunity caused more early stage death, more severe histopathological impairment in lung. CONCLUSION: A. baumannii could frustrate the immune response in immunocompromised conditions, and this reduced immune response is related to more severe lung injury and worse outcome in A. baumannii induced pneumonia.
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Infecções por Acinetobacter , Acinetobacter baumannii , Pneumonia , Animais , Humanos , Pulmão , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BLRESUMO
Although mesenchymal stem cells (MSCs) transplantation has been shown to promote the lung respiration in acute lung injury (ALI) in vivo, its overall restorative capacity appears to be restricted mainly because of low retention in the injured lung. Angiotensin II (Ang II) are upregulated in the injured lung. Our previous study showed that Ang II increased MSCs migration via Ang II type 2 receptor (AT2R). To determine the effect of AT2R in MSCs on their cell migration after systemic injection in ALI mice, a human AT2R expressing lentiviral vector and a lentivirus vector carrying AT2R shRNA were constructed and introduced into human bone marrow MSCs. A mouse model of lipopolysaccharide-induced ALI was used to investigate the migration of AT2R-regulated MSCs and the therapeutic potential in vivo. Overexpression of AT2R dramatically increased Ang II-enhanced human bone marrow MSC migration in vitro. Moreover, MSC-AT2R accumulated in the damaged lung tissue at significantly higher levels than control MSCs 24 and 72 hours after systematic MSC transplantation in ALI mice. Furthermore, MSC-AT2R-injected ALI mice exhibited a significant reduction of pulmonary vascular permeability and improved the lung histopathology and had additional anti-inflammatory effects. In contrast, there were less lung retention in MSC-ShAT2R-injected ALI mice compared with MSC-Shcontrol after transplantation. Thus, MSC-ShAT2R-injected group exhibited a significant increase of pulmonary vascular permeability and resulted in a deteriorative lung inflammation. Our results demonstrate that overexpression of AT2R enhance the migration of MSCs in ALI mice and may provide a new therapeutic strategy for ALI. Stem Cells Translational Medicine 2018;7:721-730.
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Lesão Pulmonar Aguda/terapia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/metabolismo , Receptor Tipo 2 de Angiotensina/metabolismo , Lesão Pulmonar Aguda/etiologia , Lesão Pulmonar Aguda/patologia , Animais , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/citologia , Movimento Celular , Citocinas/análise , Modelos Animais de Doenças , Contagem de Leucócitos , Lipopolissacarídeos/toxicidade , Masculino , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Neutrófilos/citologia , Receptor Tipo 2 de Angiotensina/genéticaRESUMO
BACKGROUND: Mesenchymal stem cells (MSCs) migrate via the bloodstream to sites of injury and are possibly attracted by inflammatory factors. As a proinflammatory mediator, angiotensin II (Ang II) reportedly enhances the migration of various cell types by signaling via the Ang II receptor in vitro. However, few studies have focused on the effects of Ang II on MSC migration and the underlying mechanisms. METHODS: Human bone marrow MSCs migration was measured using wound healing and Boyden chamber migration assays after treatments with different concentrations of Ang II, an AT1R antagonist (Losartan), and/or an AT2R antagonist (PD-123319). To exclude the effect of proliferation on MSC migration, we measured MSC proliferation after stimulation with the same concentration of Ang II. Additionally, we employed the focal adhesion kinase (FAK) inhibitor PF-573228, RhoA inhibitor C3 transferase, Rac1 inhibitor NSC23766, or Cdc42 inhibitor ML141 to investigate the role of cell adhesion proteins and the Rho-GTPase protein family (RhoA, Rac1, and Cdc42) in Ang II-mediated MSC migration. Cell adhesion proteins (FAK, Talin, and Vinculin) were detected by western blot analysis. The Rho-GTPase family protein activities were assessed by G-LISA and F-actin levels, which reflect actin cytoskeletal organization, were detected by using immunofluorescence. RESULTS: Human bone marrow MSCs constitutively expressed AT1R and AT2R. Additionally, Ang II increased MSC migration in an AT2R-dependent manner. Notably, Ang II-enhanced migration was not mediated by Ang II-mediated cell proliferation. Interestingly, Ang II-enhanced migration was mediated by FAK activation, which was critical for the formation of focal contacts, as evidenced by increased Talin and Vinculin expression. Moreover, RhoA and Cdc42 were activated by FAK to increase cytoskeletal organization, thus promoting cell contraction. Furthermore, FAK, Talin, and Vinculin activation and F-actin reorganization in response to Ang II were prevented by PD-123319 but not Losartan, indicating that FAK activation and F-actin reorganization were downstream of AT2R. CONCLUSIONS: These data indicate that Ang II-AT2R regulates human bone marrow MSC migration by signaling through the FAK and RhoA/Cdc42 pathways. This study provides insights into the mechanisms by which MSCs home to injury sites and will enable the rational design of targeted therapies to improve MSC engraftment.
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Angiotensina II/farmacologia , Movimento Celular/efeitos dos fármacos , Quinase 1 de Adesão Focal/metabolismo , Células-Tronco Mesenquimais/metabolismo , Receptor Tipo 2 de Angiotensina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Quinase 1 de Adesão Focal/antagonistas & inibidores , Humanos , Células-Tronco Mesenquimais/citologia , Proteína rhoA de Ligação ao GTP/antagonistas & inibidoresRESUMO
BACKGROUND: Early goal-directed therapy (EGDT) has become an important therapeutic management in early salvage stage of septic shock. However, splenic organs possibly remained hypoperfused and hypoxic despite fluid resuscitation. This study aimed to evaluate the effect of EGDT on hepatic perfusion in septic shock patients. METHODS: A prospective observational study was carried out in early septic shock patients who were admitted to Intensive Care Unit within 24 h after onset and who met all four elements of the EGDT criteria after treatment with the standard EGDT procedure within 6 h between December 1, 2012 and November 30, 2013. The hemodynamic data were recorded, and oxygen metabolism and hepatic functions were monitored. An indocyanine green clearance test was applied to detect the hepatic perfusion. The patients' characteristics were compared before treatment (T0), immediately after EGDT (T1), and 24 h after EGDT (T2). This study is registered at ClinicalTrials.org, NCT02060773. RESULTS: Twenty-one patients were included in the study; however, the hepatic perfusion data were not included in the analysis for two patients; therefore, 19 patients were eligible for the study. Hemodynamics data, as monitored by pulse-indicator continuous cardiac output, were obtained from 16 patients. There were no significant differences in indocyanine green plasma disappearance rate (ICG-PDR) and 15-min retention rate (R15) at T0 (11.9 ± 5.0%/min and 20.0 ± 13.2%), T1 (11.4 ± 5.1%/min and 23.6 ± 14.9%), and T2 (11.0 ± 4.5%/min and 23.7 ± 15.3%) (all P > 0.05). Both of the alterations of ICG-PDR and R15 showed no differences at T0, T1, and T2 in the patients of different subgroups that achieved different resuscitation goal numbers when elected (P > 0.05). CONCLUSION: There were no hepatic perfusion improvements after EGDT in the early phase of patients with septic shock. TRIAL REGISTRATION: Clinicaltrials.gov NCT02060773 (https://clinicaltrials.gov/ct2/show/NCT02060773).
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Choque Séptico/terapia , Idoso , Idoso de 80 Anos ou mais , Débito Cardíaco/fisiologia , Gerenciamento Clínico , Feminino , Hidratação , Hemodinâmica/fisiologia , Humanos , Unidades de Terapia Intensiva/estatística & dados numéricos , Masculino , Pessoa de Meia-Idade , Estudos ProspectivosRESUMO
Our previous study has demonstrated that glucagon-like peptide-1 (GLP-1) receptor agonist could protect neurons from advanced glycation end products (AGEs) toxicity in vitro. However, further studies are still needed to clarify the molecular mechanism of this GLP-1 receptor -dependent action. The present study mainly focused on the effect of GLP-1 receptor agonists against the receptor for advanced glycation end products (RAGE) signal pathway and the mechanism underlying this effect of GLP-1. Firstly the data based on the SH-GLP-1R(+) and SH-SY5Y cells confirmed our previous finding that GLP-1 receptor could mediate the protective effect against AGEs. The assays of the protein activity and of the mRNA level revealed that apoptosis-related proteins such as caspase-3, caspase-9, Bax and Bcl-2 were involved. Additionally, we found that both GLP-1 and exendin-4 could reduce AGEs-induced reactive oxygen species (ROS) accumulation by suppressing the activity of nicotinamide adenine dinucleotide phosphate-oxidase. Interestingly, we also found that GLP-1 receptor activation could attenuate the abnormal expression of the RAGE in vitro and in vivo. Furthermore, based on the analysis of the protein expression and translocation level of transcription factor nuclear factor-κB (NF-κB), and the use of GLP-1 receptor antagonist exendin(9-39) and NF-κB inhibitor pyrrolidine dithiocarbamate, we found that the effect mediated by GLP-1 receptor could alleviate the over expression of RAGE induced by ligand via the suppression of NF-κB. In summary, the results indicated that inhibiting RAGE/oxidative stress was involved in the protective effect of GLP-1 on neuron cells against AGEs induced apoptosis.
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Apoptose , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Produtos Finais de Glicação Avançada/metabolismo , Neurônios/citologia , Estresse Oxidativo , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Animais , Linhagem Celular Tumoral , Exenatida , Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Produtos Finais de Glicação Avançada/toxicidade , Humanos , Masculino , Camundongos Endogâmicos ICR , NADPH Oxidases/metabolismo , NF-kappa B/farmacologia , Neurônios/metabolismo , Peptídeos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Peçonhas/metabolismoRESUMO
There are some limitations to the therapeutic effects of mesenchymal stem cells (MSCs) on acute respiratory distress syndrome (ARDS) due to their low engraftment and differentiation rates in lungs. We found previously that noncanonical Wnt5a signaling promoted the differentiation of mouse MSCs (mMSCs) into type II alveolar epithelial cells (AT II cells), conferred resistance to oxidative stress, and promoted migration of MSCs in vitro. As receptor tyrosine kinase-like orphan receptor 2 (ROR2) is an essential receptor for Wnt5a, it was reasonable to deduce that ROR2 might be one of the key molecules for the therapeutic effect of MSCs in ARDS. The mMSCs that stably overexpressed ROR2 or the green fluorescent protein (GFP) control were transplanted intratracheally into the ARDS mice [induced by intratracheal injection of lipopolysaccharide (LPS)]. The results showed that ROR2-overexpressing mMSCs led to more significant effects than the GFP controls, including the retention of the mMSCs in the lung, differentiation into AT II cells, improvement of alveolar epithelial permeability, improvement of acute LPS-induced pulmonary inflammation, and, finally, reduction of the pathological impairment of the lung tissue. In conclusion, MSCs that overexpress ROR2 could further improve MSC-mediated protection against epithelial impairment in ARDS.
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Lesão Pulmonar Aguda/terapia , Células-Tronco Mesenquimais/citologia , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/metabolismo , Síndrome do Desconforto Respiratório/terapia , Lesão Pulmonar Aguda/genética , Lesão Pulmonar Aguda/metabolismo , Animais , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Modelos Animais de Doenças , Proteínas de Fluorescência Verde , Lipopolissacarídeos/farmacologia , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/fisiologia , Camundongos , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/genética , Síndrome do Desconforto Respiratório/genética , Síndrome do Desconforto Respiratório/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
BACKGROUND: Mesenchymal stem cells (MSCs) stabilise endothelial barrier function in acute lung injury via paracrine hepatocyte growth factor (HGF). Vascular endothelial growth factor (VEGF), which is secreted by MSCs, is another key regulator of endothelial permeability; however, its role in adjusting permeability remains controversial. In addition, whether an interaction occurs between HGF and VEGF, which are secreted by MSCs, is not completely understood. METHODS: We introduced a co-cultured model of human pulmonary microvascular endothelial cells (HPMECs) and MSC conditioned medium (CM) collected from MSCs after 24 h of hypoxic culture. The presence of VEGF and HGF in the MSC-CM was neutralised by anti-VEGF and anti-HGF antibodies, respectively. To determine the roles and mechanisms of MSC-secreted HGF and VEGF, we employed recombinant humanised HGF and recombinant humanised VEGF to co-culture with HPMECs. Additionally, we employed the RhoA inhibitor C3 transferase and the Rac1 inhibitor NSC23766 to inhibit the activities of RhoA and Rac1 in HPMECs treated with MSC-CM or VEGF/HGF with the same dosage as in the MSC-CM. Then, endothelial paracellular and transcellular permeability was detected. VE-cadherin, occludin and caveolin-1 protein expression in HPMECs was measured by western blot. Adherens junction proteins, including F-actin and VE-cadherin, were detected by immunofluorescence. RESULTS: MSC-CM treatment significantly decreased lipopolysaccharide-induced endothelial paracellular and transcellular permeability, which was significantly inhibited by pretreatment with HGF antibody or with both VEGF and HGF antibodies. Furthermore, MSC-CM treatment increased the expression of the endothelial intercellular adherence junction proteins VE-cadherin and occludin and decreased the expression of caveolin-1 protein. MSC-CM treatment also decreased endothelial apoptosis and induced endothelial cell proliferation; however, the effects of MSC-CM treatment were inhibited by pretreatment with HGF antibody or with both HGF and VEGF antibodies. Additionally, the effects of MSC-CM and VEGF/HGF on reducing endothelial paracellular and transcellular permeability were weakened when HPMECs were pretreated with the Rac1 inhibitor NSC23766. CONCLUSION: HGF secreted by MSCs protects the endothelial barrier function; however, VEGF secreted by MSCs may synergize with HGF to stabilise endothelial cell barrier function. Rac1 is the pathway by which MSC-secreted VEGF and HGF regulate endothelial permeability.
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Permeabilidade Capilar/fisiologia , Fator de Crescimento de Hepatócito/fisiologia , Células-Tronco Mesenquimais/fisiologia , Fator A de Crescimento do Endotélio Vascular/fisiologia , Proteínas rac1 de Ligação ao GTP/metabolismo , Actinas/metabolismo , Antígenos CD/metabolismo , Apoptose , Caderinas/metabolismo , Permeabilidade Capilar/efeitos dos fármacos , Caveolina 1/metabolismo , Sobrevivência Celular , Técnicas de Cocultura , Meios de Cultivo Condicionados , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/fisiologia , Fator de Crescimento de Hepatócito/antagonistas & inibidores , Humanos , Lipopolissacarídeos/toxicidade , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
INTRODUCTION: Mesenchymal stem cells (MSCs) have potent stabilising effects on vascular endothelium injury, inhibiting endothelial permeability in lung injury via paracrine hepatocyte growth factor (HGF). Recently, it has been indicated that MSCs secrete more factors by MSC-endothelial cell (MSC-EC) interactions. We hypothesised that MSC-EC interactions restore endothelial permeability induced by lipopolysaccharide (LPS) via paracrine HGF. METHODS: We investigated the endothelial permeability induced by LPS under two co-culture conditions. Human pulmonary microvascular endothelial cells (HPMECs) were added into the upper chambers of cell-culture inserts, while two different co-culture conditions were used in the lower side of the transwells, as follows: (1) MSC-EC interaction group: MSCs and HPMECs contact co-culture; (2) MSC group: MSCs only. The endothelial paracellular and transcellular permeabilities in the upper side of transwells were detected. Then the concentration of HGF was measured in the culture medium by using an enzyme-linked immunosorbent assay kit, followed by neutralisation of HGF with anti-HGF antibody in the co-culture medium. In addition, adherens junction and cytoskeleton protein expressions were measured by Western blot and immunofluorescence. HPMEC proliferation was analysed by bromodeoxyuridine incorporation assay. RESULTS: The paracellular permeability significantly increased after LPS stimulation in a dose-dependent and time-dependent manner. Meanwhile, MSC-EC interaction more significantly decreased endothelial paracellular and transcellular permeability induced by LPS. Moreover, HGF levels in the MSC-EC interaction group were much higher than those of the MSC group. However, neutralising HGF with anti-HGF antibody inhibited the role of MSC-EC interaction in improving endothelial permeability. Compared with the MSC group, MSC-EC interaction increased vascular endothelial (VE)-cadherin and occludin protein expression, reduced caveolin-1 protein expression in HPMECs, and restored remodelling of F-actin and junctional localisation of VE-cadherin. Furthermore, the proliferation ratio in the MSC-EC interaction group was higher than that of the MSC group. However, the effects of MSCs were significantly blocked by anti-HGF antibody. CONCLUSIONS: These data suggested that MSC-EC interaction decreased endothelial permeability induced by LPS, which was attributed mainly to HGF secreted by MSCs. The main mechanisms by which HGF restored the integrity of endothelial monolayers were remodelling of endothelial intercellular junctions, decreasing caveolin-1 protein expression, and inducing proliferation in HPMECs.
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Células Endoteliais/citologia , Fator de Crescimento de Hepatócito/análise , Células-Tronco Mesenquimais/citologia , Actinas/metabolismo , Antígenos CD/metabolismo , Caderinas/metabolismo , Caveolina 1/metabolismo , Técnicas de Cultura de Células , Linhagem da Célula , Células Cultivadas , Técnicas de Cocultura , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Ensaio de Imunoadsorção Enzimática , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Lipopolissacarídeos/farmacologia , Células-Tronco Mesenquimais/metabolismo , Ocludina/metabolismo , Comunicação Parácrina , Permeabilidade/efeitos dos fármacosRESUMO
INTRODUCTION: The effect of mean arterial pressure titration to a higher level on microcirculation in septic shock patients with previous hypertension remains unknown. Our goal is to assess the effect of mean arterial pressure titration to a higher level on microcirculation in hypertensive septic shock patients. METHODS: This is a single-center, open-label study. Hypertensive patients with septic shock for less than 24 hours after adequate fluid resuscitation and requiring norepinephrine to maintain a mean arterial pressure of 65 mmHg were enrolled. Mean arterial pressure was then titrated by norepinephrine from 65 mmHg to the normal level of the patient. In addition to hemodynamic variables, sublingual microcirculation was evaluated by sidestream dark field imaging. RESULTS: Nineteen patients were enrolled in the study. Increasing mean arterial pressure from 65 mmHg to normal levels was associated with increased central venous pressure (from 11 ± 4 to 13 ± 4 mmHg, P = 0.002), cardiac output (from 5.4 ± 1.4 to 6.4 ± 2.1 l/minute, P = 0.001), and central venous oxygen saturation (from 81 ± 7 to 83 ± 7%, P = 0.001). There were significant increases in small perfused vessel density (from 10.96 ± 2.98 to 11.99 ± 2.55 vessels/mm(2), P = 0.009), proportion of small perfused vessels (from 85 ± 18 to 92 ± 14%, P = 0.002), and small microvascular flow index (from 2.45 ± 0.61 to 2.80 ± 0.68, P = 0.009) when compared with a mean arterial pressure of 65 mmHg. CONCLUSIONS: Increasing mean arterial pressure from 65 mmHg to normal levels is associated with improved microcirculation in hypertensive septic shock patients. TRIAL REGISTRATION: Clinicaltrials.gov: NCT01443494; registered 28 September 2011.
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Pressão Arterial/efeitos dos fármacos , Hipertensão/tratamento farmacológico , Microcirculação/efeitos dos fármacos , Choque Séptico/tratamento farmacológico , Idoso , Idoso de 80 Anos ou mais , Feminino , Hidratação , Hemodinâmica/efeitos dos fármacos , Humanos , Unidades de Terapia Intensiva , Masculino , Pessoa de Meia-Idade , Soalho Bucal/irrigação sanguínea , Norepinefrina/administração & dosagem , Norepinefrina/farmacologia , Estudos Prospectivos , Respiração Artificial/métodos , Choque Séptico/fisiopatologia , Vasoconstritores/administração & dosagem , Vasoconstritores/farmacologiaRESUMO
INTRODUCTION: Mesenchymal stem cells (MSCs) have potential for re-epithelization and recovery in acute respiratory distress syndrome (ARDS). In a previous in vitro study, the results showed that the canonical Wnt/ß-catenin pathway promoted the differentiation of MSCs into type II alveolar epithelial cells, conferred resistance to oxidative stress, and promoted their migration, suggesting that the Wnt/ß-catenin pathway might be one of the key mechanisms underling the therapeutic effect of mouse MSCs in ARDS. METHODS: Mouse MSCs stable transfected with ß-catenin or green fluorescent protein control were transplanted intratracheally into the ARDS mice induced by lipopolysaccharide. Lung tissue injury and repair assessment were examined using haematoxylin and eosin staining, lung injury scoring, Masson's trichrome staining and fibrosis scoring. Homing and differentiation of mouse MSCs were assayed by labelling and tracing MSCs using NIR815 dye, immunofluorescent staining, and Western immunoblot analysis. The inflammation and permeability were evaluated by detecting the cytokine and protein measurements in bronchoalveolar lavage fluid using enzyme-linked immunosorbent assay. RESULTS: In this study, ß-catenin-overexpressing MSC engraftment led to more significant effects than the GFP controls, including the retention of the MSCs in the lung, differentiation into type II alveolar epithelial cells, improvement in alveolar epithelial permeability, and the pathologic impairment of the lung tissue. CONCLUSION: These results suggest that the activation of canonical Wnt/ß-catenin pathway by mouse MSCs by overexpressing ß-catenin could further improve the protection of mouse MSCs against epithelial impair and the therapeutic effects of mouse MSCs in ARDS mice.
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Lesão Pulmonar Aguda/terapia , Células Epiteliais/citologia , Alvéolos Pulmonares/metabolismo , Síndrome do Desconforto Respiratório/terapia , Mucosa Respiratória/citologia , Via de Sinalização Wnt/fisiologia , Animais , Diferenciação Celular/fisiologia , Movimento Celular , Terapia Baseada em Transplante de Células e Tecidos , Células Cultivadas , Lipopolissacarídeos , Masculino , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo/fisiologia , Alvéolos Pulmonares/patologia , Proteínas Wnt/metabolismo , beta Catenina/metabolismoRESUMO
INTRODUCTION: Prone positioning (PP) has been reported to improve the survival of patients with severe acute respiratory distress syndrome (ARDS). However, it is uncertain whether the beneficial effects of PP are associated with positive end-expiratory pressure (PEEP) levels and long durations of PP. In this meta-analysis, we aimed to evaluate whether the effects of PP on mortality could be affected by PEEP level and PP duration and to identify which patients might benefit the most from PP. METHODS: Publications describing randomized controlled trials (RCTs) in which investigators have compared prone and supine ventilation were retrieved by searching the following electronic databases: PubMed/MEDLINE, the Cochrane Library, the Web of Science and Elsevier Science (inception to May 2013). Two investigators independently selected RCTs and assessed their quality. The data extracted from the RCTs were combined in a cumulative meta-analysis and analyzed using methods recommended by the Cochrane Collaboration. RESULTS: A total of nine RCTs with an aggregate of 2,242 patients were included. All of the studies received scores of up to three points using the methods recommended by Jadad et al. One trial did not conceal allocation. This meta-analysis revealed that, compared with supine positioning, PP decreased the 28- to 30-day mortality of ARDS patients with a ratio of partial pressure of arterial oxygen/fraction of inspired oxygen ≤ 100 mmHg (n = 508, risk ratio (RR) = 0.71, 95 confidence interval (CI) = 0.57 to 0.89; P = 0.003). PP was shown to reduce both 60-day mortality (n = 518, RR = 0.82, 95% CI = 0.68 to 0.99; P = 0.04) and 90-day mortality (n = 516, RR = 0.57, 95% CI = 0.43 to 0.75; P < 0.0001) in ARDS patients ventilated with PEEP ≥ 10 cmH2O. Moreover, PP reduced 28- to 30-day mortality when the PP duration was >12 h/day (n = 1,067, RR = 0.73, 95% CI = 0.54 to 0.99; P = 0.04). CONCLUSIONS: PP reduced mortality among patients with severe ARDS and patients receiving relatively high PEEP levels. Moreover, long-term PP improved the survival of ARDS patients.
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Respiração com Pressão Positiva , Decúbito Ventral , Síndrome do Desconforto Respiratório/mortalidade , Síndrome do Desconforto Respiratório/terapia , Humanos , Ensaios Clínicos Controlados Aleatórios como Assunto , Índice de Gravidade de Doença , Fatores de TempoRESUMO
The Wnt pathways have been shown to be critical for the fate of mesenchymal stem cells (MSCs) in vitro, but their roles in MSCs in vivo remain poorly characterized due to the lack of stable alterations in their signaling. In the present study, we constructed long-term and stable mMSCs lines with activated and inactivated ß-catenin (the key molecule of the canonical Wnt signaling pathway) or ROR2 (the key molecule of the noncanonical Wnt5a/ROR2 signaling pathway) modifications with lentiviral vectors. We found that the transduction efficiencies mediated by the lentiviral vectors were 92.61-97.04% and were maintained over 20 passages of mMSCs. Transfection by lentiviral vectors not only regulated the mRNA and protein expression of ß-catenin or ROR2 but also regulated nuclear ß-catenin accumulation or the Wnt5a/JNK and Wnt5a/PKC pathways belonging to the canonical Wnt and noncanonical Wnt5a/ROR2 pathways, respectively. ß-Catenin or ROR2 gene overexpression promoted mMSC proliferation, migration and differentiation into osteoblasts, while inhibiting the adipogenic differentiation of mMSCs. In contrast, inactivation of the ß-catenin or ROR2 genes resulted in the opposite effects. Therefore, these results confirm that lentiviral vector transduction can facilitate sustained and efficient gene modification of the Wnt pathway in mMSCs. This study provides a method to investigate the effects of the Wnt pathway on the fate of mMSCs in vivo and for the further improvement of MSC-based therapies.
Assuntos
Células-Tronco Mesenquimais/metabolismo , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/metabolismo , Via de Sinalização Wnt/fisiologia , beta Catenina/metabolismo , Adipócitos/citologia , Adipócitos/fisiologia , Animais , Diferenciação Celular , Movimento Celular , Proliferação de Células , Lentivirus , Camundongos , Osteogênese/fisiologia , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/genética , Transdução de Sinais , Via de Sinalização Wnt/genética , beta Catenina/genéticaRESUMO
INTRODUCTION: Glutamine supplementation is supposed to reduce mortality and nosocomial infections in critically ill patients. However, the recently published reducing deaths due to oxidative stress (REDOX) trials did not provide evidence supporting this. This study investigated the impact of glutamine-supplemented nutrition on the outcomes of critically ill patients using a meta-analysis. METHODS: We searched for and gathered data from the Cochrane Central Register of Controlled Trials, MEDLINE, Elsevier, Web of Science and ClinicalTrials.gov databases reporting the effects of glutamine supplementation on outcomes in critically ill patients. We produced subgroup analyses of the trials according to specific patient populations, modes of nutrition and glutamine dosages. RESULTS: Among 823 related articles, eighteen Randomized Controlled Trials (RCTs) met all inclusion criteria. Mortality events among 3,383 patients were reported in 17 RCTs. Mortality showed no significant difference between glutamine group and control group. In the high dosage subgroup (above 0.5 g/kg/d), the mortality rate in the glutamine group was significantly higher than that of the control group (relative risk (RR) 1.18; 95% confidence interval (CI), 1.02 to 1.38; P = 0.03). In 15 trials, which included a total of 2,862 patients, glutamine supplementation reportedly affected the incidence of nosocomial infections in the critically ill patients observed. The incidence of nosocomial infections in the glutamine group was significantly lower than that of the control group (RR 0.85; 95% CI, 0.74 to 0.97; P = 0.02). In the surgical ICU subgroup, glutamine supplementation statistically reduced the rate of nosocomial infections (RR 0.70; 95% CI, 0.52 to 0.94; P = 0.04). In the parental nutrition subgroup, glutamine supplementation statistically reduced the rate of nosocomial infections (RR 0.83; 95% CI, 0.70 to 0.98; P = 0.03). The length of hospital stay was reported in 14 trials, in which a total of 2,777 patients were enrolled; however, the patient length of stay was not affected by glutamine supplementation. CONCLUSIONS: Glutamine supplementation conferred no overall mortality and length of hospital stay benefit in critically ill patients. However, this therapy reduced nosocomial infections among critically ill patients, which differed according to patient populations, modes of nutrition and glutamine dosages.
Assuntos
Estado Terminal/mortalidade , Estado Terminal/terapia , Infecção Hospitalar/tratamento farmacológico , Infecção Hospitalar/mortalidade , Suplementos Nutricionais , Glutamina/administração & dosagem , Infecção Hospitalar/diagnóstico , Humanos , Tempo de Internação/tendências , Mortalidade/tendências , Ensaios Clínicos Controlados Aleatórios como Assunto/métodos , Resultado do TratamentoRESUMO
The differentiation of mesenchymal stem cells (MSCs) into type II alveolar epithelial (AT II) cells in vivo and in vitro, is critical for reepithelization and recovery in acute lung injury (ALI), but the mechanisms responsible for differentiation are unclear. In the present study, we investigated the role of the canonical wnt pathway in the differentiation of mouse bone marrow-derived MSCs (mMSCs) into AT II cells. Using a modified co-culture system with murine lung epithelial-12 (MLE-12) cells and small airway growth media (SAGM) to efficiently drive mMSCs differentiation, we found that GSK 3ß and ß-catenin in the canonical wnt pathway were up-regulated during differentiation. The levels of surfactant protein (SP) C, SPB, and SPD, the specific markers of AT II cells, correspondingly increased in mMSCs when Wnt3a or LiCl was added to the co-culture system to activate wnt/ß-catenin signaling. The expression of these factors was depressed to some extent by inhibiting the pathway with the addition of DKK 1. The differentiation rate of mMSCs also depends on their abilities to accumulate and survive in inflammatory tissue. Our results suggested that the activation of wnt/ß-catenin signaling promoted mMSCs migration towards ALI mouse-derived lung tissue in a Transwell assay, and ameliorated the cell death and the reduction of Bcl-2/Bax induced by H(2) O(2), which simultaneously caused reduced GSK 3ß and ß-catenin in mMSCs. These data supports a potential mechanism for the differentiation of mMSCs into AT II cells involving canonical wnt pathway activation, which may be significant to their application in ALI.
Assuntos
Lesão Pulmonar Aguda/metabolismo , Células Epiteliais Alveolares/metabolismo , Células da Medula Óssea/metabolismo , Diferenciação Celular , Movimento Celular , Células-Tronco Mesenquimais/metabolismo , Estresse Oxidativo , Via de Sinalização Wnt , Proteína Wnt3A/metabolismo , Lesão Pulmonar Aguda/patologia , Lesão Pulmonar Aguda/cirurgia , Células Epiteliais Alveolares/efeitos dos fármacos , Células Epiteliais Alveolares/transplante , Animais , Biomarcadores/metabolismo , Células da Medula Óssea/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células , Células Cultivadas , Técnicas de Cocultura , Meios de Cultivo Condicionados/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Peróxido de Hidrogênio/farmacologia , Cloreto de Lítio/farmacologia , Masculino , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Oxidantes/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Reepitelização , Fatores de Tempo , Técnicas de Cultura de Tecidos , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/metabolismoRESUMO
OBJECTIVE: To assess the value of central venous-to-arterial carbon dioxide difference [P((cv-a))CO2] in evaluation of disease severity and prognosis in patients with septic shock. METHODS: There were 45 consecutive resuscitated septic shock patients from April 2009 to October 2010 included immediately after their admission into our ICU. The patients were divided into low P((cv-a))CO2 group and high P((cv-a))CO2 group according to a threshold of 6 mm Hg (1 mm Hg = 0.133 kPa). All patients were treated by early goal directed therapy (EGDT). The parameters of hemodynamics, lactate clearance rate, the Acute Physiology and Chronic Health Evaluation II (APACHE II) score, the sequential organ failure assessment (SOFA) score, 6 h rate of EGDT achievement, the ICU mortality and 28 days in-hospital mortality were recorded for all patients. RESULTS: There were 30 patients in the low P((cv-a))CO2 group, and 15 in the high P((cv-a))CO2 group. There were no significant differences between low P((cv-a))CO2 and high P((cv-a))CO2 patients in age, APACHE II score and SOFA score (all P > 0.05). Compared with the high P((cv-a))CO2 group, the low P((cv-a))CO2 group had higher cardiac index (CI) and 24 h CI, higher delivery O2 (DO2) and 24 h DO2, higher central venous oxygen saturation (ScvO2) [(74 ± 9)% vs (67 ± 8)%], lower lactate [(3.4 ± 2.1) mmol/L vs (5.7 ± 4.5) mmol/L] and higher ΔSOFA score [(0.7 ± 1.8) vs (-0.4 ± 1.1)], lower 24 h SOFA score [(7.8 ± 2.0) vs (9.8 ± 2.0)], higher 6 h rate of EGDT achievement (83.3% vs 53.3%) (P < 0.05), however, there were no differences in 28 days mortality and ICU mortality between the two groups (P > 0.05). CONCLUSION: P((cv-a))CO2 might be an indicator for predicting the severity of patients with septic shock and evaluating tissue perfusion.