Bradykinin/bradykinin 1 receptor promotes brain microvascular endothelial cell permeability and proinflammatory cytokine release by downregulating Wnt3a.

Stroke is a life-threatening disease with limited therapeutic options. Damage to the blood-brain barrier (BBB) is the key pathological feature of ischemic stroke. This study explored the role of the bradykinin (BK)/bradykinin 1 receptor (B1R) and its mechanism of action in the BBB. Human brain microvascular endothelial cells (BMECs) were used to test for cellular responses to BK by using the Cell Counting Kit-8 assay, 5-ethynyl-2'-deoxyuridine staining, enzyme-linked immunosorbent assay, flow cytometry, immunofluorescence, cellular permeability assays, and western blotting to evaluate cell viability, cytokine production, and reactive oxygen species (ROS) levels in vitro. A BBB induced by middle cerebral artery occlusion was used to evaluate BBB injuries, and the role played by BK/B1R in ischemic/reperfusion (I/R) was explored in a rat model. Results showed that BK reduced the viability of BMECs and increased the levels of proinflammatory cytokines (interleukin 6 [IL-6], IL-18, and monocyte chemoattractant protein-1) and ROS. Additionally, cellular permeability was increased by BK treatment, and the expression of tight junction proteins (claudin-5 and occludin) was decreased. Interestingly, Wnt3a expression was inhibited by BK and exogenous Wnt3a restored the effects of BK on BMECs. In an in vivo I/R rat model, knockdown of B1R significantly decreased infarct volume and inflammation in I/R rats. Our results suggest that BK might be a key inducer of BBB injury and B1R knockdown might provide a beneficial effect by upregulating Wnt3a.

Arterial catheterization and in-hospital mortality in sepsis: a propensity score-matched study.

BACKGROUND: Despite the extensive use of arterial catheterization (AC), clinical effectiveness of AC to alter the outcomes among patients with sepsis and septic shock has not been evaluated. The purpose of this study is to examine the association between the use of AC and in-hospital mortality in septic patients. METHODS: Adult patients with sepsis from Medical Information Mart for Intensive Care database were screened to conduct this retrospective observational study. Propensity score matching (PSM) was employed to estimate the relationship between arterial catheterization (AC) and in-hospital mortality. Multivariable logistic regression and inverse probability of treatment weighing (IPTW) were used to validate our findings. RESULTS: A total of 14,509 septic patients without shock and 4,078 septic shock patients were identified. 3,489 pairs in sepsis patients without shock and 589 pairs in septic shock patients were yielded respectively after PSM. For patients in the sepsis without shock group, AC placement was associated with increased in-hospital mortality (OR, 1.34; 95% CI, 1.17-1.54; p < 0.001). In the septic shock group, there was no significant difference in hospital mortality between AC group and non-AC group. The results of logistic regression and propensity score IPTW model support our findings. CONCLUSIONS: In hemodynamically stable septic patients, AC is independently associated with higher in-hospital mortality, while in patients with septic shock, AC was not associated with improvements in hospital mortality.

Optimal Targets of the First 24-h Partial Pressure of Carbon Dioxide in Patients with Cerebral Injury: Data from the MIMIC-III and IV Database.

BACKGROUND: It is generally believed that hypercapnia and hypocapnia will cause secondary injury to patients with craniocerebral diseases, but a small number of studies have shown that they may have potential benefits. We assessed the impact of partial pressure of arterial carbon dioxide (PaCO2) on in-hospital mortality of patients with craniocerebral diseases. The hypothesis of this research was that there is a nonlinear correlation between PaCO2 and in-hospital mortality in patients with craniocerebral diseases and that mortality rate is the lowest when PaCO2 is in a normal range. METHODS: We identified patients with craniocerebral diseases from Medical Information Mart for Intensive Care third and fourth edition databases. Cox regression analysis and restricted cubic splines were used to examine the association between PaCO2 and in-hospital mortality. RESULTS: Nine thousand six hundred and sixty patients were identified. A U-shaped association was found between the first 24-h PaCO2 and in-hospital mortality in all participants. The nadir for in-hospital mortality risk was estimated to be at 39.5 mm Hg (p for nonlinearity < 0.001). In the subsequent subgroup analysis, similar results were found in patients with traumatic brain injury, metabolic or toxic encephalopathy, subarachnoid hemorrhage, cerebral infarction, and other encephalopathies. Besides, the mortality risk reached a nadir at PaCO2 in the range of 35-45 mm Hg. The restricted cubic splines showed a U-shaped association between the first 24-h PaCO2 and in-hospital mortality in patients with other intracerebral hemorrhage and cerebral tumor. Nonetheless, nonlinearity tests were not statistically significant. In addition, Cox regression analysis showed that PaCO2 ranging 35-45 mm Hg had the lowest death risk in most patients. For patients with hypoxic-ischemic encephalopathy and intracranial infections, the first 24-h PaCO2 and in-hospital mortality did not seem to be correlated. CONCLUSIONS: Both hypercapnia and hypocapnia are harmful to most patients with craniocerebral diseases. Keeping the first 24-h PaCO2 in the normal range (35-45 mm Hg) is associated with lower death risk.

Hypercapnia Exacerbates the Blood-Brain Barrier Disruption Via Promoting HIF-1a Nuclear Translocation in the Astrocytes of the Hippocampus: Implication in Further Cognitive Impairment in Hypoxemic Adult Rats.

Hypercapnia in combination with hypoxemia is usually present in severe respiratory disease in the intensive care unit (ICU) and can lead to more severe cognitive dysfunction. Increasing evidence has indicated that the compromised blood-brain barrier (BBB) in the hippocampus in hypoxemia conditions can result in cognitive dysfunction. However, the role and underlying mechanism of hypercapnia in the BBB disruption remains poorly known. A rat model of hypercapnia was first established in this study by intubation and mechanical ventilation with a small-animal ventilator. After this, the cognitive function of the experimental rats was assessed by the Morris water maze test. The BBB permeability was evaluated by the Evans Blue (EB) test and brain water content (BWC). Western blot analysis was carried out to detect the protein expressions of total and nuclear hypoxia-inducible factor-1α (HIF-1α), matrixmetalloproteinase-9 (MMP-9) and Aquaporins-4 (AQP-4) in the hippocampus tissue. Double immunofluorescence further verified the protein expression of different biomarkers was localized in the astrocytes of the hippocampus. Hypercapnia alone did not disrupt the BBB, but it could further enhance the BBB permeability in hypoxemia. Concomitantly, up-regulation of nuclear HIF-1α, AQP-4, MMP-9 protein expression along with increased degradation of the occludin and claudin-5 proteins was found in the hypercapnia rat model, while the total HIF-1α remained unchanged. Interestingly, these changes were independent of the acidosis induced by hypercapnia. Of note, after premedication of 2-Methoxyestradiol (2ME2, an inhibitor of HIF-1α nuclear translocation), the disrupted BBB could be restored resulting in improvement of the cognitive impairment. Meanwhile, accumulation of nuclear HIF-1α, protein expression of AQP-4 and MMP-9 and protein degradation of the occludin and claudin-5 were decreased. Thus, our study demonstrated that hypercapnia can further disrupt the BBB through promoting HIF-1α nuclear translocation and up-regulation of AQP-4 and MMP-9 in hypoxemia. It is therefore suggested that the cascade of hypercapnia-induced nuclear HIF-1α protein translocation in hypoxia-activated astrocytes may be a potential target for ameliorating cognitive impairment.

Increased percentage of PD-L1+ natural killer cells predicts poor prognosis in sepsis patients: a prospective observational cohort study.

BACKGROUND: Natural killer (NK) cells play a major role in immune tolerance after sepsis, and the programmed cell death 1 (PD-1) and programmed cell death ligand 1 (PD-L1) system mediates evasion of host immunity. The correlation between PD-L1 levels in NK cells and the prognosis of patients with sepsis, however, has not been elucidated. Thus, it was hypothesized that PD-L1 in NK cells could be a novel biomarker of the mortality for sepsis patients. METHODS: A prospective, observational, cohort study in a general intensive care unit had earlier enrolled patients according to the sepsis-3 criteria, and peripheral blood samples were collected within 24 h post-recruitment. The expression of four co-signaling molecules (PD-1, CD28, PD-L1, and CD86) in NK cells was assayed, and the sequential organ failure assessment (SOFA) scores were recorded on day 1. Patients were followed up until 28 days. Multivariate regression analysis assessed the independent risk factors for 28-day mortality. The association between biomarkers and 28-day mortality was assessed by Cox regression survival analysis. The accuracy of biomarkers for mortality was determined by the area under the receiver operating characteristic (ROC) curve (AUC) analysis. RESULTS: A total of 269 patients were recruited, and 114 patients were finally included for final analysis. Of these, 30 (26.3%) patients died during 28 days. The percentage of PD-L1+ NK cells (OR 1.022; 95% CI 1.002-1.043) and SOFA scores (OR 1.247; 95% CI 1.092-1.424) were independent risk factors for 28-day mortality. The AUC of the percentage of PD-L1+ NK cells, SOFA scores, and their combination model were 0.655 (0.559-0.742), 0.727 (0.635-0.807) and 0.808 (0.723-0.876), respectively. The combination model was the indicator with the best AUC to predict mortality in 28 days (all p < 0.05). Patients with the percentage of PD-L1+ NK cells above the cutoff point 5.58% (hazard ratio (HR) 10.128 (1.372-74.772), p = 0.001), and the combination model prediction possibility above 0.1241 (HR 13.730 (3.241-58.158), p < 0.001) were the indexes that had greater discriminative capacity to predict 28 days mortality. CONCLUSIONS: The percentage of PD-L1+ NK cells at admission serves as a novel prognostic biomarker for predicting mortality and contributes to improve the predictive capacity of SOFA score in patients with sepsis.

Hypertonic saline regulates microglial M2 polarization via miR-200b/KLF4 in cerebral edema treatment.

BACKGROUND: Hypertonic saline (HS) has been used clinically for treatment of cerebral edema for decades. Previously we have demonstrated that HS alleviates cerebral edema via regulating water/ion channel protein and attenuating neuroinflammation. However, whether HS treatment triggers microglia polarization and its regulatory mechanism during this process is unclear. METHODS AND RESULTS: The Sprague-Dawley (SD) rats that underwent right-sided middle cerebral artery occlusion (MCAO) were used for assessment of neuroinflammation and microglia functions. Treatment of 10% HS not only significantly reduced infarct size and ipsilateral ischemic hemispheric brain water content (BWC) via attenuating ischemia-induction of TNF-α, IL-1ß, microglia M1 markers (iNOS, CD86) and miR-200b, but also increased neurotrophic factors such as IL-10 and IL-4, microglia M2 markers (Arg1, CD206) and Krüppel-like factor 4 (KLF4). Similar changes were confirmed in primary microglial cells subjected to hypoxia with/without HS in vitro. Importantly, overexpression of miR-200b was able to induce microglia M1 polarization via directly targeting KLF4. Restoring KLF4 expression abolished this effect. On the contrary, miR-200b inhibitor or KLF4 overexpression led to microglia M2 polarization. Mechanistically, KLF4 directly binds to promoter region of Agr1, thus inducing its transcription. Similar to treatment of HS, experimental overexpression of KLF4 in vivo exerted significant beneficial effects on ischemia-induced cerebral edema. However, knockdown of KLF4 abrogated the benefits of HS. CONCLUSIONS: Hypertonic saline regulates microglial M2 polarization via miR-200b/KLF4 during its treatment of cerebral edema. This study may provide new insights of HS-related therapy for cerebral edema.

Hypercapnia induces IL-1ß overproduction via activation of NLRP3 inflammasome: implication in cognitive impairment in hypoxemic adult rats.

BACKGROUND: Cognitive impairment is one of common complications of acute respiratory distress syndrome (ARDS). Increasing evidence suggests that interleukin-1 beta (IL-1ß) plays a role in inducing neuronal apoptosis in cognitive dysfunction. The lung protective ventilatory strategies, which serve to reduce pulmonary morbidity for ARDS patients, almost always lead to hypercapnia. Some studies have reported that hypercapnia contributes to the risk of cognitive impairment and IL-1ß secretion outside the central nervous system (CNS). However, the underlying mechanism of hypercapnia aggravating cognitive impairment under hypoxia has remained uncertain. This study was aimed to explore whether hypercapnia would partake in increasing IL-1ß secretion via activating the NLRP3 (NLR family, pyrin domain-containing 3) inflammasome in the hypoxic CNS and in aggravating cognitive impairment. METHODS: The Sprague-Dawley (SD) rats that underwent hypercapnia/hypoxemia were used for assessment of NLRP3, caspase-1, IL-1ß, Bcl-2, Bax, and caspase-3 expression by Western blotting or double immunofluorescence, and the model was also used for Morris water maze test. In addition, Z-YVAD-FMK, a caspase-1 inhibitor, was used to treat BV-2 microglia to determine whether activation of NLRP3 inflammasome was required for the enhancing effect of hypercapnia on expressing IL-1ß by Western blotting or double immunofluorescence. The interaction effects were analyzed by factorial ANOVA. Simple effects analyses were performed when an interaction was observed. RESULTS: There were interaction effects on cognitive impairment, apoptosis of hippocampal neurons, activation of NLRP3 inflammasome, and upregulation of IL-1ß between hypercapnia treatment and hypoxia treatment. Hypercapnia + hypoxia treatment caused more serious damage to the learning and memory of rats than those subjected to hypoxia treatment alone. Expression levels of Bcl-2 were reduced, while that of Bax and caspase-3 were increased by hypercapnia in hypoxic hippocampus. Hypercapnia markedly increased the expression of NLRP3, caspase-1, and IL-1ß in hypoxia-activated microglia both in vivo and in vitro. Pharmacological inhibition of NLRP3 inflammasome activation and release of IL-1ß might ameliorate apoptosis of neurons. CONCLUSIONS: The present results suggest that hypercapnia-induced IL-1ß overproduction via activating the NLRP3 inflammasome by hypoxia-activated microglia may augment neuroinflammation, increase neuronal cell death, and contribute to the pathogenesis of cognitive impairments.

Hypertonic saline attenuates expression of Notch signaling and proinflammatory mediators in activated microglia in experimentally induced cerebral ischemia and hypoxic BV-2 microglia.

BACKGROUND: Ischemic stroke is a major disease that threatens human health in ageing population. Increasing evidence has shown that neuroinflammatory mediators play crucial roles in the pathophysiology of cerebral ischemia injury. Notch signaling is recognized as the cell fate signaling but recent evidence indicates that it may be involved in the inflammatory response in activated microglia in cerebral ischemia. Previous report in our group demonstrated hypertonic saline (HS) could reduce the release of interleukin-1 beta and tumor necrosis factor-alpha in activated microglia, but the underlying molecular and cellular mechanisms have remained uncertain. This study was aimed to explore whether HS would partake in regulating production of proinflammatory mediators through Notch signaling. RESULTS: HS markedly attenuated the expression of Notch-1, NICD, RBP-JK and Hes-1 in activated microglia both in vivo and in vitro. Remarkably, HS also reduced the expression of iNOS in vivo, while the in vitro levels of inflammatory mediators Phos-NF-κB, iNOS and ROS were reduced by HS as well. CONCLUSION: Our results suggest that HS may suppress of inflammatory mediators following ischemia/hypoxic through the Notch signaling which operates synergistically with NF-κB pathway in activated microglia. Our study has provided the morphological and biochemical evidence that HS can attenuate inflammation reaction and can be neuroprotective in cerebral ischemia, thus supporting the use of hypertonic saline by clinicians in patients with an ischemia stroke.

[New Approach of Fundus Image Segmentation Evaluation Based on Topology Structure].

In view of the evaluation of fundus image segmentation, a new evaluation method was proposed to make up insufficiency of the traditional evaluation method which only considers the overlap of pixels and neglects topology structure of the retinal vessel. Mathematical morphology and thinning algorithm were used to obtain the retinal vascular topology structure. Then three features of retinal vessel, including mutual information, correlation coefficient and ratio of nodes, were calculated. The features of the thinned images taken as topology structure of blood vessel were used to evaluate retinal image segmentation. The manually-labeled images and their eroded ones of STARE database were used in the experiment. The result showed that these features, including mutual information, correlation coefficient and ratio of nodes, could be used to evaluate the segmentation quality of retinal vessel on fundus image through topology structure, and the algorithm was simple. The method is of significance to the supplement of traditional segmentation evaluation of retinal vessel on fundus image.

Hypertonic saline alleviates cerebral edema by inhibiting microglia-derived TNF-α and IL-1ß-induced Na-K-Cl Cotransporter up-regulation.

BACKGROUND: Hypertonic saline (HS) has been successfully used clinically for treatment of various forms of cerebral edema. Up-regulated expression of Na-K-Cl Cotransporter 1 (NKCC1) and inflammatory mediators such as tumor necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1ß) has been demonstrated to be closely associated with the pathogenesis of cerebral edema resulting from a variety of brain injuries. This study aimed to explore if alleviation of cerebral edema by 10% HS might be effected through down-regulation of inflammatory mediator expression in the microglia, and thus result in decreased NKCC1 expression in astrocytes in the cerebral cortex bordering the ischemic core. METHODS: The Sprague-Dawley (SD) rats that underwent right-sided middle cerebral artery occlusion (MCAO) were used for assessment of NKCC1, TNF-α and IL-1ß expression using Western blotting, double immunofluorescence and real time RT-PCR, and the model also was used for evaluation of brain water content (BWC) and infarct size. SB203580 and SP600125, specific inhibitors of the p38 and JNK signaling pathways, were used to treat primary microglia cultures to determine whether the two signaling pathways were required for the inhibition of HS on microglia expressing and secreting TNF-α and IL-1ß using Western blotting, double immunofluorescence and enzyme-linked immunosorbent assay (ELISA). The effect of TNF-α and IL-1ß on NKCC1 expression in primary astrocyte cultures was determined. In addition, the direct inhibitory effect of HS on NKCC1 expression in primary astrocytes was also investigated by Western blotting, double immunofluorescence and real time RT-PCR. RESULTS: BWC and infarct size decreased significantly after 10% HS treatment. TNF-α and IL-1ß immunoexpression in microglia was noticeably decreased. Concomitantly, NKCC1 expression in astrocytes was down-regulated. TNF-α and IL-1ß released from the primary microglia subjected to hypoxic exposure and treatment with 100 mM HS were decreased. NKCC1 expression in primary astrocytes was concurrently and progressively down-regulated with decreasing concentration of exogenous TNF-α and IL-1ß. Additionally, 100 mM HS directly inhibited NKCC1 up-regulation in astrocytes under hypoxic condition. CONCLUSIONS: The results suggest that 10% HS alleviates cerebral edema through inhibition of the NKCC1 Cotransporter, which is mediated by attenuation of TNF-α and IL-1ß stimulation on NKCC1.

PD-1 mediates microglia polarization via the MAPK signaling pathway to protect blood-brain barrier function during cerebral ischemia/reperfusion.

BACKGROUND: Cerebral ischemia is characterized by its rapid onset and high rates of recurrence, morbidity, and mortality, with blood-brain barrier (BBB) permeability playing a vital role in brain injury. Therefore, it is important to understand the molecular mechanism which regulates the BBB during cerebral ischemia. MATERIALS AND METHODS: An in vitro model of oxygen-glucose deprivation (OGD) and an in vivo model of cerebral ischemia/reperfusion (I/R) were constructed. PD-1 overexpression vectors and vectors containing si-RNA were transfected and injected into in vitro and in vivo models. Western blotting, real-time quantitative PCR (qPCR), immunofluorescence (IF) analysis, and immunohistochemical staining were employed to evaluate the expression levels of programmed cell death-1 (PD-1), microglia M1 and M2 biomarkers, and tight junction proteins. Flow cytometry and ELISA were used to measure the levels of pro-inflammatory cytokines. The BBB permeability of brain tissues was evaluated by Evans blue dye (EBD) extravasation and transendothelial electrical resistance (TEER). Brain water content was measured to assess the extent of inflammatory exudation. The infarct volume and neurological severity score (NSS) were used to assess the severity of brain injury. Brain cell apoptosis was assessed by the TUNEL assay and hematoxylin-eosin (H&E) staining. RESULTS: PD-1 helped to convert the microglia M1 phenotype to the M2 phenotype and to reduce BBB permeability both in vitro and in vivo. Overexpression of PD-1 promoted a shift of the M1 phenotype to the M2 phenotype and reduced BBB permeability via the ERK and p38 MAPK signaling pathways. PD-1 reduced inflammatory exudation, BBB permeability, cell apoptosis, and brain injury in vivo. CONCLUSION: Our present study verified that PD-1 exerts an anti-inflammatory effect by converting the microglia M1 phenotype to the M2 phenotype, reducing BBB permeability, and thereby relieves brain injury caused by cerebral ischemia. PD-1 is potential therapeutic target for brain injury caused by cerebral ischemia.

[Clinical importance of screening differential gene set of monocytes based on single-cell sequencing and digital polymerase chain reaction technology for early diagnosis of sepsis].

OBJECTIVE: To verify the specific differentiated subsets of monocytes in sepsis, and to screen and construct the differential gene set of monocytes used for early diagnosis of sepsis. METHODS: Patients with sepsis admitted to Guangdong Provincial People's Hospital from June 2020 to March 2021 were enrolled, and peripheral blood mononuclear cells (PBMC) were extracted. Single-cell sequencing technology and pseudo-time analysis were used to verify the differential subsets of monocytes. Bioinformatics methods were used to analyze the expression of genes in differential subsets of monocytes and screen out differential genes for the preliminary construction of a candidate differential gene set. The digital polymerase chain reaction (PCR) technology was used to verify the candidate differential genes in PBMC of sepsis patients and sepsis human myeloid leukemia mononuclear cells (THP-1) models, and the Venn diagram was used to construct the final differential gene set of monocytes. Gene Expression Omnibus (GEO) database was used to validate the differential gene set of monocytes. RESULTS: (1) The results of cell annotation and pseudo-time analysis showed that the differentiation of NEAT1+CD163+ monocyte occurred in the early stage of sepsis was significantly different from other subsets, which validated that NEAT1+CD163+ monocyte was the characteristic subset in the pathological process of sepsis. (2) Twenty-two differential genes related to sepsis were screened out from the gene expression of NEAT1+CD163+ monocyte. After further verification by digital PCR, basic leucine zipper ATF-like transcription factor (BATF), JUNB proto-oncogene, carcinoembryonic antigen-related cell adhesion molecule 4 (CEACAM4), chromosome 9 open reading frame 95 (C9orf95), G protein subunit alpha 15 (GNA15), complement C3a receptor 1 (C3AR1), transforming growth factor beta 1 (TGFB1) and mitochondrial carrier homolog 1 (MTCH1) were screened out to construct the final differential gene set of monocytes. (3) The external validation results showed that C9orf95 gene had no data in GSE154918 and GSE133822 from GEO, it was excluded during validation. In GSE154918, the expressions of BATF, JUNB, CEACAM4, GNA15, C3AR1, TGFB1, and MTCH1 in the sepsis group were significantly higher than those in the healthy control group (log2expression level: BATF was 12.78±0.08 vs. 11.39±0.35, JUNB was 16.88±0.07 vs. 16.04±0.03, CEACAM4 was 14.73±0.08 vs. 13.77±0.05, GNA15 was 13.16±0.06 vs. 12.30±0.04, C3AR1 was 14.62±0.13 vs. 12.87±0.05, TGFB1 was 16.95±0.05 vs. 16.57±0.36, MTCH1 was 14.80±0.02 vs. 14.61±0.15, all P < 0.05). In GSE133822, the expressions of BATF, CEACAM4, GNA15, and C3AR1 in the sepsis group were significantly higher than those in the health control group (log2expression level: BATF was 8.66±0.16 vs. 7.92±0.14, CEACAM4 was 9.20±0.16 vs. 8.36±0.20, GNA15 was 10.66±0.18 vs. 10.13±0.16, C3AR1 was 11.49±0.27 vs. 10.48±0.16, all P < 0.05), while the expressions of JUNB, TGFB1, and MTCH1 were not statistically different between two groups. The results of gene set variation analysis (GSVA) showed that the enrichment scores of monocytes differential gene set of sepsis group were significantly higher than those of the healthy control group in both GSE154918 (0.38±0.04 vs. -0.44±0.02) and GSE133822 (0.56±0.02 vs. 0.20±0.05, both P < 0.01). Receiver operator characteristic curve (ROC curve) analysis showed that the differential gene set of monocytes had a reliable diagnostic value for early sepsis with the area under ROC curve (AUC) of 0.993 [95% confidence interval (95%CI) was 0.980-1.000] in GSE154918 and 0.944 (95%CI was 0.873-1.000) in GSE133822. CONCLUSIONS: A differential gene set of monocytes (BATF, JUNB, CEACAM4, GNA15, C3AR1, TGFB1, and MTCH1) screened out by single-cell sequencing and digital PCR technology has a reliable diagnostic value for the early sepsis, and may provide a new idea for the early diagnosis of sepsis.

[Analysis of clinical features and risk factors for mortality in patients with culture-negative sepsis: a single-center retrospective cohort study based on MIMIC-IV].

OBJECTIVE: To compare the characteristics and outcomes of culture-positive sepsis (CPS) with culture-negative sepsis (CNS) patients in order to understand the impact of CNS on prognosis and explore the possible risk factors for mortality. METHODS: A retrospective cohort study was conducted. Patients with sepsis were identified from the Medical Information Mart for Intensive Care database-IV v0.4 (MIMIC-IV v0.4). Patients were divided into CPS and CNS groups according to the culture results within 24 hours before and after the diagnosis of sepsis. General information, baseline characteristics, and medical operation data between CNS and CPS groups were compared. Logistic regression analysis was used to calculate the relationship between CNS and in-hospital mortality under three regression models. Chi-square analysis and mediation analysis were used to analyze the effect of initial antibiotic and prior antibiotic use within 90 days on the in-hospital mortality of CNS. RESULTS: A total of 8 587 patients with sepsis were enrolled in the final analysis, including 5 483 patients in the CPS group and 3 104 patients in the CNS group. Compared with the CPS group, the patients in the CNS group were younger [years old: 68 (56, 79) vs. 70 (58, 81)], had higher sequential organ failure assessment (SOFA) score and higher proportion of using mechanical ventilation, renal replacement therapy and vasopressin within 24 hours after intensive care unit (ICU) admission [SOFA score: 3 (2, 5) vs. 3 (2, 4), mechanical ventilation: 48.61% (1 509/3 104) vs. 39.25% (2 152/5 483), renal replacement therapy: 13.69% (425/3 104) vs. 9.68% (531/5 483), vasopressin: 15.79% (490/3 104) vs. 13.44% (737/5 483)], longer length of ICU stay [days: 5 (3, 10) vs. 3 (2, 6)] and higher in-hospital mortality [25.00% (776/3 104) vs. 18.53% (1 016/5 483)], with significant differences (all P < 0.01). However, there was no significant difference in gender, ICU type, simplified acute physiology score II (SAPS II), and Charlson comorbidity index (CCI) score between the two groups. After adjustment for multiple confounding factors, CNS was still a risk factor for in-hospital mortality [odds ratio (OR) = 1.441, 95% confidence interval (95%CI) was 1.273-1.630, P < 0.001]. The results of Chi-square analysis and mediation analysis showed that the initial antibiotic had no significant effect on the higher in-hospital mortality of CNS, while the prior use of antibiotics within 90 days was related to higher in-hospital mortality of CNS (OR = 1.683, 95%CI was 1.328-2.134, P < 0.05). The mediating effect of CNS in prior antibiotic use within 90 days and in-hospital death was significant (Z = 5.302, P < 0.001), accounting for 7.58%. CONCLUSIONS: Compared with CPS, CNS was more severe and had a worse prognosis. Prior use of antibiotics within 90 days may be related to the higher in-hospital mortality of CNS patients, but it could not fully explain the high mortality of CNS.

Elevated intracranial pressure induces IL­1ß and IL­18 overproduction via activation of the NLRP3 inflammasome in microglia of ischemic adult rats.

Elevated intracranial pressure (ICP) is one of the most common complications following an ischemic stroke, and has implications for the clinical and neurological outcomes. The aim of the present study was to examine whether elevated ICP may increase IL­1ß and IL­18 secretion by activating the NOD­like receptor protein 3 (NLRP3) inflammasome in microglia of ischemic adult rats. Sprague­Dawley rats that underwent middle cerebral artery occlusion were used for assessment of ICP. Reactive oxygen species (ROS) production was detected, and western blotting and immunofluorescence staining were used to determine the expression levels of Caspase­1, gasdermin D­N domains (GSDMD­N), IL­1ß and IL­18 in microglial cells. ICP levels were significantly increased, which was accompanied by ROS overproduction, in the brain tissue following ischemia­reperfusion (IR) injury in rats. Treatment with 10% hypertonic saline by intravenous injection significantly reduced the ICP and ROS levels of the rats. Furthermore, high pressure (20 mmHg) combined with oxygen­glucose deprivation (OGD) treatment resulted in increased ROS production in BV­2 microglial cells compared with those subjected to OGD treatment alone in vitro. Elevated pressure upregulated the expression of Caspase­1, GSDMD­N, IL­18 and IL­1ß in IR­treated or OGD­treated microglia both in vivo and in vitro. More importantly, Caspase­1, GSDMD­N, IL­18 and IL­1ß expression in microglia was significantly downregulated when elevated pressure was reduced or removed. These results suggested that elevated ICP­induced IL­1ß and IL­18 overproduction via activation of the NLRP3 inflammasome by ischemia­activated microglia may augment neuroinflammation.

Comparison of clinical characteristics in patients with coronavirus disease and influenza A in Guangzhou, China.

BACKGROUND: This study aims to compare the epidemiological, clinical and laboratory characteristics between patients with coronavirus disease (COVID-19) and influenza A (H1N1), and to develop a differentiating model and a simple scoring system. METHODS: We retrospectively analyzed the data from patients with COVID-19 and H1N1. The logistic regression model based on clinical and laboratory characteristics was constructed to distinguish COVID-19 from H1N1. Scores were assigned to each of independent discrimination factors based on their odds ratios. The performance of the prediction model and scoring system was assessed. RESULTS: A total of 236 patients were recruited, including 20 COVID-19 patients and 216 H1N1 patients. Logistic regression revealed that age >34 years, temperature ≤37.5 °C, no sputum or myalgia, lymphocyte ratio ≥20% and creatine kinase-myocardial band isoenzyme (CK-MB) >9.7 U/L were independent differentiating factors for COVID-19. The area under curves (AUCs) of the prediction model and scoring system in differentiating COVID-19 from H1N1 were 0.988 and 0.962, respectively. CONCLUSIONS: There are certain differences in clinical and laboratory features between patients with COVID-19 and H1N1. The simple scoring system may be a useful tool for the early identification of COVID-19 patients from H1N1 patients.

Single-cell transcriptomics reveals the alteration of peripheral blood mononuclear cells driven by sepsis.

BACKGROUND: Sepsis is a serious systemic inflammatory response syndrome caused by infection, with an extremely high mortality rate. Peripheral blood mononuclear cells (PBMCs) played a key role in the immune response against infection, whose components and functions were altered radically in Sepsis. Here, we wondered to characterize the alteration of PBMCs in sepsis at the single-cell transcriptional level. METHODS: We isolated PBMCs from seven septic patients and four donors. Based on BD Rhapsody, PBMCs were generated by single-cell RNA sequencing, and cell types were clustered and named by unsupervised clustering and annotation analysis. RESULTS: PBMCs were profiled for 6 kinds of cell types, the biological properties of T cell and monocytes were shown in a detailed manner. We noticed that monocytes could be clustered into 6 subsets, with great heterogeneity in the alteration of composition, gene profile, and signaling pathways driven by sepsis. Moreover, the expression of representative genes was high associated with septic clinical indicators in clusters of monocytes, such as NEAT1. CONCLUSIONS: Although the study was preliminary, we revealed sepsis-specific alteration of PBMCs and associated pathways. These results give a panoramic picture of PBMCs in composition, genes profiles, and pathway signatures that are driven by sepsis, which offers a unique perspective to understand disease progression or treatment in clinical practice.

[Effect of hypercapnia on the clinical prognosis and severity of infection in patients with severe community-acquired pneumonia].

OBJECTIVE: To investigate the effect of hypercapnia at admission on the clinical prognosis and the severity of infection in patients with severe community-acquired pneumonia (SCAP). METHODS: The clinical data of 219 SCAP patients admitted to the department of emergency and critical care medicine of Guangdong Provincial People's Hospital from December 2017 to November 2019 were retrospectively analyzed. Based on the partial pressure of arterial carbon dioxide (PaCO2) within 1 day after admission, the patients were divided into hypocapnia group [HO group, PaCO2 < 35 mmHg (1 mmHg = 0.133 kPa)], normal carbonation group (NC group, PaCO2 35-45 mmHg) and hypercapnia group (HC group, PaCO2 > 45 mmHg). The clinical parameters of patients, such as gender, age, underlying diseases, white blood cell (WBC), procalcitonin (PCT), C-reactive protein (CRP), interleukin-6 (IL-6), pH value and lactate (Lac) within 1 day after admission were reviewed. The oxygenation index (PaO2/FiO2), pneumonia severity index (PSI) score and acute physiology and chronic health evaluation II (APACHE II) score were evaluated. The change tendencies of each index on day 1, day 3, and day 5 after admission were observed subsequently. Meanwhile, the rate of invasive mechanical ventilation (IMV), length of hospital stays and 28-day mortality among three groups were compared. Kaplan-Meier survival analysis was performed to assess the 28-day cumulative survival rate of patients with SCAP among three groups. Multivariate Logistic regression analysis was used to screen the risk factors of IMV and 28-day death in patients with SCAP. RESULTS: Compared with the HO group (n = 68) and NC group (n = 72), the HC group (n = 79) had higher proportion of preexisting comorbid chronic obstructive pulmonary disease (COPD) and PSI score, lower PCT, CRP, IL-6, and pH values. Compared with the HO group and NC group, there were smaller improvement trends on the levels of WBC, PCT, CRP, IL-6, PaO2/FiO2 and Lac at day 3 and day 5 as compared with day 1 in the HC group. On the 5th day after admission, the levels of WBC, PCT, CRP, IL-6, and Lac in the HC group were significantly higher than those in the HO group and NC group [WBC (×109/L): 18.33±1.44 vs. 10.89±2.37, 11.15±1.74; PCT (µg/L): 5.04±1.18 vs. 3.46±0.87, 3.58±0.83; CRP (mg/L): 78.43±7.17 vs. 54.24±4.97, 57.93±5.39; IL-6 (ng/L): 75.35±11.92 vs. 60.11±10.27, 57.88±12.34; Lac (mmol/L): 4.36±1.24 vs. 0.78±0.39, 0.86±0.64; all P < 0.01], and the lowest in PaO2/FiO2 was found in the HC group as compared with the HO and NC groups (mmHg: 171.31±6.73 vs. 226.68±7.36, 225.93±6.92, both P < 0.01). Compared with the HO group and NC group, the HC group had highest proportion of IMV (29.1% vs. 22.1%, 22.2%, both P < 0.01) and 28-day mortality (26.6% vs. 13.2%, 13.9%, both P < 0.01). Even when the patients with COPD were excluded from the analysis, the differences persisted among the groups. Kaplan-Meier survival analysis suggested that HC group had a higher 28-day cumulative survival rate as compared with the HO and NC groups (Log-Rank test: χ12 = 4.976, P1 = 0.026; χ22 = 4.629, P2 = 0.031). Multivariate Logistic regression analysis showed that IL-6, PSI score and hypercapnia within 1 day and PCT on the 5th day after admission were the independent risk factors of requiring IMV and 28-day death in patients with SCAP [odds ratio (OR) were 0.325, 1.229, 1.396, 1.313, respectively, all P < 0.01]. Even when patients with COPD were excluded from the analysis, the above results had not been changed. CONCLUSIONS: Hypercapnia at admission was associated with higher proportion of IMV and 28-day mortality in patients with SCAP, which may be related to its early suppression of inflammation and then increment of infection.

[Mechanism of resveratrol on ameliorating the cognitive dysfunction induced by sepsis associated encephalopathy in rats].

OBJECTIVE: To explore the mechanism of resveratrol on ameliorating the cognitive dysfunction induced by sepsis associated encephalopathy (SAE) in rats. METHODS: The 12 weeks old male Sprague-dawley (SD) male rats were randomly divided into sham group, sepsis group and resveratrol group, with 30 rats in each group. The rat model of sepsis was made by injecting LPS (10 mg/kg) into tail vein. The rats in sham group was given the same amount of normal saline (NS). After LPS injection, resveratrol (8 mg×kg-1×d-1) was intraperitoneally injected once daily for 2 days in the resveratrol group; the same amount of NS was given to the sepsis group and sham group. At 24 hours after model establishment, the cognitive function of the experimental rats was assessed by the Morris water maze test. The blood-brain barrier (BBB) permeability was evaluated by the brain water content (BWC) and Evans blue (EB) test. The protein expressions of matrix metalloproteinase 9 (MMP-9), Occludin and Claudin-5 in cortical tissue were detected by Western Blot. Double immunofluorescence was used to verify the co-localization of MMP-9 protein and the marker protein of astrocyte GFAP in the cortical tissue of rats. RESULTS: Compared with the sham group, the escape latency in the sepsis group was significantly longer [48-hour escape latency (s): 56.56±6.43 vs. 36.62±3.32, 72-hour escape latency (s): 57.72±7.23 vs. 26.46±4.24, both P < 0.01], the BWC and extravasation of EB were increased [BWC: (84.56±2.03)% vs. (76.82±2.22)%, EB (µg/g): 17.56±2.28 vs. 6.25±1.36, both P < 0.01], the expression of MMP-9 protein was increased (MMP-9/ß-actin: 0.73±0.01 vs. 0.24±0.01, P < 0.01), the protein expressions of Occludin and Claudin-5 were decreased (Occludin/ß-actin: 0.45±0.02 vs. 0.86±0.04, Claudin-5/ß-actin: 0.62±0.03 vs. 0.96±0.05, both P < 0.01). At the same time, the co-localization expression of MMP-9 protein and the astrocytes of the cortical were increased [MMP-9 fluorescence intensity (AU): 38.66±4.26 vs. 17.23±3.04, MMP-9 positive cells: (26.92±1.77)% vs. (12.82±1.46)%, both P < 0.01]. Compared with the sepsis group, the escape latency in resveratrol group was significantly shorter [48-hour escape latency (s): 41.42±6.27 vs. 56.56±6.43, 72-hour escape latency (s): 33.46±7.17 vs. 57.72±7.23, both P < 0.01], the BWC and extravasation of EB were decreased [BWC: (77.15±2.27)% vs. (84.56±2.03)%, EB (µg/g): 7.74±1.88 vs. 17.56±2.28, both P < 0.01], the expression of MMP-9 protein was decreased (MMP-9/ß-actin: 0.25±0.01 vs. 0.73±0.01, P < 0.01), the protein expressions of Occludin and Claudin-5 were increased (Occludin/ß-actin: 0.82±0.03 vs. 0.45±0.02, Claudin-5/ß-actin: 0.92±0.04 vs. 0.62±0.03, both P < 0.01). At the same time, the co-localization expression of MMP-9 protein and the astrocytes of the cortical were decreased [MMP-9 fluorescence intensity (AU): 19.44±4.37 vs. 38.66±4.26, MMP-9 positive cells: (13.11±1.29)% vs. (26.92±1.77)%, both P < 0.01]. CONCLUSIONS: Resveratrol can inhibit the expression of MMP-9 protein in the astrocytes of the cortical cortex of rats, and then reduce the degradation of tight junction proteins of Occludin and Claudin-5, thereby reducing BBB permeability and eventually ameliorate the cognitive dysfunction induced by SAE.

Hypercapnia exacerbates the disruption of the blood­brain barrier by inducing interleukin­1ß overproduction in the blood of hypoxemic adult rats.

Refractory hypoxemia is the main symptom of acute respiratory distress syndrome (ARDS). Low tidal volume ventilation is routinely applied in clinical practice to correct hypoxemia, which aims to prevent ventilator­induced lung injury. However, this ventilation strategy inevitably leads to hypercapnia. Our previous study demonstrated that hypercapnia aggravated cognitive impairment in hypoxemic rats; however, the underlying mechanism remains unclear. The aim of the present study was to investigate whether hypercapnia exacerbates the blood­brain barrier (BBB) disruption through inducing interleukin (IL)­1ß overproduction in the blood of hypoxemic rats. The BBB permeability in a rat model of hypercapnia/hypoxemia was evaluated. The levels of IL­1ß in the blood of rats and human whole­blood cultures were assessed. The expression of IL­1 receptor 1 (IL­1R1), phosphorylated IL­1R1­associated kinase (p­IRAK­1) and tight junctional proteins in cerebral vascular endothelial cells was examined in vitro and in vivo. In addition, IL­1Ra, an IL­1 receptor antagonist, was used to determine whether hypercapnia affects tight junctional protein expression in hypoxic cerebral vascular endothelial cells through inducing IL­1ß overproduction. It was observed that hypercapnia alone did not disrupt the BBB, but aggravated the damage to the BBB integrity in hypoxemic rats. Hypercapnia increased IL­1ß expression in the blood of hypoxemic rats as well as in hypoxic human whole­blood cultures. IL­1R1 and p­IRAK­1 expression was increased, while that of tight junctional proteins was reduced by hypercapnia in hypoxemic cerebral vascular endothelial cells in vitro and in vivo. Additionally, the expression of tight junctional proteins was markedly increased following treatment with IL­1Ra. These results suggest that hypercapnia­induced IL­1ß overproduction in the hypoxemic blood may decrease tight junctional protein expression in cerebrovascular endothelial cells via the IL­1R1/p­IRAK­1 pathway, further disrupting BBB integrity, and eventually resulting in increased BBB permeability.

JAK/STAT signaling pathway-mediated microRNA-181b promoted blood-brain barrier impairment by targeting sphingosine-1-phosphate receptor 1 in septic rats.

BACKGROUND: Blood-brain barrier (BBB) impairment plays a significant role in the pathogenesis of sepsis-associated encephalopathy (SAE). However, the molecular mechanisms are poorly understood. In the present study, we aimed to investigate the regulatory relationship between the Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathway, microRNA (miR)-181b and its target genes in sepsis in vivo and in vitro. METHODS: Four rat models (sham, sepsis, sepsis plus STAT3 inhibitor (Stattic), and sepsis plus miR-181b inhibitor [sepsis + anta-miR-181b]) were established. For the in vitro experiments, rat brain microvascular endothelial cells (rBMECs) and rat brain astrocytes (rAstrocytes) were cultured with 10% serum harvested from sham, sepsis, and sepsis + anta-miR-181b rats. Chromatin immunoprecipitation-quantitative polymerase chain reaction (ChIP-QPCR) analysis was carried out to detect the binding and enrichment of the JAK/STAT3 signal core transcription complex in the miR-181b promoter region. Dual-luciferase reporter gene assay was conducted to test miR-181b and its target genes. The cell adhesion rate of rBMECs was also measured. RESULTS: During our investigations, the expression levels of miR-181b, p-JAK2, p-STAT3, and C/EBPß were found to be significantly increased in the septic rats compared with the sham rats. STAT3 inhibitor halted BBB damage by downregulating the expression of miR-181b. In addition, miR-181b targeted sphingosine-1-phosphate receptor 1 (S1PR1) and neurocalcin delta (NCALD). The up-regulated miR-181b significantly decreased the cell adhesion rate of rBMECs. The administration of miR-181b inhibitor reduced damage to the BBB through increasing the expression of S1PR1 and NCALD, which again proved that miR-181b negatively regulates SIPR1 and NCALD to induce BBB damage. CONCLUSIONS: Our study demonstrated that JAK2/STAT3 signaling pathway induced expression of miR-181b, which promoted BBB impairment in rats with sepsis by downregulating S1PR1 and decreasing BBB cell adhesion. These findings strongly suggest JAK2/STAT3/miR-181b axis as therapeutic target in protecting against sepsis-induced BBB damage.