Lipopolysaccharide-induced endotoxaemia during adolescence promotes stress vulnerability in adult mice via deregulation of nuclear factor erythroid 2-related factor 2 in the medial prefrontal cortex.

RATIONALE: Sepsis is a severe inflammatory response to infection that leads to long-lasting cognitive impairment and depression after resolution. The lipopolysaccharide (LPS)-induced endotoxaemia model is a well-established model of gram-negative bacterial infection and recapitulates the clinical characteristics of sepsis. However, whether LPS-induced endotoxaemia during adolescence can modulate depressive and anxiety-like behaviours in adulthood remains unclear. OBJECTIVES: To determine whether LPS-induced endotoxaemia in adolescence can modulate the stress vulnerability to depressive and anxiety-like behaviours in adulthood and explore the underlying molecular mechanisms. METHODS: Quantitative real-time PCR was used to measure inflammatory cytokine expression in the brain. A stress vulnerability model was established by exposure to subthreshold social defeat stress (SSDS), and depressive- and anxiety-like behaviours were evaluated by the social interaction test (SIT), sucrose preference test (SPT), tail suspension test (TST), force swimming test (FST), elevated plus-maze (EPM) test, and open field test (OFT). Western blotting was used to measure Nrf2 and BDNF expression levels in the brain. RESULTS: Our results showed that inflammation occurred in the brain 24 h after the induction of LPS-induced endotoxaemia at P21 but resolved in adulthood. Furthermore, LPS-induced endotoxaemia during adolescence promoted the inflammatory response and the stress vulnerability after SSDS during adulthood. Notably, the expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2) and BDNF in the mPFC were decreased after SSDS exposure in mice treated with LPS during adolescence. Activation of the Nrf2-BDNF signalling pathway by sulforaphane (SFN), an Nrf2 activator, ameliorated the effect of LPS-induced endotoxaemia during adolescence on stress vulnerability after SSDS during adulthood. CONCLUSIONS: Our study identified adolescence as a critical period during which LPS-induced endotoxaemia can promote stress vulnerability during adulthood and showed that this effect is mediated by impairment of Nrf2-BDNF signalling in the mPFC.

Persistent viral shedding of human adenovirus type 7 in children with severe pneumonia.

To understand host-pathogen interactions and develop effective prevention and control strategies for human adenovirus (HAdV), it is essential to explore the characteristics of HAdV shedding. Hospitalized children <14 years who had severe HAdV pneumonia were tested for HAdV DNA by quantitative real-time PCR in nasopharyngeal aspirate (NPA). A total of 132 children were enrolled, including 102 patients with HAdV type 7 (HAdV-7) infection and 12 patients with HAdV type 3 (HAdV-3) infection. A total of 1372 qualified NPA samples were collected. There was a significant negative correlation between the viral load of HAdV and the course of the disease (Spearman r = -0.547, p = .000). HAdV-7 load decreased at a rate of 0.089 log10 copies/mL per day (95% CI: -0.096 to -0.081; R 2 = 0.332), and the duration of viral shedding was predicted to be 96.9 days (y = 8.624-0.089x). However, HAdV-3 load decreased more quickly (95% CI: - 0.229 to - 0.143; R 2 = 0.403), and the duration of viral shedding was 51.4 days (y = 9.558-0.186x). The median viral load of the HAdV-7 group at weeks 2 and 3, and more than 3 weeks postinfection was higher than that of the HAdV-3 group. No significant differences in the duration of viral shedding were found in different gender, age (>2 vs. ≤2 years), and with or without underlying diseases groups. Viral shedding in children with severe HAdV pneumonia persisted, among which HAdV-7 lasted longer than 3 months and the viral load decreased slowly than HAdV-3.

Effects of interleukin-6 and IL-6/AMPK signaling pathway on mitochondrial biogenesis and astrocytes viability under experimental septic condition.

OBJECTIVE: Interleukin-6 (IL-6) is a neuromodulation factor with extensive and complex biological activities. IL-6 has been reported to activate AMPK, while AMPK regulates mitochondrial biogenesis and autophagy. The aim of this study was to investigate the role of IL-6 in mitochondrial biogenesis using astrocytes under experimental septic condition and examined how IL-6/AMPK signaling pathway affected this process. METHODS: The primary cultures of cerebral cortical astrocytes were randomly allocated into six groups: control group, LPS+IFN-γ group, IL-6 group (LPS+IFN-γ+IL-6), C group (LPS+IFN-γ+IL-6+Compound C), siRNA group (LPS+IFN-γ+IL-6+IL-6R siRNA) and siRNA+C group (LPS+IFN-γ+IL-6+IL-6R siRNA+ Compound C). All groups were stimulated for 6 h. Cytokines and reactive oxygen species (ROS) analyses, detection of adenosine triphosphate (ATP), mtDNA content and cell viability, evaluation of the mitochondrial ultrastructure and volume density, western blots of proteins associated with mitochondrial biogenesis and phospho-adenosine monophosphate activated protein kinase (p-AMPK) were performed respectively. RESULTS: Compared with LPS+IFN-γ group, IL-6 group had milder ultrastructural damage of mitochondria, higher mtDNA content and mitochondrial volume density, higher expression of proteins associated with mitochondrial biogenesis (PGC-1α, NRF-1 and TFAM) and p-AMPK, and thus higher cell viability, whereas blocking IL-6/AMPK signaling pathway, the protective effect of IL-6 has been diminished, compared with IL-6 group. CONCLUSION: IL-6 enhances mitochondrial biogenesis in astrocytes under experimental septic condition through IL-6/AMPK signaling pathway.

Insulin alleviates mitochondrial oxidative stress involving upregulation of superoxide dismutase 2 and uncoupling protein 2 in septic acute kidney injury.

The aim of the present study was to explore the effects and mechanisms of insulin on mitochondrial oxidative stress in septic acute kidney injury (AKI). Male Sprague Dawley rats were divided randomly into four groups: Control group, sham surgery group, cecal ligation and puncture (CLP) group, and CLP plus insulin group. Blood specimens and kidney tissues were obtained at 12 and 24 h after surgery as separate experiments. Analyses of histology and indicators of renal injury [blood urea nitrogen (BUN) and serum creatinine (CRE) and neutrophil gelatinase-associated lipocalin (NGAL)], mitochondrial function [adenosine triphosphate (ATP) and mitochondrial membrane potential (MMP)], oxidative stress [inducible nitric oxide synthase (iNOS), reactive oxygen species (ROS) and nitric oxide (NO)], endogenous antioxidant systems [superoxide dismutase (SOD) and glutathione (GSH)] as well as the expression of uncoupling protein (UCP), PINK1 protein (a major mediator of mitophagy), PGC1α protein (a major regulator of mitochondrial biogenesis) were performed. Compared with CLP group, the CLP plus insulin group had milder histological damage, higher levels of ATP and MMP as well as lower levels of BUN, serum CRE and NGAL, intrarenal iNOS, mitochondrial ROS and total NO. Moreover, the CLP plus insulin group demonstrated increased expression of SOD2 and UCP2. In contrast, insulin administration suppressed mitophagy meanwhile did not upregulate total GSH and induce mitochondrial biogenesis following CLP. These findings indicated that the upregulation of SOD2 and UCP2 may be involved in insulin protecting against mitochondrial oxidative stress in septic AKI.

Glucose-Insulin-Potassium Alleviates Intestinal Mucosal Barrier Injuries Involving Decreased Expression of Uncoupling Protein 2 and NLR Family-Pyrin Domain-Containing 3 Inflammasome in Polymicrobial Sepsis.

Uncoupling protein 2 (UCP2) may be critical for intestinal barrier function which may play a key role in the development of sepsis, and insulin has been reported to have anti-inflammatory effects. Male Sprague-Dawley rats were randomly allocated into five groups: control group, cecal ligation and puncture (CLP) group, sham surgery group, CLP plus glucose-insulin-potassium (GIK) group, and CLP plus glucose and potassium (GK) group. Ileum tissues were collected at 24 h after surgery. Histological and cytokine analyses, intestinal permeability tests, and western blots of intestinal epithelial tight junction component proteins and UCP2 were performed. Compared with CLP group, the CLP + GIK group had milder histological damage, lower levels of cytokines in the serum and ileum tissue samples, and lower UCP2 expression, whereas the CLP + GK group had no such effects. Moreover, the CLP + GIK group exhibited decreased epithelial permeability of the ileum and increased expression of zonula occludens-1, occludin, and claudin-1 in the ileum. The findings demonstrated that the UCP2 and NLR family-pyrin domain-containing 3/caspase 1/interleukin 1ß signaling pathway may be involved in intestinal barrier injury and that GIK treatment decreased intestinal barrier permeability. Thus, GIK may be a useful treatment for intestinal barrier injury during sepsis.

Implementation of a Pediatric Emergency Triage System in Xiamen, China.

BACKGROUND: Pediatric emergency rooms (PERs) in Chinese hospitals are perpetually full of sick and injured children because of the lack of sufficiently developed community hospitals and low access to family physicians. The aim of this study was to evaluate the clinical value of a new five-level Chinese pediatric emergency triage system (CPETS), modeled after the Canadian Triage System and Acuity Scale. METHODS: In this study, we compared CPETS outcomes in our PER relative to those of the prior two-level system. Patients who visited our PER before (January 2013-June 2013) and after (January 2014-June 2014) the CPETS was implemented served as the control and experimental group, respectively. Patient flow, triage rates, triage accuracy, wait times (overall and for severe patients), and patient/family satisfaction were compared between the two groups. RESULTS: Relative to the performance of the former system experienced by the control group, the CPETS experienced by the experimental group was associated with a reduced patient flow through the PER (Cox-Stuart test, t = 0, P < 0.05), a higher triage rate (93.40% vs. 90.75%; χ2 = 801.546, P < 0.001), better triage accuracy (96.32% vs. 85.09%; χ2 = 710.904, P < 0.001), shorter overall wait times (37.30 ± 13.80 min vs. 41.60 ± 15.40 min; t = 11.27, P < 0.001), markedly shorter wait times for severe patients (2.07 [0.65, 4.11] min vs. 3.23 [1.90,4.36] min; z = -2.057, P = 0.040), and higher family satisfaction rates (94.23% vs. 92.21%; χ2 = 321.528, P < 0.001). CONCLUSIONS: Implementing the CPETS improved nurses' abilities to triage severe patients and, thus, to deliver the urgent treatments more quickly. The system shunted nonurgent patients to outpatient care effectively, resulting in improved efficiency of PER health-care delivery.

[Correlation between uncoupling protein 2 expression and myocardial mitochondrial injury in rats with sepsis induced by lipopolysaccharide].

OBJECTIVE: To investigate the correlation between uncoupling protein 2 (UCP2) expression and myocardial mitochondria injury in rats with sepsis induced by lipopolysaccharide (LPS). METHODS: The rat model of sepsis was established through an intraperitoneal injection of LPS. Forty male Sprague-Dawley rats were randomly and equally divided into control group (an intraperitoneal injection of normal saline), sepsis 6 h group (LPS-6 h group), sepsis 12 h group (LPS-12 h group), sepsis 24 h group (LPS-24 h group), and sepsis 48 h group (LPS-48 h group). The serum and heart tissues were harvested at corresponding time points and myocardial mitochondria was extracted. The microplate reader was applied to measure creatine kinase (CK), creatine kinase-MB (CK-MB), and reactive oxygen species (ROS). Flow cytometry was applied to measure the degree of mitochondrial swelling and mitochondrial membrane potential (MMP). Western blot was used to measure the expression level of UCP2. Electron microscopy was applied to observe the morphological changes in heart tissues and myocardial mitochondria. RESULTS: Compared with the control group, the LPS groups had significantly increased serum levels of CK, CK-MB, and myocardial ROS, as well as a significantly increased degree of mitochondrial swelling (P<0.05), and these values reached their peaks at 24 hours after LPS injection. The LPS groups had a significant decrease in MMP (P<0.05), which reached the lowest level at 24 hours after LPS injection. Western blot showed that the LPS groups had a significant increase in the expression level of myocardial UCP2 compared with the control group (P<0.05), which reached its peak at 24 hours after LPS injection. The results of electron microscopy showed mitochondrial swelling, partial rupture of the mitochondrial membrane, and cavity formation in rats in the LPS groups. The most severe lesions occurred in the LPS-24 h group. In rats with LPS, the ROS level in the myocardial mitochondria and the degree of mitochondrial swelling were positively correlated with the expression level of UCP2 (r=0.796 and 0.893, respectively; P<0.05), while MMP was negatively correlated with the expression level of UCP2 (r=-0.903, P<0.05). CONCLUSIONS: In the rat model of sepsis, the myocardium and myocardial mitochondria have obvious injuries, and the expression level of UCP2 is closely correlated with mitochondrial injury. Therefore, UCP2 might play an important role in myocardial mitochondrial injury in sepsis.

New learning and memory related pathways among the hippocampus, the amygdala and the ventromedial region of the striatum in rats.

BACKGROUND: The hippocampus, central amygdaloid nucleus and the ventromedial region (marginal division) of the striatum have been reported to be involved in the mechanism of learning and memory. This study aimed elucidating anatomical and functional connections among these brain areas during learning and memory. RESULTS: In the first part of this study, the c-Fos protein was used to explore functional connections among these structures. Chemical stimulation of either hippocampus or central amygdaloid nucleus results in dense expression of c-Fos protein in nuclei of neurons in the marginal division of the striatum, indicating that the hippocampus and the central amygdaloid nucleus might be functionally connected with the marginal division. In the second part of the study, the cholera toxin subunit B-horseradish peroxidase was injected into the central amygdaloid nucleus to observe anatomical connections among them. The retrogradely transported conjugated horseradish peroxidase was observed in neurons of both the marginal division and dorsal part of the hippocampus following the injection. Hence, neural fibers from both the marginal division and the hippocampus directly projected to the central amygdaloid nucleus. CONCLUSION: The results implicated potential new functional and structural pathways through these brain areas during the process of learning and memory. The pathways ran from ventromedial portion (the marginal division) of the striatum to the central amygdaloid nucleus and then to the hippocampus before going back to the marginal division of the striatum. Two smaller circuits were between the marginal division and the central amygdaloid nucleus, and between the central amygdaloid nucleus and the hippocampus. These connections have added new dimensions of neural networks of learning and memory, and might be involved in the pathogenesis of dementia and Alzheimer disease.

Atractylenolide I protects mice from lipopolysaccharide-induced acute lung injury.

Atractylenolide I (AO-I), one of the major bioactive components isolated from Rhizoma Atractylodes macrocephala, has been reported to have anti-inflammatory effects. In the present study, we investigated the protective effects of AO-I on acute lung injury (ALI) using LPS-induced ALI mouse model. Lung injury was assessed by histological study. Inflammatory cytokines TNF-α, IL-6 and IL-1ß production were detected by ELISA. TLR4 expression and NF-κB activation were measured by western blot analysis. The results showed that treatment of AO-I significantly attenuated LPS-induced lung wet-to-dry weight ratio and MPO activity. Meanwhile, treatment of AO-I significantly inhibited the production of TNF-α, IL-6, IL-1ß, IL-13, and MIF production in bronchoalveolar lavage fluid (BALF), as well as neutrophils and macrophages in BALF. AO-1 could up-regulate the production of IL-10 in BALF. Besides, LPS-induced TLR4 expression and NF-κB activation were suppressed by treatment of AO-I. In conclusion, the current study suggested that AO-I protected mice acute lung injury induced by LPS via inhibition of TLR4 expression and NF-κB activation.

[A preliminary study of long-term mitochondrial dysfunction in rat brain caused by lipopolysaccharide-induced sepsis].

OBJECTIVE: To preliminarily investigate the long-term structural and functional injuries of mitochondria in rat brain caused by sepsis. METHODS: Wistar rats were randomly assigned into sepsis and control groups. A rat model of sepsis was prepared by an intraperitoneal injection of 10 mg/kg lipopolysaccharide (LPS) of gram-negative bacteria, and the survival assay was performed. Eight rats in the sepsis group were sacrificed at 12, 24, 48, or 72 hours after LPS injection, while rats in the control group were sacrificed after an intraperitoneal injection of an equal volume of normal saline. Mitochondria were extracted from rat brain tissue. Mitochondrial membrane potential (MMP) and mitochondrial swelling level were determined by flow cytometry, and the activities of electron transport chain complexes (I-V) were measured using enzyme assay kits. Hematoxylin-eosin (HE) staining and electron microscopy were used to observe morphological changes in brain tissue and mitochondria. RESULTS: The sepsis group had a significantly lower survival rate than the control group (P<0.01). The MMP and activities of electron transport chain complexes (I-V) in the sepsis group, which were significantly lower than those in the control group (P<0.05), were reduced to the lowest levels at 48 hours and partially recovered at 72 hours. The mitochondrial swelling level in the sepsis group, which was significantly higher than that in the control group (P<0.05), increased to the peak level at 48 hours and partially recovered at 72 hours. Hematoxylin and Eosin staining revealed substantial damages in the structure of brain tissue, and electron microscopy showed mitochondrial swelling, and vacuolization in a few mitochondria. CONCLUSIONS: In the rat model of LPS-induced sepsis, both structural and functional injuries are found in cerebral mitochondria, and achieve the peak levels probably at around 48 hours.

The marginal division of the striatum and hippocampus has different role and mechanism in learning and memory.

The memory function of the hippocampal formation (Hip) and the marginal division (MrD) of neostriatum was compared. Rats with bilateral lesions of the MrD either immediate or 24 h after training in Y-maze were found to have decrease in correct runs in both groups. However, animals with transected afferent and efferent nerve bundles to isolate the Hip immediately or 24 h after training in Y-maze were found to show a decrease in correct runs only in the group injured immediately after Y-maze training but not in the 24 h group suggesting that MrD is likely involved in the entire process of long-term memory consolidation whereas the Hip only contributes to memory in the early stage. In addition, animals treated with a NMDA receptor (NMDAR) blocker, e.g. MK-801, showed decreased correct runs in Y-maze test and in expression level of phosphorylated CREB (pCREB) in neurons of the MrD but not in the Hip. Furthermore, animals treated with okadaic acid (OA), a potent protein phosphatase 1 inhibitor, showed increased correct runs in the Y-maze test. The expression level of pCREB and c-Fos and c-Jun was found increased in neurons of the MrD and the Hip in response to OA treatment. In conclusion, NMDAR and pCREB are involved in memory functions of both the Hip and the MrD. NMDAR might regulate pCREB level in neurons of the MrD but not in the Hip. Hence, the processes and mechanism of learning and memory involved in the MrD and the Hip may be different.

[Effect of UCP2-siRNA on inflammatory response of cardiomyocytes induced by septic serum].

OBJECTIVE: To study the effect of uncoupling protein 2 (UCP2)-siRNA on the inflammatory response of rat cardiomyocytes (H9C2) induced by septic serum and to investigate the possible role of UCP2 in the development of septic cardiomyopathy. METHODS: Serum samples were separately collected from normal rats and septic rats. Cultured rat cardiac cells (H9C2) were randomly divided into blank control, normal serum, 10% septic serum, UCP2-siRNA+10% septic serum and negative siRNA+10% septic serum groups. Stimulation with 10% septic serum was performed for 12 hours in relevant groups. The mRNA expression of tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1ß) was measured by RT-PCR. The expression of phosphorylated p38 mitogen-activated protein kinase (p-p38 MAPK) and nuclear factor-kappa B (NF-κB) was measured by Western blot. RESULTS: The expression levels of p-p38 and NF-κB in the UCP2-siRNA+10% septic serum group were significantly higher than in the 10% septic serum group (P<0.05). The UCP2-siRNA+10% septic serum group had a significantly higher TNF-α mRNA expression than the 10% septic serum group (P<0.01), but IL-1ß mRNA expression showed no significant difference between the two groups. CONCLUSIONS: UCP2 plays a regulatory role in the activation of p38 MAPK and NF-κB and the expression of downstream inflammatory mediators in H9C2 cells stimulated with septic serum.

Comparison of microRNA expression in hippocampus and the marginal division (MrD) of the neostriatum in rats.

BACKGROUND: MicroRNAs (miRNAs), a class of highly conserved small non-coding RNA molecules, are known to play essential roles in central nervous system (CNS) by causing post-transcriptional gene silencing. There is much evidence that miRNAs have specific temporal and spatial expression patterns in the mammal brain, but little is known about the role of the region specificity for the gene regulatory networks of the brain. This study represents the first attempt to perform a profiling analysis of the differential expression of miRNAs between hippocampus and the Marginal division (MrD) of the neostriatum in the rat brain. RESULTS: Microarray was used to detect the expression of 357 miRNAs in hippocampus and the MrD from three rats. A short-list of the most dysregulated 30 miRNAs per rat was generated for data analysis, and the miRNAs that were represented in two or three short-lists were then further analyzed. Quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) was employed to validate the aberrantly expressed miRNAs obtained from the miRNA microarray analysis. A family of 11 miRNAs demonstrated differential expression between the MrD and hippocampus in more than one rat. Amongst these, miR-383 was differentially expressed in all three rats and up-regulated to the largest degree in rat one, and the ten other miRNAs, let-7d*, miR-181b, miR-187, miR-195, miR-214, miR-382, miR-411, miR-466b, miR-592 and miR-1224 were differentially expressed in at least two rats. Of these ten, besides miR-382 and miR-411 which were up-regulated in one rat and down-regulated in another, the other eight miRNAs retained a uniform direction of regulation (up-regulation or down-regulation) between different specimens. When further examined by RT-PCR, the aberrantly expressed miRNAs, except miR-383 and let-7d*, demonstrated differential expression that significantly correlated with the microarray findings. CONCLUSION: This study reported that the miRNA expression patterns in MrD was distinct from that of Hip, suggesting the role of miRNAs in the learning and memory function of the MrD probably different from hippocampus.

[Gene cloning and immunogenicity analysis of the structural proteins VP1-VP4 of enterovirus 71].

OBJECTIVE: To clone the genes encoding the structural proteins VP1-VP4 of enterovirus 71 and investigate the immunogenicity of the expressed recombinant proteins. METHODS: The VP1-VP4 cDNAs were amplified by RT-PCR from the extracted viral RNA and cloned into pMD19-T vector. The cloned VP1-VP4 genes were then inserted into the multi-cloning sites of plasmid pQE30a and expressed in E. coli M15 with IPTG induction. After washing with 8 mol/L urea and purification with Ni-affinity chromatography, the recombinant proteins obtained were tested for immunogenicity by Western blotting and ELISA using rabbit antisera against enterovirus 71 and Coxsackie Virus A16. RESULTS: The recombinant VP1-VP4 proteins were highly expressed in E. coli M15 and the purified proteins could be specifically recognized by the rabbit sera against enterovirus 71. CONCLUSION: The expressed enterovirus 71 structural proteins show good immunogenicity and can be used for developing enterovirus 71 vaccine and detection kits.

[Adrenal mitochondrial injury in early stage of sepsis in rats].

OBJECTIVE: To study the pathogenesis of adrenal mitochondrial dysfunction in septic rats. METHODS: Thirty SPF rats were randomized into 3 groups, including a normal control group and 2 sepsis groups receiving intraperitoneal injection of 10 mg/kg lipopolysaccharide (LPS) and observed at 6 and 24 h after the injection. The adrenal mitochondria were extracted from the rats at the corresponding time points for observation by electronic microscopy. The membrane potential of the mitochondria was detected by flow cytometry. The oxidative stress levels in the mitochondria (activities of NOS and levels of MDA and NO) were assessed. RESULTS: With the progression of sepsis, the serum levels of corticosterone in LPS groups increased significantly as compared with that in the control group. Ultrastructural observation of the adrenal mitochondria showed mild mitochondrial injury in LPS groups in comparison with the control group. The mitochondrial membrane potential was lowered in the LPS groups, but all these changes appeared to be reversible. The activities of NOS and the levels of MDA and NO showed no significant difference between the sepsis groups and the control group. CONCLUSIONS: No obvious adrenal dysfunction occurs in the early stage of sepsis in rats. Mitochondrial injury, which is reversible, occurs in early sepsis without obvious evidence of oxidative stress injury in the adrenal mitochondria, suggesting a strong resistance and capacity of self-repair of the adrenal gland and the mitochondria against sepsis-induced injury.

[Protective effects of continue insulin infusion on liver mitochondrion in the early stage of septic rats].

AIM: To investigate the protective effects of continue insulin infusion on liver mitochondrion and its mechanism in the early stage of lipopolysaccharide-induced septic rats. METHODS: 24 SD rats were randomly divided into 3 groups (An external jugular vein catheterization was performed in every rat a day before intraperitoneal injection): Saline control group (n = 8), LPS group (n = 8) and insulin therapy group (n = 8). Saline control group animals received 9 g/L saline only. LPS group animals were treated with lipopolysaccharide(LPS, 10 mg/kg, i.p.), and then received an infusion of 9 g/L saline at the rate of 1 mL/h. Insulin therapy group animals received an infusion of insulin at the rate of 0.25 U/(kg x h) after being intraperitoneal injected with 10 mg/kg LPS. Blood glucose level was monitored. Blood samples were taken 2 h and 6 h later and the levels of serum TNF-alpha and IL-6 were detected by enzyme-linked immunoadsorbent assay(ELISA). The membrane potential of isolated liver mitochondrion was tested by flow cytometry. The SOD and MDA levels of isolated liver mitochondrion were tested by kit. The morphologic change of mitochondrion in liver was observed by electronic microscopy. RESULTS: In LPS group, the levels of blood glucose, TNF-alpha, IL-6 and SOD all increased significantly and liver mitochondrial membrane potential decreased significantly compared with control group(P<0.05). In insulin therapy group, the levels of blood glucose, TNF-alpha, IL-6 and SOD all decreased significantly and liver mitochondrial membrane potential increased significantly compared with LPS group(P<0.05). The MDA levels did not differ significantly in the three groups. The mitochondrial ultrastructural changes in every group were not obviously. CONCLUSION: In the early stage of septic rats, reversible liver mitochondrion injury can be observed. Continue insulin infusion can protect liver mitochondrion through attenuating inflammatory reaction and reducing the blood glucose level in septic rats.

The epidemiology of acute respiratory distress syndrome in pediatric intensive care units in China.

OBJECTIVE: To assess the incidence of, predisposing factors for, and the rates and relative risks of mortality from acute respiratory distress syndrome (ARDS) in pediatric patients. DESIGN: A prospective study in 12 consecutive months from 2004 to 2005 in 25 pediatric intensive care units (PICUs). PATIENTS AND SETTING: ARDS was diagnosed according to the 1994 American-European Consensus Conference definitions, applied to all severely ill admissions between 1 month and 14 years of age. The PICUs were in major municipalities and provincial cities, and half were university affiliated. MEASUREMENTS AND RESULTS: From a total of 12,018 admissions, 7,269 were severely ill. One hundred and five (1.44%) patients developed ARDS and 64 (61.0%) died, which accounts for 13.2%, of the total ICU death (n = 485, 6.7%) or a nine times relative risk of dying. The median age at onset of ARDS was 24 months and 40% were less than 12 month old. Median time from PICU admission to the onset of ARDS was 16 h, and in 63% <24 h. Pneumonia (55.2%) and sepsis (22.9%) were the major predisposing factors for ARDS. These were respectively 14 and 5 times as high a death rate as those of the severely ill patients without ARDS. CONCLUSIONS: ARDS has a high mortality in these Chinese PICUs, especially in those with pneumonia and sepsis, and adequate management including lung protective ventilation strategy is required.

[Application of fluorescent real-time polymerase chain reaction in analyzing the epidemic of influenza among children in Guangzhou area in 2006].

OBJECTIVE: To investigate the prevalence of influenza virus infections in children in 2006 using the real-time PCR method. METHODS: (1) Consulting the most conserved sequence NP gene of influenza virus, after comparing with the NP gene sequences of influenza virus in GenBank, one pair of specific primers and one TaqMan probe were designed for each subtype of influenza virus by the software Primer Express. The sensitivity of influenza was evaluated by testing known positive samples which had been two-fold diluted. The specificity of real-time PCR for influenza virus detection was assessed by cross testing 60 isolates of influenza A, 16 isolates of influenza B, and by testing a variety of other respiratory viruses positive samples; (2) 281 nasopharyngeal aspirate samples were detected by real-time PCR and virus isolation; (3) the 12 301 specimens from the patients of Guangzhou Children's Hospital were tested by using the real-time PCR method. Furthermore, the real-time PCR reagent was evaluated by comparing with the result of virus isolation. RESULTS: (1) The sensitivity of real-time PCR developed in this study for influenza A detection was 1:2(22) and for influenza B was 1:2(20) in two-fold serially diluted way. (2) No positive results were found in cross testing of other viruses positive specimens. (3) Influenza virus was detected from 1687 cases (13.71%) out of the 12 301 cases, including 773 cases (45.8%) positive for subtype A and 914 cases (54.2%) positive for subtype B; 455 out of 525 (86.7%) of influenza B positive specimens and 70 out of 525 (13.3%) of influenza A (H1N1) positive specimens were from patients seen during January to April; 419 out of 1118 (37.5%) specimens positive for influenza B and 699 out of 1118 (62.5%) specimens positive for influenza A (H1N1) were from patients seen from May to August. Influenza virus could be identified from 1380 samples by the methods of virus isolation, accounting for 81.80% of the 1687 positive samples detected by real-time PCR. All the influenza virus subtype A was H1N1. CONCLUSION: The real-time PCR method developed in this study was sensitive and specific for detecting influenza A and B in clinical specimens. During 2006, influenza A and influenza B co-circulated. The predominant virus was influenza B from January to April, peaking in April. Influenza A (H1N1) prevailed from May to August, with the peak in June.

Retrospective study of adenovirus in autopsied pulmonary tissue of pediatric fatal pneumonia in South China.

BACKGROUND: Adenovirus are the important pathogen of pediatric severe pneumonia. The aim of this study is to analyze the infection, subtype and distribution of adenovirus in autopsied pulmonary tissue of fatal pneumonia in infants and children, and the relationships between adenovirus infection and respiratory illness in South China. METHODS: Nested PCR was performed on DNA extracted from autopsied lung tissue from patients who died of severe pneumonia, and the positive nested PCR products were cloned and sequenced. The adenovirus in autopsied pulmonary tissue was also analyzed by immunohistochemistry assay in a blind way. RESULTS: In the 175 autopsied pulmonary tissues, the positive percentage of adenovirus was 9.14% (16/175) and 2.29% (4/175) detected with nested PCR and immunohistochemistry, respectively. There are three cases of adenovirus serotype 3, twelve cases of adenovirus serotype 4 and one case of serotype 41 determined by sequencing of the cloned positive nested PCR products. CONCLUSION: Adenovirus is an important cause of severe pneumonia, and these data suggest that adenovirus serotype 4 might be an important pathogen responsible for the fatal pneumonia in Guangzhou, South China.

Retrospective analysis of Mycoplasma pneumoniae infection in pediatric fatal pneumonia in Guangzhou, South China.

The objective of this study is to investigate the infection and distribution of Mycoplasma pneumoniae in autopsied pulmonary tissue of pediatric severe pneumonia. Mycoplasma pneumoniae nested polymerase chain reaction and immunohistochemistry were done on autopsy pulmonary tissue from 173 patients who died of severe pneumonia. Mycoplasma pneumoniae was identified in 135/173 (78.03%) and 114/173 (65.89%) samples of autopsied pulmonary tissue of lethal severe pneumonia via nested polymerase chain reaction and immunohistochemistry, respectively. The coincidence of both assays was 92.4%. Mycoplasma pneumoniae associated fatal pneumonia has showed an increasing trend from 1988 to 2005 in South China, and the fatality rate of Mycoplasma pneumoniae associated fatal pneumonia in infants, 1 to 12 months, has risen to 66.9% (97/145). Mycoplasma pneumoniae is a significant cause of severe pneumonia, it is a universal event in infants, and children have died of severe pneumonia in South China. Mycoplasma pneumoniae might be an important pathogen responsible for fatal pneumonia in Guangzhou area, South China.