[Role of blood markers in predicting the failure of prosthesis removal and antibiotic-loaded bone cement spacer implantation for treatment of periprosthetic joint infection].

Objective: To investigate the value of inflammation,coagulation and nutrition markers in predicting the failure of prosthesis removal and antibiotic-loaded bone cement spacer implantation for treatment of periprosthetic joint infection(PJI). Methods: A retrospective study was conducted on 70 patients who undertook prosthesis removal and antibiotic-loaded bone cement spacer implantation due to PJI from June 2016 to October 2020 in the Department of Orthopedics,Henan Provincial People's Hospital. There were 28 males and 42 females,aged (65.5±11.9) years (range: 37 to 88 years). Patients were divided into two groups as the successful group and the failed group depended on whether reinfection occurred after prosthesis removal and antibiotic-loaded bone cement spacer implantation at the last follow up. Patient demographics,laboratory values (C-reactive protein (CRP),erythrocyte sedimentation rate (ESR),ESR and CRP ratio (ESR/CRP),white blood cell count(WBC),platelet count(PLT),hemoglobin(HB),total lymphocyte count(TLC),albumin、fibrinogen(FIB),CRP and albumin ratio (CAR),prognostic nutritional index(PNI)),and reinfection rates were assessed. Comparison between groups was conducted by the independent sample t test or χ2test. Receiver operating characteristic (ROC) curve was plotted,and the area under the curve (AUC),optimal diagnostic threshold,sensitivity,and specificity were analyzed to predict the failure of prosthesis removal and antibiotic-loaded bone cement spacer implantation. Results: All patients were followed up for at least two years,and the follow-up time was (38.4±15.2) months (range: 24 to 66 months). Fifteen patients suffered failure after prosthesis removal and antibiotic-loaded bone cement spacer implantation,while the other 55 patients succeeded. The overall failure rate of prosthesis removal and antibiotic-loaded bone cement spacer implantation in PJI treatment was 21.4%. Level of preoperative CRP ((35.9±16.2)mg/L),PLT ((280.0±104.0)×109/L) and CAR (1.3±0.8) in successful group were lower than CRP ((71.7±47.3)mg/L),PLT ((364.7±119.3)×109/L) and CAR (2.5±2.0) in failed group (all P<0.05).Whereas,level of preoperative ESR/CRP (3.3±3.1), Albumin ((35.3±5.2)g/L) and PNI (43.6±6.2) in successful group were higher than ESR/CRP (1.6±1.4),Albumin ((31.3±4.8)g/L) and PNI (39.2±15.1) in failed group (all P<0.05). AUC of ROC curve,optimal threshold value,sensitivity and specificity of CRP,ESR/CRP, PLT, Albumin,CAR and PNI for the predicting failure of prosthesis removal and antibiotic-loaded bone cement spacer implantation were 0.776(95%CI:0.660 to 0.867),35.4 mg/L,86.7%,67.3%;0.725(95%CI:0.605 to 0.825),1.0,60.0%,78.2%;0.713(95%CI:0.593 to 0.815),253,93.3%,47.3%;0.721(95%CI:0.601 to 0.822),35.7,93.3%,49.1%;0.772(95%CI:0.656 to 0.863),1.1,86.7%,67.3%;0.706(95%CI:0.585 to 0.809),45.7,100%,41.8% respectively. Conclusion: In patients with PJI,CRP>35.4,ESR/CRP≤1.0 and CAR>1.1 could predict the failure of prosthesis removal and antibiotic-loaded bone cement spacer implantation.

[Mechanism of advanced glycation end products inhibiting the proliferation of peripheral blood mononuclear cells and osteoblasts in rats].

OBJECTIVE: To explore the mechanism of nuclear factor-kappa B (NF-κB), phosphatidylinositol 3-kinase (PI3K)/protein kinase B(PKB/Akt) and mitogen-activated protein kinase (MAPK) signaling pathways after intervention of advanced glycosylation end products (AGEs) in peripheral blood mononuclear cells (PBMCs) and osteoblasts (OB) in rats, so as to provide certain experimental basis and theoretical basis for further research on the clinical treatment of periodontal tissue inflammation caused by diabetes mellitus. METHODS: AGEs were prepared, PBMCs and OB were isolated and cultured in vitro. CCK-8 was used to detect the cell viability intervened by different concentrations and time of AGEs. Western blot and qRT-PCR were used to detect the expression changes of genes related to NF-κB, PI3K/PKB and MAPK signaling pathways. RESULTS: OB and PBMCs were successfully isolated and cultured in vitro. The activity of PBMCs and OB cells was significantly correlated with the concentration, time and interaction of AGEs. With the increase of AGEs concentration and time, the activity of PBMCs and OB cells significantly decreased (P < 0.001). AGEs stimulation significantly increased the expression of NF-κB in PBMCs and the contents of tumor necrosis factor α(TNF-α), interleukin-1ß(IL-1ß) (P < 0.01). TNF-α, IL-1ß levels were significantly reduced after inhibition of NF-κB pathway (P < 0.01). NF-κB p65, JNK, and p38 phosphorylated and non-phosphorylated proteins increased significantly after AGEs stimulation of OB (P < 0.05). The phosphorylated protein expression of IκB was significantly increased, while the expression of non-phosphorylated protein was decreased (P < 0.01).The expressions of NF-κB p65, JNK, and IκB were significantly increased at the mRNA levels, and the expressions of IκB mRNA were significantly decreased (P < 0.05). There was no difference in the expression of Akt in either phosphorylated or non-phosphorylated proteins or at the mRNA level (P>0.05). With the addition of MAPK signaling pathway inhibitors, the phosphorylation and non-phosphorylated protein expressions of NF-κB p65, p38 and JNK were significantly reduced, and the phosphorylated protein of IκB was significantly decreased and the non-phosphorylated protein was significantly increased compared with the group with AGEs alone (P < 0.05). The results of qRT-PCR showed that the expression of IκB increased significantly after the addition of the JNK pathway blocker (P < 0.05), and the expression of NF-κB p65, p38 and JNK decreased, but the difference was not significant (P>0.05). While NF-κB p65, p38 and JNK were significantly decreased and IκB was significantly increased in the AGEs group after the addition of the p38 pathway blocker (P < 0.05). At this time, there was still no significant change in the expression of Akt at the protein level and mRNA level (P>0.05). CONCLUSION: AGEs inhibit the proliferation of PBMCs and OB, and the NF-κB and MAPK pathways are likely involved in regulating this process, but not the PI3K/PKB pathway.

[Comparative study on metagenomics of esophageal flora in elderly and middle-aged esophageal squamous cell carcinoma patients].

Objective: To explore the flora characteristics and differences of esophageal tissues between elderly esophageal squamous cell carcinoma (ESCC) patients and young and middle-aged ESCC patients, so as to assist in studying the potential biomarkers of elderly ESCC patients. Methods: In this study, a retrospective study was adopted. 72 ESCC patients diagnosed in Taihe Hospital, Shiyan City, Hubei Province from July 2018 to July 2019 were selected, including 49 patients in the elderly group (≥ 60 years old, 40 males and 9 females), 23 patients in the young and middle-aged group (<60 years old, 21 males and 2 females). In the same period, 20 healthy persons without abnormal gastroscopy in endoscopy center were selected as the control group (aged 35-78 years old, median age 57 years old, 16 males and 4 females). The genomic DNA was extracted from the affected esophageal tissues of patients with ESCC and the middle esophageal samples of the control group. The V4 hypervariable region of bacterial 16SrRNA gene sequence was amplified. Illumina HiSeq sequencing technology was adopted. The flora characteristics of elderly, young and middle-aged ESCC patients was compared and analyzed. QIIME and Rstudio software were used to analyze the sequence data, and nonparametric Kruskal-Wallis test or Wilcoxon rank sum test were used for statistical methods. Results: Shannon index [5.17 (4.53, 5.95) vs. 4.79 (3.74, 5.97)], Simpson index [0.94 (0.91, 0.96) vs. 0.92 (0.83, 0.96)] and Chao1 index [343.55 (259.76, 570.59) vs. 329.16 (268.88, 648.00)] were similar in flora of two groups, and there was no significant difference (Z=-0.791, -1.057, -0.380, all P>0.05). There was no significant difference in ß-diversity between the elderly group and the young and middle-aged group (PC1=19.14%, PC2=6.95%, PPC1=0.67, PPC2=0.42). At the phyla level, the top 5 phyla in abundance were as follows: Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria and Fusobacteria in the young and middle-aged group, while the top 5 phyla in abundance were as follows: Firmicutes, Bacteroidetes, Proteobacteria, Fusobacteria and Actinobacteria in the elderly group; the significant difference between the two groups was Fusobacteria (Q=0.596, P<0.05). At the genus level, the top 5 genera in the young and middle-aged group in abundance were as follows: Prevotella, Bacteroides, Streptococcus, Selenomonas and Veillonella. In the elderly group, Prevotella, Bacteroides, Streptococcus, Selenomonas and Haemophilus were the top 5 in abundance, and there were significant difference in Fusobacterium between the two groups (Q=0.938, P<0.05). PICRUSt function prediction showed that the abundance of Aminoacyl.tRNA.biosynthesis, Nucleotide.excision.repair, RNA.polymerase, Ribosome, Clavulanic.acid.biosynthesis, Photosynthesis and Photosynthesis. proteins in the elderly group were lower than those in the young and middle-aged group (all Q=0.734, P<0.05). Conclusion: There is no significant difference in α-diversity and ß-diversity between elderly ESCC patients and young and middle-aged patients, but the abundance of Fusobacterium flora increased.

miR-144-3p increases radiosensibility of gastric cancer cells by targeting inhibition of ZEB1.

PURPOSE: This study set out to probe into the effect and mechanism of miR-144-3p on radiosensitivity of gastric cancer (GC) cells. METHODS: Cancer tissue and paracancerous tissue of GC patients admitted to our hospital were collected, their miR-144-3p expression was tested, GC cells were transfected, and survival and biological behavior of those cells under radiation were detected. RESULTS: After detection, miR-144-3p expression was down-regulated in GC tissue, while ZEB1 was up-regulated. There was no remarkable difference in the survival fraction of cells in each group before receiving radiation, but that of tumor cells decreased obviously (p < 0.05) after radiation exposure. Survival fraction of cells overexpressing miR-144-3p or silencing ZEB1 decreased more obviously, while the inhibition of miR-144-3p or overexpressing ZEB1 was weaker. Biological behavior of cells under 6 Gy radiation was detected. It was found that miR-144-3p overexpression or silencing ZEB1 dramatically inhibited the proliferation activity of GC cells under 6 Gy radiation, increased the levels of pro-apoptotic Bax and caspase-3 proteins (p < 0.05) and decreased the anti-apoptotic protein Bcl-2 level (p < 0.05), resulting in an increase in the apoptosis rate of cells. miR-144-3p was confirmed to be ZEB1 targeting site by dual luciferase report. Moreover, rescue experiments prove that it can increase the radiosensitivity of GC cells by regulating ZEB1 expression. CONCLUSION: miR-144-3p expression was down-regulated in GC, and it can increase the radiosensitivity of those cells by inhibiting ZEB1 expression.

[Sampling survey report on Chinese physicians' cognition of the guidelines on diagnosis and treatment of colorectal cancer].

Objective: To investigate and evaluate the mastery and recognition degree of Chinese clinicians on the 2015 edition of the Chinese Criteria for Diagnosis and Treatment of Colorectal Cancer in order to provide useful suggestions for updating and formulating diagnosis and treatment standards. Methods: Simple random sampling was used to conduct a questionnaire survey in 1500 colorectal cancer-related doctors in general hospitals and cancer hospitals from 115 cities in China. The study included the following guidelines: (1) Chinese Criteria for Diagnosis and Treatment of Colorectal Cancer (2015 edition); (2) Chinese Society of Clinical Oncology Colorectal Cancer Guidelines 2017 (CSCO 2017); (3) National Comprehensive Cancer Network (NCCN) Colon Cancer Guidelines 2017.v1; (4) European Society for Medical Oncology (ESMO) Rectal Cancer Guidelines. The survey was carried out in 2017 and 2019 respectively. In the first phase, the questionnaire included 4 dimensions (guideline cognition, detection and diagnosis, pathology and staging, treatment), and 1500 questionnaires were distributed. In the second phase, the questionnaire contained 3 dimensions (basic information, current treatment status of metastatic colorectal cancer, academic expectations), and 350 questionnaires were distributed. Case (%) was used to indicate the categorical variable data, and chi-square test was used for comparison between groups. P<0.05 indicated that the difference was statistically significant. Results: In the first phase, 1472 valid questionnaires were collected, and the questionnaire efficiency was 98.1% (1472/1500). In the second phase, 337 valid questionnaires were collected, and the questionnaire efficiency was 96.3% (337/350). In the survey of the first phase, doctors had some knowledge and compliance with various guidelines, but the most familiar one was the NCCN guidelines, accounting for 90.7% (1335/1472). In the dimension of detection and diagnosis, the overall correct rate was 64.1% (944/1472). The correct rate of doctors in the first-tier cities was 55.6% (148/266), which was lower than 59.1% (182/308) and 72.9% (369/506) in the second- and the third-tier cities, and the difference was statistically significant (χ(2)=42.140, P<0.001). More than 60.0% (883/1472) of doctors was clear about the specification requirements of the staging evaluation and pathological examination. However, in terms of rectal cancer local staging evaluation, the ratio of doctors who would choose rectal MRI in the first-tier cities was lower than that of those in other tier cities [51.5% (137/266) vs. 65.6% (202/308), 63.2% (320/506) and 61.2% (240/392)], and the difference was statistically significant (χ(2)=41.886, P<0.001). In the dimensions of staging evaluation and pathological examination, there were no statistically significant differences in cognition between general and specialist hospitals (P>0.05). In the treatment dimension, 79.8% (1175/1472) of doctors considered the preoperative treatment as a necessary option for patients with middle and low locally advanced (over cT3) rectal cancer. 46.3% (681/1472) of doctors, including 60.3% (433/718) of surgeons, and 31.4% (225/716) of physicians, had a vague idea that irinotecan could not be used for postoperative adjuvant treatment of colorectal cancer. In the survey of the second phase, 93.8% (316/337) of doctors approved potentially curative systemic (conversion) therapy, and 95.3% (321/337) of doctors followed the clinical guidelines in the treatment of metastatic colorectal cancer. Regarding academic expectations, the clinician's concern for surgery was more practical, and 79.2% (267/337) of doctors wanted to know the best options of conversion therapy for potentially curable metastatic colorectal cancer. In contrast, the clinician's concern for internal medicine was more exploratory, and 80.1% (270/337) of doctors focused on selecting targeted drugs and the sequence of treatment. Conclusions: This investigation has a preliminary understanding of the diagnosis and treatment of colorectal cancer in China. (1) There are many guidelines for doctors' reference, but doctors' understanding of domestic guidelines is not as good as NCCN guidelines. (2) The degree of understanding of the guidelines varies significantly among doctors in different cities. (3) The promotion of guidelines should focus on basic concepts and theories. (4) The detection, diagnosis, and treatment of colorectal cancer should be better trained and promoted. (5) The concept of conversion therapy for metastatic colorectal cancer is highly recognized.

[Analysis of pathogenic composition and clinical characteristics of viral acute gastroenteritis in children under five years old in Beijing].

Objective: To understand the epidemiological characteristics and clinical features of rotavirus-, norovirus-, adenovirus-and astrovirus-associ ated acute gastroenteritis in children under 5 years old in Beijing from Octorber, 2015 to March, 2017. Methods: In the intestinal clinic of 6 hospitals in 6 districts of Beijing, information and stool samples of the first 30 patients with acute gastroenteritis who are under the age of 5 years are collected monthly.Rotavirus, norovirus, adenovirus and astrovirus are identified by PCR.Descriptive epidemiological method was used to describe the epidemiological characteristics of diarrhea caused by rotavirus, norovirus, adenovirus and astrovirus in Beijing. One-way analysis of variance was used to analyze the Vesikari clinical severity score of of acute gastroenteritis caused by each virus. Unconditional logistic regression analysis was used to analysis the associated factors of clinical features. Results: Of the 2 052 samples, 709 (34.6%) were non-mixed infections: the positive rate of rotavirus, norovirus, adenovirus and astrovirus were 20.0%, 7.5%, 4.2% and 2.9%, respectively. A total of 135 cases (6.6%) were mixed infection. The mean and standard deviation of Vesikari clinical severity score was 8.0±3.1 for rotavirus associated acute gastroenteritis, which was significantly higher than norovirus (6.4±2.4, P<0.001), adenovirus (6.2±2.1, P<0.001) and astrovirus (6.1±2.0, P<0.001). The comparison of clinical features showed that compared with astrovirus, the children under 5 years old infected with rotavirus were more likely to have a diarrhea ≥5 days (OR=3.334), have vomiting ≥3 times within one day (OR=8.788), have vomiting≥1 day (OR=3.963), have a Vesikari clinical severity score ≥11 severe cases (OR=13.194). Norovirus infected cases were prone to have vomiting≥3 times in 1 day (OR=5.710).Adenovirus infected cases were prone to have a diarrhea≥5 days (OR=2.616). When using rotavirus as a reference, children under 5 years of age were less likely to develop fever≥38.4 ℃ after infection with norovirus (OR=0.397) or adenovirus (OR=0.280). Conclusions: The results of this study showed that the characteristics of acute gastroenteritis caused by different viruses are different. The clinical symptoms caused by rotavirus are more serious. Children under 24 months of age are at high risk of rotavirus infection. Effective preventive measures such as vaccination should be taken as soon as possible.

[Effects on the integrated treatment of colorectal cancer patients during COVID-19 epidemic in China: a cross-sectional study].

Objective: To understand the treatment status and effects on colorectal cancer (CRC) patients in China during the COVID-19 pandemic, and provide evidence for decision-making for health-care workers. Methods: The method of cross-sectional study was applied. A questionnaire survey of CRC patients in China was carried out. Their basic and treatment information during the COVID-19 were collected and associated effects on treatment, nursing, state of psychology and needs of social support were analyzed. Results: Of the 1147 participants in the study, 635 (55.4%) were male and 512 (44.6%) were female with mean age of (52.8±12.8) years. The treatment or follow up of 896 (78.1%) CRC patients were affected during the COVID-19 outbreak. A total of 253 patients (22.1%) had their treatment regiments changed, of whom 141 (12.3%) had their chemotherapy regiments postponed or changed, and 83 patients (7.2%) had their surgical treatment affected. Among the above 83 patients, 39 patients (3.4%) underwent emergency surgery. A total of 277 patients (24.1%) had their out-of-hospital care needs affected, mainly due to maintenance of PICC (49.5%, 137/277). CRC patients had poorer sleep quality and increased levels of insomnia than before (P<0.001), and were more distressed, anxious, depressed, and angry than they were before. They presented the more requirements of help (P<0.001). In addition, 376 patients (32.8%) had received telemedicine services, but only 36.4% (137/376) of them were satisfied. Conclusions: The COVID-19 epidemic has effect on the integrated treatment of CRC patients include diagnosis, treatment, examination and prognosis. While protecting CRC patients from being infected with SARS-CoV-2, health-care workers should also actively help them to receive timely and correct treatment and pay attention to their nursing, nutritional, psychological and social support needs in the COVID-19 epidemic.

[Clinicopathological characteristics of type 2 diabetes mellitus complicated with colorectal cancer].

Objective: To analyze the clinicopathological features of type 2 diabetes mellitus complicated with colorectal cancer (DCRC). Methods: A case-control study was conducted. Inclusion criteria: (1) hospitalized patients receiving fibrocolonoscopy; (2) adenocarcinoma and mucinous adenocarcinoma diagnosed by pathology; (3) with preoperative cTNM clinical staging; (4) colorectal cancer patients undergoing surgical treatment; (5) with postoperative pTNM staging; (6) no smoking or drinking habits. Exclusion criteria: (1) familial adenomatous polyposis (FAP); (2) Lynch syndrome; (3) carcinoma of anal canal and perianal carcinoma; (4) multiple primary cancer; (5) with serious cardiocerebrovascular diseases or multiple organ failure. Clinicopathlogical data of 32 DCRC patients who were diagnosed and treated in Peking University Shougang Hospital from December 2017 to December 2018 were retrospectively collected and analyzed. Forty nondiabetic colorectal cancer (CRC) patients during the same period were selected as control group according to the sex ratio and the age difference less than 5 years. Student's t test and χ(2) test were used to compare the difference between the two groups in baseline clinicopathological data, clinical test results, tumor markers and infiltration status of T cells in tumor immune microenvironment. Results: Among 32 DCRC patients, 24 were males and 8 were females with a mean age of (63.0±1.7) years; among 40 CRC patients, 30 were males and 10 were females with a mean age of (60.5±1.6) years. The duration of diabetes mellitus in DCRC patients (from the diagnosis of diabetes mellitus to the diagnosis of colorectal cancer) was (9.2±1.3) years. The body mass index (BMI) of DCRC group was significantly higher than that of CRC group [(24.8±0.6) kg/m(2) vs. (23.2±0.4) kg/m(2), t=2.372, P=0.020]. There were no significant differences in other baseline data (sex, age, primary site of tumor, R0 resection rate, pathological stage, pathological type, differentiation degree of tumor, preoperative intestinal obstruction) between the two groups (all P>0.05). Serum triglyceride level in DCRC group was higher than that in CRC group [(2.1±0.2) mmol/L vs. (1.5±0.1) mmol/L, t=3.085, P=0.003], while hemoglobin [(120.3±5.2) g/L vs. (132.7±2.8) g/L, t=-2.224, P=0.029], anti- thrombin III [(94.2±3.7)% vs. (103.5±2.4)%, t=-2.197, P=0.031], and red blood cell count [(4.2±0.1)×10(12)/L vs. (4.5±0.1)×10(12)L, t=-2.055, P=0.044] were all lower than those in CRC group. The preoperative carcinoembryonic antigen (CEA) level in DCRC group was higher than that in CRC group [(50.3±21.8) µg/L vs. (5.6±1.0) µg/L, t=2.339, P=0.022]. There were no significant differences in preoperative levels of other four tumor molecular markers (CA199, CA242, CA724 and CA125) between the two groups (all P>0.05). The expression of Foxp3 [specific markers of CD4+, CD25+ regulatory T cells (Treg)] in DCRC group was higher than that in CRC group [(82.7±6.2) cell/HPF vs. (62.6±4.9) cell/HPF, t=2.586, P=0.012]. There were no significant differences in the infiltration of CD4, CD8, PD-1 and PD-L1 positive cells between two groups (all P>0.05). Conclusions: The average diabetic history of DCRC patients is nearly 10 years. They have higher BMI and serum CEA level, and more Treg cell infiltration in the tumor. Close attention should be paid to these patients in clinical practice.

Granulocyte colony-stimulating factor improves neurological function and angiogenesis in intracerebral hemorrhage rats.

OBJECTIVE: Granulocyte colony-stimulating factor (G-CSF) plays a role in regulating phosphatidylinositol-3-kinase/serine/threonine kinase (PI3K/AKT) pathway, affecting cell proliferation and apoptosis, and inducing vascular endothelial growth factor (VEGF) expression. This study investigated the mechanism of G-CSF on angiogenesis and neural protection after intracerebral hemorrhage (ICH). MATERIALS AND METHODS: The rats were divided into four groups, including sham, ICH, ICH+G-CSF, and ICH+G-CSF+LY294002 (PI3K/AKT signaling pathway specific inhibitor). Cerebral neurological dysfunction was tested by Garcia scoring. Cell apoptosis was detected by transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) assay. Angiogenesis marker CD34 expression, PI3K/AKT signaling pathway, B-cell lymphoma-2 (Bcl-2), and VEGF expressions were compared by IHC. Rat cerebral nerve RN-c cells were divided into four groups, including control, oxygen-glucose deprivation (OGD), OGD+G-CSF, and OGD+G-CSF+LY294002. RESULTS: Neurological dysfunction was more evident; CD34+ cell number, VEGF expression, and cell apoptosis significantly increased; phosphorylated AKT (p-AKT) and Bcl-2 levels markedly reduced in ICH group compared with sham group. G-CSF apparently up-regulated p-AKT and Bcl-2 expressions, attenuated cell apoptosis, and elevated CD34+ cell number. LY294002 significantly decreased p-AKT, Bcl-2, and VEGF expressions, and alleviated the cell apoptosis protective and angiogenesis effect induced by G-CSF. OGD treatment induced RN-c cell apoptosis, down-regulated p-AKT and Bcl-2 expressions, and enhanced the tube capacity of vascular endothelial cells (VEC). G-CSF markedly elevated p-AKT and Bcl-2 contents in RN-c cells, declined cell apoptosis, increased p-AKT and VEGF levels in VEC, and enhanced tube capacity. CONCLUSIONS: G-CSF enhanced PI3K/AKT signaling pathway activity, promoted Bcl-2 and VEGF expression, reduced nerve cell apoptosis, and enhanced tube capacity of VECs, which may be the mechanism of G-CSF in improving neurological function and angiogenesis after ICH.

Research on biogenesis of mitochondria in astrocytes in sepsis-associated encephalopathy models.

OBJECTIVE: To study the structural and functional changes in mitochondria in astrocytes of the cerebral cortex of the rats in the simulated sepsis environment in vitro and the relationship between these changes and the biogenesis of mitochondria in astrocytes by establishing models of sepsis astrocytes. MATERIALS AND METHODS: The structural and functional changes in mitochondria in astrocytes of the cerebral cortex of the rats were evaluated. The ultra structural changes in the mitochondria, astrocytes, and ultrathin sections, were observed with a transmission electron microscope. The expression of the regulatory factors related to biogenesis of mitochondria in astrocytes of the cerebral cortex of the rats was evaluated in various experimental groups. RT-PCR and Western blot were used to evaluate the expression of the regulatory factors related to biogenesis of mitochondria in astrocytes of the cerebral cortex of the rats. The "point grid method" was used to evaluate the volume density of the mitochondria in the astrocytes of the cerebral cortex of the rats in various experimental groups. The Western blotting was used to evaluate the role of fusion and fission of mitochondria in the astrocytes of the cerebral cortex of the rats in various experimental groups in regulating the expression of the protein-OPAI and DRPI. RESULTS: In the sepsis astrocyte models established by co-incubation of LPS and IFN-γ and astrocytes of the cerebral cortex of the rats, the mitochondria with a minor injury in the 6 h group (2.97± 0.92) increased significantly when compared with those in the 0 h group (1.08±0.95), 12 h group (1.70±1.01), and 24 h group (1.59±0.55) (p<0.05); the concentration of adenosine triphosphate (ATP) in the astrocytes of the cerebral cortex of the rats in the 6 h, 12 h, and 24 h groups increased significantly when compared with that in the 0 h group (p<0.05). PGC-1α mRNA, NRF-1 mRNA, NRF-2α mRNA, NRF-2ß mRNA, and mitochondrial transcription factor A (TFAM) mRNA in the astrocytes of the cerebral cortex of the rats in the 6 h and 12 h groups increased significantly when compared with those in the 0 h group (p<0.05); the concentration of TFAM mRNA (1.20±0.19) increased significantly when compared with that in the 0 h group (p<0.05). The OPAI protein concentration (1.21±0.17:1.34±0.06) and DRPI protein concentration (1.04±0.05; 1.05±0.05) in the astrocytes of the cerebral cortex of the rats in the 12 h group (1.25±0.17), 24 h group (1.33±0.24), and 6 h group increased significantly when compared with that in the 0 h group (p <0.05). CONCLUSIONS: The experimental sepsis conditions can cause a minor injury of the ultrastructure of the mitochondria in the astrocytes of the cerebral cortex of the rats. The biogenesis of the mitochondria in the astrocytes of the cerebral cortex of the rats was strengthened to cater for the increased demand for energy of the astrocytes under the sepsis conditions and finally recover the ultrastructure of the mitochondria with a minor injury. In response to the increased mitochondrial biogenesis, the activities of the mitochondrial fusion and fission of the astrocytes of the cerebral cortex of the rats were increased.

The role of miR-320a and IL-1ß in human chondrocyte degradation.

OBJECTIVES: This study aimed to explore the role of miR-320a in the pathogenesis of osteoarthritis (OA). METHODS: Human cartilage cells (C28/I2) were transfected with miR-320a or antisense oligonucleotides (ASO)-miR-320a, and treated with IL-1ß. Subsequently the expression of collagen type II alpha 1 (Col2α1) and aggrecan (ACAN), and the concentrations of sulfated glycosaminoglycans (sGAG) and matrix metallopeptidase 13 (MMP-13), were assessed. Luciferase reporter assay, qRT-PCR, and Western blot were performed to explore whether pre-B-cell leukemia Homeobox 3 (PBX3) was a target of miR-320a. Furthermore, cells were co-transfected with miR-320a and PBX3 expressing vector, or cells were transfected with miR-320a and treated with a nuclear factor kappa B (NF-κB) antagonist MG132. The changes in Col2α1 and ACAN expression, and in sGAG and MMP-13 concentrations, were measured again. Statistical comparisons were made between two groups by using the two-tailed paired t-test. RESULTS: Expression of miR-320a was elevated in OA cartilage tissues and chondrocytes, and in IL-1ß-stimulated C28/I2 cells (p < 0.05 or p < 0.01). MiR-320a overexpression enhanced IL-1ß-induced down-regulation of Col2α1 and ACAN and sGAG, and increased the IL-1ß-induced overexpression of MMP-13 (p < 0.01). PBX3 was a direct target of miR-320a. PBX3 and MG132 co-transfection attenuated the effects of miR-320a on the expression of Col2α1, ACAN, sGAG and MMP-13(p < 0.01). CONCLUSION: Overexpression of miR-320a might enhance IL-1ß-induced cartilage degradation factors. These effects might be via targeting PBX3 and regulating NF-κB.Cite this article: Y. Jin, X. Chen, Z. Y. Gao, K. Liu, Y. Hou, J. Zheng. The role of miR-320a and IL-1ß in human chondrocyte degradation. Bone Joint Res 2017;6:-203. DOI: 10.1302/2046-3758.64.BJR-2016-0224.R1.

Pre-B-cell colony-enhancing factor is markedly elevated in childhood hemophagocytic lymphohistiocytosis.

Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening syndrome involving a final common pathway of hypercytokinemia, in which tumor necrosis factor (TNF)-α, interferon (IFN)-γ, and soluble interleukin 2-receptor-alpha (sIL-2Rα) are the key cytokines. Pre-B-cell colony-enhancing factor (PBEF) is an inflammatory cytokine involved in several inflammatory diseases. However, its role in HLH is unknown. In this study, we examined the role of PBEF in HLH. Plasma was collected from 22 children with HLH and 14 healthy children. The concentrations of plasma PBEF, TNF-α, IFN-γ, and sIL-2Rα were determined using an enzyme-linked immunosorbent assay. All clinical data were derived from medical records. In the acute phase, children with HLH had much higher PBEF, TNF-α, IFN-γ, and sIL- 2Rα levels than did healthy children (P < 0.05). After treatment, 13 HLH children improved and PBEF, TNF-α, and IFN-γ levels decreased to normal levels (P < 0.05); sIL-2Rα levels also decreased (P < 0.05), but remained above the normal level (P < 0.05). Two patients were lost to follow-up, while 7 patients showed a bad response to therapy and eventually died, showing high PBEF levels above those of the survivors (P < 0.01). PBEF level was significantly positively correlated with TNF-α, IFN-γ, sIL-2Rα, serum ferritin, and triglycerides (all P < 0.05), and was negatively correlated with fibrin (P < 0.05). PBEF appears to be involved in the inflammatory process of HLH, and elevated PBEF is related to disease activity. We are currently evaluating the role of PBEF as a marker for the diagnosis and management of patients.

Haploidentical hematopoietic stem cell transplantation without in vitro T-cell depletion for the treatment of hematological malignancies.

Many patients who require allogeneic hematopoietic stem cell transplantation (allo-HSCT) lack a human leukocyte antigen (HLA)-matched donor. Here, we report a protocol for haploidentical allo-HSCT that combines granulocyte-colony stimulating factor primed bone marrow (G-BM) and peripheral blood stem cells (PBSC) without in vitro T-cell depletion (TCD). In this study, 171 patients, including 86 in high-risk group, underwent transplantation from haploidentical family donors. All patients achieved sustained, full donor chimerism. One hundred and eleven patients were alive in remission at a median of 682 (253-1502) days. The cumulative incidence of grade III-IV acute graft-versus-host disease (GVHD) was 23% and that of extensive chronic GVHD, 47%; these were not influenced by HLA disparity. Patients younger than 15 years had less grade III-IV acute GVHD than older patients (P=0.044). The 2-year probability of relapse was 12% for standard-risk disease and 39% for high-risk disease. The 2-year probability of leukemia-free survival (LFS) was 68% for standard-risk patients and 42% for high-risk patients (P=0.0009). Grade III-IV acute GVHD was associated with better LFS (P=0.0017). The results require confirmation and show that G-BM combined with PBSC from haploidentical family donors, without in vitro TCD, may be used as a good source of stem cells for allo-HSCT.

Insulin regulation of beta-cell function involves a feedback loop on SERCA gene expression, Ca(2+) homeostasis, and insulin expression and secretion.

The insulin receptor signaling pathway is present in beta-cells and is believed to be important in beta-cell function. We show here that insulin directly regulates beta-cell function in isolated rodent islets. Long-term insulin treatment caused a sustained increase in [Ca(2+)](i) and enhanced glucose-stimulated insulin secretion in rat islets, but failed to increase insulin content. Chronic activation of insulin receptor signaling by IRS-1 overexpression in the beta-cell inhibited gene expression of SERCA3, an endoplasmic reticulum Ca(2+)-ATPase. Insulin gene transcription was stimulated by insulin receptor signaling and insulin mimetic compound (L-783 281) in a glucose- and Grb2-dependent manner. Thus, beta-cell SERCA3 is a target for insulin regulation, which implies that beta-cell Ca(2+) homeostasis is regulated in an autocrine feedback loop by insulin. This study identifies a novel regulatory pathway of insulin secretion at the molecular level with two main components: (1) regulation of intracellular Ca(2+) homeostasis via SERCA3 and (2) regulation of insulin gene expression.

Activation of the sphingomyelinase/ceramide signal transduction pathway in insulin-secreting beta-cells: role in cytokine-induced beta-cell death.

Activation of the sphingomyelin/ceramide pathway may mediate interleukin-1-induced beta-cell death (Welsh, N: Interleuken-1beta-induced ceramide and diacylglycerol generation may lead to activation of the c-Jun NH2-terminal kinase and the transcription factor ATF-2 in the insulin-producing cell line RINm5F. J Biol Chem 271: 8307-8312, 1996). In this report, we have examined this pathway in more detail. Culture of beta-TC3 cells with 25 micromol/l ceramide analogs (N-acetyl- and N-hexanoylsphingosine) for 72 h did not significantly affect glucose- and carbachol-induced insulin secretion. Dihydroceramide (N-acetyl- or N-hexanoylsphinganine), a structurally similar analog, had no effect on agonist-induced secretion. However, ceramide analogs both time- and dose-dependently decreased cell viability, while the dihydroceramide analog had no effect. The ceramide effect on cell viability mimicked the effect of the cytokines TNF-alpha, IL-1beta, and IFN-gamma, reported stimulators of sphingomyelin hydrolysis. Cytokines, however, failed to stimulate sphingomyelin metabolism. Furthermore, using two different methods to quantitate ceramide, cytokines failed to cause an increase in beta-cell ceramide content versus unstimulated or time-matched vehicle controls. Taken together, these data suggest that although ceramide analogs mimic the cytotoxic effect of cytokines, activation of the sphingomyelin/ceramide signaling pathway is not involved in cytokine-induced beta-cell death.

Insulin receptor substrate 1-induced inhibition of endoplasmic reticulum Ca2+ uptake in beta-cells. Autocrine regulation of intracellular ca2+ homeostasis and insulin secretion.

To understand the role of the insulin receptor pathway in beta-cell function, we have generated stable beta-cells (betaIRS1-A) that overexpress by 2-fold the insulin receptor substrate-1 (IRS-1) and compared them to vector-expressing controls. IRS-1 overexpression dramatically increased basal cytosolic Ca2+ levels from 81 to 278 nM, but it did not affect Ca2+ response to glucose. Overexpression of the insulin receptor also caused an increase in cytosolic Ca2+. Increased cytosolic Ca2+ was due to inhibition of Ca2+ uptake by the endoplasmic reticulum, because endoplasmic reticulum Ca2+ uptake and content were reduced in betaIRS1-A cells. Fractional insulin secretion was significantly increased 2-fold, and there was a decrease in betaIRS1-A insulin content and insulin biosynthesis. Steady-state insulin mRNA levels and glucose-stimulated ATP were unchanged. High IRS-1 levels also reduced beta-cell proliferation. These data demonstrate a direct link between the insulin receptor signaling pathway and the Ca2+-dependent pathways regulating insulin secretion of beta-cells. We postulate that during regulated insulin secretion, released insulin binds the beta-cell insulin receptor and activates IRS-1, thus further increasing cytosolic Ca2+ by reducing Ca2+ uptake. We suggest the existence of a novel pathway of autocrine regulation of intracellular Ca2+ homeostasis and insulin secretion in the beta-cell of the endocrine pancreas.

Mechanisms of spontaneous cytosolic Ca2+ transients in differentiated human neuronal cells.

We have studied Ca2+ homeostasis in a unique model of human neurons, the NT2N cell, which differentiates from a human teratocarcinoma cell line, NTera2/C1.D1 by retinoic acid treatment. When perifused with Krebs-HEPES buffer containing 2.5 mM CaCl2, fura-2 loaded NT2N cells produced spontaneous cytosolic Ca2+ oscillations, or Ca2+ transients. These cytosolic Ca2+ transients were not blocked by antagonists of glutamate (6-cyano-7-nitroquinoxaline-2,3-dione and D(-)-2-amino-5-phosphonopentanoic acid) or muscarinic (atropine) receptors. Omission of extracellular Ca2+ completely abolished Ca2+ oscillations and decreased the average Ca2+ level from 106 +/- 14 nM to 59 +/- 8 nM. Addition of the L-type Ca2+ channel blocker nifedipine (1 or 10 microM) or of the N-type inhibitor omega-conotoxin GVIA (5 microM) significantly, although incompletely, suppressed Ca2+ oscillations, while omega-conotoxin MVIIC (5 microM), a selective antagonist of P- and Q-channels, had no effect. Ni2+, at 100 microM, a concentration selective for T-type channels, did not inhibit Ca2+ transients. Non-specific blockage of Ca2+ channels by higher concentrations of Ni2+ (2-5 mM) or Co2+ (1 mM) abolished Ca2+ oscillations completely. The endoplasmic reticulum Ca2+-ATPase inhibitor, thapsigargin (1 microM), slightly decreased Ca2+ oscillation frequency, and induced a small transitory increase in the average cytosolic Ca2+ concentration. The mRNAs of L- (alpha1D subunit) and N-type (alpha1B subunit) Ca2+ channel were present in NT2N cells, while that of a T-type Ca2+ channel (alpha1-subunit) was not present in the NT2N cells as shown by reverse transcription-polymerase chain reaction. In conclusion, NT2N neuronal cells generate cytosolic Ca2+ oscillations mainly by influx of extracellular Ca2+ through multiple channels, which include L- and N-type channels, and do not require activation of glutamate or muscarinic receptors.

Regulation of amyloid precursor protein secretion by glutamate receptors in human Ntera 2 neurons.

The amyloid precursor protein (APP) can be cleaved by a beta-secretase to generate a beta-amyloid peptide, which has been implicated in the pathogenesis of Alzheimer's disease. However, APP can also be cleaved by an alpha-secretase to form a non-amyloidogenic secreted form of APP (APP-S). APP-S secretion can be physiologically regulated. This study examined the glutamatergic regulation of APP in the human neuronal Ntera 2 (NT2N) cell line. Metabotropic glutamate receptor subtypes 1alpha/beta and 5alpha were identified in the NT2N neurons by reverse transcription-polymerase chain reaction. Stimulation of these phosphatidylinositol-linked receptors with glutamate or specific receptor agonists resulted in a dose- and time-dependent increase in the secretion of the amyloid precursor protein (APP-S), measured by the immunoprecipitation of APP-S from the medium of [35S]methionine-labeled NT2N neurons. The glutamate-induced APP-S secretion was maximal at 30 min and at a concentration of 1 mM glutamate. Glutamate-induced APP-S secretion required activation of phospholipase C, which resulted in inositol 1, 4,5-trisphosphate production, as shown by the rapid glutamate-induced accumulation of inositol 1,4,5-trisphosphate. Glutamate also caused an increase in intracellular Ca2+. The protein kinase C activator phorbol 12-myristate 13-acetate, a phorbol ester, as well as 1-oleoyl-2-acetoyl-3-glycerol, a cell-permeable diacylglycerol analog, also stimulated APP-S secretion. These findings suggest that APP-S secretion from NT2N neurons can be regulated by the activation of phosphatidylinositol-linked metabotropic glutamate receptor signaling pathway.

Retinoic acid induction of calcium channel expression in human NT2N neurons.

Ca2+ channel expression and regulation of intracellular Ca2+ homeostasis were studied during retinoic acid (RA)-induced differentiation of the human teratocarcinoma cell line Ntera 2/C1.D1 (NT2- cells) into NT2N neurons, a unique model of human neurons in culture. The cytosolic Ca2+ level of undifferentiated NT2- cells was low (75 +/- 5 nM) and stable under basal conditions, and it was only marginally decreased (by 9%) upon removal of extracellular Ca2+. After 10 microM RA treatment, NT2- cells were irreversibly differentiated into a phenotype of neuron-like NT2N cells. Cytosolic Ca2+ level of NT2N neurons was higher (106 +/- 14 nM) than that of NT2- cells and spontaneously fluctuated (0.208 +/- 0.038 transients/min) under basal conditions. Although K+ increased 86Rb fluxes in both NT2- cells and NT2N neurons, it only increased cytosolic Ca2+ level in NT2N neurons. The K+-induced increase in cytosolic Ca2+ in NT2N neurons was antagonized by 0.1-10 microM nifedipine or verapamil, 5 microM omega-CgTx GVIA, but not by 1 microM omega-agatoxin IVA, 1 microM omega-agatoxin TK, 1 microM FTX-3.3, or 100 microM Ni+ implicating L- and N-type voltage-dependent Ca2+ channels. In L- and N-type channels, but not in P- and Q-types, mRNAs were expressed in NT2N neurons as well as NT2- cells. Quantitative analysis of L- and N-type Ca2+ protein levels showed major differences between NT2- cells and NT2N neurons. In NT2- cells, N-type Ca2+ channels were undetectable while L-type channels levels were fivefold lower compared to NT2N neurons. Our findings show that L- and N-type channels are expressed during differentiation of NT2- cells into neurons, and that these voltage-dependent Ca2+ channels have a major role in regulating intracellular Ca2+ homeostasis and neuronal excitability.

Cytotoxic effect of beta-amyloid on a human differentiated neuron is not mediated by cytoplasmic Ca2+ accumulation.

The effects of synthetic beta-amyloid (A beta1-42) on cell viability and cellular Ca2+ homeostasis have been studied in the human neuron-like NT2N cell, which differentiates from a teratocarcinoma cell line, NTera2/C1.D1, by retinoic acid treatment. NT2N viability was measured using morphological criteria and fluorescent live/dead staining and quantified using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide metabolism. A beta1-42 dose-dependently caused NT2N cell death when it was present in the cell culture for 14 days but had no effect on viability when it was present for 4 days. The lowest effective concentration was 4 microM, and the strongest effect was produced by 40 microM. Control NT2N cells produced spontaneous cytosolic Ca2+ oscillations under basal conditions. These oscillations were inhibited dose-dependently (0.4-40 microM) by A beta1-42 that was present in the cell culture for 1 or 4 days. Ca2+ wave frequency was decreased from 0.21 +/- 0.02 to 0.05 +/- 0.02/min, amplitude from 88 +/- 8 to 13 +/- 4 nM, and average Ca2+ level from 130 +/- 8 to 58 +/- 3 nM. The Ca2+ responses to 30 mM K+ and 100 microM glutamate were not different between control and A beta-treated cells. Thus, the results do not support the hypothesis that cytosolic early Ca2+ accumulation mediates A beta-induced NT2N cell death.