[The study of increased immunogenicity of UCMSC Stimulated by TLR7 agonist CL264].

OBJECTIVE: To study the change of immune status of umbilical cord mesenchymal stem cells (UCMSC) stimulated by toll like receptor 7 (TLR7) agonist CL264. METHODS: TLR7 specific ligand CL264 was used to stimulate the UCMSC. Flow cytometry was conducted to assay the expression of co-stimulators [human leukocyte antigen (HLA)-E, CD80 and CD86] and surface markers of stem cells (CD29, CD59 and CD90). Quantitative PCR was applied to measure the expression variation of immune-related molecules and stem cell markers. Cell differentiation experiment was used to study the change of differentiation ability of UCMSC upon CL264 stimulation. Peripheral blood mononuclear cells (PBMC) were isolated from healthy human and then cocultured with UCMSC in the presence of CL264. Cytotoxicity assay was used to measure the attack of PBMC to UCMSC. RESULTS: Expression of cotimulatory molecules CD86 and HLA-E were enhanced in UCMSC upon CL264 stimulation. Real-time PCR indicated that many pro-inflammatory molecules [interleukin (IL)-1beta, IL-6, IL-8, IL-10, interferon (IFN)-beta, IFN-gamma, nuclear factor-KB (NF-kappaB), transforming growth factor-beta (TGF-beta)] were induced in the presence of CL264 while the expression of stem cells markers were inhibited [Kruppel-like factor-4 (Klf4), Nestin, SRY-related high-mobility-group-box protein-2 (Sox2), Lin28]. Activation of TLR7 also increased the immune attack of PBMC on UCMSC. Our study also indicated that the treatment of CL264 did not influence the differentiation ability of UCMSC. CONCLUSION: TLR7 agonist CL264 could increase the immunogenicity of UCMSC.

[The Correlation Analysis between the Titer of IgG Anti-A/B Erythrocyte Antibody In Vivo of the Neonate and the Severity of Hemolytic Disease of Newborn].

OBJECTIVE: To investigate the titer of IgG anti-A/B erythrocyte antibody in vivo of the neonate with hemolytic disease of newborn(HDN), and explore its clinical valua in evaluating the severity of HDN. METHODS: 300 neonates with HDN, 50 neonates with neonatal hyperbilirubinemiain and 50 healthy neonates were selected as research object and Microtubes Gel Test was used to detect the titer of IgG anti-A/B erythrocyte antibody in vivo. Their clinical data and their mothers' prenatal examination data were retrospectively analyzed. Three hemolysis tests (direct antiglobulin test, free antibody test and release test), irregular antibody screening, and the titer of IgG anti-A/B blood group antibody was determined by serological method. Red blood cells(RBC), hemoglobin(Hb), reticulocytes(Ret) and nucleated red cells were detected by hematology analyzer. Indirect bilirubin and albumin(Alb) were detected by biochemical analyzer. The relationship between the titer of IgG anti-A/B erythrocyte antibody in vivo and the severity of HDN was analyzed. RESULTS: There were six serological diagnosis modes in the HDN group,the difference between modes was statistically significant (P<0.05). The antibody titer relationship between HDN neonates and pregnant women was positive correlation(r=0.8302). The highest antibody titer of release test and free antibody test were 1∶32 and 1∶2, and the difference was statistically significant（P<0.05）. RBC, Hb and Alb in HDN patients were lower than those in neonatal hyperbilirubinemia patients and healthy neonates (P<0.05), and were negatively relevant with antibody titer in vivo (r=-0.8016). Bilirubin content in HDN patients were higher than those in neonatal hyperbiliru binemia patients and healthy neonates group(P<0.05), and was positively relevant with antibody titer in vivo (r=0.8731). The hospital day in HDN patients was significantly relevant with the antibody titer in vivo (r=0.8547), but not with the age, sex, weight and ABO blood types (P>0.05). CONCLUSION: The detection of antibody titer in HDN patients can be used to evaluate the antibody concentration in vivo, predict the ability of antibody to induce erythrocyte hemolysis, and help to judge the serenrity and prognosis of HDN.

B-type natriuretic peptide, vascular endothelial growth factor, endothelin-1, and nitric oxide synthase in chronic mountain sickness.

The pathogenesis of chronic mountain sickness (CMS) may involve vasoactive peptides. The aim of this study was to investigate associations between CMS and levels of B-type natriuretic peptide (BNP), vascular endothelial growth factor (VEGF), endothelin-1 (ET-1), and endothelial nitric oxide synthase (eNOS). A total of 24 patients with CMS and 50 control subjects residing at 4,300 m participated in this study. Mean pulmonary arterial pressure (mPAP) was measured by echocardiography. Serum BNP, VEGF, ET-1, and eNOS were measured. Receiver operator characteristic curves to assess the balance of sensitivity and specificity for CMS were constructed. As a result, patients with CMS had significantly greater mPAP compared with controls and had lower arterial O(2) saturation (Sa(O(2))). Both BNP and ET-1 correlated positively with mPAP and negatively with Sa(O(2)), whereas serum VEGF levels were inversely correlated with Sa(O(2)); eNOS correlated negatively with mPAP and positively with Sa(O(2)). Median concentrations of BNP were greater in patients with CMS compared with those without CMS: 369 pg/ml [interquartile range (IQR) = 336-431] vs. 243 pg/ml (IQR = 216-279); P < 0.001. Similarly, concentrations of VEGF [543 pg/ml (IQR = 446-546) vs. 243 pg/ml (IQR = 216-279); P < 0.001] and ET-1 [14.7 pg/ml (IQR = 12.5-17.9) vs. 11.1 pg/ml (IQR = 8.7-13.9); P = 0.05] were higher in those with CMS compared with those without, whereas eNOS levels were lower in those with CMS [8.90 pg/ml (IQR 7.59-10.8) vs. 11.2 pg/ml (9.13-13.1); P < 0.001]. The areas under the receiver operator characteristic curves for diagnosis of CMS were 0.91, 0.93, 0.77, and 0.74 for BNP, VEGF, ET-1, and eNOS, respectively. In age- and biomarker-adjusted logistic regression, BNP and VEGF were positively predictive of CMS, whereas eNOS was inversely predictive. In conclusion, severe chronic hypoxemia and consequent pulmonary hypertension in patients with CMS may stimulate release of natriuretic peptides and angiogenic cytokines. These vasoactive peptides may play an important role in the pathogenesis and clinical expression of CMS and may indicate potential prognostic factors in CMS that could serve as targets for therapeutic trials or clinical decision making.

Hyperhomocysteinemia as a metabolic disorder parameter is independently associated with the severity of coronary heart disease.

OBJECTIVES: To study the associations between hyperhomocysteinemia (HHcy) and the severity of coronary heart disease (CHD). METHODS: We retrospectively analyzed metabolic parameters, anthropometric variables, and life style habits in 292 CHD patients of different categories, and 100 controlled non-CHD patients with chest pain symptoms who were hospitalized in the Department of Cardiovascular Medicine, Sichuan Academy of Medical Sciences and Sichuan Provincial People's Hospital, Chengdu, China between October 2013 and September 2014. RESULTS: The prevalence of HHcy in CHD patients was 79.1%, while only 5% of non-CHD patients had elevated serum homocysteine (Hcy) concentrations. The prevalence of HHcy significantly increased from 5% in non-CHD controls to 66% in the stable angina pectoris (SAP) group, to 81.9% in the unstable angina pectoris group, and to 93.15% in the acute myocardial infarction (AMI) group (p less than 0.001). After adjusting for confounding factors, multivariate logistic regression analysis showed that HHcy was independently associated with CHD category (AMI versus SAP, odds ratio [6.38], 95% confidence interval; 1.18-34.46). The Hcy was negatively correlated with folic acid (r=-0.67, p less than 0.001) and vitamin B12 (r=-0.56, p less than 0.001). Of the CHD patients with HHcy, 51.1% had low folic acid and 42% had low vitamin B12, 7 or 5 times higher than that of CHD patients with normal-low Hcy concentrations (p less than 0.001). CONCLUSION: Hyperhomocysteinemia is independently associated with the severity of CHD, and significantly correlated with low status of folic acid and vitamin B12 in CHD patients.