Effect of Toll-like receptor 4 deficiency on clinical severity and expression of Th1/Th2/Th17-associated cytokines in a murine model of experimental autoimmune neuritis.

Introduction: The aim was to observe the effect of Toll-like receptor 4 (TLR4) deficiency on clinical severity and expression of Th1/Th2/Th17-associated cytokines in experimental autoimmune neuritis (EAN). Material and methods: We selected C57BL/10 wild type (WT) mice and TLR4 knockout (KO) mice with the C57BL/10 background for induction of the EAN model by immunizing mice twice (days 0 and 8) via subcutaneous injection of 180 µg P0 peptide 180-199 emulsion in 25 µl of PBS and 0.5 mg Mycobacterium tuberculosis (Difco, USA) in 25 µl of Freund's incomplete adjuvant into the back of mice. The concentrations of serum cytokines (IL-2, IL-4, IL-6, IL-10, IL-17A, IFN-γ and TNF) were determined using the Ms Th1/Th2/Th17 CBA kit. Results: We found that TLR4 deficiency could attenuate the clinical severity and delay the onset of EAN. Moreover, our data showed that the sera levels of IFN-γ, TNF, IL-6 and IL-17A were elevated in the WT mice with EAN when compared with the naive WT mice, but only the production of IL-17A was significantly lower in the TLR4 KO mice with EAN than in their WT counterparts. Conclusions: Based on these findings, TLR4 may contribute to the pathogenesis of EAN by regulating Th17 cells and the production of Th17-associated factors. However, the exact mechanism remains unclear and more evidence is needed to elucidate its role in EAN.

Association of PTTG1 polymorphism rs1895320, rs2910200 and rs6882742 with non-functioning pituitary adenomas in Chinese Han population: a case-control study.

Due to absence of clinical manifestations of hormonal hyper secretion, the treatment of Nonfunctioning pituitary adenoma (NFPA) was always delayed. PTTG1 was reported to be overexpressed in most of pituitary tumors, however, the polymorphism of PTTG1 rs1895320, rs2910200 and rs6882742 with NFPA were still not fully elucidated in NFPA. Thus, a hospital based case control study which included 79 patients and 142 healthy control participants were conducted. DNA was extracted from peripheral blood samples and genotyped by Mass Array methods. In addition, a meta-analysis of rs2910200 was also employed to further testify the conclusion. Significant difference were observed between patients and healthy controls under rs2910200 locus between allelic genotype (p = 0.0219). However, no other significant difference was observed in rs1895329 and rs6882742. In addition, a logistic regression analysis showed that the dominant model of rs2910200 were closely correlated with the NFPA susceptibility (OR = 1.951, 95% CI:1.075-3.542, p = 0.028). While no significant difference was observed in the rs1895320 and rs6882742 under dominant model, recessive model and additive model The meta-analysis results showed that the dominant model and heterozygote model can significantly increase the risk of PA (p = 0.007, OR = 1.57, 95% CI:1.14-2.18; p = 0.009, OR = 1.57, 95% CI:1.12-2.19). Whereas no significant difference were observed under the homozygous model and recessive model. In conclusion, the polymorphism of PTTG1 rs2910200 dominant model and T allelic might increase the risk of NFPA.

Factors interfering with the accuracy of five blood glucose meters used in Chinese hospitals.

BACKGROUND: The prevalence of diabetes is increasing in China. Glucose control is very important in diabetic patients. The aim of this study was to compare the accuracy of five glucose meters used in Chinese hospitals with a reference method, in the absence and presence of various factors that may interfere with the meters. METHODS: Within-run precision of the meters was evaluated include Roche Accu-Chek Inform®, Abbott Precision PCx FreeStyle®, Bayer Contour®, J&J LifeScan SureStep Flexx®, and Nova Biomedical StatStrip®. The interference of hematocrit level, maltose, ascorbic acid, acetaminophen, galactose, dopamine, and uric acid were tested in three levels of blood glucose, namely low, medium, and high concentrations. Accuracy (bias) of the meters and analytical interference by various factors were evaluated by comparing results obtained in whole blood specimens with those in plasma samples of the whole blood specimens run on the reference method. Impact of oxygen tension on above five blood glucose meters was detected. RESULTS: Precision was acceptable and slightly different between meters. There were no significant differences in the measurements between the meters and the reference method. The hematocrit level significantly interfered with all meters, except StatStrip. Measurements were affected to varying degrees by different substances at different glucose levels, e.g. acetaminophen and ascorbic acid (Freestyle), maltose and galactose (FreeStyle, Accu-Chek), uric acid (FreeStyle, Bayer Contour), and dopamine (Bayer Contour). CONCLUSIONS: The measurements with the five meters showed a good correlation with the plasma hexokinase reference method, but most were affected by the hematocrit level. Some meters also showed marked interference by other substances.

Association analysis of USF1 gene polymorphisms and total unstable carotid plaque area in atherosclerotic stroke patients.

Polymorphisms of the upstream stimulatory factor 1 (USF1) have been associated with carotid artery intima-media thickness and coronary atherosclerotic lesions. Unstable carotid plaque is an atherosclerotic change of vascular morphology that has been correlated with cerebrovascular ischemic symptoms. Associations of three single nucleotide polymorphisms of the USF1 gene with total unstable carotid plaque area (CPA) were investigated in Chinese atherosclerotic stroke patients. We recruited 668 atherosclerotic stroke patients and 602 controls. Total unstable CPA values were measured by ultrasound. Genotypes were analyzed using polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP) or mismatched PCR-RFLP. A significant difference in total unstable CPA was found for rs2516838 and rs2516839 genotypes (P = 0.039 and 0.046, respectively) in atherosclerotic stroke patients with unstable carotid plaque. Furthermore, in multiple logistic regression analysis adjusted by age, sex, BMI, hypertension, smoking status, glucose, total cholesterol, triglycerides, high-density lipoprotein-cholesterols, low-density lipoprotein-cholesterols and high-sensitivity C-reactive protein, significant associations were seen between the total unstable CPA values and genotypes of the rs2516838 or the rs2516839 in these patients. The rare allele C of rs2516838 or rare allele A of rs2516839 could predict relative low total unstable CPA values. The rs2516838 and rs2516839 polymorphisms of USF1 influence total unstable CPA in atherosclerotic stroke patients, which might be new markers to predict the risk of recurrence for this disease.

Oxidized low-density lipoprotein and ß-glycerophosphate synergistically induce endothelial progenitor cell ossification.

AIM: To investigate the ability of ox-LDL to induce ossification of endothelial progenitor cells (EPCs) in vitro and explored whether oxidative stress, especially hypoxia inducible factor-1α (HIF-1α) and reactive oxygen species (ROS), participate in the ossific process. METHODS: Rat bone marrow-derived endothelial progenitor cells (BMEPCs) were cultured in endothelial growth medium supplemented with VEGF (40 ng/mL) and bFGF (10 ng/mL). The cells were treated with oxidized low-density lipoprotein (ox-LDL, 5 µg/mL) and/or ß-glycerophosphate (ß-GP, 10 mmol/L). Calcium content and Von Kossa staining were used as the measures of calcium deposition. Ossific gene expression was determined using RT-PCR. The expression of osteocalcin (OCN) was detected with immunofluorescence. Alkaline phosphatase (ALP) activity was analyzed using colorimetric assay. Intercellular reactive oxygen species (ROS) were measured with flow cytometry. RESULTS: BMEPCs exhibited a spindle-like shape. The percentage of cells that expressed the cell markers of EPCs CD34, CD133 and kinase insert domain-containing receptor (KDR) were 46.2%±5.8%, 23.5%±4.0% and 74.3%±8.8%, respectively. Among the total cells, 78.3%±4.2% were stained with endothelial-specific fluorescence. Treatment of BMEPCs with ox-LDL significantly promoted calcium deposition, which was further significantly enhanced by co-treatment with ß-GP. The same treatments significantly increased the gene expression of core-binding factor a-1 (cbfa-1) and OCN, while decreased the gene expression of osteoprotegerin (OPG). The treatments also significantly enhanced the activity of ALP, but did not affect the number of OCN(+) cells. Furthermore, the treatments significantly increased ROS and activated the hypoxia inducible factor-1α (HIF-1α). In all these effects, ox-LDL acted synergistically with ß-GP. CONCLUSION: Ox-LDL and ß-GP synergistically induce ossification of BMEPCs, in which an oxidizing mechanism is involved.

Susceptibility of vertilmicin to modifications by three types of recombinant aminoglycoside-modifying enzymes.

The susceptibilities of vertilmicin and seven reference aminoglycosides to modifications by six recombinant aminoglycoside-modifying enzymes, AAC(6')-Ie, APH(2'')-Ia, AAC(6')-Ie-APH(2'')-Ia, ANT(2'')-Ia, AAC(6')-Ib, and AAC(6')-Ib-cr, were studied by coupled spectrophotometric assays in microtiter plates. In comparison to other aminoglycosides, the susceptibility of vertilmicin was 45.8- to 250.0-fold lower for AAC(6')-Ie acetylation, 39.2- to 116.7-fold lower for AAC(6')-Ie-APH(2'')-Ia acetylation, and 1.8- to 7.5-fold lower for ANT(2'')-Ia adenylation (except that shown by amikacin) while relatively comparable for AAC(6')-Ib acetylation, AAC(6')-Ib-cr acetylation, APH(2'')-Ia phosphorylation, and AAC(6')-Ie-APH(2'')-Ia phosphorylation.

[Protective effect and mechanism of hepcidin in rats with alcoholic liver damage].

OBJECTIVE: To study the mechanism of how iron-regulatory protein (hepcidin) affect iron overload in alcoholic liver disease (ALD). METHODS: Thirty male wistar rats were randomly divided into 3 groups: Lieber-Decarli liquid without alcohol group (control group), Lieber-Decarli liquid with alcohol (alcohol group) and hepcidin intraperitoneally injected group (hepcidin group), each rat was fed for 6 weeks. The Serum concentration of Alanine Aminotransferase (ALT), Aspartate Amino Transferase (AST), Iron, Total Iron Binding capacity (TIBC), Ferritin, Malonyl Dialdehyde (MDA) and Hepcidin were determined. Hepatic tissue was examined by hematoxylin and eosin staining, prussian blue iron staining and immunohistochemistry staining. RESULTS: (1) Serum concentration of ALT in control group, alcohol group and hepcidin group were (25.2 ± 4.6) U/L, (37.9 ± 14.3) U/L and (40.9 ± 14.1) U/L (F = 4.907, P < 0.05), respectively. Serum AST among three groups were (32.3 ± 13.4) U/L, (55.0 ± 18.6) U/L and (48.3 ± 26.0) U/L (F = 3.742, P < 0.05), respectively. The secretions of ferritin were (224.72 ± 85.49) ng/ml, (345.59 ± 124.75) ng/ml and (339.47 ± 138.47) ng/ml (F = 3.539, P < 0.05). The serum concentrations of TIBC were (147.30 ± 31.98) µmol/L, (148.04 ± 58.74) µmol/L and (143.28 ± 37.38) µmol/L (F = 1.209, P > 0.05), respectively. The serum concentrations of iron were (55.64 ± 13.32) µmol/L, (60.37 ± 25.89) µmol/L and (49.77 ± 17.64) µmol/L (F = 0.651, P > 0.05), respectively. The serum concentration of MDA were (5.84 ± 2.17) nmol/ml, (6.51 ± 2.23) nmol/ml and (4.27 ± 2.68) nmol/ml (F = 2.782, P > 0.05), respectively. The serum concentration of Hepcidin were (155.96 ± 44.91)ng/ml, (124.11 ± 31.98) ng/ml and (114.96 ± 25.81) ng/ml (F = 3.839, P < 0.05), respectively. (2) Significant fat change observed in the liver of alcohol group. The positive granulations of iron staining were (0.8 ± 1.0), (1.2 ± 1.6) and (1.1 ± 1.1) (F = 0.254, P > 0.05), respectively. No differences found of liver iron express among the three groups. Intraperitoneal injection of hepcidin increased hepcidin expression in liver which was inhibited by alcohol (F = 4.139, P < 0.05). CONCLUSIONS: ALD rats with lower hepcidin expression in liver can result in iron metabolism disorder. Ectogenic hepcidin can protect liver against alcohol damage by inhibiting lipid peroxidation.

Chemokine CXC Ligand 16 serum concentration but not A181V genotype is associated with atherosclerotic stroke.

BACKGROUND: Serum chemokine CXC Ligand 16 (CXCL16) concentration is associated with atherosclerosis and CXCL16 expression may be influenced by the polymorphism, A181V. We established whether serum CXCL16 concentration or the A181V genotype is more strongly associated with atherosclerotic stroke and its associated risk factor, carotid atherosclerosis. METHODS: PCR-RFLP was used to genotype 244 atherosclerotic stroke patients (AS group), 153 stroke-free controls (patient controls) and 167 healthy controls. Serum CXCL16 concentration was determined for a subset of patients (n=135) and all controls. The same subset of patients was then examined using ultrasound to evaluate their carotid atherosclerotic lesions, including intima-media thickness (IMT), plaque stability and carotid plaque area (CPA). RESULTS: Compared with the patient controls and healthy controls, serum CXCL16 concentration was significantly increased in the AS group (P<0.05, and 0.01). It was also strongly associated with increased IMT, vulnerable plaque and increased CPA (P<0.05, <0.001, and <0.01). However, the CXCL16 A181V genotype distribution and allele frequencies showed no differences between AS and control groups, nor did it influence serum CXCL16 concentration. CONCLUSION: Serum CXCL16 concentration is significantly associated with atherosclerotic stroke and carotid atherosclerosis, suggesting that this biochemical test may be useful to identify patients at increased risk of atherosclerosis.

[Study on the association of the CRP gene +1444C/T polymorphism with symptomatic carotid artery stenosis].

OBJECTIVE: To investigate the potential association of the C-reactive protein (CRP) gene +1444C/T polymorphism with symptomatic carotid artery stenosis. METHODS: Polymerase chain reaction-restriction fragment length polymorphism was used for the detection of CRP +1444C/T genotypes in 192 patients with symptomatic carotid artery stenosis and 197 healthy controls. Serum high sensitivity-CRP (hs-CRP) levels were measured by routine method. RESULTS: No TT genotype was detected in this study. Patients with >70% stenosis had higher CC genotype compared with those with <70% stenosis after adjusting for major cerebrovascular risk factors (OR: 2.958; 95% CI: 1.198 - 7.305; P=0.019). CRP levels were significantly higher in patients than in controls. Subgroup analysis according to clinical characteristics (single or double stenosis; >70% or <70% stenosis) did not show difference in CRP levels. There was no significant difference in the prevalence of CT genotype between patients and controls, or between single and double stenosis (P>0.05). CONCLUSION: The CRP +1444 CC genotype is a risk factor for >70% carotid artery stenosis. The serum CRP level is associated with the presence of carotid stenosis. However, it is not associated with the number and severity of stenosis.

Analysis of translocation of the CagA protein and induction of a scattering phenotype in AGS cells infected with Helicobacter pylori.

OBJECTIVE: To investigate whether the presence of structured CagA proteins in Western- and Eastern-type Helicobacter pylori (H. pylori) induces different incidences of gastric diseases. METHODS: CagA and phosphorylated CagA were expressed in AGS gastric epithelial cells infected with wild type and mutant strains. The ability of individual CagA was determined by immunoprecipitation and Western blot assay. Morphological changes of these cells were observed under microscope to evaluate the appearance of elongation hummingbird phenotype. RESULTS: The sizes of CagA proteins in different strains were different, and no phosphorylated CagA proteins were detected in wild-type strains. Meanwhile, the kinetics of CagA status in AGS infected with H. pylori was detected. The molecular weight of phosphorylated CagA with the same size of CagA proteins in H. pylori was different in infections with different wild-type strains. CagA and phosphorylated CagA increased in a time-dependent manner after the infection. The hummingbird phenotype with H. pylori for time-course was observed under microscope. Instead of HPK5 strain, the wild-type 26695 strain induced hummingbird phenotype in a time-dependent manner. CONCLUSION: Translocation and phosphorylation of CagA are necessary, but not sufficient, for the induction of hummingbird phenotype in AGS cells.

Polymorphism in the human C-reactive protein (CRP) gene, serum concentrations of CRP, and the difference between intracranial and extracranial atherosclerosis.

BACKGROUND: C-reactive protein, a proinflammatory factor, is involved in the development of atherosclerosis. The CRP 1059G>C polymorphism appeared to be a susceptive marker for atherosclerosis. We investigated the relationship of the distribution of cerebral atherosclerosis with triggered serum CRP concentrations following acute ischemic stroke/transient ischemic attack (IS/TIA) and CRP 1059G>C polymorphism. METHODS: We recruited 222 IS/TIA patients (122 with only intracranial atherosclerotic lesions and 100 with isolated extracranial atherosclerotic lesions) and 227 controls. Intra- and extracranial atherosclerotic lesions were determined by digital subtraction angiography. Serum CRP concentrations were measured by particle-enhanced immunonephelometry assay. CRP 1059G>C genotypes were obtained through PCR amplification and restriction enzyme digestion. RESULTS: CRP concentrations were significantly higher in intra- and extracranial groups than in controls. No significant difference was found in CRP concentrations between intra- and extracranial groups. The CRP 1059G>C single-nucleotide polymorphism did not influence CRP serum concentrations. CRP genotype and allele frequencies did not differ significantly between patients and controls. However, the frequencies of GC genotype and C allele were significantly higher in extracranial group than that in intracranial group. The GC individuals showed a higher risk of extracranial atherosclerosis compared with GG individuals (OR 3.41; 95%CI, 1.124-10.347; P=0.030). CONCLUSIONS: Serum CRP is associated with cerebral atherosclerotic disease. CRP 1059G>C polymorphism is one possible genetic determinant for the difference between intra- and extracranial atherosclerosis.

[Therapeutic effect of brain-specific angiogenesis inhibitor 1 on glioblastoma: an animal experiment].

OBJECTIVE: To study the therapeutic effects of brain-specific angiogenesis inhibitor 1 (BAI1) on human glioblastoma and relevant mechanism. METHODS: Recombinant adenovirus carrying human BAI1 cDNA, AdeBAI1 and recombinant adenovirus carrying LacZ, AdeMock, were constructed with the COS-TPC method. The successful construction of AdeBAI1 and expression of AdeBAI1 was verified using RT-PCR. Glioblastoma cells of the line U87MG were transplanted into the mice brain using stereotactic technique. AdeBAI1 and AdeMock were injected into the tumors after the tumors were developed. The survival of the mice was observed. Human glioblastoma cells of the lines SW1783, U87MG, and U373MG were cultured and transfected with AdeBAI1 or AdeMock, and then collected 48 hours later and counted using MTT method. The total RNA was extracted using Trizol agent. The mRNA of BAI1 and other angiogenesis related genes were detected using RT-PCR. RESULTS: The mean survival time of the AdBAI1-treated mice was 26 +/- 4.6 d, significantly longer than that of the AdMock-treated mice (17.3 +/- 2.3 d, P < 0.05). RT-PCR showed that BAI1 mRNA was expressed only in the glioblastoma cells transfected with AdeBAI1. The number of AdeBAI1 treated glioblastoma cells was 2.12 +/- 0.18 x 10(5), significantly less than that of the AdeMock treated cells (4.23 +/- 0.18 x 10(5), P < 0.05). The mRNA expression of angiostatin of the AdeBAI1 treated cells was 0.66 +/- 0.08, significantly less than that of the AdMock-treated cells (0.95 +/- 0.12, P < 0.05). The mRNA expression of vascular endothelial growth factor (VEGF) of the AdeBAI1 treated cells was 0.68 +/- 0.07, significantly less than that of the AdMock-treated cells (1.02 +/- 0.14, P < 0.05). The mRNA expression of VEGF-B of the AdeBAI1 treated cells was 1.11 +/- 0.10, significantly more than that of the AdMock-treated cells (0.77 +/- 0.18, P < 0.05). The mRNA expression of thrombospondin of the AdeBAI1 treated cells was 1.16 +/- 0.16, significantly more than that of the AdMock-treated cells (0.60 +/- 0.22, P < 0.05). CONCLUSION: Intratumor injection of AdeBAI1 can inhibit the tumor growth. The anti-tumor effect of BAI1 may arise from both anti-angiogenesis and anti-proliferation effects.