Tanshinone IIA reduces AQP4 expression and astrocyte swelling after OGD/R by inhibiting the HMGB1/RAGE/NF-κB/IL-6 pro-inflammatory axis.

This study aimed to investigate the role of tanshinone IIA (TSO IIA) in astrocytic swelling caused by ischemia-reperfusion-like injury in an in vitro model and the molecular mechanisms underlying this effect. Primary brain astrocytes were cultured under conditions of glucose and oxygen deprivation and reoxygenation (OGD/R). The study explored the effects of TSO IIA treatment on cell swelling and injury and the protein levels of aquaporin 4 (AQP4) in the plasma membrane. It then examined the involvement of the high-mobility group box protein 1 (HMGB1)/receptors for advanced-glycation end products (RAGE)/nuclear factor-kappa B (NF-κB)/interleukin-6 (IL-6) pro-inflammatory axis in TSO IIA-mediated protection. The treatment with TSO IIA alleviated OGD/R-induced astrocytic swelling and the overclustering of AQP4 protein in the plasma membrane. In addition, TSO IIA significantly reduced the overexpression of HMGB1 and the high levels of the NF-κB protein in the nucleus and of the IL-6 protein in the cytoplasm and extracellular media induced by OGD/R. The combination of TSO IIA and recombinant HMGB1 reversed these effects. The inhibition of the RAGE, the receptor of HMGB1, induced results similar to those of TSO IIA. In addition, exogenous IL-6 reversed TSO IIA-mediated effect on AQP4 overclustering and cell swelling. TSO IIA significantly reduced astrocyte swelling after OGD/R injury in vitro, via blocking the activation of the HMGB1/RAGE/NF-κB/IL-6 pro-inflammatory axis and thereby decreasing the expression of AQP4 in the plasma membrane.

A Novel Tree Shrew Model of Chronic Experimental Autoimmune Uveitis and Its Disruptive Application.

Background: Previous studies have established several animal models for experimental autoimmune uveitis (EAU) in rodents without the fovea centralis in the human retina. This study aimed to develop and explore the application of a novel EAU model in tree shrews with a cone-dominated retina resembling the human fovea. Methods: Tree shrews were clinically and pathologically evaluated for the development and characteristics of EAU immunized with six inter-photoreceptor retinoid-binding proteins (IRBPs). IRBP-specific T-cell proliferation and serum cytokine of tree shrews were evaluated to determine the immune responses. Differentially expressed genes (DEGs) were identified in the eyes of tree shrews with EAU by RNA-sequencing. The disruptive effects of the DEG RGS4 inhibitor CCG 203769 and dihydroartemisinin on the EAU were investigated to evaluate the potential application of tree shrew EAU. Results: IRBP1197-1211 and R14 successfully induced chronic EAU with subretinal deposits and retinal damage in the tree shrews. The immunological characteristics presented the predominant infiltration of microglia/macrophages, dendritic cells, and CD4-T-cells into the uvea and retina and pathogenic T helper (Th) 1 and Th17 responses. The subretinal deposits positively expressed amyloid ß-protein (Aß), CD8, and P2Y purinoceptor 12 (P2RY12). The crucial DEGs in R14-induced EAU, such as P2RY2 and adenylate cyclase 4 (ADCY4), were enriched for several pathways, including inflammatory mediator regulation of transient receptor potential (TRP) channels. The upregulated RGS4 in IRBP-induced EAU was associated with mitogen-activated protein kinase (MAPK) activity. RGS4 inhibition and dihydroartemisinin could significantly alleviate the retinal pathological injuries of IRBP1197-1211-induced EAU by decreasing the expression of CD4 T-cells. Conclusion: Our study provides a novel chronic EAU in tree shrews elicited by bovine R14 and tree shrew IRBP1197-1211 characterized by retinal degeneration, retinal damage with subretinal Aß deposits and microglia/macrophage infiltration, and T-cell response, probably by altering important pathways and genes related to bacterial invasion, inflammatory pain, microglial phagocytosis, and lipid and glucose metabolism. The findings advance the knowledge of the pathogenesis and therapeutics of the fovea-involved visual disturbance in human uveitis.

Parental uveitis causes elevated hair loss in offspring of C57BL/6J mice.

Our previous study demonstrated that parental uveitis in a susceptible population can cause hair loss and increase the susceptibility to experimental autoimmune uveitis (EAU) in offspring. However, it is unclear whether parental uveitis affects the development of offspring in an EAU-moderate-susceptible population. Herein, moderate-susceptible C57BL/6J mice were immunized with inter-photoreceptor retinoid binding protein (IRBP) 651-670 to develop EAU and were kept together for mating. Gross examination and histopathological changes of the offspring gestated with parental uveitis were observed to evaluate the impact of parental uveitis on the development of the offspring. Differentially expressed genes (DEGs) were screened by RNA sequencing in the affected skin and eyeball of the offspring on postnatal day 27. Adult offspring were injected 75 µg IRBP651-670 to evaluate their susceptibility to EAU. Gross examination in the offspring revealed hair loss on postnatal days 11-31. Histopathological observation showed increased melanin granules and hair follicles of skin in the affected offspring with hair loss. Gene Ontology (GO) analysis in the skin revealed differential expression of genes involved in the mitotic cell cycle, response to endogenous stimulus, hair follicle development, and hair cycle. The DEGs in the skin were predominately associated with the cell cycle and peroxisome proliferator-activated receptor (PPAR) signaling pathway. The GO enrichment analysis in the eyeball showed differential expression of genes involved in the nervous system development, camera-type eye photoreceptor cell differentiation, neuron projection morphogenesis, axon development, and calcium-induced calcium release activity; enriched pathways included the circadian entrainment and glutamatergic synapses. No increased susceptibility to EAU in offspring gestated from parental remitting EAU was observed at a low-dose 75 µg IRBP induction. These results suggested that parental uveitis in a moderate-susceptible population could affect the skin development and DEG profiles of skin and eyeball related to the response to endogenous stimulus, the PPAR signaling pathway, and glutamatergic synapse, which provides the molecular evidence to explain the influence of parental uveitis on offspring development.

Short chain fatty acids inhibit endotoxin-induced uveitis and inflammatory responses of retinal astrocytes.

Short chain fatty acids (SCFAs) are produced by gut microbiota as fermentation products of digestion-resistant oligosaccharides and fibers. Their primary roles are functioning as major energy sources for colon cells and assisting in gut homeostasis by immunomodulation. Recent evidence suggests that they affect various organs both at cellular and molecular levels, and regulate functions in distance sites including gene expression, cell proliferation, cell differentiation, apoptosis and inflammation. In this study, we examined whether SCFAs are present in the mouse eye and whether SCFAs affect inflammatory responses of the eye and retinal astrocytes (RACs). We observed that intra-peritoneal injected SCFAs were detected in the eye and reduced intraocular inflammation induced by lipopolysaccharide (LPS). Moreover, SCFAs displayed two disparate effects on LPS-stimulated RACs - namely, cytokine and chemokine production was reduced, but the ability to activate T cells was enhanced. Our results support the existence of gut-eye cross talk and suggest that SCFAs can cross the blood-eye-barrier via the systemic circulation. If applied at high concentrations, SCFAs may reduce inflammation and impact cellular functions in the intraocular milieu.

AKT/GSK-3ß/ß-catenin signaling pathway participates in erythropoietin-promoted glioma proliferation.

PURPOSE: Although erythropoietin (EPO) has been proven to significantly promote the proliferation of cancer cells, the mechanism for promoting glioma proliferation is poorly understood. Here, we examined the functional role of the AKT/GSK-3ß/ß-catenin signaling pathway in the EPO-mediated proliferation of glioma. METHODS: The distribution of EPO and Ki-67 among clinical samples with different WHO grades was plotted by Immunological Histological Chemistry analysis. U87 and U251 glioma cell lines were treated with short hairpin RNA targeting (shEPO), recombinant human erythropoietin (rhEPO) and/or AKT-specific inhibitor (MK-2206). The changes in phosphorylated AKT, nuclear ß-catenin, cyclin D1 and p27kip1 expression were detected. Cell cycle distributions and glioma proliferation in vitro and in vivo were analyzed. RESULTS: The expression level of EPO was significantly elevated with the increase of WHO grade and Ki67 in clinical glioma specimens. In vitro, knockdown of endogenous EPO in U87 and U251 cells effectively block the phosphorylation of AKT and GSK-3ß and the expression of nuclear ß-catenin. shEPO treatment also significantly decreased the expression of cyclin D1 and increased the expression of p27kip1. The cell cycle transition then slowed down and the proliferation of glioma cells or mouse xenograft tumors both decreased. Treatment of cells or tumors with extra rhEPO reversed the above biological effects mediated by shEPO. rhEPO-induced activation of the AKT/GSK-3ß/ß-catenin pathway and proliferation were abolished by MK-2206. CONCLUSIONS: Our study identified the AKT/GSK-3ß/ß-catenin axis as a critical mediator of EPO-induced glioma proliferation and further provided a clinically significant dimension to the biology of EPO.

[Blocking ERK signaling pathway lowers MMP-9 expression to alleviate brain edema after traumatic brain injury in rats].

OBJECTIVE: To investigate the effects of blocking the activation of ERK pathway on the expression of matrix metalloproteinase-9 (MMP-9) and the formation of cerebral edema in SD rats after brain injury. METHODS: Ninety SD rats were randomly divided into 3 equal groups, including a sham-operated group, modified Feeney's traumatic brain injury model group, and ERK inhibition group where the ERK inhibitor SCH772984 (500 µg/kg) was injected via the femoral vein 15 min before brain trauma. At 2 h and 2 days after brain trauma, the permeability of blood-brain barrier was assessed by Evans blue method, the water content of the brain tissue was determined, and the phosphorylation level of ERK and the expression level of MMP-9 mRNA and protein were measured by RT-PCR and Western blotting. RESULTS: Compared with the sham-operated group, the rats with brain trauma exhibited significantly increased level of ERK phosphorylation at 2 h and significantly increased expression of MMP-9 mRNA and protein 2 days after the injury (P < 0.01). Treatment with the ERK inhibitor significantly decreased the phosphorylation level of ERK after the injury (P < 0.01), suppressed over-expression of MMP-9 mRNA and protein 2 days after the injury (P < 0.01). The permeability of blood-brain barrier increased significantly 2 h after brain trauma (P < 0.05) and increased further at 2 days (P < 0.01); the water content of the brain did not change significantly at 2 h (P > 0.05) but increased significantly 2 d after the injury (P < 0.01). Treatment with the ERK inhibitor significantly lowered the permeability of blood-brain barrier and brain water content after brain trauma (P < 0.01). CONCLUSIONS: Blocking the activation of ERK pathway significantly reduced the over-expression of MMP-9 and alleviates the damage of blood-brain barrier and traumatic brain edema, suggesting that ERK signaling pathway plays an important role in traumatic brain edema by regulating the expression of MMP-9.

Anti-CD40 monoclonal antibody ameliorates experimental autoimmune uveoretinitis in mice.

OBJECTIVE: To investigate the effects of CD40 on ocular inflammation in experimental autoimmune uveoretinitis (EAU) in B10.RIII mice. ANIMALS STUDIED: EAU-susceptible B10.RIII mice were subcutaneously immunized with interphotoreceptor retinoid-binding protein (IRBP) 161-180 in complete Freund's adjuvant and evaluated clinically and pathologically on days 7, 14, 21, 28, and 35 postimmunization. Anti-CD40 antibody was intraperitoneally injected into mice every other day from days 7 to 14 postimmunization. Phosphate-buffered saline (PBS)-injected EAU mice were used as the controls. PROCEDURES: The frequencies of CD11c+ CD40+ dendritic cells (DCs), CD11c+ MHC-II+ DCs, and CD11c+ CD40+ MHC-II+ DCs in splenocytes were evaluated by flow cytometry on days 0, 7, 14, and 21 after immunization. Tumor necrosis factor (TNF)-α and interleukin (IL)-6 production in CD11c+ DCs was assessed by ELISA. IRBP-specific lymphocyte proliferation was assessed using a modified MTT cell proliferation assay. RESULTS: The number of CD11c+ CD40+ DCs, CD11c+ MHC-II+ DCs, and CD11c+ CD40+ MHC-II+ DCs increased at the onset of EAU, peaked at the height of disease severity, and was sustained at a high level until day 21. Treatment with anti-CD40 antibody significantly alleviated clinical and pathological activities related to EAU. Compared with the control mice, antibody-treated EAU mice showed few CD11c+ CD40+ DC and CD11c+ CD40+ MHC-II+ DC frequencies in splenocytes. The anti-CD40 antibody significantly suppressed IRBP-specific lymphocyte proliferation and TNF-α and IL-6 production by DCs in EAU mice. CONCLUSIONS: The increased expression of CD40 and major histocompatibility complex (MHC) class II molecules in the splenocytes of EAU mice were correlated with inflammatory activity. Anti-CD40 treatment can significantly attenuate EAU activity by inhibiting systemic IRBP-specific immune responses.

Parental Uveitis Influences Offspring With an Increased Susceptibility to the Experimental Autoimmune Uveitis.

Purpose: Previous studies have shown that parental abnormal physiological conditions such as inflammation, stress, and obesity can be transferred to offspring. The purpose of this study was to investigate the impact of parental uveitis on the development and susceptibility to experimental autoimmune uveitis (EAU) in offspring. Methods: Parental male and female B10RIII mice were immunized with interphotoreceptor retinoid binding protein (IRBP) 161-180 in complete Freund's adjuvant and were immediately allowed to mate. Gross examination of the offspring gestated with EAU was performed to determine the influence of parental uveitis on offspring development after birth. Gene expression profiles were analyzed in the affected eyes of offspring under EAU to identify differentially expressed genes (DEGs). Adult offspring were given 5, 25, and 50 µg IRBP161-180 to compare their susceptibility to EAU. Immunized mice were clinically and pathologically evaluated for the development of EAU. Ag-specific T-cell proliferation and IL-17 production from spleens and lymph nodes were evaluated on day 14 or 35 after immunization. Results: Hair loss, delay of eye opening, and swollen spleens in the offspring from parents with uveitis were observed from day 14 to 39 after birth. DEGs were involved in the immune system process, muscle system process, and cell development. The altered antigen processing and presentation, cell adhesion molecules, and phagosome in the eyes of the offspring from uveitis-affected parents were enriched. Offspring gestated with EAU showed a susceptibility to EAU and an earlier onset and higher severity of EAU compared to the control group mice. IRBP-specific lymphocyte proliferation and IL-17 production were observed in the EAU offspring with exposure to parental uveitis. Conclusions: The results suggest that mouse parents with uveitis can increase their offspring's susceptibility to EAU, probably through altering cell adhesion molecules and antigen processing and presentation related to the T-cell proliferation and Th17 response.

EDA2R mediates podocyte injury in high glucose milieu.

EDA2R is a member of the large family of tumor necrosis factor receptor (TNFR). Previous studies suggested that EDA2R expression might be increased in the kidneys of diabetic mice. However, its mRNA and protein expression in kidneys were not analyzed; moreover, its role in the development of diabetic kidney disease was not explored. Here we analyzed the mRNA and protein expressions of EDA2R in diabetic kidneys and examined its role in the podocyte injury in high glucose milieu. By analysis with real-time PCR, Western blotting, we found that both the mRNA and protein levels of EDA2R were increased in the kidneys of diabetic mice. Immunohistochemical studies revealed that EDA2R expression was enhanced in both glomerular and tubular cells of diabetic mice and humans. In vitro studies, high glucose increased EDA2R expression in cultured human podocytes. Overexpression of EDA2R in podocytes promoted podocyte apoptosis and decreased nephrin expression. Moreover, ED2AR increased ROS generation in podocytes, while inhibiting ROS generation attenuates EDA2R-mediated podocyte injury. In addition, EDA2R silencing partially suppressed high glucose-induced ROS generation, apoptosis, and nephrin decrease. Our study demonstrated that high glucose increases EDA2R expression in kidney cells and that EDA2R induces podocyte apoptosis and dedifferentiation in high glucose milieu partially through enhanced ROS generation.

Patient-derived xenografts of different grade gliomas retain the heterogeneous histological and genetic features of human gliomas.

BACKGROUND: Gliomas account for the major part of primary brain tumors. Based on their histology and molecular alternations, adult gliomas have been classified into four grades, each with distinct biology and outcome. Previous studies have focused on cell-line-based models and patient-derived xenografts (PDXs) from patient-derived glioma cultures for grade IV glioblastoma. However, the PDX of lower grade diffuse gliomas, particularly those harboring the endogenous IDH mutation, are scarce due to the difficulty growing glioma cells in vitro and in vivo. The purpose of this study was to develop a panel of patient-derived subcutaneous xenografts of different grade gliomas that represented the heterogeneous histopathologic and genetic features of human gliomas. METHODS: Tumor pieces from surgical specimens were subcutaneously implanted into flanks of NOD-Prkdcscid ll2rgnull mice. Then, we analyzed the association between the success rate of implantation with clinical parameters using the Chi square test and resemblance to the patient's original tumor using immunohistochemistry, immunofluorescence, short tandem repeat analysis, quantitative real-time polymerase chain reaction, and whole-exome sequencing. RESULTS: A total of 11 subcutaneous xenografts were successfully established from 16 surgical specimens. An increased success rate of implantation in gliomas with wild type isocitrate dehydrogenase (IDH) and high Ki67 expression was observed compared to gliomas with mutant IDH and low Ki67 expression. Recurrent and distant aggressive xenografts were present near the primary implanted tumor fragments from WHO grades II to IV. The xenografts histologically represented the corresponding patient tumor and reconstituted the heterogeneity of different grade gliomas. However, increased Ki67 expression was found in propagated xenografts. Endothelial cells from mice in patient-derived xenografts over several generations replaced the corresponding human tumor blood vessels. Short tandem repeat and whole-exome sequencing analyses indicated that the glioma PDX tumors maintained their genomic features during engraftments over several generations. CONCLUSIONS: The panel of patient-derived glioma xenografts in this study reproduced the diverse heterogeneity of different grade gliomas, thereby allowing the study of the growth characteristics of various glioma types and the identification of tumor-specific molecular markers, which has applications in drug discovery and patient-tailored therapy.

PRKCQ rs4750316 is associated with Vogt-Koyanagi-Harada syndrome in a Han Chinese population.

Purpose: The PRKCQ and REL genes are said to be associated with multiple autoimmune diseases. This study investigated the association between these genes and Vogt-Koyanagi-Harada (VKH) syndrome in Han Chinese. Methods: A two-stage case-control study was performed on three single nucleotide polymorphisms ([SNPs] rs4750316, rs11258747, and rs947474) of the PRKCQ gene and three SNPs (rs842647, rs702873, and rs13031237) of the REL gene using PCR-restriction fragment length polymorphism (PCR-RFLPs) in a total of 859 patients with VKH syndrome and 1,542 healthy controls. Variables such as extraocular presentations were assessed. The data were analyzed using chi-square analysis, and corrected for multiple comparisons with the Bonferroni method. Results: We found a decreased frequency of the GC genotype and the C allele of rs4750316 in patients with VKH syndrome when the GG genotype or G allele was used as a reference, respectively (GC genotype: P =2.45e-10, odds ratio [OR]=0.37, 95% confidence interval [CI]=0.28-0.51; C allele: P=8.79e-10, OR=0.41, 95% CI=0.31-0.55). The genotypic and allelic frequencies of rs11258747, rs947474, rs842647, rs702873, and rs13031237 were not statistically significantly different between patients with VKH syndrome and controls. Stratification analysis indicated that the PRKCQ rs4750316 polymorphism was associated with patients with VKH syndrome experiencing headache, alopecia, poliosis, tinnitus, and dysacusia, but no statistically significant association of the other five SNPs was found. Conclusions: The PRKCQ rs4750316 polymorphism may be a susceptibility factor for VKH syndrome pathogenesis and extraocular presentations, indicating that PRKCQ may be involved in the pathogenesis and extraocular presentations of VKH syndrome through the T-cell receptor (TCR) signaling pathway.

[Erythropoietin accelerates the proliferation of glioma cells via activating Akt pathway].

OBJECTIVE: To determine whether erythropoietin (EPO) promotes rapid proliferation of glioma through Akt pathway. METHODS: We detected the expression of EPO in human glioma tissues using immunohistochemistry. A nude mouse model bearing human glioma U87 cell xenograft was established and given intraperitoneal injection of EPO or saline every other day, and the tumor growth was observed. In the in vitro experiment, U87 cells were treated with PBS (control), EPO, or EPO with Akt inhibitor, and the expression of p-Akt and cyclin D1 was detected using Western blotting; the cell proliferation rate was determined using cell counting kit-8 and clone formation assay, and the cell cycle changes were analyzed with flow cytometry. RESULTS: Compared with low-grade glioma tissues, high-grade glioma tissues exhibited a significantly increased EPO expression (P=0.0002). In the tumor-bearing mice, EPO treatment significantly increased the expression of EPO (P=0.0006) and p-Akt (P=0.0003) in the tumor and obviously increased the tumor volume (P<0.0001) and weight (P=0.0003). In U87 cells cultured in vitro, EPO treatment obviously accelerated the cell proliferation (P=0.020 on day 3 and 0.028 on day 5), promoted clone formation (P=0.0010), and increased proliferation index (P=0.0028); EPO significantly enhanced the protein expression of p-Akt (P=0.0020) and cyclin D1 (P=0.0022). The application of Akt inhibitor significantly suppressed the effect of EPO in enhancing cyclin D1 and p-Akt expression (both P<0.0001) and promoting cell proliferation. CONCLUSION: EPO can significantly accelerate the proliferation of glioma through Akt pathway.

Clinical and Pathologic Features of a Suspected Selenium Deficiency in Captive Plains Zebras.

Previous studies have shown that selenium (Se) deficiency is associated with nutritional myopathy, known as white muscle disease (WMD), in horses. However, correlations between Se deficiency and clinical findings, such as hematologic biochemical values and pathological features, have not been evaluated in captive plains zebras. The purpose of the present study was to investigate the clinical and pathologic features that may be caused by a Se deficiency in the captive plains zebra. Clinical findings, feed analyses, hematologic biochemical analyses, response to treatment, and pathologic examination were assessed in six affected plains zebras. The dietary concentration of Se in feed was also tested. Sudden death occurred in two cases during the first day of the onset of symptoms. Two zebras died at 4 days and two zebras survived after treatment. The clinical signs in affected animals were characterized by general weakness, astasia, and abnormal postural positions. The Se concentration in hay from the breeding stable was low, based on the reference value. Glutathione peroxidase (GSH-Px) activity was lower compared with the equine reference value. Multiple areas of subcutaneous steatitis and pale skeletal muscle and myocardium were revealed at gross necropsy. Degeneration and necrosis of myocardial and skeletal muscles, as well as congestion of the liver, lung, and kidney were found via histopathological examination. No suspected bacterial infections were found. Feed analyses, response to treatment, serum GSH-Px activity, and pathological features suggest that Se deficiency may have caused the disease in the six affected captive plains zebra.

Genetic Variation in the REL Gene Increases Risk of Behcet's Disease in a Chinese Han Population but That of PRKCQ Does Not.

Genome-wide association studies (GWAS) and candidate gene studies have identified the REL and PRKCQ genes as risk loci for various autoimmune diseases. The purpose of the present study was to investigate the association of the REL and PRKCQ genes with Behcet's disease (BD) in a Chinese Han population. A case-control study was conducted on three single nucleotide polymorphisms (SNPs), rs13031237, rs702873, and rs842647 of the REL gene and three SNPs (rs4750316, rs11258747, and rs947474) of the PRKCQ gene using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in a total of 623 BD patients and 1,074 healthy controls. Multiple variables were assessed, including age, sex distribution, and extra-ocular findings. In the present study, the frequencies of rs842647 GG genotypes and rs842647 G alleles were significantly higher in patients than in controls and those of the rs842647 AG genotypes were lower in patients than in controls [GG genotype: Bonferroni corrected P-value for gender adjustment (Pc(a)) = 0.0074, odds ratio (OR) = 1.63; G allele: Pc(a) = 0.0072, OR = 1.57; AG genotype: Pc(a) = 0.024, OR = 0.63, respectively]. No statistically significant differences in the frequencies of rs702873, rs13031237, rs4750316, rs11258747, and rs947474 between BD patients and controls were observed. Stratification analysis indicated that the REL rs842647 polymorphism was associated with BD patients with skin lesions. No significant association of the other five SNPs between BD patients with other extra-ocular findings, including genital ulcer, arthritis, and positive pathergy test results was found. The REL rs842647 polymorphism may be a susceptibility factor for BD pathogenesis and skin lesions, which indicate that c-Rel may be involved in the pathogenesis and skin lesions of BD through the NF-κB pathway.

[Establishment of an HBV chronic hepatitis B infection mouse model by vivo transduction of HBV cccDNA].

OBJECTIVE: To generate a mouse model of chronic hepatitis B (CHB) infection by performing in vivo transduction of hepatitis B virus (HBV) covalently closed circular (ccc)DNA. METHODS: Nude mice were injected with HBV cccDNA at doses of 1.5, 1.0 or 0.5 mug/ml. A control group was generated by giving equal injection volumes of physiological saline. The serum levels of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) on post-injection days 1 and 3, weeks 1-6, 8 and 10 were assayed by reflection immunoassay. At post-injection week 10, all animals were sacrificed and liver tissues were collected. Copies of HBV DNA in serum and liver tissue were detected by real-time PCR. HBV antigens in liver tissue were detected of by immunohistochemistry. Pathological analysis of liver tissue carried out with hematoxylin-eosin staining. Linear correlation of data was determined by statistical analysis. RESULTS: HBsAg and HBeAg were detected in sera from all three groups of cccDNA-injected mice staring at post-injection day 1 and lasting through week 10. The levels of HBsAg over the 10-week period showed two patterns of increase-decrease;the lowest level was detected at week 4 and the highest level was detected at week 8. In contrast, the levels of HBeAg over the 10-week period showed three patterns of increase-decrease; the lower levels were detected at weeks 2 and 4 and the higher levels at weeks 3 and 6. HBV DNA copies in liver tissues showed a cccDNA dose-dependent descending trend over the 10-week study period (1.5 mug/ml:1.14E+07 ± 6.51E+06 copies/g, 1.0 mug/ml:9.81E+06 ± 9.32E+06 copies/g, and 0.5 mug/ml:3.72E+06 ± 2.35E+06 copies/g; Pearson's r =0.979). HBV DNA copies in sera showed the pattern of 1.0 mug/ml cccDNA more than 1.5 mug/ml cccDNA more than 0.5 mug/ml cccDNA, and in general were higher than those detected in the liver tissues. Liver tissues from all cccDNA-injected mice showed positive immunohistochemistry staining for both HBsAg and HBeAg. HE staining showed that the liver tissues of all cccDNA-injected mice had severe fatty and vacuolar degeneration and less obvious structure of liver lobules (compared to the liver tissues from control mice). CONCLUSION: The CHB mouse model successfully established in this study by in vivo transduction of HBV cccDNA may represent a useful tool to study the pathogenic mechanisms and potential antiviral treatments of human CHB.

Replication study confirms the association between UBAC2 and Behçet's disease in two independent Chinese sets of patients and controls.

INTRODUCTION: The purpose of this study was to replicate genetic factors associated with the susceptibility to Behçet's disease (BD). We conducted a two-stage candidate genes association and functional study, involving 477 BD patients and 1,334 normal controls of Chinese Han descent. METHODS: The genotyping of five candidate genes/loci, including LOC100129342, KIAA1529, CPVL, UBASH3B and UBAC2, were performed using TaqMan single nucleotide polymorphism (SNP) assays. Real-time PCR and luciferase reporter assay were performed to test the function of the identified promoter polymorphism. The main outcome measures were genotype frequencies and expression levels in BD patients. RESULTS: The first-stage study results showed that UBAC2 (rs9513584, Pc = 0.018, OR = 1.4), but not LOC100129342, KIAA1529, CPVL, UBASH3B was associated with the susceptibility to BD in Chinese Han. The fine-mapping association study of UBAC2 identified six risk SNPs for BD in the Chinese cohort; three of them were verified in validation study (rs3825427, first-stage Pc = 2.2 × 10-3, second-stage Pc = 9.3 × 10-3, combined Pc = 6.9 × 10-6; rs9517668, first-stage Pc = 1.7 × 10-3, second-stage Pc = 0.03, combined Pc = 3.3 × 10-4; rs9517701, first-stage Pc = 5.1 × 10-3, second-stage Pc = 9.0 × 10-3, combined Pc = 2.9 × 10-5; respectively). Functional analysis showed that the risk T allele of the promoter polymorphism rs3825427 had a significantly lower promoter activity than the non-risk G allele (P = 0.002) and a decreased expression of UBAC2 transcript variant 1 in peripheral blood mononuclear cells (PBMCs) and skin of normal controls carrying the risk T allele than that in individuals with the G allele (P = 0.045, P = 0.025; respectively). The mRNA expression of UBAC2 transcript variant 1 was significantly decreased in PBMCs and skin of BD patients as compared with controls (P = 0.025; P = 0.047, respectively). The mRNA expression of UBAC2 transcript variant 2 was significantly increased in skin of BD patients as compared with controls (P = 0.004). CONCLUSIONS: This study replicates a predisposition gene to BD, UBAC2, and suggests that UBAC2 may be involved in the development of BD through its transcriptional modulation.

CD40 gene polymorphisms confer risk of Behcet's disease but not of Vogt-Koyanagi-Harada syndrome in a Han Chinese population.

OBJECTIVES: Recent genetic surveys including a genome-wide association study have identified CD40 as a susceptibility gene for several autoimmune diseases. This study was designed to investigate the association of CD40 gene polymorphisms with Behçet's disease (BD) and Vogt-Koyanagi-Harada (VKH) syndrome in a Han Chinese population. METHODS: Two single nucleotide polymorphisms (SNPs), rs4810485 and rs1883832, were genotyped using polymerase chain reaction-restriction fragment length polymorphism in 373 BD patients, 519 VKH patients and 402 controls. A binary logistic regression analysis was applied to assess the influence of gender on the association of CD40 polymorphisms with BD. RESULTS: Our results showed significantly increased frequencies of the homozygous rs4810485 TT and rs1883832 TT in BD patients as compared with the controls [Bonferroni-corrected P-value for gender adjustment (Pac) = 0.006, odds ratio (OR) = 1.98, 95% confidence interval (CI) 1.38, 2.83; = 0.012, OR = 1.73, 95% CI 1.22, 2.46, respectively]. A markedly decreased frequency of the heterozygous rs4810485 GT was observed in BD patients as compared with the controls ( = 0.042, OR = 0.68, 95% CI 0.51, 0.90). The genotype and allele frequencies of rs4810485 and rs1883832 were not different between VKH patients and controls. Stratification analysis did not find any association between the tested SNPs and extra-ocular manifestations of both diseases. CONCLUSIONS: The results suggest that TT genotypes of rs4810485 and rs1883832 may be predisposing genotypes for BD, and that the rs4810485 GT genotype may be a protective genotype for BD. The two tested CD40 gene polymorphisms are not associated with VKH syndrome in the investigated Han Chinese population.

CD40 polymorphisms in Han Chinese patients with Fuch uveitis syndrome.

PURPOSE: Polymorphisms of the cluster of differentiation 40 (CD40) gene have recently been identified to be associated with the risk to several immune diseases. The aim of this study was to determine the potential association of CD40 polymorphisms with Fuch uveitis syndrome (FUS). METHODS: A total of 131 Han Chinese patients with FUS and 402 healthy controls were genotyped using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Genotype counts in patients and controls were analyzed by the χ(2) test. RESULTS: All genotypic and allelic frequencies of the tested two CD40 polymorphisms were in Hardy-Weinberg equilibrium. The genotypic and allelic frequencies of rs4810485 and rs1883832 were not different between patients with FUS and controls. No influence of sex could be found following stratification analysis according to gender. CONCLUSIONS: Our results suggest that the two investigated single-nucleotide polymorphisms (SNPs), rs4810485 and rs1883832, in CD40 are not associated with FUS in the Han Chinese population.

Polymorphisms of IL23R and Fuchs' syndrome in a Chinese Han population.

PURPOSE: The aim of the study was to investigate the association of polymorphisms of the interleukin-23 receptor (IL23R) gene with Fuchs' syndrome in a Chinese Han population. METHODS: Three single-nucleotide polymorphisms (SNPs), rs7517847, rs11209032 and rs17375018 of IL23R were genotyped in 138 Chinese Han patients with Fuchs' syndrome and 407 healthy controls by using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Data were analyzed by χ(2) analysis. RESULTS: All genotype and allele distributions in patients with Fuchs' syndrome and healthy controls were in Hardy-Weinberg equilibrium. The frequency of the rs11209032 AA genotype was significantly increased in patients with Fuchs' syndrome as compared to controls (corrected p [pc]=0.036, OR 1.86, 95%CI 1.21 to 2.86). There were no statistically significant differences between patients and healthy controls concerning the other two tested SNPs (rs17375018 and rs7517847). The haplotypes of the tested SNPs were not different between patients and controls. Additionally, analysis according to gender did not show any influence of sex on the association of IL23R with Fuchs' syndrome. CONCLUSIONS: Our results suggested that the rs11209032 AA genotype of the IL23R gene may predispose for Fuchs' syndrome in Chinese patients.

Human malignant glioma cells expressing functional formylpeptide receptor recruit endothelial progenitor cells for neovascularization.

Endothelial progenitor cells (EPCs) are involved in tumor neovascularization with undefined mechanisms. In this study, we explored the role of formylpeptide receptor, a G protein-coupled receptor, expressed by human malignant glioma cells in neovascularization of malignant glioma. EPCs were isolated from human umbilical cord blood and their migratory capability and tubulogenesis induced by the supernatant of U87 glioblastoma (GBM) cell line were examined. We also assessed the recruitment and incorporation of EPCs into orthotopic intracranial tumors formed by implanted U87 GBM cells. The supernatant of control U87 cells induced high levels of migration and tubule-formation in vitro by EPCs. In contrast, the chemotactic and tubule-stimulating activities on EPCs in the supernatant of U87 cells with FPR knocking down by small interference (si) RNA were significantly attenuated. In addition, the number of EPCs recruited and incorporated into intracranial glioma xenografts was significantly higher in tumors formed by control U87 cells than tumors formed by U87 cells containing FPR-siRNA. Our results suggest that expression of functional FPR in glioma cells plays an important role in regulating vasculogenesis by EPCs, which constitute a novel target for anti-angiogenic therapy in gliomas.