[Investigation and research on ticks carrying spotted fever group rickettsia in the border area of Tumen River Basin].

Objective: To understand the situation and genotype distribution of spotted fever group rickettsia (SFGR) in the border area of Tumen River Basin in free ticks in Yanbian Korean Autonomous Prefecture (Yanbian Prefecture), Jilin Province. Methods: From April to September, 2017, ticks were collected using flagging method from Hunchun, Tumen, Helong and Longjing cities in the Tumen River basin of Yanbian Prefecture. Outer membrane protein A (ompA) was detected by Polymerase Chain Reaction (PCR), then, the species were identified by gene sequencing and analyzed systematically. The positive rate of pools and MIR(minimum infection rate per 100 ticks,MIR) of SFGR were calculated, and the difference of positive rate of pools among ticks with different characteristics was compared by Chi-square test. Results: A total of 3 079 ticks were collected and divided into 536 pools. The positive rate of pools of SFGR nucleic acid was 39.7% (213 pools). The MIR of SFGR was 6.9%.The positive rate of pools of SFGR in Dermacentor silvarum, Haemaphysalis concinna, Haemaphysalis japonica, Haemaphysalis longicornis and Ixodes persulcatus were 80.4% (41/51), 14.0% (25/179), 20.2% (18/89), 78.9% (101/128) and 25.9% (21/81), and the difference was statistically significant (P<0.001). There was statistical difference in the positive rate of pools of SFGR in developmental stages of ticks (P<0.001); the positive rate of pools of female adults, male adults, nymph and larvae were 36.4% (95/261), 34.2% (67/196), 56.3% (40/71) and 7/8, and the MIR was 7.9%, 7.7%, 4.9% and 3.5%. The five genotype was detected which was Candidatus Rickettsia longicornii, Rickettsia raoultii, Rickettsia heilongjiangensis, Candidatus Rickettsia tarasevichiae,Rickettsia monacensis and have 98%-100% homology with known gene sequences. Candidatus Rickettsia longicornii, Rickettsia raoultii, Rickettsia heilongjiangensis and Candidatus Rickettsia tarasevichiae showed close evolutionary relationship with known specie (have 98%-100% homology with known gene sequences); Rickettsia monacensis showed Far from evolutionary relationship with known species (have 98% homology with known gene sequences). Conclusion: SFGR infection of ticks is common in the border areas of the Tumen River Basin. There was high diversity in SFGR species and tick species in the areas surveyed.

Hepatitis C virus genotype diversity in Ningxia Hui autonomous region, Northwestern China.

Hepatitis C virus (HCV) genotypes vary greatly in different regions. The aim of this study is to investigate the distribution of HCV genotypes in HCV infected patients, in Ningxia Hui Autonomous Region. Nucleic acid extraction and amplification were performed with test kits on 153 HCV infected patients serum samples. The HCV viral load was measured using reverse transcriptase PCR (RT-PCR) and HCV genotypes were determined. Among the 153 HCV-infected patients, 56 had genotype (GT)1b (36.60%), 45 had GT2a (29.40%), 23 had GT3a (15.00%), 14 had GT3b (9.20%),13 had GT6a (8.50%), 1 had GT1g (0.70%), 1 had GT6xa (0.70%). In GT1b, 21.40% were female and 78.60% were male; in GT2a, 42.20% were female and 57.80% were male;Males were most prevalent in genotypes 1b(39.30%), while female were most prevalent in genotype 2a(46.30%). Rare GT1g and GT6xa were also detected in males. The 41-50 year age group had the highest HCV prevalence of 32.00%. HCV GT1b is the predominant HCV genotype in Ningxia Hui Autonomous Region.

IL-21 modulates CD4+ CD25+ regulatory T-cell homeostasis in experimental autoimmune encephalomyelitis.

The homeostasis of CD4+ CD25+ regulatory T cells (Tregs) depends on the cytokine interleukin (IL)-2. As IL-21 shares sequence homology with IL-2 and the IL-21 receptors contain a gamma-chain common to IL-2, we hypothesized that IL-21 could also affect the homeostasis of Tregs. We tested this hypothesis in experimental autoimmune encephalomyelitis (EAE), an animal model of relapsing-remitting human multiple sclerosis. We show that blockade of IL-21 in SJL/J mice before and after the induction of EAE enhances the influx of inflammatory cells into the central nervous system (CNS). The blockade of IL-21 leads to proliferation of proteolipid peptide (PLP(139-151))-autoreactive CD4+ T cells, which are capable to cause severe EAE in adoptively transferred recipient mice. Conversely, Tregs from mice where IL-21 was blocked, lose their capacity to prevent EAE induced PLP(139-151)-reactive T cells. Notably, direct effects of IL-21 on Tregs are confirmed by studies of blockade of IL-21 in mice expressing a green fluorescent protein 'knocked' into a Foxp3 allele, in which a reduction of the number of Tregs and a downregulation of their frequency and expression of Foxp3 are observed. These data suggest a role of the IL-21/IL-21R axis in the homeostasis of Tregs in CNS autoimmunity.

Induction of G(2)/M phase arrest and apoptosis by a new synthetic anti-cancer agent, DW2282, in promyelocytic leukemia (HL-60) cells.

We studied the effect of DW2282-,[(S)-(+)-4-phenyl-1-[N-(4-aminobenzoyl)-indoline-5-sulfonyl-4,5-dihydro-2-imidazolone].hydrochloride], a newly developed anti-cancer agent, on cell proliferation, cell cycle progression, and induction of apoptosis in human promyelocytic leukemia (HL-60) cells. DW2282, a diarylsulfonylurea compound, was cytotoxic to HL-60 cells, with an IC(50) of 1.0 microg/mL. Treatment with DW2282 fragmented DNA in a concentration- and time-dependent manner, suggesting that these cells underwent apoptosis. Flow cytometric analysis further confirmed that DW2282-treated HL-60 cells were hypodiploid, in terms of DNA content, and were arrested at the G(2)/M phase. The cell cycle arrest was reversible upon the removal of DW2282. HL-60 cells also underwent distinct morphological changes in response to DW2282 treatment, including the appearance of elongated cells with conical tails and other apoptotic characteristics. G(2)/M phase cell cycle arrest was accompanied by a decrease in the levels of cdc2, a protein that plays a critical role for progression through the G(2)/M phase. Treatment of HL-60 cells with DW2282 was also associated with decreased levels of the anti-apoptotic protein Bcl-2, activation of caspase-3, and proteolytic cleavage of poly(ADP-ribose) polymerase. Taken together, these results demonstrate that DW2282 dramatically suppressed HL-60 cell growth by inducing apoptosis after G(2)/M phase arrest. These findings are consistent with the possibility that G(2)/M phase arrest was mediated by the down-regulation of cdc2 levels in HL-60 cells. The data also suggest that DW2282 triggered apoptosis by decreasing Bcl-2 levels and activating caspase-3 protease. These results provide important new information towards understanding the mechanisms by which DW2282 and other diarylsulfonylureas mediate their therapeutic effects.

Hyper-methylation of RIZ1 tumor suppressor gene is involved in the early tumorigenesis of hepatocellular carcinoma.

The retinoblastoma protein-interacting zinc finger gene RIZ1 is a putative tumor suppressor gene, and the inactivation of the RIZ1 is frequently found in tumors through a loss of mRNA expression. In order to understand the role of RIZ1 inactivation in the tumorigenesis of hepatocellular carcinoma (HCC), we detected the RIZ1 promoter methylation status in 39 HCCs using a methylation specific PCR (MSP) method, and carried out LOH study with marker P704. We also assessed the associations between the methylation status and clinicopathological parameters, tumor size, tumor differentiation, and fractional allelic loss (FAL). The results showed that the RIZ1 promoter methylated both in advanced tumors (>3 cm), (18/31, 58.0%) and in early tumors (<3 cm), (4/8, 50.0%). There were 54.6% (12/22) tumors with hyper-methylation in the low FAL group and 45.5% (10/22) in the high FAL group. Moreover, the DNA methylation of the RIZ1 promoter was found not only in the poorly differentiated tumors (12/22, 54.6%), but also in the well differentiated tumors (10/22, 45.5%). Among the 22 HCCs (22/39, 56.4%) that showed hyper-methylation at the RIZ1 promoter region, 3 cases showed biallelic methylation. Interestingly, one case showed hyper-methylation on one allele and a loss of heterozygosity (LOH) on the other allele. In other words, 4 HCCs showed the biallelic inactivation of the RIZ1. These results suggest that the inactivation of the RIZ1 by DNA methylation at its promoter region is involved in the tumorigenesis of HCC, particularly in the early stage of disease.

CD4(+)CD25(+) regulatory T cells contribute to the therapeutic effects of glatiramer acetate in experimental autoimmune encephalomyelitis.

CD4(+)CD25(+) regulatory T cells (Tregs) are potent immunosuppressors that are pivotal in the maintenance of self-tolerance. The involvement of Tregs in therapies for immune-mediated diseases has been proposed, but direct supporting evidence is still lacking. While investigating mechanisms underlying the clinical benefits of glatiramer acetate (GA) in an animal model of multiple sclerosis (MS), i.e., experimental autoimmune encephalomyelitis (EAE), we recently demonstrated that GA can protect mice deficient in the Th(2) cytokines IL-4, IL-10 and IL-4/IL-10 from acquiring EAE, suggesting that mechanisms other than Th(2) cells may be responsible for the therapeutic effects of GA. Here we demonstrate that GA treatment boosts the expression of Foxp3 on Tregs during EAE. Furthermore, adoptive transfer of purified Tregs from GA-treated EAE mice is more effective in preventing EAE development than Tregs from untreated EAE controls. Thus, our current data provide evidence that Tregs may be the major contributor to GA's therapeutic action in EAE and, possibly, MS. Further mechanistic studies to reveal the molecular events linking GA with Tregs may optimize GA treatment and lead to the development of new, even more effective therapies that utilize this mechanism of action.

Dynamic analysis of hepatitis B virus DNA and its antigens in 2.2.15 cells.

The 2.2.15 cells-derived from HepG2 cells transfected with a plasmid containing hepatitis B virus (HBV) DNA secrete surface antigen (HBsAg) particles, nucleocapsids and virions (Proc Natl Acad Sci U S A 1987; 84: 1005-1009). The latter elicit acute hepatitis in chimpanzees (Proc Natl Acad Sci U S A 1987; 84: 4641-4644). We studied the presence of intracellular and extracellular HBV covalently closed circular (ccc) DNA in this culture system by polymerase chain reaction (PCR), kinetically analysed HBsAg and hepatitis B e antigen (HBeAg) released in the culture media by quantitative enzyme-linked immunosorbent assay and quantitated by real-time PCR but HBV DNA from intracellular and extracellular HBV-DNA. HBV cccDNA was found both intracellularly and extracellularly. A significant correlation was seen between the extracellular HBV DNA levels and virus antigens (r = 0.833; P = 0.01 and r = 0.939; P < 0.01 for HBsAg and HBeAg, respectively), whereas there was no statistical correlation between intracellular HBV DNA levels and virus antigen levels (r = 0.024; P = 0.955 and r = 0.177; P = 0.625 for HBsAg and HBeAg, respectively). These data would be valuable in studies of the HBV life cycle and of potential anti-viral agents.