Behavior of the dengue virus in solution

The dengue virus consists of four antigenically related but distinct viruses, termed Dengue virus 1-4 (DEN 1-4). We have established that the dengue virus loses infectivity over time in solution in an exponentially declining manner. The four strains examined (one from each serotype) have half-lives that range from 2.5 to 7.5 hr in defined medium. The half-life is temperature and pH-dependent and is affected by the nature of the host cell in which it is produced, but is not dependent upon the presence of either Mg(2+) ions or chelating agents. Electron microscopy (EM) of solutions of the dengue virus show almost complete virus aggregation after 24 hr at room temperature, while RT-PCR shows an intact RNA genome. These results show that the solution environment of the dengue virus is an important determinant of dengue virus infectivity.     

角膜胶原交联术治疗圆锥角膜的疗效和安全性研究

目的：研究角膜胶原交联术治疗圆锥角膜的疗效和安全性。

方法：对2015-04/2018-08在泰国朱拉隆功国王纪念医院行角膜胶原交联术的圆锥角膜患者病历进行回顾性分析。评估术前和术后1a的视力、屈光度、角膜地形图、高阶像差(HOA)、地形图参数和角膜密度。根据患者年龄是否小于24和30岁、基线角膜最大曲率(Kmax)是否小于55 D、基线最佳矫正视力(BCVA)是否小于0.3 LogMAR分组评估年龄、基线Kmax和BCVA对手术疗效的影响。分析术前Kmax、Kmean、平均等效球镜度数(MRSE)、视力、角膜最薄点厚度值、Kmax的变化以及相关参数的变化与角膜密度测量值变化之间的关系。P<0.05具有统计学意义。

结果：共155例患者185眼纳入研究,其中119例男性,36例女性。根据Amsler-Krumeich进行分类,1期和2期占优势(分别为37.84%和35.14%)。术后1a,平均裸眼视力(UCVA)提高0.1 LogMAR(P<0.05)。与基线BCVA较好组(术前BCVA<0.3 LogMAR)相比,基线BCVA较差组(术前BCVA≥0.3 LogMAR)术后BCVA改善大于0.2 LogMAR的眼数较多(78.26% vs 21.74%,P<0.05)。平均Kmax比基线下降2.36 D(P<0.05)。术前Kmax≥55 D的患眼术后Kmax下降超过2.0 D的眼数占比73%。距角膜顶点6 mm处角膜HOA下降0.40(P<0.05)。术后1mo～1a,0～6 mm区角膜密度测量值持续增加。术后1a,角膜密度的增加与最薄点厚度的减少呈线性相关。表面变异指数、高度非对称性指数、圆锥角膜指数、高度轴偏心指数在术后1a时下降(P<0.05)。术后1a,手术成功率为90.24%。术后1wk、1、3、6mo、1a角膜混浊发生率分别为11.35%、30.27%、15.67%、10.27%、2.16%。无角膜水肿发生,但有1例无菌性角膜炎患者。

结论：角膜胶原交联术可有效治疗圆锥角膜,使角膜变平、重塑,提高视力、HOA和角膜形态指数,晚期圆锥角膜Kmax也明显降低。     

Glycyrrhizin inhibits highly pathogenic H5N1 influenza A virus-induced pro-inflammatory cytokine and chemokine expression in human macrophages

Hypercytokinaemia is thought to contribute to highly pathogenic H5N1 influenza A virus disease. Glycyrrhizin is known to exert immunomodulatory and anti-inflammatory effects and therefore a candidate drug for the control of H5N1-induced pro-inflammatory gene expression. Here, the effects of an approved parenteral glycyrrhizin preparation were investigated on H5N1 virus replication, H5N1-induced pro-inflammatory responses, and H5N1-induced apoptosis in human monocyte-derived macrophages. Glycyrrhizin 100 μg/ml, a therapeutically achievable concentration, impaired H5N1-induced production of CXCL10, interleukin 6, and CCL5 and inhibited H5N1-induced apoptosis but did not interfere with H5N1 replication. Global inhibition of immune responses may result in the loss of control of virus replication by cytotoxic immune cells including natural killer cells and cytotoxic CD8⁺ T-lymphocytes. Notably, glycyrrhizin concentrations that inhibited H5N1-induced pro-inflammatory gene expression did not affect cytolytic activity of natural killer cells. Since H5N1-induced hypercytokinaemia is considered to play an important role within H5N1 pathogenesis, glycyrrhizin may complement the arsenal of potential drugs for the treatment of H5N1 disease.     

Comparison of pro-inflammatory cytokine expression and cellular signal transduction in human macrophages infected with different influenza A viruses

Influenza A virus infection of macrophages and virus-induced pro-inflammatory gene expression are regarded to contribute to severity of influenza A virus-caused diseases. Although some data are available on cytokine production by influenza A virus-infected macrophages, systematic comparisons of the virus types are currently considered to be of high relevance in humans (pandemic H1N1/2009, seasonal H1N1, seasonal H3N2, highly pathogenic avian influenza H5N1) on pro-inflammatory potential, and relevant underlying cellular signalling events are missing. Here, we show that the infection of human monocyte-derived macrophages with pandemic H1N1/2009 (A/HH/01/2009), seasonal H1N1/1999 (A/New Caledonia/20/99), seasonal H3N2/2004 (A/California/7/2004) or highly pathogenic H5N1/2004 (A/Thailand/1(Kan-1)/04) results in similar infection rates. However, the investigated H1N1 strains caused delayed and decreased apoptosis in comparison with H3N2/2004 or H5N1/2004. Moreover, human macrophage infection with H3N2/2004 or H5N1/2004 but not with H1N1 viruses was associated with pronounced pro-inflammatory cytokine production and activation of relevant mitogen-activated protein kinase pathways as indicated by phosphorylation of p38, JNK and ERK 1/2. These findings are in line with clinical observations indicating enhanced disease severity in H3N2- or H5N1-infected patients compared to individuals infected with pandemic H1N1/2009 or seasonal H1N1.     

Interaction of folate-conjugated human serum albumin (HSA) nanoparticles with tumour cells

Folic acid has been previously demonstrated to mediate intracellular nanoparticle uptake. Here, we investigated cellular uptake of folic acid-conjugated human serum albumin nanoparticles (HSA NPs). HSA NPs were prepared by desolvation and stabilised by chemical cross-linking with glutaraldehyde. Folic acid was covalently coupled to amino groups on the surface of HSA NPs by carbodiimide reaction. Preparation resulted in spherical HSA NPs with diameters of 239 ± 26 nm. As shown by size exclusion chromatography, 7.40 ± 0.90 μg folate was bound per mg HSA NPs. Cellular NP binding and uptake were studied in primary normal human foreskin fibroblasts (HFFs), the human neuroblastoma cell line UKF-NB-3, and the rat glioblastoma cell line 101/8 by fluorescence spectrophotometry, flow cytometry, and confocal laser scanning microscopy. Covalent conjugation of folic acid to HSA NPs increased NP uptake into cancer cells but not into HFFs. Free folic acid interfered with cancer cell uptake of folic acid-conjugated HSA NPs but not with uptake of folic acid-conjugated HSA NPs into HFFs. These data suggest that covalent linkage of folic acid can specifically increase cancer cell HSA NP uptake.     

Detection of Babesia bovis in cattle by PCR-ELISA

We established a new highly sensitive method, PCR-ELISA, for the dectection of Babesia bovis in cattle for farms in Thailand. The detection of around 2.4 x 10(-8)% parasitemia (equivalent to 1 infected erythrocyte per 2 ml) was achieved by PCR amplification followed by the ELISA detection of a biotin tagged gene. When comparing the sensitivity of PCR-ELISA with the microscopic method, our PCR-ELISA method is more sensitive than thin blood smears by at least 1,000 times. The established PCR-ELISA also showed high specificity to B. bovis with no cross reaction to other endemic parasites except for A. marginale. Regarding the detection threshold for B. bovis, the PCR-ELISA method could detect parasites inoculated into splenectomized calves at least 1 week earlier than the thin blood microscopic method. The PCR-ELISA method is a valuable screening technique for B. bovis and applicable for the routine detection of carrier states and automated analysis.