Studies on the integration of hepatitis B virus DNA sequence in human sperm chromosomes

Aim: To study the integration of hepatitis B virus (HBV) DNA into sperm chromosomes in hepatitis B patients and the features of its integration. Methods: Sperm chromosomes of 14 subjects (5 healthy controls and 9 HB patients, including 1 acute hepatitis B, 2 chronic active hepatitis B, 4 chronic persistent hepatitis B, 2 HBsAg chronic carriers with no clinical symptoms) were prepared using interspecific in vitro fertilization between zona-free hamster oocytes and human spermatozoa. Fluorescence in situ hybridization (FISH) to sperm chromosome spreads was carried out with biotin-labeled full length HBV DNA probe to detect the specific HBV DNA sequences in the sperm chromosomes. Results: Specific fluorescent signal spots for HBV DNA were seen in sperm chromosomes of one patient with chronic persistent hepatitis B. In 9(9/42) sperm chromosome complements containing fluorescent signal spots, one presented 5 obvious FISH spots and the others 2 to 4 signals. The fluorescence intensity showed significant diffe     

Fertilization of hamster ova by human spermatozoa in relation to other semen parameters

Different indices of the zona-free hamster ovum test system were interrelated. High correlations were found between the fertilization percentage and the average number of penetrated spermatozoa/ovum (r = 0.99) and between the fertilization percentage and the number of spermatozoa attached to the ova surfaces (r = 0.72). Fertilization percentages from 63 subfertile patients were correlated with different semen factors assessed by multiple exposure photography (MEP). Low correlations were found between sperm concentration and fertilization percentage (r = 0.29) and between fertilization percentage and motile sperm count (r = 0.33). Spermatozoa progressing with high average velocity had low fertilization rates compared to specimen with moderate average velocity. Effects of sperm washing on the in vitro fertilizing capacity were studied in a group of 40 subfertile men. The fertilization percentages of 8 specimens -with visual seminal plasma abnormalities- increased significantly when immediate dilution of semen was applied.     

The occupational hazard of human immunodeficiency virus and hepatitis B virus infection

We have conducted a postal survey of members of the Association of Anaesthetists to ascertain perceived risks and preventive measures adopted with regard to the occupational hazard of Human Immunodeficiency Virus and Hepatitis B Virus infection. Despite recognition of the infection risk and the adoption of appropriate measures when managing known infected patients, the majority of anaesthetists have not implemented simple precautions in their daily routine work. Less than 16% of respondents routinely wear gloves and more than one in three still resheath needles. It would appear that the recommendations of the Association with regard to universal safety precautions have not been implemented by the majority of its members.     

The occupational hazard of human immunodeficiency virus and hepatitis B virus infection

The effect of grade, age, sex and region of employment on the attitude of anaesthetists to the possible risk of Human Immunodeficiency Virus (HIV) and Hepatitis B Virus (HBV) infection and the measures adopted to minimise the risk were assessed. As a group, anaesthetists in training were more concerned than consultants about the risk of HIV or HBV infection and, as a consequence, were more likely to adopt protective measures. A similar variation was seen with age, younger anaesthetists being more concerned about the risk of infection and adopting preventive measures in greater numbers than their older colleagues. The sex of the anaesthetist had minimal effect on their attitude. Despite the marked variation in the incidence of both HIV and HBV, the attitude of anaesthetists to the risk of infection and the numbers adopting simple preventive measures did not vary significantly on a regional basis throughout the country. However, there was a significant inter-regional variation in the availability and uptake of HBV immunisation (p < 0.01) and knowledge of the existence of local policy guidelines for the management of known HIV or HBV positive patients (p < 0.01).     

Sperm ultrastructure and meiotic segregation in a group of patients with chronic hepatitis B and C

Little is known about the effect of chronic hepatitis B and hepatitis C on sperm quality. In this study we analysed sperm quality from selected patients with chronic hepatitis B virus (HBV) or hepatitis C virus (HCV) infections. Semen samples were examined by light and transmission electron microscopy (TEM). TEM data were elaborated with a mathematical formula able to indicate a fertility index and the presence of the three main sperm pathologies: apoptosis, immaturity and necrosis. Meiotic chromosome segregation was investigated by fluorescence in situ hybridisation carried out on sperm nuclei, using probes for chromosomes 18, X and Y. Despite normal sperm concentration, we observed reduced motility. TEM analysis highlighted that 35.7% of patients showed generally good semen quality. However, significantly higher values of apoptosis and necrosis, compared with controls, were observed, demonstrating spermatogenetic alterations. Regarding meiotic segregation, we found an incidence of disomies similar to that observed in control samples, whereas diploidy resulted higher in HCV patients, without reaching statistical significance. In conclusion, sperm quality in the studied group was not impaired, however, apoptosis and necrosis resulted out of normal range and the fertility index was significantly lower in HCV and HBV infected patients versus controls.     

核苷（酸）类似物应答不完全慢性乙型肝炎患者HBV多聚酶区耐药基因分析

核苷（酸）类似物的应用开启了慢性乙型肝炎（CHB）治疗新的里程碑,使大多数久治不愈CHB患者的病情得到了有效的控制。然而,随着核苷（酸）类似物疗程的延长,病毒变异和耐药成为临床棘手的问题。笔者对初次治疗应用恩替卡韦（ETV）12个月以上,或拉米夫定（LAM）初治失败后加用或改用其他核苷（酸）类似物序贯或联合治疗病毒学应答不充分的21例患者的HBV多聚酶区（P区）的耐药基因进行了研究和分析,以期进一步了解和研究核苷（酸）类似物治疗HBV变异和耐药的相关问题,为探索减少HBV变异和耐药的有效方法提供依据。     

The existence and significance of hepatitis B virus DNA in glomerulonephritis

Summary: In order to investigate the role of hepatitis B virus (HBV) in the pathogenesis of glomerulonephritis 50 cases of glomerulonephritis with HBV antigenaemia and/or hepatitis B antigen (HBAg) detected by immunohistochemistry in renal tissue were collected. the distribution and localization of HBV DNA were observed by using in situ hybridization. In addition, Southern blot analysis was performed on 23 of the 50 cases in order to reveal the state of renal HBV DNA. Thirty-six cases (72%) were found to be HBV DNA positive by in situ hybridization, which localized in the nucleus of tubular cells. In 26 cases HBV DNA was detectable in the nucleus of glomerular mesangial and epithelial cells as well as the mesangial matrix. Seventeen of the 23 cases were proved to be HBV DNA positive in Southern blot analysis (82%). Three of these cases were identified with non-replicating free HBV DNA, while 14 cases were the integrated form. the results of this study showed that the renal tissue was infected with HBV; however, it was considered that it may be possible that the HBAg deposited on glomeruli was not only from circulation but also from the HBV infected glomerular cells although the evidence of this is not conclusive. In addition to the humoral immune injury mediated by HBAg-hepatitis B antibody (HBAb) immune complexes the cellular immune injury mediated by target antigen (hepatitis B core antigen; HBcAg) might be also involved in the pathogenesis of HBV glomerulonephritis (GN) associated GN.     

Separation of human spermatozoa with hyaluronic acid induces, and Percoll® inhibits, the acrosome reaction

The effect on the acrosome reaction of human spermatozoa of two widely used sperm separation media, hyaluronic acid (Sperm Select®) and Percoll®, was studied. Viable and highly motile fractions of human spermatozoa were separated from seminal plasma using self-migration on a Percoll® gradient. After translocation of separated spermatozoa from the Percoll® solution to a culture medium, serum, Percoll® or hyaluronic acid (Sperm Select®) was added to aliquots of the spermatozoa containing culture medium. At increasing time intervals, the influx of ⁴⁵Ca²⁺ into spermatozoa was measured and the concentration of viable spermatozoa that had undergone the acrosome reaction was analysed using the triple stain technique. Serum was found to be necessary to support sperm motility and viability. Compared to culture medium with serum only, addition of hyaluronic acid induced influx of ⁴⁵Ca²⁺ and the acrosome reaction, whilst Percoll® inhibited both of these actions. Hyaluronic acid (Sperm Select®) added to spermatozoa separated by a 'swim-up' method induced, and the addition of Percoll® inhibited, influx of ⁴⁵Ca²⁺ when compared to the addition of culture medium with serum only. This study demonstrates that both hyaluronic acid (Sperm Select®) and Percoll® affect the acrosome reaction and the prerequisite for Ca²⁺ influx in human spermatozoa. These effects should be taken into consideration when using these media for preparation of spermatozoa for insemination or for fertilization in vitro.     

原位杂交检测乙肝病毒携带者精子中乙肝病毒-DNA的研究

目的通过检测乙肝病毒携带者经辅助生育技术洗涤后的精子是否携带有乙型肝炎病毒,从而判断是否存在经精子传播乙型肝炎的可能性。方法用地高辛标记,针对乙型肝炎病毒DNA(HBV-DNA)设计的探针,对来我院要求行辅助生育治疗的20例乙肝病毒携带者的精子,经洗涤离心上游后进行检测。结果发现8例携带者精子中有乙肝病毒DNA,分布于精子的头部,阳性精子计数占每片精子总数的3‰～10‰。结论经辅助生育技术洗涤后的乙肝病毒携带者的精子仍可传播HBV-DNA。     

Transplantation of human cells in the peritoneal cavity of immunodeficient mice for rapid assays of hepatitis B virus replication

Kumar M, Bandi S, Cheng K, Gupta S. Transplantation of human cells in the peritoneal cavity of immunodeficient mice for rapid assays of hepatitis B virus replication. Xenotransplantation 2011; 18: 380–389. © 2011 John Wiley & Sons A/S. Abstract: Background: Studies of natural hepatitis B virus infection must be restricted to humans or primates due to viral species‐specificity. Alternative hepadnavirus animal models, e.g., woodchuck hepatitis virus in captive woodchucks, are not convenient, while in transgenic mice hepatitis B virus or viral proteins are expressed permanently through integrated genomes. Availability of small animal models that are easily produced and permit rapid assays will be quite helpful. Aims: We examined whether transplantation of human cells in the peritoneal cavity of mice will generate an appropriate mass of cells with hepatitis B virus replication. Methods: HepG2 2.2.15 cells were transplanted intraperitoneally into NOD/SCID mice. Replication of hepatitis B virus and viral gene expression was determined by analysis of blood and transplanted tissues with viral DNA and hepatitis B core antigen expression. Interruption of viral replication was examined. Results: After intraperitoneal transplantation with microcarrier scaffolds, 2.2.15 cells engrafted and proliferated in the peritoneal cavity of NOD/SCID mice. Hepatitis B virus replicated in transplanted 2.2.15 cells as shown by hepatitis B core antigen expression. Moreover, viral particles were secreted into the blood. Hepatitis B virus replication was susceptible to conventional antiviral drug therapy, such as lamivudine, as well as experimental antiviral gene therapy with a synthetic mimic of an antiviral cellular microRNA. Conclusions: Intraperitoneal transplantation of human cells rapidly provided reservoirs of hepatitis B virus in mice. This simple xenotransplantation approach will be effective and convenient for studies of hepatitis B and other human viruses in vivo.     

The prevalence of serological markers for hepatitis B virus infection amongst anaesthetists in the Oxford region

A total of 125 anaesthetists from nine hospitals within the Oxford region were surveyed to study the prevalence of serological markers for hepatitis B virus (HBV) infection. No anaesthetists were positive for Hepatitis B Surface Antigen (HBsAg) and only four (3.2%) were positive for HBsAg antibody (anti-HBsAg). This result is in marked contrast to other studies and suggests that anaesthetists in the United Kingdom do not constitute a high risk population. The reasons for this are discussed.     

Sperm head ultramorphology and chromatin stability of males with unexplained infertility who fail to fertilize normal human ova in vitro

Summary. An in vitro nuclear chromatin decondensation test, and quantitative nuclear ultramorphology analysis, were performed on 18 males judged to be infertile, by two failures in in vitro fertilization, and 16 fertile males. These two clinical groups only differed significantly in (1) the direction of their chromatin stability change, which took place 30–120 min post-ejaculation while stored in the seminal plasma, and (2) in the incidence of the hypoelongated sperm-head. Generally, the fertile male group exhibited positive chromatin stability change after prolonged storage, and low incidence of hypoelongated sperm heads, and vice versa in the unexplained infertile group. When the nuclear chromatin decondensation test and quantitative ultramorphology analysis were performed in step-wise fashion, it was possible to correctly classify 94% of the fertile cases with 6% of false-negative, and 89% of the unexplained infertile cases with 11% of false-positive. Therefore, it appears that these tests might be of benefit clinically for identifying functional properties of sperm-cells in unexplained infertile males, which cannot be detected by routine semen analysis.     

免疫组化方法观察乙肝病毒感染肝星状细胞的研究

目的 研究HBV能否体外感染LX-2细胞,以期为阐明慢性乙肝的致病机制提供新的实验依据.方法 体外培养LX-2细胞细胞数目达到106/培养瓶时,用HBV感染者的血清进行感染,HBVDNA终浓度分别为104、105、106、107拷贝/mL,同一浓度的感染时间均为24、48和72 h.按照观察时间点收取细胞,免疫组织化学法(DAB显色)测定HBV在细胞内是否表达乙肝表面抗原(HB-sAg)和乙肝核心抗原(HBcAg).结果 用免疫组化方法标记HBsAg和HBcAg时,LX-2细胞中未发现阳性颗粒;HepG2.2.15细胞中有少量棕黄色颗粒,位于胞质中;慢性乙肝患者肝组织中可见大量棕黄色颗粒.结论 在体外培养中HBV不能感染LX-2细胞和进行抗原表达.     

Supplementation of the dilution medium after thawing with reduced glutathione improves function and the in vitro fertilizing ability of frozen-thawed bull spermatozoa

In this study, we evaluated the effects of glutathione (l-gamma-glutamyl-l-cysteinylglycine; GSH) supplementation of the thawing extender on bull semen parameters to compensate for the decrease in GSH content observed during sperm freezing. To address these questions fully, we used a set of functional sperm tests. These included tests of sperm motility assayed by computer-assisted semen analysis, membrane lipid packing disorder, spontaneous acrosome reaction, free radical production [reactive oxygen species (ROS) generation], sperm chromatin condensation, DNA fragmentation by terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling and acridine orange staining measured by flow cytometry. Finally, the in vitro penetrability of in vitro matured oocytes and the in vitro production of embryos were evaluated. The main findings emerging from this study were that addition of GSH to the thawing medium resulted in: (i) a higher number of non-capacitated viable spermatozoa; (ii) a reduction in ROS generation; (iii) lower chromatin condensation; (iv) lower DNA fragmentation; (v) higher oocyte penetration rate in vitro and (vi) higher in vitro embryo production compared with control group. Nevertheless, GSH had no significant effect on motion parameters or the occurrence of the spontaneous acrosome reaction. Addition of GSH to the thawing extender could be of significant benefit in improving the function and fertilizing capacity of frozen bull spermatozoa.     

乙型肝炎病毒感染者血清干扰素诱导基因20 kDa蛋白表达水平及其临床意义

目的观察乙型肝炎病毒感染者血清干扰素诱导基因20 kDa蛋白（ISG20）蛋白表达变化及其临床意义。 方法选取2017年3月至2018年6月汉中市中心医院收治的HBV感染所致慢性乙型肝炎患者（CHB组）67例、肝硬化患者（LC组）58例、肝癌患者（HCC组）54例和同期体检47例健康者（对照组）。采用酶联免疫吸附法检测各组研究对象血清ISG20蛋白水平。比较不同组患者血清ISG20水平差异。采用Spearman相关分析探讨血清ISG20水平与各组患者年龄、性别、红细胞计数、白细胞计数、血小板计数、天门冬氨酸氨基转移酶（AST）、丙氨酸氨基转移酶（ALT）、总胆红素（TBil）、直接胆红素（DBil）、白蛋白、凝血酶原、甲胎蛋白（AFP）、HBV DNA的相关性。 结果对照组患者血清ISG20水平为9.4（0.82～25.72）ng/ml,显著低于CHB组[18.6（1.87～56.32）ng/ml,Z =－1.567、P = 0.034]和HCC组[28.2（3.09～81.42）ng/ml,Z = －1.854、P = 0.021]。HCC组患者血清ISG20水平显著高于CHB组（Z =－1.431、P = 0.041）和LC组[12.9（2.81～77.54）ng/ml,Z =－1.987、P = 0.029）。HCC组不同Child-Pugh分级患者血清ISG20水平差异有统计学意义（H = 6.976、P = 0.031）。HCC组患者血清ISG20水平与AST（r = 0.323、P < 0.001）、ALT（r = 0.248、P = 0.036）、TBil（r = 0.221、P = 0.031）、DBil（r = 0.215、P = 0.043）、AFP（r = 0.176、P = 0.044）呈正相关,与白蛋白呈负相关（r =－0.239、P = 0.019）。 结论HBV感染者,尤其是HBV感染所致HCC患者,血清ISG20水平升高。血清ISG20水平与HBV感染疾病进展和临床参数有一定的相关性。     

RhoGDIα在人睾丸、精子中表达及其与体外受精成功率相关性的初步分析

目的:观察Rho特异性的GDP解离抑制因子α(RhoGDIα)在人睾丸、精子中的表达及定位并比较RhoGDIα在正常生育男性和体外受精(IVF)不育患者精子中的表达差异。方法:通过免疫组化方法观察RhoGDIα在人睾丸中的定位;通过免疫荧光方法观察RhoGDIα在人精子获能前、获能后、顶体反应后的定位;收集正常男性精液标本(10例),高受精率(≥60%,12例)和低受精率(<60%,13例)的IVF不育患者的精液标本,Percoll细胞分离液分离精液标本,排除生精细胞和白细胞,分别通过免疫荧光和Western印迹方法,检测RhoGDIα的表达。结果:免疫组化结果显示RhoGDIα存在于人睾丸各级生精细胞中,并在长形精子细胞高表达。免疫荧光结果显示RhoGDIα在人精子的顶体和尾部有较强表达,并且随着获能的发生,在顶体上的表达减弱,当顶体反应发生后,顶体上的表达完全消失。Western印迹结果显示不易受精的IVF患者精子中RhoGDIα的表达(0.66±0.18)显著低于正常组(1.13±0.21)和易受精的IVF患者组(0.97±0.17)。结论:RhoGDIα定位于人精子的顶体和尾部,可能参与了精子运动,获能及顶体反应过程。RhoGDIα在受精率低下患者精子中表达显著降低,提示RhoGDIα可能成为一个新的男性不育的诊断指标,并且可能成为IVF供精选择的一个指标。     

Use of telbivudine in kidney transplant recipients with chronic hepatitis B virus infection: A preliminary experience

Telbivudine is a relatively novel oral nucleoside analogue with favourable efficacy and tolerability in treatment‐naïve chronic hepatitis B virus (HBV) infection, but its data in kidney transplant recipients (KTRs) was lacking. The efficacy and tolerability of telbivudine in four treatment‐naïve HBsAg‐positive KTRs were reviewed (treatment duration 54 (36–72) months) HBV DNA declined from 2.6 × 10⁵(7.8 × 10³–1.5 × 10⁷) copies/mL at baseline to 170 (0.0–3.2 × 10⁴) copies/mL at 12 months, and became undetectable at 24 and 36 months (P = 0.060, 0.118 and 0.005 compared with baseline). Alanine aminotransferase levels dropped from 46.5 (30–48) IU/mL at baseline to 28 (13–45) IU/mL, 34.5 (15–71) IU/mL and 26 (12–41) IU/mL at 12, 24 and 36 months, respectively (P = 0.109, 0.715 and 0.068 compared with baseline). Serum creatinine level and estimated glomerular filtration rate (eGFR) remained stable after 36 months of treatment (P all > 0.05 compared with baseline). No virological breakthrough, cirrhosis or hepatocellular carcinoma occurred. Our pilot data suggests that telbivudine has favourable efficacy and renal safety profiles in HBsAg‐positive KTRs.     

Impact of human herpesvirus-6 on the frequency and severity of recurrent hepatitis C virus hepatitis in liver transplant recipients

A role of tumor necrosis factor-alpha (TNF-alpha) In the immunopathogenesis of hepatitis C virus (HCV) infection has been proposed. The novel herpes virus, human herpes virus-6 (HHV-6), is amongst the most potent inducers of cytokines, including TNF-alpha. The impact of HHV-6 viremia on the progression of recurrent HCV hepatitis was assessed in 51 HCV-positive liver transplant recipients. The frequency of recurrent HCV hepatitis did not differ between patients with HCV viremia (47.6%, 10/21) as compared with those without HCV viremia (46.7%, 14/30, p = 0.9). However, the patients with HHV-6 viremia had a significantly higher fibrosis score upon HCV recurrence than those without HHV-6 viremia (mean 1.5 vs. 0.3, p = 0.01). An association between cytomegalovirus (CMV) viremia and HCV recurrence was not documented; 50% (15/30) of the patients with CMV viremia and 42.8% (9/21) of those without CMV viremia had recurrent HCV hepatitis (p > 0.5). Receipt of ganciclovir (administered upon the detection of CMV viremia) was associated with lower total Knodell score (mean 5.2 vs. 6.9, p = 0.05) and a trend towards lower fibrosis score (mean 0.44 vs. 1.00, p = 0.12) in patients with recurrent HCV hepatitis. Thus, HHV-6 viremia in HCV-positive liver transplant recipients identified a subgroup of patients at increased risk for early fibrosis upon HCV recurrence.     

原发性肝癌与乙型肝炎病毒基本核心启动子和前C区基因变异的相关性

目的 研究原发性肝癌（PLC）与HBV基本核心启动子（BCP）和前C区基因变异的相关性。方法对144例HBsAg（＋）PLC患者的血清进行HBV标志物和HBVDNA检测,对HBeAg（-）但HBVDNA（＋）的样本再采用实时荧光定量PCR进行前C区和BCP区基因变异检测。随机选取120例慢性乙型肝炎（CHB）患者作为对照组。结果PLC患者血清HBV标志物中HBeAg（＋）46例,占31．94％,HBeAg（-）98例,占68．06％。在98例HBeAg（-）患者的血清中,HBVDNA（＋）56例,占57．14％,其中前C1896变异43例,占76．79％,BCP1762／1764基因变异50例,占89．29％,共同变异38例,占67．86％。PLC患者中的前C1896和BCP1762／1764基因变异率高于CHB患者,且差异有统计学意义（X^2值分别为9．36和5．77,P值均〈0．05）。结论PLC的发生可能与前C区和BCP区基因变异有关。     

Sperm count and motility influence the results of human fertilization in vitro

In order to select sperm characteristics that can predict the outcome of in-vitro fertilization-embryo transfer (IVF-ET), semen samples delivered in conjunction with this treatment were studied carefully. We have analysed these data retrospectively in relation to the outcome of treatment. Ninety-one couples were treated for tubal infertility by IVF-ET. Fifteen women became pregnant. Sperm were isolated from semen using a swim-up technique and in most cases 40-80 x 10(3) (range 20-120 x 10(3)) motile sperm per ovum were used for insemination. The couples were divided into three groups: group A who achieved pregnancies, group B who achieved cleaved ova but no pregnancies, and group C who achieved no ova that were cleaved 48 h after oocyte recovery. Comparisons between these groups showed that some characteristics of the native semen samples and the swim-up preparations were significantly different: the sperm concentration (P = 0.001) and total sperm count (P = 0.003) in the native sample, the number of sperm recovered during 30 min of swim-up (P = 0.001), and the specific progressive motility of sperm in the swim-up preparation, both at the time of insemination and on each day, up to 5 days thereafter (P = 0.002-0.028). No pregnancy was achieved with a sperm concentration below 26 x 10(6) ml-1 in the native sample. Some of the sperm characteristics studied in this paper may be of value in the pretreatment evaluation for IVF treatment.