SEROPREVALENCE OF HUMAN HERPESVIRUS-6 IN HEALTHY POPULATION IN TWO PROVINCES OF NORTH CHINA

Baekground. Human herpvsvirus-6 (HHV-6) infection is ubiquitous in selected population with seroprevalence of 60 ～ 80%. Little is known for that in China, except few sporadic studies. To understand prevalenoe of HHV-6 antibody in Chinese population ,this seroepideraiological study was conducted. Methods. Sera were collected from 430 healthy persons and donors living in North China,and tested for HHV-6 antibodies using 1FA with HHV-6 GS strain passaged on HSB-2 oells as antigen, and titer equal or higher than 1 : 10 was regarded as seropositive. Results. Of the 430 serum samples tested, 297 (69.1%) were positive for HHV-6 IgG. Both seropositive rate and GMT in females were significantly higher than those in males, with X^2 = 7.05 ,P<0. 01 and F=7.23,P<0. 01,respectively. Statistically significant difference in prevalence of HHV-6 antibody among various age groups was observed in both sexes, with X^2= 20. 08 and 20.28, P = 0. 04, respectively, and young children had a higher titer than adults. But,no significant difference in prevalence was observed in blood donors between various age groups or both sexes. Conclusions. This study suggests that HHV 6 infection with seropositixe IgG is ubiquitous in North China ,and its imvortance should be further studied.

Establishment of a humanized ST6GAL1 mouse model for influenza research

Background:This study aimed to construct and characterize a humanized influenza mouse model expressing hST6GAL1.Methods:Humanized fragments,consisting of the endothelial cell-specific K18 promoter,human ST6GAL1-encoding gene,and luciferase gene,were microinjected into the fertilized eggs of mice.The manipulated embryos were transferred into the oviducts of pseudopregnant female mice.The offspring were identified using PCR.Mice exhibiting elevated expression of the hST6GAL1 gene were selectively bred for propagation,and in vivo analysis was performed for screening.Expression of the humanized gene was tested by performing immunohistochemical(IHC)analysis.Hematologic and biochemical analyses using the whole blood and serum of humanized hST6GAL1 mice were performed.Results:Successful integration of the human ST6GAL1 gene into the mouse genome led to the overexpression of human SiaT ST6GAL1.Seven mice were identified as carrying copies of the humanized gene,and the in vivo analysis indicated that hST6GAL1gene expression in positive mice mirrored influenza virus infection characteristics.The IHC results revealed that hST6GAL1 was expressed in the lungs of humanized mice.Moreover,the hematologic and biochemical parameters of the positive mice were within the normal range.Conclusion:A humanized influenza mouse model expressing the hST6GAL1 gene was successfully established and characterized.

DNA N^6-methyladenine demethylase ALKBH1 enhances osteogenic differentiation of human MSCs

ALKBH1 was recently discovered as a demethylase for DNA N6-methyladenine （N6-mA）, a new epigenetic modification, and interacts with the core transcriptional pluripotency network of embryonic stem cells. However, the role of ALKBH1 and DNA N6-mA in regulating osteogenic differentiation is largely unknown. In this study, we demonstrated that the expression of ALKBH1 in human mesenchymal stem cells （MSCs） was upregulated during osteogenic induction. Knockdown of ALKBH1 increased the genomic DNA N6-mA levels and significantly reduced the expression of osteogenic-related genes, alkaline phosphatase activity, and mineralization. ALKBHl-depleted MSCs also exhibited a restricted capacity for bone formation in vivo. By contrast, the ectopic overexpression of ALKBH1 enhanced osteoblastic differentiation. Mechanically, we found that the depletion of ALKBH1 resulted in the accumulation of N6-mA on the promoter region of ATF4, which subsequently silenced ATF4 transcription. In addition, restoring the expression of ATP by adenovirus-mediated transduction successfully rescued osteogenic differentiation. Taken together, our results demonstrate that ALKBH1 is indispensable for the osteogenic differentiation of MSCs and indicate that DNA N6-mA modifications area new mechanism for the epigenetic regulation of stem cell differentiation.

The Seropositivity Rate of Human Herpesvirus Type 6 among Infants in Diyala Province, Iraq

Background: Human herpesvirus type 6 (HHV-6) has been shown to infect almost all children by 4 years of age. Primary infection causes an undifferentiated febrile illness, with approximately 30% of children exhibiting the classic clinical manifestations of roseola infantum. Objectives: The current study was carried out to explore the anti-HHV-6 IgG positivity rate as a marker of past infection among apparently healthy infants and to figure out the effect of certain infant and family characteristics on the infectivity rate. Materials and methods: This is a cross sectional study conducted in Diyala province during the period from August 2017-July 2018. A total of 180 apparently healthy infants were included, their ages ranged between 6 - 24 months. They consist of 100 males with mean age ± SD 15.05 ± 6.42 months and 80 females with mean age ± SD 15.56 ± 6.66 months. Human privacy was respected by obtaining parental consent. Venous blood samples were collected aseptically from each participant. Sera were separated and tested for the anti-HHV6 IgG (Sunlong Biotech, China) by Enzyme Linked Immunosorbant assay (ELISA) technique. Statistical analysis was done using SPSS version 23 and P value Conclusion: About one half of apparently healthy infants aged up to two years of Diyala population have anti-HHV6 IgG antibodies and the presence of intrafamilial primary HHV-6 positive case is markedly associated with increased rate of anti-HHV6 IgG among siblings.

REGULATORY EFFECTS OF HUMAN RECOMBINANT IL-6 ON NATURAL KILLER CELL ACTIVITY OF HUMAN FETAL SPLEENS

In the present study it was proved first that human recombinant interleukin-6(HrIL-6) significantly augmented natural killer(NK) cell activity derived from human fetal spleens against K562 target cells in a 4 hours 51Cr release assay. The enhancement of NK activity with 24 hours preincubation in HrlL-6 was dose-dependent, and significantly higher than that of fresh NK cells and controls cultured with RPMI-1640 medium alone (P<0.001). We also found that IL-6 was able to augment NK activity from different fetal spleens at 20 to 40 weeks of gestation (up to 2.24 to 2.78 times), and no difference of NK activity of fetal splenocytes treated by HrIL-6 was observed between different fetal age (32.3% to 45.4%, P>0.05). Furthermore, IL-6-augmented NK activity of fetal splenocytes was very similar to adult levels (P>0.05). These finding strongly indicated that IL-6 plays an important role in the development of NK cell function during the gestational period, suggesting that IL-6 may be of importance in the regulation of host defense mechanisms against malignancies and viral diseases.

HEXOKINASE, GLUCOSE-6-PHOSPHATASE DEHYDROGENASE AND ALEOSE REDUCTASE IN HUMAN FETAL LENSES

The lens HK, G6PD, AR activity and its relationship with fetal age was determined.There is a positive correlation between the age of fetus and the activity(IU/mg pro.) of HK and G6PD(r=0.8069, 0.8204, P<0.01) and a negetive correlation between the age of fetus and activity of AR(r=-0.810 1,0.05>P>0.01).

The role of TRPC6 in HGF-induced cell proliferation of human prostate cancer DU145 and PC3 cells

Hepatocyte growth factor （HGF） is a glycoprotein that induces prostate cancer cell proliferation, migration and invasion. The activation of transient receptor potential canonical 6 （TRPC6） channels is considered important in promoting prostate cancer cell proliferation. In this study, we assessed the role of endogenous TRPC6 channels in the HGF-induced cell proliferation of prostate cancer. Reverse transcription-PCR and Western blotting were used to investigate TRPC6 expression. Electrophysiological techniques （whole-cell patch clamp configuration） and Ca^2＋ imaging analysis were used to investigate the channel activity in cells. The effects of TRPC6 channels on cell cycle progression, cell apoptosis and cell growth were also examined. TRPC6 and c-MET were expressed in DU145 and PC3 cells. In addition, functional TRPC6 channels were present in DU145 and PC3 cells, and TRPC6 knockdown suppressed TRPC-Iike currents evoked by oleoyl-2-acetyl-sn-glycerol （OAG）. Inhibition of TRPC6 channels in DU145 and PC3 cells abolished OAG- and HGF-induced Ca^2＋ entry. Furthermore, inhibition of TRPC6 channels arrested DU145 and PC3 cells at the G2/M phase and suppressed HGF-induced cell proliferation. Collectively, our results indicate that TRPC6 has an important role in HGF-induced DU145 and PC3 cell proliferation.

Impact of human herpes virus 6 in liver transplantation

Human herpes virus 6 (HHV-6) infects > 95% of humans.Primary infection which occurs mostly during the f irst 2 years of life in the form of roseola infantum,non-spe cif ic febrile illness,or an asymptomatic illness,results in latency.Reactivation of latent HHV-6 is common after liver transplantation.Since the majority of human beings harbor the latent virus,HHV-6 infections after liver transplantation are most probably caused by end ogenous reactivation or superinfection.In a minority of cases,primary HHV-6 infection may occur when an HHV-6-seronegative individual receives a liver allograft from an HHV-6-seropositive donor.The vast major ity of HHV-6 infections after liver transplantation are asy-mptomatic.Only in a minority of cases,when HHV-6 causes a febrile illness associated with rash and mye- losuppression,hepatitis,gastroenteritis,pneumonitis,and encephalitis after liver transplantation.In addition,HHV-6 has been implicated in a variety of indirect effects,such as allograft rejection and increased predis- pos ition to and severity of other infections,includingcytomegalovirus,hepatitis C virus,and opportunistic fungi.Because of the uncommon nature of the clinical illnesses directly attributed to HHV-6,there is currently no recommended HHV-6-specific approach prevention after liver transplantation.Asymptomatic HHV-6 infection does not require antiviral treatment,while treatment of established HHV-6 disease is treated with intravenous ganciclovir,foscarnet,or cidofovir and this should be com plemented by a reduction in immunosuppression.

Contributions of neurotropic human herpesviruses herpes simplex virus 1 and human herpesvirus 6 to neurodegenerative disease pathology

Human herpesviruses （HVs） have developed ingenious mechanisms that enable them to traverse the defenses of the central nervous system （CNS）. The ability of HVs to enter a state of latency, a defining char- acteristic of this viral family, allows them to persist in the human host indefinitely. As such, HVs represent the most frequently detected pathogens in the brain. Under constant immune pressure, these infections are largely asymptomatic in healthy hosts. However, many neurotropic HVs have been directly connected with CNS pathology in the context of other stressors and genetic risk factors. In this review, we discuss the potential mechanisms by which neurotropic HVs contribute to neurodegenerative disease （NDD） patholo- gy by highlighting two prominent members of the HV family, herpes simplex virus 1 （HSV-1） and human herpesvirus 6 （HHV-6）. We （i） introduce the infectious pathways and replicative cycles of HSV-1 and HHV-6 and then （ii） review the clinical evidence supporting associations between these viruses and the NDDs Alzheimer＇s disease （AD） and multiple sclerosis （MS）, respectively. We then （iii） highlight and dis- cuss potential mechanisms by which these viruses exert negative effects on neurons and glia. Finally, we （iv） discuss how these viruses could interact with other disease-modifying factors to contribute to the initiation and/or progression of NDDs.

TM6SF2 E167K variant predicts severe liver fibrosis for human immunodeficiency/hepatitis C virus co-infected patients, and severe steatosis only for a non-3 hepatitis C virus genotype

AIM To evaluate the impact of the Glu167Lys(E167K) transmembrane 6 superfamily member 2(TM6SF2) variant on the biochemical and morphologic expression of liver lesions in human immunodeficiency virus(HIV)/hepatitis C virus(HCV) co-infected patients.METHODS The study comprised 167 consecutive patients with HIV/HCV coinfection and biopsy-proven chronic hepatitis. A pathologist graded liver fibrosis and necroinflammation using the Ishak scoring system, and steatosis using Kleiner's scoring system. Patients were genotyped for TM6SF2 E167K(rs58542926) by real-time Polymerase chain reaction. The 167 patients, 35 therapy-naive and 132 receiving ART, were prevalently males(73.6%), the median age was 40.7 years and the immunological condition good(median CD4+ cells/mm3 = 505.5).RESULTS The 17 patients with the TM6SF2 E167 K variant, compared with the 150 with TM6SF2-E/E, showed higher AST(P = 0.02) and alanine aminotransferase(P = 0.02) and higher fibrosis score(3.1 ± 2.0 vs 2.3 ± 1.5, P = 0.05). In a multivariate analysis, TM6SF2 E167 K was independently associated with severe fibrosis. The same analysis showed that HCV-genotype 3, present in 42.2% of patients was an independent predictor of severe steatosis. The association of TM6SF2 E167 K with severe steatosis, absent for the whole group of 167 patients, was re-evaluated separately for HCVgenotype 3 and non-3 patients: No factor was independently associated with severe steatosis in the HCV-genotype-3 subgroup, whereas an independent association was observed between severe steatosis and TM6SF2 E167 K in non-3 HCV genotypes. No association between the TM6SF2 E167 K variant and severe liver necroinflammation was observed.CONCLUSION In HIV/HCV coinfection the TM6SF2 E167 K variant is an independent predictor of severe fibrosis, but appears to be independently associated with severe steatosis only for patients with a non-3 HCV genotype.

Expression of Messenger RNA of Oncoproteins E6 and E7 of Human Papilomavirus in Women with Negative Oncotic Cytologies, Epithelial Squamous Atypias of Undefined Significance and Low-Grade Intraepithelial Lesions

Objectives: To verify the prevalence of E6/E7 RNAm expression of HPV in patients with negative cervicovaginal cytology, ASC-US and LSIL;to correlate with negative anatomopathological exams and/or squamous intraepithelial neoplasy grade I (SIN 1) of the lower genital tract (LGT);to relate the RNAm expression with viral infection types;to assess the genotyping in single infections. Methods: Findings from 825 women submitted to E6/E7 RNAm survey and 422 women submitted to LGT biopsies were analyzed. Results: A larger percentage of E6/E7 in ASC-US and LSIL cytologies occurred. Negative results of RNAm expression were in accordance with negative cytologies and negative anatomopathological exam. In positive cases, the infection by a single HPV type was most prevalent, with the type 16 being the most common. Conclusions: the expression of mRNA was most prevalent in ASC-US and LSIL cytologies, comparing with the negative ones. The findings of SIN 1 biopsies were related to the positive expression of mRNA and negative cytologies;the negative expression was in agreement with negative anatomopathological exam. The infection by a HPV type was more frequent in cases of positive expression, the HPV type 16 being most frequently found. Patients with low grade intraepithelial lesion cytologies had a higher percentage of multiple infections.

Competition between TRAF2 and TRAF6 Regulates NF-κB Activation in Human B Lymphocytes

Objective To investigate the role of TNF receptor-associated factor 2 (TRAF-2) and TRAF6 in CD40-induced nuclear factor-κB (NF-κB) signaling pathway and whether CD40 signaling requires TRAF2. Methods Human B cell lines were transfected with plasmids expressing wild type TRAF2 or dominant negative TRAF2,TRAF2-shRNA,or TRAF6-shRNA. The activation of NF-κB was detected by Western blot,kinase assay,transfactor enzyme-linked immunosorbent assay (ELISA),and fluorescence resonance energy transfer (FRET). Analysis of the role of TRAF-2 and TRAF-6 in CD40-mediated NF-κB activity was examined following stimulation with recombinant CD154. Results TRAF2 induced activity of IκB-kinases (IKKα,IKKi/ε),phosphorylation of IκBα,as well as nuclear translocation and phosphorylation of p65/RelA. In contrast,TRAF6 strongly induced NF-κB activation and nuclear translocation of p65 as well as p50 and c-Rel. Engagement of CD154-induced nuclear translocation of p65 was inhibited by a TRAF6-shRNA,but conversely was enhanced by a TRAF2-shRNA. Examination of direct interactions between CD40 and TRAFs by FRET documented that both TRAF2 and TRAF6 directly interacted with CD40. However,the two TRAFs competed for CD40 binding. Conclusions These results indicate that TRAF2 can signal in human B cells,but it is not essential for CD40-mediated NF-κB activation. Moreover,TRAF2 can compete with TRAF6 for CD40 binding,and thereby limit the capacity of CD40 engagement to induce NF-κB activation.

Seizure-related 6,a brain-specific expression gene,is highly expressed in the human cerebellum

Epilepsy is a complex, Mendelian disease, and most cases are sporadic. Genomic comparisons of tissue from identified monogenic epilepsies with multigenic and acquired syndromes could ultimately reveal crucial molecular neuropathology for an epileptic phenotype. In the present study, a novel gene, human seizure-related （hSEZ）-6, was isolated from a human brain cDNA library. hSEZ-6 comprises 17 exons and spans a region of at least 55.6 kb, which was localized to 17q 12 by radiation hybridization, hSEZ-6 exhibits two isoform types, hSEZ-6A and hSEZ-6B, which encode 996 and 995 amino acids, respectively. The two putative hSEZ-6 proteins contain similar motifs and share 82% and 84% identity with mouse SEZ-6A protein, whose expression level increased in mouse cerebral cortex-derived cells treated with a convulsant drug, pentylentetrazole. Northern blot analysis demonstrated that hSEZ-6 is expressed highly in the cerebellum and in nucleus of the extrapyramidal system, such as the caudate nucleus and putamen. Reverse transcription polymerase chain reaction revealed that hSEZ-6 is expressed in neurons rather than gliocytes, which suggests that hSEZ-6 is a seizure-related gone.

Up-regulation interleukin-6 and interleukin-8 by activated protein C in lipopolysaccharide-treated human umbilical vein endothelial cells

Objective: To investigate the effect of activated protein C (APC) on inflammatory responses in human umbilical vein endothelial cells (HUVEC) stimulated with lipopolysaccharide (LPS). Methods: The second passage of collagenase digested HUVEC was divided into the following groups: serum free medium control group (SFM control), phosphate buffer solution control group (PBS control), LPS group with final concentration of 1 μg/ml (LPS group), APC group with final concentration of 7 μg/ml, Pre-APC group (APC pretreatment for 30 min prior to LPS challenge), and Post-APC group (APC administration 30 min after LPS challenge). Supernatant was harvested at 0, 4, 8, 12 and 24 h after LPS challenge. Interleukin-6 (IL-6) and Interleukin-8 (IL-8) levels were analyzed with ELISA. Cells were harvested at 24 h after LPS challenge, and total RNA was extracted. Mes-senger RNA levels for IL-6 and IL-8 were semi-quantitatively determined by RT-PCR. Results: Compared with control group, IL-6 and IL-8 levels steadily increased 4 to 24 h after LPS stimulation. APC treatment could increase LPS-induced IL-6 and IL-8 production. The mRNA levels of IL-6 and IL-8 exhibited a similar change. Conclusion: APC can further increase the level of IL-6 and IL-8 induced by LPS. The effect of these elevated cytokines is still under investigation.

Correlation between human seizure-related gene 6 variants and idiopathic generalized epilepsy in a Southern Chinese Han population

This study sought to analyze the genotype and gene mutations of human seizure-related gene 6 in 98 patients with idiopathic generalized epilepsy （non-febrile seizures）, who were selected from three generations of the Chinese Han population living in Shanghai, Zhejiang Province, Wuxi of Jiangsu Province, and Jiangxi Province of Southern China. Twenty-six patients＇ parents were available as a first-degree relatives group and 100 biologically unrelated healthy controls were collected as the control group. Based on the age of onset and seizure type, the patients were divided into six subgroups. Polymerase chain reaction and DNA direct sequencing analysis showed that the most frequent mutations c. 1249dupC （p.Gly418Argfx31 ） and c.1636A 〉 G （p.Thr546Ala） were detected in some idiopathic generalized epilepsy patients and tl^eir asymptomatic first-degree relatives （30.6% vs. 19.2% and 11.2% vs. 26.9%）. A novel mutation c.1807G 〉A （p.Val603Met） was found in a patient with late-onset idiopathic generalized epilepsy. There was no significant difference in the incidence of these three mutations among the different subgroups of idiopathic generalized epilepsy and controls. Thus, further analysis of a larger population is needed to confirm the assumption that human seizure-related gene 6 is a susceptibility gene for idiopathic generalized epilepsy with various sub-syndromes.

Effectof the Local Strain CN5 of Human Herpesvirus 6 on CD Molecule Expression and PHA-Induced Proliferation of Cord Blood or Peripheral Blood Mononuclear Cells

Cord blood mononuclear cells (CBMC's) and adult peripheral blood mononuclear cells (PBMCs), cultured with RPMI 1640 medium containing 20% fetal calf serum plus 5 mg/L PHA and 10 mg/L IL-Z, were inoculated with CN5 strain of human herpesvirus 6 (HHV 6 ) isolated from a Chinese patient with exanthem subitum and the CN5 infected cell lysates were added to cultures of CBMCs and PBMCs, so as to observe the effects of the local strain CN5 on expression of CD molecule or on proliferation of mononuclear cells by the methods of APAAP staining and MTT assay.The results were as follows: ①Expressions of some CD antigens of CBMCs and PBMCs could change after CN5 strain infection. In both cases, CD3 expresion was down-regulated while CD4 expression was up-regulated. There were no significant differences of CD2, CD8 and CD45RA expressions between the two groups with and without CN5 infection. But the ratio of CD4 to CD8 sigmificantly rose because of the increasing of CD4 positiveity. ②The lysates of CB5-infected CBMCs inhibited the liferation of PBMCs, not of CBMCs, in a protein concentration-dependent pattern. This inhibition was partially neutralized by specific antiserum to CN5, not by antisera to INF-α and TNF-α.

Sequence analysis of human papillomavirus type 16 E6E7 gene from cervical carcinoma biopsies of Chinese patients in Shandong province

Objective To study the structure specificity of HPV16E6E7 gene from cervical carcinoma biopsies of patients in Shandong province. Methods The tissue DNAs were extracted from cervical carcinoma biopsies of 14 patients of Chinese from Shandong province. By PCR method using HPV multiple primers, the HPV types were identified in cervical carcinoma tissues, Using the tissue DNA of the 2 cases with the infection of HPV16 type only as templates, HPV16E6E7 gene was amplified by PCR respectively, then inserted the HPV16E6E7 gene into pALTER I vector, and obtained the recombinant plasmid pALTER HPV16E6E7. The constructs were sequenced using Sanger method, and then compared with the sequence of HPV16E6E7 gene of the prototype. Results Sequencing results showed that HPV16E6E7 gene in the 2 cases had sequence diversity from that of the prototype, and a total of five nucleotide exchanges were detected, four of these led to amino acid exchanges. Conclusion There are structure differences between the HPV16E6E7 of Chinese and that of the prototype.

Expression of LL-37, Human beta Defensin-2, and CCR6 mRNA in Patients with Psoriasis Vulgaris

To investigate whether LL-37 and human beta defensin-2 (hBD-2) is related to the patients with psoriasis seldom having skin infections and explore the role of the two peptides and CCR6 (the receptor of hBD-2) in the pathogenesis of psoriasis, the expression levels of mRNA of LL-37, hBD-2, and CCR6 in skin lesions of patients with psoriasis vulgaris were detected by using RT-PCR. The results showed that the mRNA expression levels of the two peptides and CCR6 in psoriatic lesions all increased compared with the normal skin (P<0.001). It was suggested that up-regulated expression of LL-37 and hBD-2 might be the main reason that result in the the skin of patients with psoriasis being seldom infected, and the two peptides and CCR6 might play crucial roles in the pathogenesis of psoriasis.