Reasonable selection of antiviral agents for herpes zoster / 中华皮肤科杂志

Antiviral treatment is the core part in the treatment of herpes zoster. Based on the latest studies, consensus and guidelines, this article aims to provide a basis and reference for clinicians to make a reasonable choice of types and doses of antiviral agents. Valacyclovir, a precursor of acyclovir with high oral bioavailability and great convenience of administration, is generally the first choice of oral antiviral agents; for some special cases, such as immunocompromised patients, intravenous drips of acyclovir should be selected when appropriate. Brivudine is often a better choice for patients with severe renal insufficiency; famciclovir or other antiviral agents should be considered for patients resistant to acyclovir; for immunocompromised patients resistant to acyclovir, intravenous drips of foscarnet sodium can be an option. Oral antiviral agents should be administered at adequate doses. Selecting appropriate antiviral agents and their doses can effectively relieve acute symptoms of patients and reduce the probability of postherpetic neuralgia.

Effects of autophagy on viral structures and expression of functional proteins in dorsal root ganglia in a guinea pig model of varicella-zoster virus infection / 中华皮肤科杂志

Objective:To investigate effects of the autophagy inducer rapamycin and autophagy inhibitor 3-methyladenine on viral structures and biosynthesis of functional proteins in dorsal root ganglia in a guinea pig model of varicella-zoster virus (VZV) infection, and to explore their possible mechanisms.Methods:VZV was cultured and proliferated in human embryonic lung fibroblasts (HELFs) , and peripheral blood mononuclear cells (PBMCs) were isolated from guinea pigs. VZV-HELFs and PBMCs were co-cultured for 18-20 hours, and VZV-PBMCs were collected by centrifugation. Thirty-two guinea pigs were randomly and equally divided into 4 groups (8 mice in each group) : blank control group was injected with autologous PBMCs via the medial canthal venous plexus; autophagy inhibition group, autophagy induction group, and VZV infection group were intraperitoneally injected with 3 mg/kg 3-methyladenine solution, 0.5 mg/kg rapamycin solution, and the same volume of 0.9% NaCl solution respectively, followed 2 hours later by injections with 50 μl of VZV-PBMCs via the medial canthal venous plexus. Fourteen days later, the guinea pigs in each group were sacrificed, and dorsal root ganglion tissues were collected. The transmission electron microscope was used to observe the morphology of virus particles, as well as the morphology and number of autophagic vesicles, Western blot analysis was performed to determine the expression of VZV nucleocapsid protein (NCP) , immediate-early protein 62 (IE62) , and autophagy-related proteins Beclin-1 and p62, and immunohistochemical study to determine the expression of anti-VZV antibodies in VZV-infected dorsal root ganglia. Statistical analysis was carried out by using two-independent-sample t test, one-way analysis of variance, least significant difference- t test or Kruskal-Wallis H test. Results:Nucleocapsid-containing virions and scattered autophagosomes were seen in the dorsal root ganglia in the VZV infection group under the transmission electron microscope. The number of autophagic vesicles significantly differed among the blank control group, VZV infection group, autophagy induction group and autophagy inhibition group ( M[ Q1, Q3]: 0, 5[4, 6], 7[5, 9], 0, respectively; H = 135.60, P < 0.01) , and was significantly higher in the VZV infection group than in the blank control group and autophagy inhibition group (both P < 0.05) , as well as in the autophagy induction group than in the autophagy inhibition group ( P<0.05) , but there was no significant difference between the VZV infection group and autophagy induction group ( P>0.05) . Western blot analysis showed that the expression level of IE62 protein was significantly higher in the VZV infection group (1.49 ± 0.06) than in the blank control group (0.50 ± 0.09, t = 9.17, P < 0.05) ; the expression of anti-VZV antibodies was significantly lower in the autophagy inhibition group than in the autophagy induction group and VZV infection group ( t = 9.24, 7.78, respectively, both P < 0.01) , while there was no significant difference between the autophagy induction group and VZV infection group ( P > 0.05) . Conclusion:Autophagy occurred in the dorsal root ganglia of guinea pigs after VZV infection; the inhibition of autophagy could affect the structure of VZV and decrease the expression of VZV functional proteins in the dorsal root ganglia of guinea pigs.

Correlation between interferon-γ rs2430561 single nucleotide polymorphisms and susceptibility to herpes zoster / 中华皮肤科杂志

Objective:To investigate the correlation of interferon (IFN) -γ rs2430561 single nucleotide polymorphisms (SNPs) and serum IFN-γ levels with susceptibility to herpes zoster.Methods:Blood samples were collected from 74 patients with herpes zoster and 40 healthy controls in General Hospital of Southern Theatre Command and the Fifth People Hospital of Hainan Province from November 2019 to November 2020. PCR and Sagner resequencing were conducted to detect the IFN-γ rs2430561 SNPs in the subjects, fluorescence-based quantitative PCR was performed to determine the copy number of varicella-herpes zoster virus (VZV) DNA in the serum of the patients with herpes zoster, and enzyme-linked immunosorbent assay was conducted to detect the serum IFN-γ level. Measurement data were compared by using t test or non-parametric test, and enumeration data by using chi-square test or Fisher′s exact test. Results:As rs2430561 genotyping showed, there were 4 patients with AA genotype, 37 with TT genotype and 33 with TA genotype in the herpes zoster group, as well as 13 subjects with TA genotype and 27 with TT genotype in the healthy control group, and the frequency of A allele of rs2430561 was significantly higher in the herpes zoster group (27.70%) than in the control group (16.25%, P=0.036) . The serum IFN-γ level ( M[ P25, P75]) was significantly lower in the herpes zoster group (33.45[0.80, 95.01]pg/ml) than in the control group (67.83[2.74, 318.35]pg/ml, U=1 822, P=0.028) . The VZV DNA copy number (expressed as a logarithm to the base of 10) per milliliter was 3.23 ± 0.71 in the serum of the patients with herpes zoster, and the serum IFN-γ level was negatively correlated with the VZV DNA copy number ( r=-0.302, P=0.009) . Conclusion:The carriage of rs2430561 A allele may affect the expression of IFN-γ, leading to higher susceptibility to herpes zoster.

Detection of autophagy in peripheral CD4+ T lymphocytes and skin lesions of patients with herpes zoster / 中华皮肤科杂志

Objective@#To observe changes in expression of autophagy proteins in peripheral CD4+ T lymphocytes and the epidermis of skin lesions, as well as generation of autophagy vesicles in epidermal cells in skin lesions of patients with herpes zoster, and to explore the relationship between varicella-herpes zoster virus (VZV) infection and autophagy.@*Methods@#Totally, 35 patients with herpes zoster were enrolled from Department of Dermatology, General Hospital of Southern Theater Command of PLA between December 2017 and December 2018, including 20 males and 15 females. Their age ranged from 18 to 79 (59.23 ± 9.27) years, pain duration was 5.14 ± 2.28 days, and lesion duration (from the onset of the lesion to the clinic visit) was 3.45 ± 1.77 days. Flow cytometry was performed to determine the expression of autophagy proteins including microtubule-associated protein 1 light chain 3B (LC3B) , Beclin-1 and p62 in peripheral blood CD4+ T lymphocytes of these patients. Thirty healthy adults served as control group. Lesional skin tissues were obtained from 12 patients with herpes zoster, and perilesional normal skin tissues of the same patient served as the control. Immunohistochemical study was conducted to determine the expression of autophagy proteins LC3B, Beclin-1 and p62 in epidermal tissues, and transmission electron microscopy to observe the generation of autophagy vesicles in epidermal cells. Two independent-sample t-test was carried out for intergroup comparison.@*Results@#The expression rates of autophagy proteins LC3B and Beclin-1 in peripheral CD4+ T lymphocytes were significantly higher in the herpes zoster group (61.23% ± 7.61%, 35.84% ± 4.22%, respectively) than in the control group (36.56% ± 4.27%, 15.34% ± 1.89%, respectively; t = 15.75, 24.56 respectively, both P < 0.01) , while the expression rate of p62 (5.75% ± 0.67%) was significantly lower in the herpes zoster group than in the control group (10.03% ± 1.15%, t = 18.65, P < 0.01) . Among the 12 patients with herpes zoster, the expression levels of LC3B and Beclin-1 in the epidermis were significantly higher in the skin lesions than in the perilesional normal skin tissues (t = 2.86, 4.58, P < 0.05) , but the expression level of p62 was significantly lower in the skin lesions than in the perilesional normal skin tissues (t = 2.43, P < 0.05) . Transmission electron microscopy showed formation of autophagy vesicles containing virus particles in epidermal cells in the skin lesions of 12 patients with herpes zoster, and vesicle counts were significantly higher in the skin lesions than in perilesional normal skin tissues (t = 9.67, P < 0.01) .@*Conclusion@#The autophagy level was elevated in peripheral CD4+ T lymphocytes and epidermis of skin lesions of patients with herpes zoster.

Detection of autophagy in peripheral CD4+ T lymphocytes and skin lesions of patients with herpes zoster / 中华皮肤科杂志

Objective To observe changes in expression of autophagy proteins in peripheral CD4+ T lymphocytes and the epidermis of skin lesions,as well as generation of autophagy vesicles in epidermal cells in skin lesions of patients with herpes zoster,and to explore the relationship between varicella-herpes zoster virus (VZV) infection and autophagy.Methods Totally,35 patients with herpes zoster were enrolled from Department of Dermatology,General Hospital of Southern Theater Command of PLA between December 2017 and December 2018,including 20 males and 15 females.Their age ranged from 18 to 79 (59.23 ± 9.27) years,pain duration was 5.14 ± 2.28 days,and lesion duration (from the onset of the lesion to the clinic visit) was 3.45 ± 1.77 days.Flow cytometry was performed to determine the expression of autophagy proteins including microtubule-associated protein 1 light chain 3B (LC3B),Beclin-1 and p62 in peripheral blood CD4 + T lymphocytes of these patients.Thirty healthy adults served as control group.Lesional skin tissues were obtained from 12 patients with herpes zoster,and perilesional normal skin tissues of the same patient served as the control.Immunohistochemical study was conducted to determine the expression of autophagy proteins LC3B,Beclin-1 and p62 in epidermal tissues,and transmission electron microscopy to observe the generation of autophagy vesicles in epidermal cells.Two independent-sample t-test was carried out for intergroup comparison.Results The expression rates of autophagy proteins LC3B and Beclin-1 in peripheral CD4 + T lymphocytes were significantly higher in the herpes zoster group (61.23％ ± 7.61％,35.84％ ± 4.22％,respectively) than in the control group (36.56％ ± 4.27％,15.34％ ± 1.89％,respectively;t =15.75,24.56 respectively,both P ＜ 0.01),while the expression rate of p62 (5.75％ ± 0.67％) was significantly lower in the herpes zoster group than in the control group (10.03％ ± 1.15％,t =18.65,P ＜ 0.01).Among the 12 patients with herpes zoster,the expression levels of LC3B and Beclin-1 in the epidermis were significantly higher in the skin lesions than in the perilesional normal skin tissues (t =2.86,4.58,P ＜ 0.05),but the expression level of p62 was significantly lower in the skin lesions than in the perilesional normal skin tissues (t =2.43,P ＜ 0.05).Transmission electron microscopy showed formation of autophagy vesicles containing virus particles in epidermal cells in the skin lesions of 12 patients with herpes zoster,and vesicle counts were significantly higher in the skin lesions than in perilesional normal skin tissues (t =9.67,P ＜ 0.01).Conclusion The autophagy level was elevated in peripheral CD4+ T lymphocytes and epidermis of skin lesions of patients with herpes zoster.

Aberrant expression of Epstein-Barr virus genes in children with systemic lupus erythematosus / 中华皮肤科杂志

Objective To investigate the expression and significance of Epstein-Barr virus (EBV) genes in children with systemic lupus erythematosus (SLE).Methods Peripheral blood mononuclear cells (PBMCs) were isolated from 20 children with SLE and 12 healthy human controls.Enzyme-linked immunosorbent assay (ELISA) was conducted to detect anti-EBV viral capsid antigen (VCA) IgG/IgM antibodies.The culture supernatants of cells from patients with anti-EBV VCA IgG/IgM antibodies were collected,and PBMCs from the patients and controls were co-cultured with the supernatants respectively for 12 days.RNA was extracted from PBMCs before and after the coculture,and reverse transcription-PCR was performed to detect the expression of EBV genes,including LMP1,LMP2,EBNA1,BCRF1,BLLF1 and BILF1 genes.Results LMP1 gene was detected in fresh PBMCs from 10 out of 20 patients and 1 out of 12 controls (P ＜ 0.05).No significant differences were observed between the patients and controls in the detection rate of LMP2 gene (4/20 vs.1/12),EBNA1 gene (13/20 vs.3/12),BCRF1 gene (3/20 vs.1/12) or BLLF1 gene (5/20 vs.2/12) in fresh PBMCs.After co-culture with the supernatants of cells from patients with anti-EBV VCA IgG/IgM antibodies,the expressions of EBV genes in these PBMCs were increased to different degrees,and there was a significant difference in the expressions of EBV latent genes LMP1,LMP2 and EBNA-1 as well as EBV replicative genes BCRF1 and BLLF1 between the patient-derived and control-derived PBMCs (all P ＜ 0.05).Conclusions There is an aberrant expression of EBV genes in children with SLE,and EBV genes may contribute to the development of SLE.

Expression of EB virus in peripheral blood mononuclear cells in children with systemic lupus erythematosus / 中华实用儿科临床杂志

Objective To discuss the role of EB virus (EBV)in the pathogenesis of systemic lupus erythematosus(SLE) in children through investigating the copies of EBV DNA and expression of EBV genes in peripheral blood mononuclear cells(PBMCs).Methods (1)PBMCs were isolated from 30 patients with SLE and 12 healthy normal controls respectively and DNA was extracted from PBMCs.(2) PBMCs were co-cultured with EBV for 12 days and RNA was extracted from PBMCs.(3)Real-time fluorescence quantitative PCR(Real-time PCR) was applied to detect the copies of EBV DNA in PBMCs.(4)Reverse transcription PCR was applied to detect expression of EBV genes.Results (1) Compared with the healthy control group [(40.1 ± 11.6) copies/μg],a significant increase of EBV DNA copies was observed in SLE group[(658.6 ± 183.6) copies/μg] (P ＜0.05).The EBV DNA copies in the active SLE group [(785.2 ± 179.2) copies/μg] were significantly higher than those in the non-active SLE group [(586.0 ± 193.1) copies/μg] (P ＜ 0.05).(2)There was no correlation between EBV DNA copies and systemic lupus erythematosus disease activity index (r =0.03,P ＞ 0.05).(3) After PBMCs got co-cultured with EBV,expression of latent EBV genes and lytic genes were both increased in the patients and healthy controls.The latent EBV genes including latent membrane protein 1 (LMP1),LMP2,EBV nuclear antigen 1 and the lytic genes including BCRF1,BLLF1 were all increased significantly in the patients compared with the healthy controls (all P ＜ 0.05).Conclusions There is a significant increase of EBV DNA copies and aberrant expression of EBV genes in SLE patients,which suggests that EBV may contribute to the pathogenesis of SLE.

Transcript levels of the Treponema pallidum protein Tp0751 in skin lesions of a rabbit model of early syphilis / 中华皮肤科杂志

Objective To trace changes in the transcript level of the Treponema pallidum(Tp)protein Tp0751 in skin lesions of a rabbit model of early syphilis. Methods Three New Zealand white rabbits were intracutaneously injected with 0.1 ml of Tp (Nichols Seattle strains)suspensions (107 treponemes/ml)at 10 sites on the shaved back to establish a model of early syphilis. All the rabbits received a single injection with the total amount of treponemes being 107. Then, skin changes at injection sites were observed, and the size of skin rashes was recorded on a daily basis. Skin specimens sized 0.4 cm × 0.4 cm were excised from an injection site and a non-injection site(negative control)separately every 3 days for the detection of Tp0751 and Tp0574 mRNAs. The whole experiment lasted 30 days, and a total of 11 skin biopsies were carried out. Fluorescence-based quantitative PCR was performed to measure the mRNA expressions of Tp0751 and Tp0574 continuously and dynamically during the development of chancre. Results After intracutaneous injection of Tp suspensions, red papules occurred on the back of rabbits on day 6, and reached maximum size on day 19 with the formation of ulcer and chancre. On day 25, disseminated secondary syphilides gradually appeared all over the body surface of the rabbits. The mRNA expression levels of Tp0574 and Tp0751 increased at the early stage, peaked onday 15 (compared with the other time points, all P < 0.05), thereafter rapidly declined, but rose slightly on day 27. The standardized expression level of Tp0751 mRNA increased gradually after day 15, and peaked on day 24 (compared with the other time points, all P < 0.05). Conclusion The transcript level of Tp0751 was high in rabbits at the late stage of Tp clearance when generalized disseminated secondary syphilides had not appeared, suggesting that Tp0751 may be involved in the systemic spread of Tp.

Preparation of a dendritic cell vaccine modified by the adenovirus-mediated HSV-2 gD gene / 医学研究生学报

Objective Up to now, there has been no sure cure for genital herpes (GH), and vaccine seems a most promis-ing approach to the prevention and treatment of herpes simplex virus Ⅱ(HSV-2) infection.In this study, we investigated the feasibili-ty of preparing a dendritic cell ( DC) vaccine modified by the adenovirus-mediated HSV-2 gD gene. Methods We subcloned the HSV-2 gD gene into the vector Shuttle-2 and constructed the recombinant adenovirus pAdeno-HSV-2 gD following identification by en-zyme digestion and DNA sequence analysis .We isolated DCs from the mouse bone marrow , analyzed their phenotypes by flow cytome-try after transfection with the recombinant adenovirus pAdeno-HSV-2 gD, and determined the expression of HSV-2 gD by immunohisto-chemistry, RT-PCR, SDS-PAGE, and Western blot. Results Based on HSV-2 DNA, the corresponding target fragments were am-plified with the gD gene primers.Agarose gel electrophoresis showed the correct size of the PCR product (1182 bp) as predicted.The recombinant adenovirus pAdeno-HSV-2 gD was obtained by transfecting the 293 cells with pAdeno-gD DNA, which had an activity of 4 ×1010 IU/mL.The contents of CD40, CD80, and CD86 were (74.2 ±3.9), (73.9 ±4.1), and (76.1 ±5.5) % in the mature DCs and (81.3 ±3.1), (80.4 ±2.9), and (83.7 ±3.9) % in the pAdeno-HSV-2 gD DCs, significantly increased as compared with those in the immature DCs ([9.7 ±0.5], [7.5 ±1.2], and [5.2 ±1.1] %) (P0.05).RT-PCR and immunohistochemistry confirmed the expression of HSV-2 gD in DCs.SDS-PAGE and Western blot of the expressed protein showed a new band with an apparent molecular mass corresponding to the predicted size (43000). Conclusion The results of our study have paved the ground for the successful preparation and identification of a dendritic cell vaccine modified by the adenovirus-mediated HSV-2 gD gene.

An experimental study on the immune response induced by a dendritic cell-based vaccine carrying the herpes simplex virus type 2 glycoprotein D gene in mice / 中华皮肤科杂志

Objective To evaluate the specific immune response induced by a dendritic cell-based adenovirus-mediated vaccine carrying the herpes simplex virus type 2 glycoprotein D gene (pAdeno-HSV-2 gD-DC) in BALB/c mice.Methods Forty BALB/c mice were equally divided into four groups:blank control group receiving no treatment,pAdeno-DC group immunized with pAdeno-DC,pAdeno-HSV-2 gD-DC group immunized with the previously constructed vaccine pAdeno-HSV-2 gD-DC,DC group immunized with DCs only.Totally,three rounds of vaccination were conducted at a 7-day interval.Ten days after the last vaccination,serum samples were collected and spleen cells were isolated from these mice.Enzyme-linked immunosorbent assay (ELISA) was performed to measure the level of IgG antibody against HSV-2 gD in the serum samples.Some spleen cells were stimulated with HSV-2 gD protein (10 mg/L) for 72 hours; then,ELISA was carried out to determine the levels of interferon (IFN)-γand interleukin (IL)-4 in the supernatant,and 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT) assay to estimate the proliferative activity of these cells.The cytotoxicity of spleen cells was also evaluated based on the measurement of lactate dehydrogenase (LDH) release.Results The serum level of IgG antibody against HSV-2 gD (given in the absorbance value at 450 nm) was 0.313 ± 0.034 in the pAdeno-HSV-2 gD-DC group,significantly higher than that in the pAdeno-DC group,DC group and blank control group (0.034 ± 0.009,0.028 ± 0.009 and 0.026 ± 0.010 respectively,all P ＜ 0.05).Increased proliferative activity and cytotoxicity were observed in spleen cells from the pAdeno-HSV-2 gD-DC group compared with those from the pAdeno-DC group,DC group and blank control group (cell stimulation index:1.600 ± 0.215 vs.1.063 ± 0.070,1.056 ± 0.063 and 1.020 ± 0.051,all P ＜ 0.05; percentage of cytotoxicity:37.1％ vs.16.0％,14.9％ and 15.7％,all P ＜ 0.05).The levels of IFN-γ and IL-4 (both given in the absorbance value at 450 nm) were 0.568 ± 0.031 and 0.544-± 0.043 respectively in the supernatant of spleen cells from the pAdeno-HSV-2 gD-DC group,compared to 0.266 ± 0.021 and 0.278 ± 0.037 respectively in the pAdeno-DC group (bothP＜ 0.05),0.271 ± 0.023 and 0.275 ± 0.044 respectively in the DC group (bothP＜ 0.05),and 0.252 ± 0.012 and 0.245 ± 0.051 respectively in the blank control group (both P＜ 0.05).Conclusion The vaccine pAdenoHSV-2 gD-DC could induce a specific and strong immune response in BALB/c mice.

Effects of microRNA hsa-mir-634 on the proliferation and apoptosis of Vero cells / 中华皮肤科杂志

Objective To investigate the function and possible action mechanisms of microRNA hsa-mir-634 in Vero cells.Methods The binding sites for hsa-mir-634 in the 3' UTR of cyclin D1 (CCND1) were predicated by bioinformatics methods.Then,the 3'UTR sequence of CCND1 containing the binding sites for hsamir-634 was amplified by PCR.Site-directed mutagenesis was used to create mutations in the binding sites.The wild and mutant 3' UTR sequences of the CCND1 gene were ligated into the psi-CHECK2 vector separately to construct dual-luciferase reporter vectors,including CHECK2-CCND1 wild,CHECK2-CCND1 mut 1,CHECK2-CCND1 mut 2 and CHECK2-CCND1 mut 3.Then,293T cells were transfected with the four constructed plasmids,and luciferase activity was measured 48 hours after the transfection.Vero cells were transfected with hsa-mir-634 mimics and negative control separately,and harvested after additional culture for different durations; the Vero cells remaining untreated served as the blank control.Subsequently,fluorescence-based quantitative PCR and Western blot were performed to detect the mRNA and protein expressions of CCND1 respectively in,3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt (MTS) assay to evaluate the proliferation of,and flow cytometry to detect the apoptosis in,Vero cells.Results The binding sites for hsa-mir-634 in the 3'UTR of CCND1 were successfully predicated.Sequencing results showed the successful construction of dual-luciferase reporter vectors.As the luciferase assay revealed,the overexpression of hsa-mir-634 could significantly inhibit the CCND1 3'UTR-mediated luciferase activity.Compared with the negative control,the hsamir-634 mimics markedly decreased the protein expression of CCND1,but had no obvious effect on the mRNA expression of CCND1 in Vero cells.The proliferation of Vero cells transfected with hsa-mir-634 mimics was significantly restrained compared with those transfected with the negative control,and the strongest restraining effect was observed on day 4 after the transfection.In addition,the overexpression of hsa-mir-634 also induced the apoptosis of Vero cells,with the apoptosis rate being 8.03％,7.96％ and 17.33％ in the blank control group,negative control group and mimics group respectively.Conclusion Hsa-mir-634 may regulate the proliferation and apoptosis of Vero cells via influencing the expression of CCND1.

Expression of herpes simplex virus type 2 latency associated transcript ORF1 and its anti-apoptotic function / 生物工程学报

To study the expression of herpes simplex virus type 2 latency-associated transcript (LAT) open reading frame 1 (ORF1) and its anti-apoptosis function induced by actinomycin D in Vero cells. The recombinant plasmid pEGFP-ORF1 was constructed and transfected into Vero cells, and the expression of ORF1 was identified by RT-PCR. The changes of Vero cells morphology induced by actinomycin D were observed by fluorescence microscopy, Hochest33258 fluorescence staining. Cells viability was evaluated by MTT assay and cells apoptosis rate was detected by flow cytometry. Double digestion and sequencing confirmed the pEGFP-ORF1 was constructed successfully, RT-PCR showed that the target gene was highly expressed in Vero cells. Hochest33258 staining reaveals that Vero cells transfected with pEGFP-ORF1 and induced apoptosis by actinomycin D had no changes in morphology. MTT assay showed that the viabilities of Vero cells transfected with recombinant plasmid pEGFP-ORF1 and induced apoptosis by actinomycin D has no statistically significant difference compared with the untreated normal control group (P > 0.05), but remarkable higher than Vero cells transfected with empty plasmid pEGFP-C2 and induced apoptosis by actinomycin D, the difference was statistically significant (P < 0.05). Flow cytometry assay shows that the cells apoptosis rate had no significant difference between pEGFP-ORF1 group and the normal group, but the cells apoptosis rate ofpEGFP-ORF1 was lower than the pEGFP-C2 group. HSV-2 LAT ORF1 gene can be expressed in Vero cells and can protect Vero cells from apoptosis induced by actinomycin D.

Effects of herpes simplex virus 2 latency-associated transcript open reading frame 3 on the apoptosis in Vero cells / 中华皮肤科杂志

Objective To explore the effects of herpes simplex virus 2 (HSV-2) latency-associated transcript open reading frame 3 (LAT ORF3) gene on Vero cells against cisplatin-induced apoptosis.Methods Recombinant plasmid enhanced green fluorescent protein-open reading frame 3 (named pEGFP-ORF3) was constructed and transfected into Vero cells; then,reverse transcription (RT)-PCR was performed to detect the expression of the target gene.Cisplatin of 3 mg/L was selected to induce the apoptosis in Vero cells.Cultured Vero cells were transfected with empty plasmid and induced by cisplatin (pEGFP-C2 group),transfected with recombinant plasmid pEGFP-ORF3 and induced by cisplatin (pEGFP-ORF3 group),only induced by cisplatin (cisplatin-induced control group),or remained untreated (normal control group).Subsequently,fluorescence microscopy was conducted to observe apoptotic bodies,Giemsa stain to observe the morphology of cell nuclei,methyl thiazolyl tetrazolium (MTT) assay to evaluate cell proliferation,and flow cytometry to assess cell apoptosis.Data were assessed by using SPSS 13.0 software,and statistical analysis was carried out by one-way ANOVA and t test.Results HSV-2 333 LAT ORF3 gene was successfully cloned.The eukaryotic expression plasmid for LAT ORF3 was constructed,and the expression of LAT ORF3 gene in Vero cells was confirmed by RT-PCR.Giemsa stain showed blue-staining nuclei and pale cytoplasm in recombinant plasmid-transfected and cisplatin-induced Vero cells with a normal shape.The value of cell proliferation (absorbance at 490 nm) by MTT assay was 2.56 ± 0.21 in pEGFP-ORF3 group,similar to that in the normal control group (2.66 ± 0.13,P ＞ 0.05),but significantly higher than cisplatin-induced control group (1.65 ± 0.11,P ＜ 0.05) and pEGFP-C2 group (1.56 ± 0.18,P ＜ 0.05).As far as the apoptosis rate was concerned,no significant difference was observed between pEGFP-ORF3 group and normal control group (4.03％ ± 1.04％ vs.2.13％ ± 0.09％,P ＞ 0.05),but pEGFP-ORF3 group was statistically lower than pEGFP-C2 group (19.45％ ± 2.05％,P ＜ 0.05).Conclusion The transfected HSV-2 LAT ORF3 gene could protect Vero cells from cisplatin-induced apoptosis.

Changes of microRNA expression profiles in Vero cells induced by HSV-2 LAT overexpression / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the changes in the microRNA expression profile of Vero cells induced by HSV-2 LAT overexpression.</p><p><b>METHODS</b>The full-length open reading frame of HSV-2 LAT was synthesized and cloned into pRetroQ- AcGFP1-C1 vector, and the recombinant retrovirus expressing HSV-2 LAT was packaged. Using a microRNA microarray, the microRNA expression profile changes in Vero cells were analyzed after infection with the recombinant retrovirus.</p><p><b>RESULTS</b>In Vero cells infected with the recombinant retrovirus for stable HSV-2 LAT overexpression, 5 microRNAs (hsa-miR-23a*, kshv-miR-K12-3, hsa-miR-943, hsa-miR-634, and hsa-miR-1270) were up-regulated and 5 (hsa-miR-181a-2*, hsa-miR-450b-5p, hsa-miR-31, hsa-miR-24, and kshv-miR-K12-12*) were down-regulated.</p><p><b>CONCLUSION</b>The expression of HSV-2 LAT can induce changes in microRNA expression profile in Vero cells.</p>

Differential expression of mRNAs associated with low-dose UVA irradiation-induced adaptation reaction in cultured human melanocytes / 中华皮肤科杂志

Objective To screen for genes associated with low-dose UVA irradiation-induced adaptation reaction in cultured human melanocytes, and to explore the molecular mechanism of the adaptation.Methods Cultured human melanocytes of fifth to tenth passage were divided into two groups, experimental group was subjected to an irradiation with UVA at 7.2 J/cm2 once daily for 4 times and an additional irradiation at a lethal dose of 86.4 J/cm2 6 hours after the above 4-session irradiation, and control group subjected to a single irradiation with UVA at 86.4 J/cm2. A human genome-wide oligonucleotide chip was used to screen for differentially expressed genes between the two groups of cells followed by a functional classification based on international standard. Moreover, a part of these genes were analyzed and identified by real-time fluorescent quantitative PCR. Results In the adaptation reaction induced by low-dose UVA irradiation in cultured melanocytes, there were 129 differentially expressed genes, including 81 up-regulated genes and 48 down-regulated genes. These genes were found to be mainly involved in metabolism, transport, signal transduction, apoptosis, DNA synthesis and repair, and some of them were oncogenes or anti-oncogenes. Real-time PCR confirmed some of the differentially expressed genes. Conclusions The whole genome-wide oligonucleotide chip could screen with high efficiency for differentially expressed genes in low-dose UVA irradiation-induced adaptation reaction in cultured melanocytes.

Combination of 308 nm excimer laser with topical pimecrolimus cream for vitiligo in childhood / 中华皮肤科杂志

Objective To assess the efficacy and safety of 308 nm excimer laser plus topical pimeevaluated after 15 and 30 times of laser therapy respectively.ResultsExcept for one patient,all patients were able to be evaluated for effiicacy.After 30 times of laser therapy,the response and excellent response rates were 89.6%and 77.1%respectively,in group A，5.0%and 52.1%respectively in group B;both rates were significantly higher in group A than in group B(both P＜0.05).Also,a highler repigmentation rate was obtained in facial lesions in group A compared with group B.Conclusions The 308 nm excimer laser is safe,erective and well-tolerated for vitiligo in childhood,and the combination with topical imecrolimus cream may improve its efficacy in facial vitiligo.

Updated functions of the herpes simplex virus Us3 gene / 医学研究生学报

The herpes simplex virus(HSV) Us3 gene encodes a ser/thr protein kinase(PK).As an accessory gene,it plays an important role in the regulation of the apoptosis of infected cells and virus release.Us3-induced alterations in the host cytomorphology are associated with enhanced intercellular virus spread,suggestive of a previously undescribed aspect of alphaherpesvirus spread.

Prophylactic Effects of a HSV-2DNA Vaccine in Guinea Pigs / 中华皮肤科杂志

Objective To observe the prophylactic effects of a HSV-2gD2DNA vaccine in guinea pigs challenged with HSV-2strains.Methods Female guinea pigs were divided into3groups with10each,which was immunized intramuscularly with100?g of pc-gD plasmids(recombinant HSV-2DNA vac-cine),or with pcDNA3blank plasmids,with normal saline as control,respectively.Two booster injections were given on day7and day21.Sera were collected for virus neutralization test on day0,day28,and day56.The animals were challenged with HSV-2strain sav intravaginally,and lesions induced on the external genital skin were scored between day1and day21after challenge.Results The titer of neutralizing anti-body to HSV-2was much higher in the sera from animals immunized by pc-gD plasmids than that from ani-mals immunized by pcDNA3blank plasmids or normal saline.Furthermore,the lesion scores on external genital skin were significantly decreased in pc-gD group than those in other two groups with either primary or recurrent infections.Conclusion The constructed gD2vaccine can efficiently protect guinea pigs from genital infection and reduce recurrent infection induced by latent herpes simplex virus.

TCR/CD3 complex-mediated signal transduction in T cells from patients with systemic lupus erythematosus / 中华风湿病学杂志

Objective To investigate whether systemic lupus erythematosus (SLE) T cell function disorder is related to abnormal biochemical pathways.Methods After cross linking of anti CD3 mAbs to sheep anti mouse IgG and stimulating T cells,the changes of free calcium ion within T cells and these changes under interference of Thapsigargin and EGTA were observed respectively for 10 minutes with an adhesion cytometry.The relation between [Ca 2+ ]i response in SLE T cells and expression of CD3 molecules,or InsP 3 levels was evaluated.Results The base [Ca 2+ ]i response in T cells of SLE patients was similar to that of normal control ( P =0 105).Peak and plateau [Ca 2+ ]i responses were significantly higher in the group of SLE patients ( P

Clinical Feature and Glycoprotein D Gene Sequence Analysis of a Wild Strain of HSV-2 Isolated from One Relapsed Patient with Genital Herpes / 中华皮肤科杂志

Objective To study the relation between the clinical feature and glycoprotein D gene sequence analysis of a wild strain of HSV 2 isolated from one relapsed patient with genital herpes. Methods The partial glycoprotein D gene sequence of the above mentioned strain was amplified and cloned with PCR. Results The comparison of the amino acid sequence of gD gene between the wild strain and HSV 2G strain published showed that there was a mutation at site80 and site159. Conclusion In order to develop effective vaccine which is suitable for China, it is necessary to investigate the differences of the gene structure and function of gD among HSV isolated from China and other countries.