参芪止疱颗粒抗HSV-2的体外实验研究

目的:探讨参芪止疱颗粒体外抗HSV-2的作用。方法:利用HFF感染HSV-2,用参芪止疱颗粒予以干预,以ACV做对照药,采用CPE法和MTT法进行实验。结果:实验发现HSV-2的TCID50为104;参芪止疱颗粒对HFF的CC50值大于10mg/ml,表明其对HFF的毒性作用小;在药物直接抗病毒复制实验中,参芪止疱颗粒对感染细胞EC50为0.30mg/ml,TI>36表明其对HSV-2感染HFF具有一定保护作用;在HSV-2感染HFF的治疗实验中,参芪止疱颗粒EC50值为0.60mg/ml,TI>25,提示其对病毒感染后复制有一定的抑制作用;其对HSV-2复制的抑制作用时相主要在感染后0h、2h、4h。但是,其对HSV-2感染细胞无预防作用。结论:参芪止疱颗粒在体外试验中具有较好的抗HSV-2的作用,值得临床进一步推广。

A Study of Monocyte Excretion of TNF- α and IL-6 and Monocyte Expression of HLA Class Ⅱ Antigen In Genital Herpes

Objective: To study the role of monocytes in the pathogenesis of genital herpes. Methods: TNF- α and IL-6 levels in 27 cases of genital herpes were detected by enzyme linked immunosorbant assay (ELISA). HLA class Ⅱ antigen expression on monocytes were detected by an alkaline phosphatase anti-alkaline phosphatase method. Results: Compared with normal controls, levels of TNF- a and IL - 6 secreted by monocytes responding to LPS mitogen in vitro were significantly decreased [(3.13 ± 0.44ng/ml) vs (4.68 ± 0.54ng/ml), P<0.05 and (3.32 ± 1.06ng/ml) vs (6.46 ± 1.94ng/ ml), P<0.05, respectively]. HLA class Ⅱ antigen expression on monocytes in the genital herpes group was also significantly decreased [HLA-DR (67.48% ± 1.51%) vs (81.03% ± 1.32%), P<0.01 and HLA-DQ (29.54% ± 1.15%) vs (37.63% ± 1.79%), P <0.01 respectively]. Conclusion: These findings suggest that the decreased monocyte function may contribute to the pathogenesis of genital herpes. Augmenting or inducing monocyte function may be important in the prevention, treatment, and reduction of genital herpes cases.

Comparison of Multiplex Fluorescent PCR with Serum Type-specific Antibody Detection in Diagnosis of Genital Herpes

Objectives: To compare multiplex fluorescent PCRwith serum type-specific antibody detection in thediagnosis of herpes simplex virus (HSV) infection andto evaluate its significance in the diagnosis of genitalherpes.Methods: We detected HSV infection in 121 speci-mens collected from patients with genital herpesusing both multiplex fluorescent PCR and serum type-specific antibody detection. HSV viral isolation wasused as the standard control.Results: When compared with the viral isolation, thesensitivity and specificity for multiplex fluorescentPCR were 100% and 88.89%, respectively afterdiscrepant analysis. The sensitivity and specificity fortype-specific antibody detection was 77.68 % and77.78 %, respectively. However, the type-specificantibody detected HSV in two asymptomatic patientswhile the multiplex fluorescent PCR couldn’t detectany HSV DNA from those specimens.Conclusions: Multiplex fluorescent PCR is a verysensitive and specific method for detection and typingof HSV in the lesion of genital herpes, it failed todetect HSV DNA from the asymptomatic patients.Serum type-specific antibody detection was a lesssensitive and specific test but could detect the specificantibody from some asymptomatic patients. Thecombination of these two techniques would allow rapid,sensitive and accurate detection and typing of HSVand help clinical diagnosis and epidemiologic survey-ing of genital herpes.

Association Between Genital Ulcer Disease and HIV Seroprevalence among STD Patients in Guangzhou

Objectives: To understand genital ulcer disease(GUD) among patients attending sexually transmitteddisease (STD) clinics in Guangzhou, China, and itsassociation with HIV infection.Methods: Between September 9th, 1997 and Octo-ber 30th, 2002, 8 962 patients with STDs wereevaluated. 285 patients were diagnosed with GUD basedon clinical manifestations and microbiologic evalua-tions including dark field microscopy and serologytest for syphilis (RPR, TPPA). Swabs of each genitalulcer were processed in a multiplex PCR assay (M-PCR) for simultaneous detection of Herpes simplexvirus (HSV), Treponema pallium, and Haemophilusducreyi. Other STDs were classified by routine diag-nostic criteria, including microscopy or culture forNeisseria gonorrhoeae, Chlamydia trachomatis,Urea- plasma urealyticum, Human papillomavirus,Trichomonas, etc.Results: Of the 8 962 patients with STDs, the HIVseroprevalence in patients with and without GUD was1.75% (5/285) and 1.53% (133/8677), respectively,with no statistically significant difference (χ 2=0.09,P>0.05; OR=1.15, 95%CI=0.47-2.81) . HIVseroprevalence in patients with syphilis, genital her-pes and other STDs was 2.81% (22/784), 0.74% (6/814) and 1.49% (110/7 364), respectively. Prevalencein patients with syphilis was significantly higher thanthat in patients with genital herpes and other STDs,(χ 2=9.92, P<0.005, OR=3.89, 95%CI=1.67-9.05;χ 2=7.66, P<0.001, OR=1.90, 95%CI=1.21-3.00).Conclusions: The study shows that the HIV sero-prevalence in this population of patients with GUDis very low. The results also indicate an associationbetween syphilis and HIV infection. The relationshipbetween genital herpes and HIV infection needsfurther research.

Long Dan Xie Gan Formula Granule Promotes Pro-Inflammatory Cytokine Expression in Female Guinea Pigs with Recurrent Genital Herpes

Objective To explore the effects of Long Dan Xie Gan formula granule(LDXGFG)on regulation of pro-inflammatory cytokines in female guinea pigs with recurrent genital herpes(herpes simplex virus 2,HSV-2).Methods Levels of pro-inflammatory cytokines in blood of HSV-2-infected guinea pigs,including IL-6,IL-8,IL-10,IL-12,IFN-α,IFN-β,IFN-γ,and TNF-α,were detected by ELISA;corresponding gene expression levels in tissues were detected by real-time PCR.Results IL-6,IL-10,IL-12,IFN-α,IFN-γand TNF-αdecreased significantly in both blood and diseased tissues after infection with recurrent genital herpes.Upon feeding LDXGFG to HSV-2-infected guinea pigs,IL-6,IL-10,IL-12,IFN-α,IFN-γand TNF-αdemonstrated significant increases,similar to the effects of acyclovir(ACV).LDXGFG promoted the expression of pro-inflammatory cytokines in blood and tissue,with a stronger effect than ACV.Moreover,LDXGFG demonstrated broader effects than ACV.Conclusion The present results suggest that LDXGFG can serve as an alternative,inexpensive,and long-term treatment for HSV-2 infection.

Interleukin-2 and Interleukin-6 in Recurrent Genital Herpes Patients

Objective:To study the cellular immunity status of patientswith recurrent genital herpes. Methods: Serum levels of interlukin-2 and its solublereceptor and interlukin-6 were measured by ELISA in 34patients with recurrent genital herpes. Results: Serum levels of IL-2 and IL-6 were significantlydecreased in patients compared to healthy controls (P<0.01),and the level of sIL-2R was significantly increased in patientswith recurrent genital herpes (P<0.01). There were nosignificant differences in all variables amongst patientsregarding relapse stage and remission stage (P>0.05). Conclusion: There was a cellular immune deficiency inpatients with recurrent genital herpes.

Asymptomatic Herpes Simplex Virus Shedding in STI Patients

Objective: This study examined Herpes Simplex Virus (HSV) subclinical shedding in the genital tract of patients withgenital herpes (GH) or non-gonoccal urethritis (NGU). Method Swabs were collected after exposure to rash andgenital tract during GH relapse or remission on a weekly basisfor four to six weeks. NGU patients with negative chlamydiaand mycoplasma tests were also swabbed for a similarduration. All swabs underwent HSV DNA detection withquantitative PCR. Result: There was a significant difference in the rate ofasymptomatic HSV shedding in urinary tracts comparing GHand the control group and comparing NGU and the controlgroup (P<0.05). The rate of HSV shedding was 22%, 9.8%and 3.3% for GH, NGU and control groups respectively. Therate of HSV shedding was 21.7% (20/92) for patients withactive GH and 23% for those in remission. The HSV positiverate was significantly higher in the group with patients whohad more than six relapses within one year compared to thegroup of patients with less than six relapses.Conclusion: There is HSV subclinical shedding in theirgenital tract during active GH and remission. SubclinicalHSV shedding is more common in patients with more than sixGH relapses per year compared to GH patients with fewerrelapses. Approximately 9.9% of NGU patients with negativechlamydia mycoplasma testing was found to have subclinicalHSV infection.

Clinical Presentation of Atypical Genital Herpes

Objective: To make a clinical analysis on the basis of 36 cases of atypical genital herpes (GH) patients. Methods: Thirty-six cases of atypical GH were diagnosedclinically, and their case histories, symptoms and signs wererecorded in detail and followed up. Polymerase chain reaction(PCR) was adopted for testing HSV2-DNA with cotton-tippedswabs. Enzyme-linked immuno sorbent assay (ELISA) forserum anti-HSV2-IgM was done to establish a definitivediagnosis. Other diagnoses were excluded at the same time bytesting for related pathogens including fungi, Chlamydia,Mycoplasma, Treponema pallidum, gonococci, Trichomonas,etc. Results: The main clinical manifestations of atypical GHwere: (1) small genital ulcers; (2) inflammation of urethralmeatus; (3) nonspecific genital erythema; (4) papuloid noduleson the glands; (5) nonspecific vaginitis. Twenty-three cases(64%) tested by PCR were HSV2-DNA sera-positive, and 36cases (100%) anti-HSV2-1gM sera-positive by ELISA. Conclusion: atypical HSV is difficult to be dinosed. But the combination of PCR and ELIAS will be helpful to thediagnosis of atypical HSV.

Effect of Traditional Chinese Medicine Antiviral Capsules On Animal Model Genital Herpes and HSV-2 in Cell Culture

Objective: To study the effect of traditional Chinese medicine antiviral capsules in the treatment of genital herpes. Methods: Using female guinea pig genital herpes as the animal model, this study used oral administration of two formulations of antiviral capsules (AC) and observed the effect on vaginal HSV-2 titers and vulvar symptoms. Cell cultures were also used to examine the direct inactivation of HSV-2 by the antiviral capsules and the suppression of HSV-2 via three drug administration methods. Results: There was no significant difference of mean vaginal virus titers between the antiviral capsule groups and that of the positive acyclovir (ACV) control (P>0.05). Mean vulvarsymptom scores of the two antiviral capsule groups were also significantly lower than that of the saline negative control group on days 2, 3, 5, 7 and 8 (P<0.05) and similar to that of the ACV control (P>0.05). Cell culture showed the minimum inhibitory concentrations of antiviral capsules No. 1 and No. 2 were 0.390625 mg/ml and 1.5625 mg/ml, respectively. Conclusion: The traditional Chinese medicine antiviral capsules had suppressive effects on HSV-2 in both animal model GH and in vitro cell culture.

T Lymphocytes and Th1/Th2 Cytokines in Peripheral Blood of Patients with Recurrent Genital Herpes

Objective: This study analyzed the T lymphocytes and Th1/Th2 type cytokine profile shift in the peripheral blood ofpatients with recurrent genital herpes (RGH). Methods: Immunofluorescent staining or cell surface antigenand intracellular cytokines(IL-2、IL-4、IL-12、IFN-r)inperipheral blood from 20 RGH patients and 10 controls wereanalyzed using flow cytometric techniques. Results: RGH patients had signiflcantly lower levels ofCD3^+T cells, CD4^+T cells and CD4^+ T/ CD8^+ T cells ratiocompared to control levels (P<0.001), and IL-2-producing,IFN-r-producing and IL-12-producing T cells were increasedin RGH patients (CD4^+T: P<0.001, CD8^+T: P<0.05respectively), whereas IL-4-producing T cells were increased inRGH patients compared to controls (CD4^+T: P<0.05; CD8^+T:P<0.001 respectively). Conclusions: RGH patients have T lymphocyte subsetvariations and Th1/Th2 cytokine changes. The increase in Th2cells Th1/Th2 imbalance may have important implications forRGH pathogenesis.

Detection of Intracellular Cytokine Production in Peripheral Blood CD_^+ T Cells of Patients with Recurrent Genital Herpes

Objective: To study the role of Th1/Th2 cytokines in thepathogenesis of recurrent genital herpes (RGH), and tobetter understand the relationship between them. Methods: A two-color immunofluorescent staining of cellsurface antigen and intracellular cytokines for flowcytometric analysis was used for CD_3, IL-2, IL-10, IL-12,IFN-γ and TNF-α in CD_3^+ T-lymphocytes in activatedperipheral blood mononuclear cells of patients with RGH. Results: Compared to controls, patients with RGHshowed fewer CD_3^+ T cells (P<0.05) and IL-2 producing andIFN-γ producing T cells (P<0.02 and P<0.001, respectively)after in vitro stimulation with PMA and ionomycin in thepresence of a protein transport inhibitor. More IL-10producing and IL-12 producing T cells were found inpatients with RGH (P<0.01). There was no significantdifference in the number of TNF-α producing cells betweenRGH patients and controls (P<0.05). Conclusion: RGH patients showed relatively more Th2cytokines. The imbalance between Th1 and Th2 cytokinesresults in inhibitory effects on a series of cell-immuneresponses, which may play an important role in thepathogenesis of RGH.

Detection of Treponema pallidum,Herpes Simplex Virus,and Haemophilus ducreyi from Genital Ulcers by Multiples Polymerase Chain Reaction

Objective: To evaluate the clinical application of multiplex PCR inthe detection of Treponema pallidum, Herpes simplex virus (HSV), andHaemophilus ducreyi. Method: Three standard strains were used to set up a multiplexPCR (MPCR) for detecting syphilis, herpes genitalis, and chancroidsimultaneously. Samples from 122 patients with genital ulcer disease(GUD) were subjected to MPCR and the results were compared withthose of dark-field microscopy and TP serology, HSV antigen ELISA,and H. ducreyi culture. Results: In the 122 patients with GUD, MPCR identified 34 casesof T. pallidum infection, 40 cases of HSV infection, and 2 cases of mixedinfection of T. pollidum and herpes. No positive results of H. ducreyiwere found. The sensitivity of MPCR to T. pallidum and herpes was100% and 93.3%, respectively. The sensitivities of dark-field mi-croscopy and TP serology, HSV antigen ELISA, and H. ducreyi cul-ture was 35.3%, 50% and 100%, respectively. Conclusion: MPCR showed a relatively higher sensitivity for T.pallidum as compared with the routine techniques. Although its sensi-tivity for HSV was not as good as that of antigen ELISA, it also yieldeda high detection rate. MPCR can detect more than one pathogen. It issimple, quick, sensitive, and suitable for clinical use or epidemiologicalinvestigation.