The Clinical And Experimental Research On The Treatment Of Genital HPV Infection With 5-aminolevulinic Acid Photodynamic Therapy

Background:Genital warts are one of the most common sexually transmitted diseases and are caused by certain types of human papillomavirus(HPV)infection.The overall HPV prevalence was 26.8%among US females aged 14 to 59 years[1]and the overall genital HPV infection prevalence was 45.2%among men aged 18 to 59 years[2].5-aminolevulinic Acid photodynamic therapy(ALA-PDT)has demonstrated good clinical cure rates and low recurrence rates in the treatment of genital warts.Human papillomavirus(HPV)genotypes and viral load assays can reflect the status of persistent or latent infection and serve as a predictor of infection clearance.But the mechanism of this is not completely clear and there were still no objective indicators to evaluate the treatment effect of ALA-PDT.Purpose:1.Collecting the clinical data of HPV genotypes and viral loads of patients with Condyloma acuminatum and latent or subclinical HPV infection who received photodynamic therapy(PDT)treatment.2.To analyze the expression of TAP-1 and MHC class I molecules in the tissue of condyloma acuminata.3.To investigate the proliferation level of Siha/Hela and the expression of HPV E6/E7,TAP,MHC class I molecules after ALA-PDT.Methods:1.The clinical research on the treatment of HPV external anogenital infection1.1 ALA-PDT treatment for patients with condyloma acuminatum1.1.1 Patients who were diagnosed as condyloma acuminatum and met the inclusion criteria in our department from December 2015 to October 2017 were recruited.1.1.2 HPV DNA testing was performed before each PDT session.Patients underwent one cycle of PDT treatment every week until the results of the HPV DNA test were negative for two consecutive times.1.1.3The adverse reactions were recorded during the treatment of ALA-PDT.1.1.4 All patients were follo wed-up for at least six months.1.2 ALA-PDT treatment for patients with asymptomatic HPV infection1.2.1 Patients who were positive for HPV detection and were not simultaneous other sexually transmitted diseases were recruited during December 2015 to October 2017.1.2.2 At each patient visit,polymerase chain reaction was used to identify HPV genotypes and measure the viral loads,which reflect the status of HPV infection.We defined active infections as those with increasing viral loads between at least two consecutive tests.By contrast,we defined regressing infections as those with gradually decreasing viral loads between two consecutive tests.ALA-PDT was performed every week in patients with active infection and no treatment was applied in patients with regression infection.1.2.3 The negative rate of HPV test after three times of PDT treatment was analyzed.2.The expression of TAP-1 and MHC-I molecules in the tissues of condyloma acuminataImmunohistochemical,Western Blot and RT-PCR methods were used to detect the expression of TAP-1 and MHC-1 molecules in the tissues of condyloma acuminatum.3.Experiments in vitro3.1 Siha/Hela cell lines were obtained from Experimental study of clinical medicine in our hospital.Cells were incubated in Dulbecco's modified Eagle medium(DMEM)supplemented with 10%fetal bovine serum and 1%penicillin-streptomycin in an atmosphere of 5%CO2 at 37?.3.2 Siha/Hela cells were divided into following four groups:Control,ALA,Laser and ALA-PDT.3.3 Drug concentration screening:Average fluorescence intensity(MFI)was analysed using confocal laser scanning microscope.Cell proliferation activity was detected by CCK-8 Kit.3.4 Red light irradiation and dose calculation:cells were irradiated with 635nm red light(20 mW/cm2);light dose = power × time(s).3.5 Cell proliferation activity was detected by CCK-8 Kit.3.6 Detection of HPV E6/E7 protein:Western Blot assay was used to detect the expression of HPV E6/E7 protein after ALA-PDT treatment.3.7 Transporter associated antigen processing mRNA detection:RT-PCR assay was used to detect the level of TAP-1,TAP-2 and tapasin mRNA after ALA-PDT treatment.3.8 Detection of TAP-1 protein:Western Blot assay was used to detect the expression of TAP-1 after ALA-PDT treatment.3.9 MHC class I molecular detection:flow cytometry was used to detect the average fluorescence intensity of MHC-I molecule after ALA-PDT treatment.Results:1.The Clinical research on the treatment of HPV infection with ALA-PDT1.1 HPV viral loads decreased significantly after PDT treatment in patients with Condyloma Acuminata1.1.1 As for patients with single type of HPV infection,HPV viral loads were found to be significantly higher before treatment compared to three times of PDT treatment(p<0.05).A similar significant difference was observed for six times of PDT treatment.There was no significant difference between three and six times of PDT treatment.1.1.2 There were significant differences in HPV viral loads between pretherapy and after six rounds of PDT treatment in both the LR-HPV and HR-HPV types(p<0.001)in patients with multiple type of HPV infection.1.1.3 After six rounds of PDT treatment,HPV viral loads were negative in 27 samples(27/31,87.1%)with single type HPV infection,which was significantly higher than that of samples with multiple type HPV infection(11/21,52.4%)after six cycles of PDT treatment.Multiple type HPV infection samples were more likely to have a higher viral load and less likely to be cleared of infection.1.1.4 The most common side effect experienced during irradiation and after treatment is pain.None of the patients developed systemic adverse effects.1.1.5 A total of five patients(14.28%,5/35)experienced infection recurrence.1.2 ALA-PDT cleared the latent or subclinical HPV infectionHPV viral load was significantly decreased after 3 times of ALA-PDT treatment in patients with latent or subclinical HPV infection.The negative rate of HPV test in patients with latent or subclinical HPV infection was higher than condyloma acuminata after one times of ALA-PDT treatment.It seems that the latent or subclinical HPV infection may be more easily cleared.2.The inhibitory effect of HPV infection on the expression of TAP-1 and MHC-I molecules.The results of immunohistochemical revealed that the expression of TAP-1 and MHC class I molecules in the condyloma acuminatum was significantly lower than that in the control group.The results of RT-PCR showed that the expression of TAP-1,TAP-2 and tapasin in condyloma acuminatum was significantly lower than that of the control group.Consistently,the results of Western-blot showed that the expression of TAP-1 protein in condyloma acuminata decreased significantly.3.The experimental research on the treatment of ALA-PDT3.1 We chose a concentration of 0.lmmol/L ALA and a light fluence of 0.6 J/cm2 as the condition for subsequent experiments.The proliferation activity decreased in O.lmmol/L ALA group,but compared with the control group,the difference was not statistically significant.On the contrary,the proliferation activity decreased significantly in 0.25,0.5,1 and 2mmol/L group(p<0.001).When cells were exposed to light fluences of 0 and 0.6 J/cm2,there was no noticeable change in cell growth.However,the growth rates all drastically decreased when the cells were subjected to the optical dose of 1.2,2.4,3.6 and 4.8 J/cm2(P<0.001).3.2 ALA-PDT suppressed the expression of E6/E7 protein of HPV virus.Western blot results showed that the expression of E6/E7 protein in ALA-PDT group was significantly lower than that in control group,and the difference was statistically significant.3.3 ALA-PDT increased the expression of transporter associated antigen processing significantly.RT-PCR results showed that the level of TAP-1,TAP-2,tapasin mRNA in the ALA-PDT group increased significantly.3.4 ALA-PDT induced the expression of TAP-1 protein.Western blot results showed that the expression level of TAP-1 in ALA-PDT group increased significantly compared with the control group.3.5 ALA-PDT increases the expression of MHC-I moleculesThe results of flow cytometry showed that the average fluorescence intensity of MHC class I molecule in ALA-PDT group was higher than that of control group.Conclusion:In summary,we observed that 5-aminolevulinic acid photodynamic therapy(ALA-PDT)can reduce the HPV viral loads and the recurrence rate in patients with condyloma acuminatum,which may be associated with the ability of ALA-PDT to reduce HPV E6/E7 protein and increase the expression of TAP and MHC class I molecules.Human papillomavirus(HPV)genotype and viral load assay can reflect the status of persist or latent infection as well as a predictor of infection clearance,so dynamic monitoring HPV genotype and viral loads might be help to guide PDT treatment to eliminate virus effectively.