INK4A immunostaining. Nonsmoking and nondrinking patients had more HPV-positive tumors than smoking and drinking patients . All HPV-positive tumors showed p16INK4A overexpression, and 1 HPV-negative tumor had p16INK4A overexpression, (P < 0.001). Overall survival and disease-specific survival were higher for HPV-positive compared to HPV-negative cases . In conclusion, HPV is strongly associated with OSCC of nonsmoking and nondrinking patients. Specific diagnostic and therapeutic actions should be considered for these patients to achieve a better prognosis. We aimed to determine the role of HPV in the pathogenesis and outcome of oropharyngeal squamous cell carcinoma (OSCC) in lifelong nonsmoking and nondrinking patients. A case-case analysis was performed to compare the presence of HPV-DNA in tumor cells of 16 nonsmoking and nondrinking with 16 matched smoking and drinking patients . HPV was detected using 2 PCR tests, FISH analysis, and p16 The most important risk factors for developing head and neck squamous cell carcinoma in the Western countries are consumption of tobacco and alcohol . HoweverIt is estimated that up to 15–20% of all head and neck squamous cell carcinomas are associated with high-risk HPV infection –10. ThisIn the pathogenesis of HPV-related cancer, integration of the viral genome into the cellular DNA and, as a result, upregulation of the viral oncoproteins E6 and E7 seem to be crucial events. These oncoproteins subsequently cause dysfunction of amongst others tumor suppressor proteins, p53 and pRb, respectively, leading to cell proliferation, impaired apoptosis, and ultimately chromosome instability . INK4A overexpression, a product of tumor suppressor gene CDKN2A, has been associated with HPV-related head and neck squamous cell carcinoma and in some studies used as a surrogate biomarker for HPV detection [INK4A overexpression predicts the presence of oncogenic HPV infection and identifies those with a better prognosis [INK4A have been detected in head and neck squamous cell carcinoma without a relationship with HPV infection [Immunohistochemical detection of p16etection , 15, 16.rognosis , 18. Mornfection , 20. HPV-positive head and neck squamous cell carcinomas are predominantly poorly differentiated and show a characteristic basaloid morphology in comparison with HPV-negative tumors , 6. FurtThe correct determination of HPV's involvement in the pathogenesis and prognosis of OSCC is dependent on several patient- and tumor-related cofactors, such as tobacco and alcohol use, TNM stage, and treatment modality. Although most investigators have found a trend between HPV and lesser amount of tobacco and alcohol use, the definitions of the used amounts are not always clear. Furthermore, to date, no matched analysis with smoking and drinking patients has been performed. In addition, previous studies have often used only one assay to determine the biological association of HPV infection with tumorigenesis. INK4A. The presence of HPV DNA was analysed using three different methods, that is, fluorescence in situ hybridization (FISH) and two polymerase-chain-reaction- (PCR-) based assays (Amplicor and Linear Array HPV detection kits). In this study, we aimed to determine the role of HPV in carcinogenesis and disease outcome for nonsmoking and nondrinking patients with OSCC. Therefore, we performed a case-case study of a well-defined population of 16 nonsmoking and nondrinking and 16 matched, smoking and drinking patients with OSCC for the presence of HPV DNA and overexpression of biomarker p16n = 198), when they had no history of smoking tobacco and alcohol consumption. Patients were classified as smoking and drinking (n = 2181), when they actively smoked tobacco and consumed alcohol. Former smokers or drinkers were not included. All patients were treated according to institutional protocols, and final decision was made in consultation with the patient. Follow-up time (in months) was considered from the date of diagnosis  to the date of death or date of last followup . Seventeen nonsmoking and nondrinking patients with a primary head and neck squamous cell carcinoma located in the oropharynx  were found in the database of which 16 were selected because of absence of tumor tissue in 1 case. These patients were matched with smoking and drinking patients on gender, age (±5 years), sublocation of tumor, and tumor stage. A case-case analysis was performed to compare the prevalence of HPV DNA and overexpression of p16INK4A in both groups. Patients were selected from a database at the University Medical Center Utrecht, in which all patients with newly diagnosed head and neck squamous cell carcinoma are prospectively registered. Between 1980 and 2004, 4607 patients were entered in the database. This database contains information on patient characteristics, risk factors, tumor classification, treatment modalities, and follow-up data including number of recurrences and subsequent primary tumors. Patients were classified as nonsmoking and nondrinking  tumor tissue blocks from either biopsy or surgical resection specimens were obtained. Two experienced head and neck pathologists  examined H&E-stained slides to select the areas in which tumor cells were present and evaluated the morphological appearances. Both pathologists were blinded to the smoking and drinking status. Tumor grade was recorded as well, moderate, or poor according to the criteria of the World Health Organization . In addiμL 50 mM Tris/HCL (pH 8.0) 0,5% (v/v) Tween-20 with proteinase K . After 1 hour incubation at 56°C, the lysates were boiled to inactivate the proteinase K and subsequently centrifuged. Supernatants were transferred into clean eppendorf tubes and directly used for PCR. PCR was performed using the Amplicor HPV Test kit  as well as the Linear Array HPV Genotyping Test . Both tests were carried out according to the manufacturer's recommended protocol including positive and negative controls. The Amplicor test is a qualitative in vitro test which uses amplification of target DNA by PCR and nucleic acid hybridization for the detection of high-risk HPV DNA genotypes . It uses primers to define a sequence of nucleotides within the L1 region of the HPV genome that is 150 base-pair (bp) long. This test also features a concurrent isolation and amplification of the human β-globin gene to assess DNA integrity for each tested specimen. The Linear Array test uses the same detection technique; however, it targets an HPV genome sequence of 450 bp and is able to detect high-risk (same types as mentioned above) as well as low-risk HPV-DNA . For DNA extraction tumor, areas from FFPE slides were isolated by microdissection. After deparaffinization, the tissue fragments were digested in 150 μm thick tissue sections as described previously [2O2, 1 M NaSCN, and 4 mg/mL pepsin in 0.02 M Hcl, postfixed in 1% formaldehyde in PBS, dehydrated in an ethanol series, and hybridized with a digoxigenin-labeled HPV 16-specific probe  according to the manufacturer's instructions. After hybridization, the preparations were washed stringently in 50% formamide, 2×SSC, pH 7.0 at 42°C (2 times 5 min). The probes were detected by application of mouse antidigoxin , peroxidase-conjugated rabbit antimouse IgG, and peroxidase-conjugated swine antirabbit IgG  and visualized by a peroxidase reaction using rhodamine-labeled tyramide. Preparations were mounted in Vectashield  containing 4,6-diamidino-2-phenyl indole . Microscope images were recorded with the Metasystems Image Pro System  mounted on top of a Leica DM-RE fluorescence microscope equipped with DAPI and rhodamine filters. Evaluation of nuclear hybridization signals was performed by two investigators (FF and EJMS) according to the previously described criteria [FISH was performed on 4 eviously , 15. Briμm thick tissue sections were deparaffinized with xylene and rehydrated by serial ethanol dilutions. Endogenous peroxidase activity was blocked by incubation for 30 minutes with 0.3% (v/v) H2O2 in methanol followed by antigen retrieval by boiling in 0.01 M sodium citrate buffer pH 6 for 15 minutes in a microwave oven. Slides were then incubated with a p16INK4A-specific primary mouse monoclonal antibody  and diluted 1 : 160 for one hour at room temperature followed by a secondary visualisation reagent for 45 minutes . After each incubation step, slides were washed in phosphate-buffered saline containing 3% (w/v) BSA. Peroxidase activity was visualized by incubation with diaminobenzidine/H2O2, and cell nuclei were counterstained with hematoxylin. All p16INK4A-positive cases were assessed for nuclear and/or cytoplasmic staining pattern. The staining patterns were scored semiquantitatively for the percentage of p16INK4A-positive tumor cells. The sections were graded as positive (+) when at least 75% of the tumor cells showed p16INK4A positivity and as negative (−) when no staining was visible. Only one case  method as patients were matched on possible confounding factors. Estimated survival curves were compared using log-rank test. A P value ≤0.05 was considered statistically significant.The association between HPV status and other variables was tested using Chi-square and Fisher's exact test. Disease-specific survival  and overall survival  were determined for HPV-positive and HPV-negative cases, nonsmoking and nondrinking and smoking and drinking groups, and for cases with and without p16n = 10), 5–9 units of alcohol/day (n = 4), and >9 units of alcohol/day (n = 2); ≤20 cigarettes/day (n = 5) and >20 cigarettes/day (n = 11). For 2 nonsmoking and nondrinking patients, the best possible match was disease stage IVA instead of III. The incidence dates ranged from 1980 to 2005. Sixteen nonsmoking and nondrinking patients with OSCC were matched with 16 smoking and drinking patients according to the above-mentioned criteria. The smoking and drinking patients used the following amounts of tobacco and alcohol at the time of diagnosis: 2–4 units of alcohol/day , and 19 cases without a detectable signal and 1 case which was inconclusive due to insufficient tissue material. Eight of the FISH-positive cases showed punctate signals in the tumor cell nuclei indicating integrated HPV DNA, and 4 showed granular nuclear staining. Based on these outcomes , we determined the HPV status as follows: 12 of 16 nonsmoking and nondrinking cases (75%) had a positive HPV status versus 2 of 16 smoking and drinking controls  was found in 14 cases (44%), in 1 case (1B) 25% of cells stained positive (3%) and 17 cases (53%) were negative. All positive cases had strong nuclear as well as cytoplasmic staining except case 16B which showed predominantly cytoplasmatic staining. All HPV-positive cases had p16INK4Aoverexpression, whereas 17 of 18 HPV-negative cases had no detectable p16INK4A  and more often basaloid features , other causes  of which 5 cardial and 2 pulmonary disease, recurrent disease , and second primary tumor (n = 3 smoking and drinking). For HPV-positive and HPV-negative cases, the 5-year overall survival was 71% and 42%, and 5-year disease-specific survival was 76% and 57%, respectively. Overall and disease-specific survival were both significantly higher for HPV-positive compared to HPV-negative cases  and for cases with p16INK4A overexpression compared to those without detectable p16INK4A overexpression . Follow-up time ranged from 5.9 to 182.1 months. Median follow-up time was 61.1 months. The 5-year overall and disease-specific survival for all cases was 53% and 64%, respectively. Cause of death in 20 deceased patients was as follows: due to primary tumor , the β-globin gene could not be amplified by both PCR tests; hence, the FISH data were used to determine the HPV status, which corresponded with the presence of p16INK4A overexpression in case of HPV positivity. Nevertheless, also some discrepancies were found between the different tests used. In cases 5A and 2A, the Amplicor test was positive for HPV, whereas the Linear Array test was negative, probably due to the large fragments that need to be amplified in the latter assay. As a consequence, the Amplicor and the FISH results were used to proof HPV positivity for these cases. In case 1A, PCR revealed the presence of HPV DNA of types 33/52, 33, 35, and 58 with corresponding p16INK4A overexpression, which explains the negative outcome of the HPV type 16-specific FISH analysis. Only in cases 1B and 4B, FISH analysis did not correlate with PCR and p16INK4A immunostaining, and in these cases, we decided to consider a positive p16INK4A and PCR status as signs for HPV positivity. However, the opposite may also be true as one considers the very high sensitivity of HPV DNA PCR, which may lead to false-positive results [INK4A can be overexpressed without the presence of HPV, for example, case 16B and a study by Hafkamp et al. [INK4A staining pattern in case 16B was purely cytoplasmic in contrast to the other p16INK4A-positive cases in which cytoplasmic and nuclear pattern was seen. This may point to other reasons than HPV for upregulation of this biomarker. Furthermore, the results as mentioned in Some controversy exists concerning the most reliable way to determine biologically relevant HPV infection in FFPE tissue. Therefore, it has been proposed to use at least more than one method to identify a firm association of the virus with the tumor cells. Most studies agree upon the use of the surrogate marker p16 DNA PCR , HPV E6  DNA PCR , or HPV  DNA PCR , 33. We  results , as wellp et al. . On the We conclude that HPV is strongly associated with oropharyngeal tumors, especially in lifelong nonsmokers and nondrinkers. With better and more valid detection techniques, it is likely that these patients will be recognized as a specific entity within the heterogeneous group of head and neck cancer. Diagnostic and therapeutic actions will then be more focussed on this distinct group and may lead to better prognosis.


Cartilage oligomeric matrix protein (COMP) is a major non-collagenous component of cartilage. Earlier, we developed a new mouse model for rheumatoid arthritis using COMP. This study was undertaken to investigate the epitope specificity and immunopathogenicity of COMP-specific monoclonal antibodies (mAbs).B cell immunodominant regions on the COMP molecule were measured with a novel enzyme-linked immunosorbent assay using mammalian expressed full-length mouse COMP as well as a panel of recombinant mouse COMP fragments. 18 mAbs specific to COMP were generated and the pathogenicity of mAbs was investigated by passive transfer experiments.B cell immunodominant epitopes were localized within 4 antigenic domains of the COMP but with preferential response to the epidermal growth factor (EGF)-like domain. Some of our anti-COMP mAbs showed interactions with the native form of COMP, which is present in cartilage and synovium. Passive transfer of COMP-specific mAbs enhanced arthritis when co-administrated with a sub-arthritogenic dose of a mAb specific to collagen type II. Interestingly, we found that a combination of 5 COMP mAbs was capable of inducing arthritis in naive mice.We have identified the specificities of mAbs to COMP and their contribution to the development of arthritis. These findings will further improve our understanding of the autoantibody mediated immunopathologies occurring widely in rheumatoid arthritis (RA), as well as in other autoimmune disorders. Rheumatoid arthritis (RA) is believed to be an autoimmune disease involving an antibody response to citrullinated proteins (ACPA) ,2 and IgCartilage oligomeric matrix protein (COMP) is a major glycoprotein in the extracellular matrix (ECM) of cartilage and synovium . Its bio+ T cells but not cytotoxic CD8+ T cells. Furthermore, arthritis can be transferred from arthritic mice to healthy recipients with affinity purified COMP-specific polyclonal antibodies . The mouse COMP recombinant fragment cDNAs were amplified using 5'-phosphorylated upstream primers in combination with downstream primers adding a stop codon followed by an Nhe I site. The PCR products were digested with Nhe I and inserted into the Pvu II and Nhe I sites of the expression vector pCEP4-TAGZyme. This plasmid was constructed by inserting the sequence 5'-GGTACCGCCTGCCGCCTGCCT GCCTGCCACTGAGGGTTCCCAGCACCATGAGGGCCTGGATCTTCTTTCTCCTTTGCCTGGCCGGGAGGGCTCTGGCAGCCCGACATCACCATCACCATCACCAGCTGAAGCTT-3' between the Kpn I and HinDIII sites of plasmid pCEP4, yielding an expression vector with a BM40 signal peptide followed by a hexahistidine tag compatible with the TAGZyme removal system . All the PCR-generated fragments and their cloning sites were confirmed by nucleotide sequencing.The corrected cDNA for mouse COMP  served aGibco, Life Technologies Inc, Camarillo, CA, USA). Secretion of recombinant proteins into the medium was confirmed by SDS-PAGE of samples from transfected and non-transfected cells. Serum-free culture medium was harvested, and his-tagged recombinant mouse COMP proteins were purified using Ni2+-metal chelating and MonoQ ion exchange chromatography. Purity and size of all the proteins were assessed by SDS-PAGE and colloidal Coomassie Brilliant Blue G250 staining with or without prior reduction of disulfide bonds.The resulting recombinant COMP plasmids were transfected into 293-c18 cells (ATCC CRL-10852) using the FuGene6 reagent  and transfected cells were selected with hygromycin and grown to confluence in Dulbecco's modified Eagle's medium with 10% fetal calf serum , COMP-containing fractions were evaluated by SDS-PAGE. Pooled fractions were then exchanged to 150 mM NaCl, 10 mM Tris, and 2 mM EDTA, pH 7.4 buffer. Pooled fractions were concentrated and negatively purified using HiTrap heparin-Sepharose column, HiTrap Q Sepharose column, heparin column  and Q column. The purity and size of COMP fractions were assessed by SDS-PAGE under reducing and non-reducing conditions.Purification of COMP from the Swarm rat chondrosarcoma was performed as previously described . BrieflyRVKEITFLK (in coiled-coil repeats); P2: PGLSVRPVALCAPGSCF (in EGF1); P3: SCFPGVVCTETATGARC (in EGF1); P4: GVGLTFAKTNKQVCTDI (in EGF2); P5: QPGFVGDQRSGCQRRGQ (in EGF3); P6: PSPCHEKADCILERDGSRS (in EGF4); P7: GQEDVDRDRIGDACDPD (in TIII2); P8: NPDQRNSDKDKWGDAC D (in TIII3); P9: DACDNCRSQKNDDQKDT (in TIII4); P10: NDDQKDTDRDGQG DACDDDI (in TIII4); P11: DTIPEDYERHRLRRA . Peptide identities were confirmed by mass spectrometry and fast protein liquid chromatography was used to maintain purity at > 98% .Sequence alignment identified 15 amino acid differences between the mature peptide sequences of rat and mouse COMP. The following peptides, corresponding to the rat COMP sequence and containing the amino acids differing from mouse COMP (underlined), were synthesized using methods previously described in detail . P1: DVR-/-Ncf1*/* and COMP+/+Ncf1*/* mice were immunized at the base of the tail with 100 μg of rat COMP emulsified in complete Freund's adjuvant , followed by an i.p. booster with 50 μg of rat COMP in PBS. Three to five days after the booster dose, spleen or inguinal lymph cells were fused with SP2/0 or NSO-bcl2 myeloma cell lines. Two weeks after the fusion, all the primary plates were screened for antibody production by ELISA using rat COMP. Cells from positive wells were sub-cloned 5 times by limiting dilution and expanded for further use. Monoclonal antibodies were purified from the supernatants by affinity chromatography using protein G column  and bound IgG was eluted in 0.1 M glycine (pH 2.8) and neutralized with one-sixth volume of 1 M Tris-HCl (pH 8.5). The eluted fractions were dialyzed against PBS buffer extensively. Monoclonal antibodies were quantified by measuring absorbance at 280 nm and the purity of the antibodies was assessed by SDS-PAGE. The endotoxin content in all mAbs preparations was found to be below detection limit (less than 1 EU/mg) as analyzed with the Limulus amebocyte lysate (Pyrochrome) method .Hybridomas producing anti-COMP mAbs were generated as described earlier . BrieflyEnzyme immunoassays were performed as described earlier . BrieflyEscherichia coli serotype O26:B6  intraperitoneally to enhance the severity of the arthritis developed. Arthritis development was monitored daily in a blinded fashion for 16 days using an extended scoring protocol [The anti-CII mAb (M2139) binding to the MPGERGAAGIAGPK epitope of the CB10 fragment aa 551-564) of the CII molecule used in this study was produced as described earlier 4 of the . The cocprotocol . Brieflyin vivo, neonatal mice were injected with 100 μg biotinylated mAbs against COMP intraperitoneally. After 24 h, knee joints were snap frozen in isopentane on dry ice and stored at -70°C. Joint sections (7 μm) were fixed in 4% paraformaldehyde for 5 minutes, rinsed in PBS, incubated with Extravidin peroxidase  for 30 minutes and developed with diaminobenzidine  for 8 to 9 minutes. To assess direct binding of anti-COMP mAbs to the tissue sections, limbs from naïve neonatal mice were harvested and snap frozen, cryo-sectioned, and sections of 7 μm were subjected to 5 μg/ml biotinylated mAbs against COMP for 40 minutes. Extravidin peroxidase and DAB were used for the detecting system.For histological assessments, knee joint and paws from adult mice were dissected, decalcified, dehydrated, and then paraffin-embedded, as described previously . SectionU-test. Analysis of variance (ANOVA) was used to analyze polyclonal Ab response statistics. Differences with P-values less than 0.05 were considered statistically significant.Quantitative data are expressed as mean ± standard error of the mean (SEM). Arthritis incidence was analyzed using chi-square or Fisher's exact test  and comparison of severity was performed using the non-parametric Mann-Whitney To perform epitope mapping of COMP, we expressed recombinant his-tagged full-length mouse COMP and a panel of overlapping COMP fragments in HEK 293-c18 cells Figure . All theTo ascertain whether antibodies show reactivity towards individual full-length and truncated form of COMP proteins, microtiter plates were coated with either recombinant COMP molecules or COMP purified from rat chondrosarcoma, and used in enzyme immunoassays with sera obtained at 50 days after primary immunization. As shown in Figure +/+Ncf1*/* mice, the incidence of arthritis was 80% with the day of disease onset at 36 to 38 days [In COMP 38 days . Since cNcf1 mutation immunized with the native form of rat COMP during secondary immune response. A total of 21 IgG mAbs from four separate fusions were developed and sub-cloned to obtain stable single clones. None of the COMP antibodies cross-reacted to CII, which might be present as a minor contaminant with the rat COMP during protein preparation from the Swarm rat chondrosarcoma. Isotype analysis revealed that all of the COMP mAbs generated were of IgG isotype, containing IgG1, IgG2a, and IgG2b subtypes, indicating that they were indeed produced by T cell-dependent follicular B cells  mAb M2139 staining was restricted to the cartilage surface Figure . The 1D1in vitro binding capacity of mAbs to cartilage, joint sections from untreated neonatal wild-type or COMP-/- mice were incubated with the individual biotinylated mAbs and binding of the antibody detected by indirect immunohistochemistry  F1 mice were first injected with single COMP-specific mAbs in combination with a sub-arthritogenic dose of M2139, a CII specific IgG2b mAb. This experimental design was chosen because most of the COMP-specific mAbs are of the poorly complement-fixing IgG1 isotype, and complement activation is one of the critical factors for development of arthritis ,26. We hTo further test whether a combination of antibodies to the most dominant epitopes of COMP was arthritogenic, anti-COMP mAbs 1B8, 1D10, 3F8, 15A11 and 16B5, which bind sites located at different domains of COMP, were mixed at equal concentrations and injected intravenously into 2-month-old mice. Clinical symptoms of arthritis developed in four out of six mice characterized by a rapid onset albeit with a low disease severity Figure . No signCOMP is expressed predominantly in cartilage and synovium . An immuin vivo [To analyze the epitope specificity of autoantibodies to COMP, we injected mice with COMP and investigated the ensuing polyclonal antibody response at different time points. Furthermore, we generated monoclonal antibodies from the responding B cells to characterize the fine specificity of various COMP antigenic determinants. Interestingly, the polyclonal antibody response was dominated by the reactivity to the EGF-like domain compared to the TSP3 and C-terminal globular domains at all the time points tested in our experiments. Similar to the anti-COMP polyclonal response, the majority of the mAb (12 out of 18) binding sites are located on the EGF-like domain. These results indicate that the EGF-like domain is the highly immune-dominant region in the B cell immune response to COMP. In the present study, we produced full-length mouse COMP, as well as a panel of overlapping COMP fragments in mammalian cells to preserve the three-dimensional structure of the protein. These recombinant COMP molecules allowed us to select mAbs that are specifically binding to conformational COMP epitopes, which are highly dependent on the three-dimensional structure. Although some investigators have proposed that any surface patch of a protein can be an epitope ,28, in min vivo ,30. In tIncrease in synovial fluid and serum COMP level is used as a bio-marker for cartilage turnover in human joint diseases ,32. Seve-/- mice, were immunized with COMP, 80% COMP+/+ mice developed severe arthritis, whereas COMP-/- animals exhibited no clinical abnormalities (our unpublished data). These differences in arthritis severity are very likely a result of the absence of COMP as a target for the anti-COMP immune response. In this study, we provide evidence to show that mAbs to defined epitopes on COMP are indeed pathogenic, thereby emphasizing a link to COMP-mediated autoimmunity. Current observations of binding of COMP-specific mAbs directly to the cartilage COMP in its native conformation, and the passive transfer of COMP-specific mAbs leading to arthritis, have important implications for the understanding of the ensuing immune responses against COMP in arthritis. It has been reported that antibodies play a pivotal role in triggering inflammation in arthritis [Earlier, we reported that the affinity-purified COMP-specific polyclonal antibodies can induce arthritis in healthy recipients . Here, wrthritis ,8,37. Itrthritis -40. It hrthritis ,42.In RA patients, joint-specific autoantigens are considered to play an important role in the induction of synovial B cell expansion . A sign We elucidated specificities of autoantibodies to COMP and its contribution to the development of arthritis. These findings will further improve our understanding of the autoantibody-mediated immunopathologies occurring widely in RA as well as in other autoimmune disorders.ACPA: anti-citrullinated protein antibodies; ADAMTS: a disintegrin and metalloprotease with thrombospondin motifs; COMP: cartilage oligomeric matrix protein; COMPIA: COMP-induced arthritis; pCOMP: pentameric COMP; mCOMP: monomeric COMP; CG: C-terminal globular domain; CII: collagen type II; EGF: epidermal growth factor; ELISA: enzyme-linked immunosorbent assay; ECM: extracellular matrix; LPS: lipopolysaccharide; MED: multiple epiphyseal dysplasia; PSACH: pseudoachondroplasia; PBS: phosphate-buffered saline; RA: rheumatoid arthritis; RF: rheumatoid factor; TSP: thrombospondin; TIII: thrombospondin type 3 repeat.The authors declare that they have no competing interests.RH and HG had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. RH, AA and RM designed the study. Experiments were performed by HG, KSN and PA. HG and KSN performed statistical analyses. HG prepared the first draft of the manuscript. All authors contributed to the interpretation of the study findings, revised and approved the manuscript.


For practice, the factors influencing the choice of vaccine included strength of vaccine protection (61.1%), long-lasting immunity (56.8%) and good antibody response (55.6%). The most significant barriers to prescribe HPV vaccines consisted of parental refusal due to safety concerns (48.2%), and their practice of advising vaccination was mostly affected by local Governmental recommendations (78.7%). A substantial proportion of physicians had recommended HPV vaccines for their female clients/patients aged 18–26 years for protection of cervical cancer (83.8%) or both cervical cancer and genital warts (85.5%). The knowledge on HPV infection was low among physicians in Hong Kong. Prescription of HPV vaccine was hindered by the perceived parental concerns and was mostly relied on Governmental recommendations. Educational initiatives should be targeted towards both physicians and parents, and the Government should consider full subsidy to enhance vaccine uptake rate.This study explored the knowledge, attitude, practice and barriers to prescribe human papillomavirus (HPV) vaccines among private primary care physicians in Hong Kong. A self-administered questionnaire survey was conducted by sending letters to doctors who had joined a vaccination program for school girls. From 720 surveys sent, 444 (61.7%) completed questionnaires were returned and analyzed. For knowledge, few responded to questions accurately on the prevalence of cervical HPV (27.9%) and genital wart infection (13.1%) among sexually active young women in Hong Kong, and only 44.4% correctly answered the percentage of cervical cancers caused by HPV. For attitude, most agreed that HPV vaccination should be fully paid by the Government (68.3%) as an important public health strategy. Vaccination against HPV was perceived as more important than those for genital herpes (52.2%) and Chlamydia (50.1%) for adolescent health, and the majority selected adolescents aged 12–14 years as the ideal group for vaccination. Gardasil Human papillomavirus (HPV) infection is the most common sexually transmitted disease worldwide. It has been estimated that at least 50% of sexually active people had acquired genital HPV infection during their lifetime However, in order for the vaccination programmes to be successful, a high uptake rate is needed. Recommendation of HPV immunization by primary care physicians (PCPs) has been recognized as one the most influential factors in the individual’s willingness to receive the vaccine. Updated knowledge and positive attitude towards the vaccine among PCPs, as well as the removal of any barriers to prescription are the main determinant factors Nevertheless, most of the existing studies were conducted among physicians in Western countries The objectives of this study were to evaluate the knowledge on HPV infection, and the attitude towards and perceived barriers of HPV vaccination among PCPs in Hong Kong. Their practice of prescribing HPV vaccine according to recommendations and patient characteristics was also explored.http://www.youitv.com/partner/hps/cervicalcancer/). The contents of the online platform include the prevalence of cervical cancer, causes, symptoms, preventive measure and information of the vaccine. Schools were invited to join a special seminar conducted by the Chinese University of Hong Kong for knowledge enrichment. Schools were given an option to join the voluntary HPV vaccination programme, and participated students had the first dose (Cervarix®) free of charge on site at their schools. The acceptance of the 1st free-of-charge dose of Cervarix at school was over 80%, and there has been no local recommendations regarding interchangeable use of two HPV vaccines. Physicians were not recommended to prescribe Gardasil after an initial dose of Cervarix. The second and third doses were then administered in the clinics of PCPs at market price determined by individual physicians. The PCPs in Hong Kong consist of general practitioners, family physicians, and specialists in the private sector who practice in the community setting including medical internists, gynecologists, pediatricians and oncologists. All 720 PCPs who participated in the school HPV vaccination programme received an invitation letter to complete an anonymous questionnaire designed for the current study.As HPV is not under universal coverage in Hong Kong, a Family-School-Doctor Model was developed by university academics in 2011 to improve the initiation of HPV vaccine among adolescent girls by tackling the socio-cultural barriers and also the barriers related to cost and convenience Since part of this survey assessed the knowledge of the doctors on the research topic, the surveys were kept anonymous and did not require participants’ signatures. The completion of the survey by the study participants implied their consent. In terms of the measures that were taken to document the process, the reseachers invited the participants to return their completed surveys in an enclosed envelope after which they could sign their name in an attendance sheet to obtain Continuous Medical Education (CME) points. The return of their surveys was recorded to evaluate the response rate. The Survey and Behavioral Research Ethics Committee of The Chinese University of Hong Kong has approved this study and the consent procedure. The study did not involve minors/children participants, thus the concerns related to minors/children participants were not applicable in this study.The survey used in this study was designed by an academic physician, and validated by an expert panel consisting of epidemiologists, public health specialists, family physicians, microbiologists and academic professionals. The survey was pilot-tested by 20 physicians for face-validity and comprehensibility before study commencement. In addition, the information provided in the online platform was not related to the questions in the survey to ensure accurate assessment of the PCPs’ knowledge. We used a five-point Likert scale for survey items which assessed attitude towards the HPV vaccine. These questions are close-ended with possible responses including “extremely important”, “very important”, “important”, “somewhat important”, and “not at all”. The same applied to questions on their perceived barriers and likelihood to follow recommendations on administration of HPV vaccines. The questionnaire consists of 10 pages with 33 questions, and English was used in the survey as all PCPs in Hong Kong know English. For assessment of PCPs’ knowledge on HPV vaccine, the correctness of an answer was established from a thorough literature search and validation by the expert panel who designed the survey. For comparison of importance among different vaccines, one question item was phrased as “Compared to the following other vaccines that are in use or development, how would you rank the HPV vaccine in terms of importance for adolescent health?” According to our pilot study, the PCPs will need an average of 10 minutes to complete the questionnaire.® vs. Gardasil® in their routine practice were compared according to the reasons of vaccine choice. P values <0.05 were regarded as statistically significant.The Statistical Package for Social Sciences  version 16.0 was used for all data entry and analyses. All descriptive statistics were presented as proportions. Chi square tests of heterogeneity were used to compare proportions. As an exploratory analysis, the proportions of physicians choosing CervarixOf the 720 questionnaires distributed, 444 completed ones were collected . The aveOnly 27.9% and 13.1% of physicians correctly responded to questions on the prevalence of cervical HPV infection and genital wart infection, respectively, among sexually active young women in Hong Kong . Only 44The participants were asked about the importance of different public health strategies for promoting HPV vaccination . They weThe attitudes of participating PCPs towards the ideal age range for HPV vaccine administration were explored. Most physicians indicated that 12–14 years (45.6%), 10–11 years (31.6%), and 15–17 years (20.3%) were the ideal age groups. Very few regarded ages 18 years or older as ideal for HPV vaccination.® (30.9%) and Cervarix® (28.0%) were approximately equally preferred. The major reason for choosing a vaccine regarded by the participants as “extremely important” or “very important” included its strength of protection (61.1%), immunity being long-lasting (56.8%), good antibody response (55.6%), more HPV types covered (53.5%), cross-protection for other cancer-associated HPV types (53.4%) and vaccine safety (51.2%). Factors which were less considered included vaccine price (27.9%), credibility of manufacturers (28%) and comprehensive service of manufacturers (29.2%)   as important reasons for the choice  (41.1% of participants did not express any preference to the two available vaccines . Among t (29.2%) . Among p p<0.05) . Alterna p<0.05) .The most frequent factors perceived as “extremely likely” or “somewhat likely” barriers to advise HPV vaccination among adolescents included parental refusal due to safety concerns (78.6%), parental reluctance to discuss sexuality and sexually transmitted diseases (70.4%), and physicians being regarded as hard selling an expensive vaccine (65.0%) . Most peA majority of the physicians reported that the recommendations to prescribe HPV vaccine to adolescents were “extremely likely” or “somewhat likely” influenced by the local Government  (95.8%), the respective colleges of their specialties (93.8%), local University academics (90.7%) and overseas authorities or colleges (86.8%) . Very fe® and Gardasil® were almost equally preferred, with the major factors for vaccine choice being their strength of protection, duration of immunity, cross-protection for other cancer-related HPV types, types of HPV covered and safety. Parental refusal based on vaccine safety and reluctance to discuss sexually transmitted diseases as perceived by physicians represented the most frequently encountered barriers. Most physicians recommended HPV vaccination among females aged 18–26 years, and followed guidelines from the Government and their respective colleges.This study found that the knowledge on HPV prevalence was low yet the knowledge on the role of different HPV types in cervical cancer and anogenital warts was high among PCPs in Hong Kong. They regarded HPV vaccine relatively important for adolescent health, and should preferably be administered at the age range of 12–14 years. CervarixA survey among 214 Vaccine For Children (VFC) providers in Georgia reported high levels of knowledge about the epidemiology of HPV infection, the causative role of HPV for cervical cancer, and the status of FDA approval for quadrivalent HPV vaccines for use in adolescents aged 9–26 years (range 76.7% to 97.2%) Turning to physicians’ attitude towards HPV vaccine, a high degree of overall acceptance for HPV vaccine use directed against cervical cancer and genital warts was consistently reported by several large-scale studies, including primary care providers like family physicians, gynecologists and pediatricians For the ideal age of adolescent girls to receive HPV vaccine from the PCPs’ perspective, the present study reported 12–14 years as the range most commonly selected by our study participants. One study reported that 89% of pediatricians in the US recommended HPV vaccines to girls aged 16–18 years, whilst only 46% recommended vaccines for adolescent girls aged 10–12 years From a recent systematic review This study has several important implications. Firstly, more educational initiatives should be organized for PCPs working in the private sector so as to enhance their perceived importance of prescribing HPV vaccine to their clients and patients. The focus should be on the epidemiology of HPV infection and genital wart, as well as proportion of cervical cancer attributed to HPV types covered by current vaccines. This should be well received as PCPs strongly agreed that HPV vaccine has a higher priority in improving adolescent health as compared with other vaccines. Several aspects in these educational initiatives should be emphasized, including the ability of HPV vaccines to achieve strong protection with long-lasting immunity, some degrees of cross-protection against the important oncogenic types of HPV, and their good safety profiles. Secondly, since parental refusal was mainly on the vaccine’s safety, parents should be particularly reassured about its low rates of adverse effects in lieu of its many health benefits. The parents’ concerns on discussing sexuality and sexually transmitted diseases signify the crucial role played by PCPs in adopting a holistic approach in their consultations, lending higher levels of attention to discuss sensitive issues. Consultations with prospective clients and patients eligible to receive HPV vaccine should be cautiously conducted with good communication skills taking possible embarrassment to girls and their parents into account. Also, since the Governmental recommendations and the PCPs’ respective academic colleges were perceived as the most influential in affecting their practice of prescribing HPV vaccine, they should be the primary agents to deliver educational messages. That a high proportion of PCPs suggesting full subsidy of the vaccine by the Government also implied that the vaccine might raise an affordability issue. At the early stage to enhance vaccine uptake rate among adolescent girls, the Government should consider at least partial subsidy to eligible clients to overcome vaccine cost which is one of the important barriers.In light of the present findings, future research should evaluate interventions which could effectively enhance PCPs’ knowledge of HPV and its vaccination in a sustainable manner; remove the barriers of offering immunization, and optimize proper practice of HPV vaccines in suitable subjects.To our knowledge this study is the first-ever evaluation among Chinese PCPs on their prescriptions of HPV vaccination. The validity of survey instrument, the high response rate and the sampling methodology enhanced the generalizability of the findings. However, several limitations should be addressed here. Firstly, this is a cross-sectional study and one could not establish causal relationships between PCPs’ knowledge or attitude and their HPV prescriptions. In addition, we had invited PCPs working in the private sector only, and the attitudes and barriers of HPV vaccine prescription might differ to a certain extent with physicians working in the public sector and also doctors in the academic field; the PCPs were heterogeneous with respect to their specialties and we have adopted an aggregate analysis. Also, since the demographic and practice characteristics of all PCPs in Hong Kong are not known, the generalizability of the present findings could not be ascertained. Furthermore, this is a descriptive analysis and there could be potential confounders affecting clinical practice, like the choice between Gardasil and Cervarix. Lastly, this cross-sectional study was conducted in 2011–2012, five years after the licensure of the HPV vaccines in Hong Kong, and it is anticipated that the perceptual variables pertinent to the vaccine will change with time as more promotional materials and media communications emerge. A time series of cross-sectional surveys is needed to reflect the trends of changing knowledge, attitude and perceived barriers.There is still room to equip PCPs the knowledge on HPV epidemiology infection and skills in motivating behavioural change in discussing sex related issues not directly related common presentations by adolescents. Public health education on safety and efficacy of the HPV vaccination is still needed. Recommendation by Government including subsidy would be an important factor to enhance vaccination rates.Human papillomavirus (HPV) infection could be effectively prevented by vaccination, and physician recommendation has been known to be the most influential factor for patients’ uptake of the vaccine. However, few studies explored the knowledge, attitude, barriers and practice of HPV vaccination among primary care physicians.The knowledge of 444 physicians on HPV infection was low. Vaccination against HPV was perceived as more important than other vaccines, and Government subsidy was regarded as an important public health strategy. The most significant barriers to prescribe HPV vaccines consisted of parental refusal due to safety concerns. Public health education on safety and efficacy of vaccination is needed, and support by Governmental funding would be an important factor to enhance vaccination rates.


Laboratory data is a rich source of information that can be used to estimate adherence to physician guidelines and motivate improvement in clinical practice. Human papillomavirus (HPV) testing is an important component of cervical cancer screening programs with established screening guidelines. The purpose of this study was to develop methods to estimate concordance with published guidelines for HPV testing in order to provide clinicians and payors specific feedback about overscreening.This retrospective analysis of laboratory test ordering patterns evaluated 454,532 HPV tests ordered from September 2003 to October 2009 from 110 facilities and performed at ARUP laboratories. We used laboratory data including patient demographics, ordering frequency, timestamps and results to examine the proportion of HPV tests ordered on women under 21 years, ordered on women between 21 and 29 years apparently before cytological examination, repeated less than 1 year after a positive HPV result in women over 30 years, and repeated less than 3 years after a negative HPV result in women over 30 years.The absolute number and proportion of HPV tests performed on women under 21 years declined from 20% in 2005 to 5% in October 2009. The proportion of HPV tests performed women between 21 and 29 years also declined during this period. Approximately one-third of HPV tests performed on women between 21 and 29 years arrived for HPV testing before cervical screening had presumably been completed. The most common follow-up intervals for HPV testing on women over 30 years were 6 months following a positive HPV result and 12 months following a negative HPV result. Only 6% of repeat HPV testing in women over 30 years followed a negative HPV result by 3 years or more. Approximately one-fourth of HPV tests ordered the year ending October 2009 were unnecessary based on the American Society for Colposcopy and Cervical Pathology guideline.We demonstrate simple methods to evaluate appropriate utilization of HPV testing using laboratory data. Our data illustrates that some aspects of HPV test ordering have become more consistent with guidelines over time. However, a large portion of HPV testing in the United States is unnecessary. This highlights opportunities for optimization of a rational cancer prevention strategy to reduce unnecessary screening, colposcopy and biopsies. As the US healthcare system moves toward greater promotion of evidence-based practice, it is critical to develop better measurement systems to evaluate concordance between practice and evidence. Laboratory data is one of the richest sources of such information. This paper illustrates how a laboratory can take advantage of multiple attributes from laboratory data, including patient demographics, ordering frequency, timestamps (i.e. database records of when samples are collected and arrive in lab), and results to estimate adherence to an evidence-based guideline.Testing for human papillomavirus (HPV) is a widely used component of cervical cancer screening programs, and may become more important as studies continue to support its effectiveness. In the U2We analyzed HPV test ordering patterns at a US national referral laboratory to estimate concordance with published guidelines. The American Society for Colposcopy and Cervical Pathology (ASCCP) has published a widely cited guideline for cervical cancer screening which has been reiterated by other professional organizations including the American College of Obstetricians and Gynecologists and the American Society for Clinical Pathology.–8 These 4The ASCCP guideline identifies two specific situations in which HPV testing should be repeated in less than 3 years. In patieIn order to evaluate physician compliance with the ASCCP guideline with regard to HPV testing, we used laboratory information to calculate four measures: 1) Proportion of HPV tests ordered on women under 21 years; 2) Proportion of women between 21 and 29 years for whom HPV tests appear to have been ordered before cytological examination; 3) Proportion of repeat HPV tests performed less than 1 year after a positive HPV result in women over 30 years; 4) Proportion of repeat HPV tests performed less than 3 years after a negative HPV result in women over 30. We then show how these values can be combined to estimate the total proportion of unnecessary HPV tests ordered at an individual laboratory.ARUP, a national reference laboratory affiliated with the University of Utah Department of Pathology, provides HPV testing for several hundred hospitals and regional laboratories across the US. As a component of our laboratory service we provide analyses of test ordering practices, in which we identify potential overuse and underuse of laboratory tests in order to help health care facilities improve the quality and lower the cost of their services. In order to further develop our analyses of HPV ordering practices we evaluated data from 454,532 HPV tests ordered September 2003, when HPV testing was initiated at ARUP, to October 2009 at 110 facilities that each averaged over 20 HPV test orders per month during the study period. We limited evaluation to facilities which averaged over 20 HPV tests per month as to evaluate only facilities which perform routine HPV testing at ARUP and to exclude facilities that only send quality control or confirmation samples to ARUP. Data was stored as discrete data elements in a translational SQL database, retrieved using Crystal Reports v11.5 , and validated with queries using SQL Server 2008 Management Studio v10.0 . All analysis was performed using Microsoft Excel 2007 (Microsoft).We calculated the proportion of HPV tests performed on women under 21 years and under 30 years during the study period. To estimate the proportion of women of 21-29 years for whom cytology had been performed before HPV testing we evaluated data from selected months, May 2005, May 2007 and May 2009. Results were similar for each of these 3 months, so we did not evaluate this measure the entire study period. We used a comparison of time from sample collection to time of sample arrival at the laboratory with the median turnaround time for cytological examination at the University of Utah, Department of Pathology, which was approximately 7 days, to estimate the proportion of tests that had undergone previous cytological review. Discussions with cytologists revealed that it would be highly unlikely for an abnormal sample to be collected, sent to a pathology laboratory for review, reviewed by a cytology technician, reviewed by a pathologist and referred to our laboratory in less than 5 days. Thus, time from collection to time of arrival in lab can serve as an easily accessible surrogate that could obviate the need for detailed review of cytology records to determine the appropriateness of test orders on women between 21 and 29 years.To study follow-up times, we identified 12,523 HPV tests on patients who were over 30 years at the time of initial HPV testing and for whom repeat testing had been performed during the 1-year period between November 2008 and October 2009. Although we limited our evaluation to testing repeated the last year of the study, initial testing, which determined the appropriateness of the repeat test, was performed any time between September 2003 and the end of the study. We separated these paired tests into groups based on the outcome of the initial HPV test.We used the results of each of these analyses to identify the number of apparently inappropriate tests performed for between November 2008 and October 2009 . We combThis project was approved by the University of Utah IRB_00039168.Proportion of HPV tests ordered on women less than 21 years.There was both an absolute and relative reduction in testing on women under 21 years and increased testing on women over 30 years Figures .Proportion of women between 21 and 29 years for whom HPV tests were presumably ordered before cytological examination.Although the absolute number of HPV tests ordered for women of age 21-29 years, the proportion of HPV tests ordered for women of age 21-29 years declined from 30% in 2004 to 18% in October 2009 [Figures Proportion of repeat HPV tests performed less than 1 year after a positive HPV result in women over 30 yearsBetween November 2008 and October 2009 the most common observed follow-up intervals for HPV testing on women over 30 years following a positive HPV test result was 6 months . For theProportion of repeat HPV tests performed less than 3 years after a negative HPV result in women over 30 years.The most common observed follow up intervals for HPV testing on women over 30 years after a negative test HPV results was 12 months . For theTotal proportion of unnecessary HPV tests during 1 calendar year.Between November 2008 and October 2009 7.1% of all HPV tests were ordered on women less than 21 years, all of which were inappropriate and unnecessary according to ASCCP guideline . During The proportion and absolute number of young women receiving unnecessary HPV testing has decreased since 2005. Likewise, the proportion of women 21-29 years receiving HPV testing decreased since 2005, but the absolute number of tests on this population did not change. Throughout the study period approximately one-third of HPV tests performed on women between 21 and 29 years arrived at the laboratory less than 5 days after collection, suggesting screening rather than follow-up evaluation of abnormal cytology. Retesting for HPV was performed more frequently than expected, with most tests performed 6 months after positive and 12 months after negative HPV results, even though clinical indications for these screening intervals are very rare. HPV posiThe accuracy of our retrospective evaluation of test ordering patterns may be limited by lack of both cytology results and clinical information about the patients on whom these tests were ordered. However, clinical laboratories that wish to evaluate overuse of HPV testing for their clients are unlikely to have the resources to perform detailed reviews of cytology and clinical information to determine the appropriateness of tests. Our methods illustrate rapid, automatable methods that we believe produce results sufficiently accurate to indicate where improvement in clinical practice is necessary and possible.Previous studies evaluating utilization of cervical cancer screening have used surveys or billing data to evaluate utilization of cervical cancer screening.15 SurveyCombining data from all of our endpoints, we estimate that approximately one-fourth of HPV tests ordered in the US in the year ending October 2009 were unnecessary based on the ASCCP guideline. Overuse of HPV tests does not have a place in a rational cancer prevention strategy and leads to additional clinical and financial costs in the form of unnecessary follow-up screening, colposcopy and biopsies. Our data highlights opportunities for laboratories to use informatics tools in the optimization of a rational cancer prevention strategy in the US.


To date, there have been only few studies that investigated integration of anal Human Papillomavirus (HPV). Most of them were conducted on HIV-infected individuals and mainly analyzed samples from high-grade lesions and invasive cancer. We aimed to investigate HPV physical status in HIV-negative men who have sex with men (MSM) with a detectable anal HPV infection, irrespective of the presence of lesions. We also sought to explore the presence of other circular DNA viruses in the anal region. Study participants were attendees of an STI screening program, which were also screened for anal HPV infection and cytological abnormalities. HPV physical status was assessed using multiply-primed RCA. HPV16-positive samples were also analyzed using E2/E6 multiplex PCR, qRT-PCR and APOT assay. RCA and virus-specific PCR were employed to investigate the presence of other DNA viruses. Anal HPV infection was detected in 76.9% of the 230 MSM enrolled. The anal cytological reports were: 129 NILM, 37 ASC-US and 28 L-SIL (36 samples were inadequate for interpretation). HPV physical status was evaluated in the 109 anal specimens that harbored one or two different HPV genotypes. Integration was observed only in one HPV16-positive sample (0.9%), in which integrate-derived viral transcripts of type B were detected. Integration occurred in chromosome 14 q. In 22 of the 53 (41.5%) mucosal HPV-negative samples, RCA restriction results would seem to indicate the presence of circular DNA viruses. Indeed, cutaneous HPV (4 samples), MCPyV (5 samples) and TTV (4 samples) were detected. In conclusion, anal HPV integration was rarely evidenced in HIV-uninfected MSM with no or mild anal cytological abnormalities, although the integration rate may have been underestimated because of the limitations of the employed assays. Other DNA viruses were detected in the anal samples of these individuals, although the significance of this occurrence needs to be assessed. Human Papillomavirus (HPV) is responsible for 90–99% of cervical cancer HPV-driven mechanisms of cervical carcinogenesis are largely known. E6 and E7 viral oncoproteins, de-regulating cellular proliferation and inducing genetic instability, are the main responsible for the neoplastic process At present, the available data mainly concern HPV physical status in cervical samples, while only a limited number of studies have ascertained HPV integration in anal infections. In addition, most of them have been conducted in HIV-infected individuals.The main aim of this study was to analyze HPV physical status in HIV-uninfected MSM with anal infection, irrespective of the presence of lesions. Moreover, we aimed to investigate the anal samples for the presence of other DNA viruses.The study was approved by the San Gallicano Dermatological Institute Ethics Committee (Prot. CE/564/11) and performed in compliance with the Helsinki Declaration. A written informed consent was obtained from all participants.Study participants were selected among the HIV-uninfected MSM attending the STI Unit of the San Gallicano Dermatological Institute  for a large STI-HIV screening program (COROH project), which includes evaluation for anal HPV infections and cytological abnormalities A sterile Dracon swab was used to collect anal epithelial cells, then dispersed in PreservCyt  Mucosal HPV were detected and genotyped utilizing the Linear Array HPV Genotyping Test  according to the manufacturer's instructions. HPV genotype risk classification has been specified elsewhere RCA was performed using the Illustra TempliPhi100 Amplification Kit  following the manufacturer's instructions with 450 µM extra dNTPs. To resolve the concatemers, RCA products were digested with BamHI, EcoRI or HindIII , according to the HPV genotype/s present in the samples. Digestion was performed for 3 h at 37°C  or 30°C (BamHI) in a total volume of 20 µl. Restriction results were verified by 0.8% agarose gel electrophoresis.A multiplex PCR for the simultaneous amplification of HPV16 E2 and E6 was performed as described SYBR Green-based quantitative Real time PCR was performed using the iCycler iQ detection system , according to the procedures and primers described by Nagao et al Total nucleic acids were treated with DNase (Invitrogen) for 15 min at room temperature and, after EDTA addition, 10 min at 65°C. Total RNA was reverse transcribed using the Advantage RT for PCR kit  and an oligo(dT)17 primer coupled to a linker sequence (p3) Cutaneous HPV DNA was amplified using CP65/70 primer set in the first PCR and CP66/69 in the nested PCR, as described elsewhere MCPyV DNA detection was performed using the LT1primer set in the first PCR and M1/M2 in the nested PCR (nPCR), as previously described The presence of TTV was investigated by PCR using RCA products as a template and the NG054/NG147 primer set in the first PCR and the NG132/NG133 primer set in the nPCR The first 230 consecutive HIV-uninfected MSM of the COROH project, screened for anal HPV infection and morphological changes, were included in the present study. The median age of these individuals was 32 (IQR 26–39) and 96.9% of them were Caucasian. The other socio-demographic and behavioral characteristics of the study participants were similar to those of the larger cohort, which have been described elsewhere In regard to anal cytology, 36 samples (15.6%) were inadequate for the evaluation. The cytological reports for the remaining 194 participants were: 129 NILM (66.5%), 37 ASC-US (19.1%) and 28 L-SIL (14.4%).Concerning anal HPV infection, 177 out of the 230 MSM  were positive: 64 (36.2%) were infected by a single HPV genotype, 51 (28.8%) by 2 genotypes and the remaining 62 (35.0%) by more than 2 genotypes . Thus, with the exception of one sample, all specimens harbored episomal viral DNA. The physical status of the viral genome in the 24 HPV16-positive samples was further analyzed by multiplex E2/E6 PCR. Apart from sample 132, the E2/E6 ratio of all the other HPV16-positive samples confirmed the episomal status. Differently, sample 132 E2/E6 ratio was <1.53, which would seem to suggest a possible mix of HPV16 episomal and integrated genomes , which was HPV16-positive . Differently, the remaining 22 samples (41.5%) showed several restriction products . As these results would seem to suggest the presence of circular DNA molecules, possibly viral genomes, several attempts to characterize them were made. Given that contamination with cutaneous HPV genotypes from the perianal area is possible during anal sample collection, these 22 specimens were screened for cutaneous HPV genotypes. Four samples were positive , possibly deriving from small circular DNA viruses. Since TTV is an ubiquitous virus detected in multiple human tissues Finally, Bam HI restriction of RCA products evidenced in a few cases a clear band ranging in size between 2 and 3.5 Kb , a highly lethal form of skin cancer This is the first report showing the presence of TTV in anal samples. The 2–3.5 Kb RCA restriction bands observed in four samples might correspond to full-length genomes and intragenomic rearranged TTV subviral molecules In conclusion, RCA, together with E2/E6 multiplex PCR, qPCR and APOT assay used on HPV16-positive samples, did not evidence a high rate of anal HPV integration in HIV-uninfected MSM with no or mild cytological abnormalities. However, considering the limitations of the RCA and the use of more sensitive methods exclusively for HPV16-positive samples, the integration rate might have been underestimated. Importantly, RCA allowed to evidence that a variety of circular DNA viruses are present in the anal canal. Further studies are needed to ascertain the significance of these infections.


Johannes Bogaards and colleagues use mathematical models to investigate whether vaccinating females only, males only, or both sexes is the best way to achieve the most effective reduction in the population prevalence of sexually-transmitted infections Sex-specific differences regarding the transmissibility and the course of infection are the rule rather than the exception in the epidemiology of sexually transmitted infections (STIs). Human papillomavirus (HPV) provides an example: disease outcomes differ between men and women, as does the potential for transmission to the opposite sex. HPV vaccination of preadolescent girls was recently introduced in many countries, and inclusion of boys in the vaccination programs is being discussed. Here, we address the question of whether vaccinating females only, males only, or both sexes is the most effective strategy to reduce the population prevalence of an STI like HPV.We use a range of two-sex transmission models with varying detail to identify general criteria for allocating a prophylactic vaccine between both sexes. The most effective reduction in the population prevalence of infection is always achieved by single-sex vaccination; vaccinating the sex with the highest prevaccine prevalence is the preferred strategy in most circumstances. Exceptions arise only when the higher prevaccine prevalence is due to a substantially lower rate of natural immunity, or when natural immunity is lifelong, and a prolonged duration of infectiousness coincides with increased transmissibility. Predictions from simple models were confirmed in simulations based on an elaborate HPV transmission model. Our analysis suggests that relatively inefficient genital transmission from males to females might render male vaccination more effective in reducing overall infection levels. However, most existing HPV vaccination programs have achieved sufficient coverage to continue with female-only vaccination.Increasing vaccine uptake among preadolescent girls is more effective in reducing HPV infection than including boys in existing vaccination programs. As a rule, directing prophylactic immunization at the sex with the highest prevaccine prevalence results in the largest reduction of the population prevalence.Please see later in the article for the Editors' Summary About 10% of cancers in women occur in the cervix, the structure that connects the womb to the vagina. Every year, more than a quarter of a million women (85% of them in developing countries) die because of cervical cancer, which only occurs after the cervix has been infected with a human papillomavirus (HPV) through sexual intercourse . There are many types of HPV, a virus that infects the skin and the mucosa . Most people become infected with HPV at some time during their life, but most never know they have been infected. Some HPV types cause harmless warts on the skin or around the genital area, and several—in particular HPV16 and HPV18, so-called high-risk HPVs—can cause cervical cancer . HPV infections are usually cleared by the immune system, but about 10% of women infected with a high-risk HPV develop a long-term infection that puts them at risk of developing cervical cancer.Screening programs have greatly reduced cervical cancer deaths in developed countries by detecting the cancer early, when it can be treated. However, it would be better to prevent cervical cancer ever developing. Moreover, most women in developing countries do not have access to screening. Because infection with specific HPV types can cause the development of some types of cervical cancer, vaccination of girls against HPV before the onset of sexual activity might be one way to prevent cervical cancer. Scientists recently developed a vaccine that prevents infection with HPV16 and HPV18, and HPV vaccination programs have been introduced in several countries. These programs are currently directed only at girls because HPV-related illness and death are higher among women than men, but should boys also be included in HPV vaccination programs? Men would benefit directly from immunization against HPV-related diseases, but, in addition, vaccination of boys might help to reduce the circulation of HPV in the population, thereby indirectly improving the protection of women through so-called “herd immunity.” In this study, the researchers used mathematical models to investigate whether vaccinating girls only, boys only, or both sexes is the most effective way to reduce the population prevalence of HPV infection (the proportion of the population infected with HPV).The researchers first used a range of standard two-sex mathematical models of infection and transmission in heterosexual populations to identify general criteria for allocating an HPV vaccine between the sexes. They found that the most effective reduction in the population prevalence of HPV infection was always achieved by single-sex vaccination and that, in most situations, the preferred strategy was to vaccinate the sex with the highest prevaccine prevalence of HPV infection. The researchers confirmed these predictions using a more elaborate HPV transmission model that incorporated differences among individuals in age and level of sexual activity. Importantly, this second analysis also suggested that for existing girl-only vaccination programs, increasing coverage of vaccination among girls would bolster herd immunity more effectively than switching to a policy of vaccinating both sexes.The findings of this study suggest that increasing vaccine uptake among preadolescent girls is a more effective way to reduce HPV infection than including boys in existing vaccination programs. They also suggest that directing HPV vaccination at the sex with the highest prevaccine prevalence of infection will reduce the population prevalence of HPV most effectively. Although the accuracy of these findings is dependent on the assumptions included in the mathematical transmission models used by the researchers, these findings support a policy of increasing female HPV vaccine coverage as far as possible, within the limits set by vaccine acceptance and economic constraints. More generally, these findings suggest that single-sex preventative interventions might be the best way to reduce heterosexual transmission of other sexually transmitted infections and that targeting the sex with the highest prevalence of infection might achieve the most effective reduction in the population prevalence of these common diseases.http://dx.doi.org/10.1371/journal.pmed.1001147.Please access these websites via the online version of this summary at cervical cancer for patients and for health professionals, including information on HPV vaccines (in English and Spanish)The US National Cancer Institute provides information about cervical cancer and HPVThe US Centers for Disease Control and Prevention also has information about cervical cancer and HPV vaccination The UK National Health Service Choices website has pages on European Cervical Cancer AssociationThe PREHDICT project investigates health-economic modeling of prevention strategies for HPV-related diseases in European countries; information about this project is available from the cervical cancer and HPV vaccination is available from Macmillan Cancer SupportMore information about Personal stories about cervical cancer are available through the charity Healthtalkonlinecervical cancer and other sexually transmitted infections (in English and Spanish)MedlinePlus provides links to additional resources about  Key issues in the allocation of limited public health resources for the control of sexually transmitted infections (STIs) are (a) whether interventions are as effective for males as for females; and (b) whether directing interventions at both males and females adds to the population-level impact of directing interventions at one sex alone. These topics have been addressed in relation to gonorrhea and chlamydia prevention strategies In many countries, vaccination against infection with the two most common oncogenic papillomavirus types, HPV16 and HPV18, was recently introduced or will be introduced soon. Among women without previous exposure to these types, vaccination against HPV16 and HPV18 has shown high, sustained efficacy against persistent type-specific infections and precancerous lesions of the cervix, vulva, and vagina The primary target for HPV vaccination currently is girls in age groups when HPV16/18 infection is not yet common, i.e., before or just after initiation of sexual activity. In the US, the Advisory Committee on Immunization Practices has recommended HPV vaccination for routine use in preadolescent girls and young women since 2006, and is currently considering inclusion of males into the vaccination program Two recent studies have calculated the cost-effectiveness of extending HPV programs in the US to include boys Here, we address the question of whether increasing protection of females only, of males only, or of both males and females, is the most effective strategy for reducing the prevalence of an STI in a heterosexual population. In addressing this question, we allow for differences between the sexes in the transmissibility, the course of infection, the degree of natural immunity, or any combination thereof. We do not consider sex-related differences in disease-associated mortality. Throughout we restrict ourselves to prophylactic interventions that are applied before girls or boys become sexually active, which precludes the targeting of highly sexually active individuals.We use mathematical models of infection and transmission in heterosexual populations. These transmission models allow us to investigate how prophylactic vaccine is best distributed between males and females in order to lower the population prevalence of infection. The central idea is that immunization benefits not only the individual but also the population at large, because vaccination confers indirect protection to nonvaccinated individuals by lowering transmission of vaccine-preventable disease (herd immunity). This is especially important for STIs, as immunization of individuals of a single sex offers indirect protection against infection to members of the opposite sex. In principle, vaccinating a substantial proportion of one sex may suffice to eliminate infection from the entire heterosexual population S), infectious (I), and resistant (R) to infection, resistance being due to natural immunity or to vaccination. In the heterosexual transmission model, each compartment is split in two sexes . Taken together, the change in the proportion of susceptible, infectious, and resistant individuals of either sex is described by the following set of ordinary differential equations:d in males and females, each sex constitutes half of the heterosexual population. The parameter kv denotes the effective vaccine coverage among individuals of sex k, i.e., the fraction vaccinated times the probability that the vaccinee is protected against infection by vaccine types. The parameter λk denotes the sex-specific force of infection, which is the product of the rate c at which sexual contacts are made, the probability βk that infection is transmitted from the opposite sex k′, and the probability that a sexual partner is infectious:k as well as in the rate αk at which they recover from being infectious. Throughout, we assume that the duration of infectiousness corresponds to the duration of infection. The parameter kf denotes the sex-specific fraction of individuals who become immune following infection; such immunity is assumed to be lifelong. Note that the model is generic in the sense that individuals may intermittently go through susceptible and infectious stages (with ff = fm = 0), as in susceptible-infected-susceptible (SIS) models, or go through the susceptible, infectious, and immune stages only once (with ff = fm = 1), as in susceptible-infected-resistant (SIR) models. The susceptible-infected model, without recovery from infection (αf = αm = 0), is also a special case of this model.To derive general rules for allocating prophylactic vaccine between two sexes, we first use a standard model of heterosexual transmission. The standard transmission model partitions the population into fractions that are susceptible  is greater than one over two generations of transmission—from men to women and back to men vR, is related to the basic reproduction number R0 without vaccination as follows vf denotes the immunization coverage among females, and vm denotes the immunization coverage among males. This equation implies that it makes no difference whether the fraction of susceptible males or females is diminished in order to reduce the basic reproduction number. Indeed, the critical immunization coverage vc needed to achieve vR<1 is the same whether only males or only females are vaccinated:R0,f is the basic reproduction number for heterosexual transmission from women to men and R0,m is the basic reproduction number for heterosexual transmission from men to women. Note that reducing either sex-specific reproduction number below one may neither be necessary nor sufficient to achieve vR<1. Also note that low or waning vaccine efficacy may cause even complete coverage of a single sex to be insufficient for elimination. We refer to others for an analysis of conditions in which vaccination of both sexes may be needed to achieve vR<1 Sustained transmission of an infectious disease in heterosexual populations requires that the basic reproduction number vR more effectively than male-only or female-only vaccination , the steady-state prevalence of infection prior to the introduction of vaccine is highest in sex k either if transmissibility is lower in this sex given equal recovery rate α, or if recovery is slower in this sex given equal transmission probability β . The remaining set of parameters was split into equal-sized subsets on the condition that the highest recovery rate and transmission probability occurred in the same sex or not. Next, we determined which allocation scheme minimizes the total prevalence of infection at a certain vaccine coverage v, taken either close to the prevaccine situation or close to the critical immunization coverage.Vaccinating the sex with the highest prevaccine prevalence of infection may not achieve the largest reduction in population prevalence if there is a trade-off along the lines αInterestingly, vaccinating the sex with the highest prevaccine prevalence always yielded the largest reduction in heterosexual infection levels in a SIS system , but notWe evaluated the impact of a small proportion of men who have sex with men (MSM) in the general population on the performance of the rule of vaccinating the sex with higher prevaccine prevalence . Performff = fm). It appears that the strategy of vaccinating the high-prevalence sex always remains the most effective strategy, whenever the probability of developing natural immunity in this sex is larger than in the other sex , one should continue the existing single-sex program. But at very low immunization coverage (close to the prevaccine situation), one should switch to a vaccination program directed only at the other sex. Between these extremes lies some threshold value below which switching to a two-sex vaccination strategy might be considered. Numerical analyses demonstrate that this value is well below 50% immunization coverage for almost all possible parameter combinations  lowers the total prevaccine prevalence of infection and changes the relative effectiveness of vaccinating girls or boys  raises the total prevaccine prevalence of infection and causes vaccination of boys to become the most effective strategy . If the By exploring various two-sex transmission models, we demonstrate that directing prophylactic intervention at a single sex more effectively reduces heterosexual STI transmission than any allocation that includes both sexes. In addition, we demonstrate that a strategy of protecting the sex with the highest endemic prevalence generally achieves the largest reduction in the population prevalence. The implication of our finding is that the prevaccine prevalence of infection might be a good proxy to determine which individuals should be vaccinated in order to achieve the highest impact of vaccination at the population level.Our results provide a justification, under most circumstances, for the intuitively plausible strategy of targeting intervention at the subgroups that harbor most infections and that act as a reservoir for transmission. An alternative strategy that uses allocation rules defined on the basis of sex-specific reproduction numbers would also be intuitively plausible but performs poorly in minimizing the population prevalence of infection. Our results can be viewed as a generalization of a recently formulated argument for prioritization of vaccination to groups with the highest product of incidence and force of infection The allocation that achieves the largest reduction in the population prevalence of infection for a fixed amount of vaccine is not necessarily the most attractive from an economic point of view. The cost per vaccine dose delivered is subject to logistics, and universal vaccination could sometimes be a cost-effective alternative to single-sex vaccination. For example, the variable costs of vaccine purchase and delivery could be low compared to the total costs of running a vaccination program. In addition, the marginal cost of increasing vaccine uptake might depend on the coverage already achieved and might be different between the sexes. Males and females, or in the case of preadolescent vaccination, their parents, likely have different perceptions of the risk from HPV infection and different attitudes towards vaccination, although more research is needed to reliably measure vaccine acceptability Our analysis adds new arguments to the ongoing debate about whether males should also be offered HPV vaccination We have focused on a heterosexual population. Often, bisexuality acts as a bridge for transmission between heterosexual and homosexual subpopulations. This bridging phenomenon is especially important for the persistence of STIs, such as hepatitis B virus The free availability of quadrivalent HPV vaccine to young Australian women has led to a reduced morbidity of genital warts in STI clinics since 2007, among women as well as heterosexual men Rules for achieving the most effective reduction in the population prevalence of infection are relevant both for developed and for developing countries. Given that the worldwide burden of HPV-related cancer is concentrated in low-resource settings, HPV vaccines have the potential to dramatically aid global cancer control We have focused on HPV, but our findings are also applicable to other infections. Sex-specific differences in the transmissibility and in the course of infection are the rule rather than the exception in the epidemiology of STIs. These differences have been demonstrated to have implications for the effectiveness of control strategies directed at either sex, with regard to contact tracing to prevent secondary transmission Our analysis extends previous modeling work on the topic of male HPV vaccination Text S1Equilibrium conditions of the two-sex transmission model.(DOC)Click here for additional data file.Text S2Marginal reductions in the equilibrium prevalence of infection.(DOC)Click here for additional data file.Text S3Global minimization of the equilibrium prevalence of infection.(DOC)Click here for additional data file.Text S4Extending the two-sex transmission model with homo- and bisexuality.(DOC)Click here for additional data file.


Prevalence of an abnormal Papanicolaou smear was significantly increased in lupus patients in cross-sectional studies, associated with a higher prevalence of high-risk human papillomavirus (HPV) infection. The nucleic acid-specific Toll-like receptors (TLRs) locate at the endolysosomal compartments and trigger the induction of cytokines for the innate immune response. This study evaluated whether abnormal host innate immune response in lupus patients may enhance HPV persistence.Ex vivo productions of cytokines and interferon-gamma (IFN-γ) upon TLR ligands stimulations were subsequently measured using cytometric bead array and ELISA.Protein levels of TLRs 3, 7, 8 and 9 in cervical epithelial cells of lupus patients and controls with or without HPV infection were assessed using flow cytometry. Characteristics associated with the differential expression of TLRs in systemic lupus erythematosus (SLE) were elucidated. The effect and interferon-stimulated genes (ISGs) (ISG15 and Mx-1) gene expressions were then measured in oncogenic HeLa (HPV18), CaSki (HPV) and C33A (HPV negative) cell lines using flow cytometry and quantitative real-time PCR. For subjects with HPV infection, levels of TLR3 and TLR7 were significantly lower in lupus patients compared with controls. Significantly decreased TLRs 7, 8 and 9 levels were observed in HPV-negative SLE compared to healthy controls. For SLE with and without HPV infection, TLR7 and 9 levels were significantly lower in infected SLE than those in HPV-negative patients. Independent explanatory variables associated with down-regulation of TLR7 level included HPV infection and a higher cumulative dose of prednisolone; while a higher cumulative dose of hydroxychloroquine and HPV infection were associated with down-regulation of TLR9 level. In cervical cell lines, TLRs 3, 7, 8, 9 protein levels and antiviral ISG15 and Mx-1 gene expressions were inhibited in two oncogenic HPV types. Functional data showed that the induction of pro-inflammatory cytokines by TLR ligands  was greatly impaired in CaSki and HeLa than C33A cells.In conclusion, prednisolone and TLR antagonist (hydroxychloroquine) may down-regulate protein levels of TLR7 and TLR9 in lupus patients, thereby decreasing the innate immune response against HPV infection. Upon infection, HPV further down-regulate TLR7 and 9 levels for viral persistence. Furthermore, reduction of nucleic acid-sensing TLRs 7, 8 and 9 in carcinogenic HPVs ensures that the expression of inducible pro-inflammatory cytokines is minimized to prevent the expression of antiviral ISGs (ISG15 and Mx-1) on a biologically relevant antiviral response. Persistent infection with an oncogenic human papillomavirus (HPV)16 or 18 is thought to be necessary for the development of invasive cervical cancer, particularly among immunocompromised patients ,3. IndepInnate immune recognition of viral infection triggers antiviral immune responses ,7. ViralType I interferons (IFNs) are key effector molecules of the innate immune system and are essential for the antiviral response towards a plethora of viruses . NucleicThe aims of this cross-sectional study included the following: 1) To compare the protein levels of TLRs 3, 7, 8 and 9 in cervical epithelial cells of SLE patients and controls with or without HPV infection; 2) To ascertain the potential explanatory clinical and laboratory variables associated with TLR protein levels in cervical epithelial cells; 3) To investigate whether the reduced TLR protein levels has functional consequences for cytokine induction. Our findings showed that prednisolone and hydroxychloroquine may down-regulate levels of TLR7 and TLR9, respectively. Interestingly, upon infection, HPV further down-regulates TLR7 and TLR9 levels in lupus patients, which is consistent with previous reports showing that oncogenic HPV16 may suppress the host immune response by down-regulating the TLR9 transcript and, subsequently, limit its ability to induce the transcription of pro-inflammatory genes for antiviral immune responses .One hundred and fifty consecutive female patients attending the lupus clinic at The Prince of Wales Hospital of The Chinese University of Hong Kong, who fulfilled the 1997 American College of Rheumatology revised criteria for the classification of SLE , and parn = 15); Group 2 - SLE with negative HPV DNA (n = 31); Group 3 - control subjects with positive HPV DNA  (n = 7); and Group 4 - healthy controls with negative HPV DNA  (n = 22)Subjects were divided into four groups based on the following criteria: Group 1 - SLE patients with positive HPV DNA  or worse) or positive HPV DNA were referred for colposcopy and cervical biopsy to obtain histological diagnosis. Ethics approval has been obtained from the Ethics Committee of The Chinese University of Hong Kong, and informed consent was obtained from all participants according to the Declaration of Helsinki.The study visit included a structured interview with a standardized questionnaire. Clinical assessment and blood collection for the determination of disease activity, gynecologic examination for the collection of specimens for a Pap test, and HPV testing were performed. Medication history for the past 15 years was retrieved from case notes. Physical examinations and laboratory investigations, including complete blood count, liver and renal functions, anti-dsDNA titer, serum complements C3 and C4 levels, were performed at study visit. The SLEDAI and Systemic Lupus International Collaborating Clinics/American College of Rheumatology damage index (SLICC) were evaluated during the clinical assessment ,28. MajoThe method of cervical sample collection, detection and identification of HPVs has been described previously . Briefly6 cells) were resuspended and blocked with 2% human pooled serum for blocking the non-specific Fc receptors. Leukocytes were phenotyped by staining with anti-CD45 antibody using R-phycoerythin (PE) mouse IgG1κ isotypic control  as background control. The cells were then fixed and permeabilized using Fix/Perm solution . Purified mouse anti-cytokeratin antibody  and mouse IgG1κ isptypic control (BD Biosciences) were used for intracellular staining, together with a fluorescein iso-thiocyanate (FITC) secondary antibody . The side scatter (SSC), forward scatter (FSC) and florescence signal of 10,000 epithelial cells were then acquired and analysed by flow cytometer using CellQuest™ software .Cervical cell pellets  and corresponding FITC-conjugated IgG isotypic control (BD Biosciences) and PE-conjugated mouse IgG1, κ (Biolegend) were incubated with cells for 30 minutes at 4°C. The levels of TLRs in 10,000 epithelial cells were assessed using flow cytometer and the results were expressed as mean fluorescence intensity (MFI).5 cells) were incubated with or without TLR ligand Poly (I:C)  and ODN2395  at 10 μg/ml each and R837/Imiquimod (TLR7 ligand) and ssRNA (TLR8 ligand) at 5 μg/ml each for 24 h at 37°C in a 5% CO2 atmosphere. The cell-free supernatant was harvested and stored at -70°C for subsequent assays of IFN-γ and cytokines.HeLa  and C33A cells  were purchased from American Type Culture Collection  and maintained in MEM-Earle medium , supplemented with 10% FBS, 60 mg/l gentamycine and 1% non-essential amino acid . Sodium pyruvate (5%) (Sigma) was added for C33A cells. CaSki cells  obtained from ATCC was grown in RPMI1640 medium (Sigma) containing 10% FBS, 60 mg/ml gentamycine and 1% non-essential amino acid. Cells . All RNA samples were treated with DNase I (Invitrogen) and reversely transcribed to cDNA with TaqMan Reverse Transcription Reagents . The mRNA ISG15, Mx1 and GAPDH (endogenous control) was quantified by real-time PCR using SYBR Green probe  with the use of Applied Biosystems' 48-well StepOne™ Real Time PCR System. The primers of human ISG15, Mx1 and GAPDH were as follows : ISG15 fT-3') and reverse (5'-GGCGATGGCATTCTGGGCTTTAT-3'); GAPDH forward (5'-ATGGGGAAGGTGAAGGTCG-3') and reverse (5'-GGGGTCATTGATGGCAACAATA-3'). Real-time PCR was performed in a 25ul reaction mixture containing primers, FastStart Universal SYBR Green master (ROX) reagent (Roche) and cDNA sample in duplicate. The real-time PCR reaction was performed as previously described .The concentration of cytokines, including interleukin (IL-6), IL-8, IL-10 and IL-12p70, tumor necrosis (TNF)-α and IL-1β in culture supernatant with equal cell number loading, were measured simultaneously using human inflammatory cytokine Cytometric Bead Array  with flow cytometry  . Human It test or Mann-Whitney U test was used where appropriate. Association between the potential explanatory variables and TLRs level were tested using Chi-squared tests for categorical variables and Pearson's or Spearman's correlation for continuous variables with normal and skewed distribution, respectively. Variables with P < 0.05 in the univariate analysis were entered into linear regression analysis (enter). Variables that were skewed were logarithmically transformed before entering the regression analysis. All hypotheses were two-tailed, and P-value < 0.05 were considered significant. The Statistics Package for Social Sciences  was used for the analysis.Results were expressed as mean ± S.D. for normally distributed data. Non-normally distributed data were expressed as median . Chi-squared tests were used for categorical variables. For continuous variables, the Student's P <0.05)  .The protein levels of TLRs 3, 7, 8 and 9 in gated cytokeratins Figure  of lupuss Figure . For subP < 0.05) (Table P < 0.05) (Table Tables 5) Table . Regardi5) Table . Moreove5) Table . The TLR5) Table .P < 0.05) (Table P < 0.05) . Binding to IFNAR then stimulate more than 300 ISGs, which subsequently induce an antiviral state. The antiviral state is a collective term for limitation of viral replication, viral resistance of neighboring cells, and apoptosis of virally infected cells . In thisIn conclusion, prednisolone and TLR antagonist (hydroxychloroquine) may down-regulate expression levels of TLR7 and TLR9 in lupus patients, thereby decreasing the innate immune response to HPV infection. Furthermore, upon infection, HPV further down-regulate TLR7 and 9 levels which may favor viral persistence. Reduction of nucleic acid-sensing TLRs 7, 8 and 9 in carcinogenic HPVs ensures that the expression of inducible pro-inflammatory cytokines is minimized in order to suppress the expression of antiviral ISGs (ISG15 and Mx-1) of a biologically relevant antiviral response. Further studies to elucidate the role of immunosuppressants and HPV infectious on TLR expression in SLE, and whether topical TLR agonists may be useful as therapeutic tools for clearing oncogenic HPV infection in the cervical epithelial cells are warranted.ASCUS: atypical squamous cells of undetermined significance; CBA: Cytometric Bead Array; CsA: cyclosporine A; DMEM: Dulbecco's modified Eagle's minimal essential medium/F12 medium; dsDNA: double-stranded DNA; dsRNA: double-stranded RNA; ELISA: enzyme-linked immunosorbent assay; FBS: fetal bovine serum; FC: flow cytometry; FITC: fluorescein iso-thiocyanate; FSC: forward scatter; HPV: human papillomavirus; IFN: interferon; IFNAR: interferon-α/β receptor; IL: interleukin; ISG: interferon-stimulated gene; MFI: mean fluorescence intensity; MMF: mycophenolate mofetil; Mx: myxovirus resistance; Pap: papanicolaou; PBMC: peripheral blood mononuclear cells; PBS: phosphate buffered saline; PCR: polymerase chain reaction; PE: phycoerythin; PRR: pattern-recognition receptor; RPMI: Roswell Park Memorial Institute medium; SIL: squamous intraepithelial lesion; SLE: systemic lupus erythematosus; SLEDAI: systemic lupus erythematosus disease activity index; SLICC: Systemic Lupus International Collaborating Clinics/American College of Rheumatology damage index; SSC: side scatter; ssRNA single-stranded RNA; TLR: toll-like receptor; TNF: tumor necrosis factor.The authors declare that they have no competing interests.PKSC, C-KW, C-CS, SCH, KS, MMYY, S-FY, T-HC, MCSW, EKL and L-ST contributed to the study design. S-LY, JLKC and ACMY contribute to the analysis and interpretation of data. S-LY, C-KW and L-ST contributed to manuscript preparation. S-LY contributed to statistical analysis. All authors read and approved the final manuscript.