Development of HPV-associated cancers not only depends on efficient negative regulation of cell cycle control that supports the accumulation of genetic damage, but also relies on immune evasion that enable the virus to go undetected for long periods of time. In this way, HPV-related tumors usually present MHC class I down-regulation, impaired antigen-processing ability, avoidance of T-cell mediated killing, increased immunosuppression due to Treg infiltration and secrete immunosuppressive cytokines. Thus, these are the main obstacles that immunotherapy has to face in the treatment of HPV-related pathologies where a number of different strategies have been developed to overcome them including new adjuvants. Although antigen-specific immunotherapy induced by therapeutic HPV vaccines was proved extremely efficacious in pre-clinical models, its progression through clinical trials suffered poor responses in the initial trials. Later attempts seem to have been more promising, particularly against the well-defined precursors of cervical, anal or vulvar cancer, where the local immunosuppressive milieu is less active. This review focuses on the advances made in these fields, highlighting several new technologies . The most promising immunotherapies used in clinical trials are also summarized, along with integrated strategies, particularly promising in controlling tumor metastasis and in eliminating cancer cells altogether.After the early promising clinical results, the development of therapeutic HPV vaccines need to be implemented and applied to the users in order to eradicate HPV-associated malignancies, eradicating existing perception  that we have already solved the problem. Cervical cancer is the third most common cancer in women and the fifth most common overall cancer worldwide as age standardized incidence rate in both sexes combined . The priCervical carcinogenesis is a multi-step process, which starts with viral infection and requires the establishment of persistent HPV infection. Papillomavirus persistence is favored by at least three major factors: the virus life cycle that takes place away from dermal immune cells, the absence of virus-induced cell lysis with, in turn, no or weak inflammatory response and finally the altered immune response induced by viral proteins. Thus, cancer development depends not only on efficient negative regulation of cell cycle control supporting the accumulation of genetic damage, but also on immune evasion that enables the virus to lie undetected for a long time. Many studies confirm that persistent HPV infection is the most important risk factor, mainly with high-risk subtypes . StudiesA number of escape factors may affect the natural immune response against HPV proteins, together with the loss of correct signals from immune system to activate adaptive immune system. Indeed, optimal activation of adaptive immunity and generation of specific CD4 T helper 1 type immunity supporting development of CD8 cytotoxic T cells against viral early proteins, like E2, E6, E7, is critical for virus clearance in basal epithelial cells. T helper cells also support optimal activation of B cells, with secreting HPV capsid type specific neutralizing antibodies, which can protect against subsequent infections at mucosal and systemic levels . Spontanin vitro[In the early phases of carcinogenesis the three oncogenes of the virus E5, E6 and E7 play an important role in immune evasion. In particular, the E5 protein  seems toin vitro. This doin vitro,21. Sevein vitro. In thisin vitro.In addition, therapeutic T cell effector mechanisms are limited due to the following: changes in local immunity; the production of cytokines such as Interleukin (IL)-10 or transforming growth factor (TGF-beta); increased number of regulatory T cells (Tregs) and to immunosuppressive myeloid cells that are commonly observed in HPV related cancers. Moreover, frequent mutational events in cancer include HLA loss of expression, with subsequent escape of tumor cells ,24.In this manner, HPV-related tumors usually present MHC class I down-regulation, impaired antigen-processing ability, avoidance of T-cell mediated killing, increased immunosuppression due to Treg infiltration, and secrete immunosuppressive cytokines .adjuvare, which means to aid or to help. Historical boosting humoral immune response was the goal of the developed adjuvants and, as a result, many commonly used adjuvants are effective in elevating serum antibody titers, but do not elicit significant Th1 responses or cytotoxic T lymphocytes (CTLs). However, certain adjuvants have recently been demonstrated to be able to induce cellular immunity which are summarized in Table These are the main obstacles faced when achieving a valid immunotherapy against HPV-related pathologies where a number of different strategies have been developed in order to overcome them including adjuvants. Adjuvant is a term derived from the Latin word, Conceptually, immunity can be utilized in a therapeutic setting in two ways: first, by using specific natural o synthetic antibodies against defined targets, or second, by inducing an immune response in the organism against specific antigens (preventive and therapeutic vaccines). In particular, for HPV-induced lesions and cancer viral antigens or/and virus-induced host antigens can be targeted by these approaches. Indeed, once a patient is infected with HPV there is no effective way to cure persistent HPV infection which is the first step towards the development of pre-cancerous lesions. It was estimated that even with mass vaccination through commercially highly effective preventive quadrivalent or bivalent HPV vaccines -31, it wThus, there is great need for the development of novel therapeutic intervention to control HPV-associated disease and cancers. Given that existing treatments -34 are pThe following paragraphs summarize these issues and focus on the treatment  of already established infections Table  or canceThe use of intracellular antibodies (intrabodies) to inhibit protein function holds promise for the treatment of human diseases. The difficulties associated with the development of intrabody-based therapies are similar to those that hamper the development of protein inhibitors in general, and reflect problems in generating reagents that are effective and specific. This effectiveness and specificity can be achieved by using intrabodies to combat intracellular parasites like viruses. Indeed, viruses express proteins that are directly involved in causing disease and are clearly diverse from those of the host cell. For viruses that cause only local infections, such as HPV, the intrabody approach may be more appropriate. Furthermore, not only the infected cells but also the transformed cells require the continuous expression of some HPV proteins, particularly the oncogenes E6 and E7. This is most evident in Hela cells, derived from an HPV-associated malignancy in the 1950s, which still almost 50 years later require the expression of these oncogenes for their growth in tissue culture ,36.in vitro cancer cell models. More recently, an intrabody against the E7 of HPV 16 was proved to block tumor growth in animal models [Intrabodies against the E6  and E7  of HPV hl models . Given tInstead of using intrabodies, the utilization of monoclonal antibodies against membrane-expressed antigens is suggested, that may be induced by the HPV, i.e. Epidermal Growth Factor Receptor (EGFR). Monoclonal antibodies anti EGFR are already in clinical use and have been fully reviewed elsewhere . HoweverTherapeutic vaccines aim to eradicate or reduce already infected cells by stimulating cytotoxic T cells against target infected cells and up-regulating MHC Class I expression. Vaccine-mediated immune strategies could be directed toward at least two different stages of the oncogenic infection: firstly, infection and then secondly, the established infection. By eliciting neutralizing antibody responses the prophylactic vaccines challenge the first infection by inhibiting the HPV to bind to the cell or the early phases of viral entry. These vaccines are already in clinical use and will not be discussed in this review.The therapeutic vaccines should fight already infected cells, and could be tailored based on the presence of episomal replicating virus or integrated viral sequences. In the first case, the vaccine targets could be all the early proteins, in the second case only the E6-E7 proteins appear to be a realistic target of intervention . In addiIn the experimental model an effective immunotherapy administered before tumor challenge includes an antigen-specific component, whereas an effective immunotherapy after tumor challenge can be achieved through the enhancement of either innate or adaptive immunity, and seems to be optimal with both. Therefore, valid therapeutic vaccines must achieve this goal. Trials of immunotherapy in patients with HPV associated pre-malignancy are expected to be more effective than in cancer patients, since the impaired antigen presentation by cervical cancer cells due to mutations in MHC and TAP genes may render the immunotherapy less effective. Although there are potential immune-evasive mechanisms that are attributable to the HPV infection itself .Examples of those therapeutic vaccines, which have reached phases I and II clinical trials, are presented in the subsequent paragraphs according to the different formulations.To date, several protein or peptide-based vaccines are either undergoing clinical evaluation or are in development. A major limitation to peptide-based vaccines is the HLA restriction that can be overcome by the use of whole protein-based vaccines, which harbor multiple immunogenic epitopes, binding various allelic HLA molecules. On the other hand, protein-based vaccines are generating predominantly antibody responses rather than CTL responses because proteins are processed through the endocytic/MHC class II pathway. In addition, both peptides and proteins are poorly immunogenic. Therefore, most of the research in this area was focused on the co-administration of adjuvant immune-enhancing agents such as chemokines, cytokines, and co-stimulatory molecules to enhance the potency of the vaccine. In particular saponin-based  or liposPeptide-based vaccines need to increase not only the poor immunogenicity level but also the obstacle of MHC restriction. TLR agonists have also been explored as adjuvants for peptide-based HPV vaccines because of their capability to activate both innate and adaptive immunity. Vaccines consisting in CTL and or TH epitope adjuvated with TLR 9 , TLR4 orHowever, among the protein-based vaccine candidates, SGN-00101 vaccine, a fusion protein consisting of Hsp from Mycobacterium bovis and HPV 16 E7, has generated considerable interest. As a single-agent therapy, in both phase I and phase II clinical trials, fusion protein was able to induce regression of lesions in anal high-grade squamous intraepithelial lesions , recurreSince peptide-based vaccines are stable, easy to produce and have a high safety profile, many clinical trials have been reported. Research has focused on addressing the main limitations of peptide-based vaccines, namely their low immunogenicity, and the enhancement of the immunogenicity of peptide-based vaccines has been explored in clinical studies. The PADRE universal T-helper peptide was utilized to increase the activity of CTL epitopes encoding HPV 16 E7 that was presented by HLA-A*0201 . These vaccines failed to mount a valid immune response in women with late stage cervical cancer ,66. MoreThe presence of these Foxp3+ T cells is linked to impaired immunity in malignancies. The efficacy of this vaccine was also demonstrated in a placebo-controlled randomized Phase II study showing an increased number of HPV 16-specific T cells in patients with HPV 16+ high squamous intraepithelial lesion (HSIL) .Plant molecular pharming represents a well-established biotechnology area that includes the production of protein biopharmaceuticals such as enzymes, hormones, antibodies, and vaccine antigens in plant systems. Plant-produced proteins represent a significant fraction of pharmaceuticals in advanced preclinical and clinical trial status. However, plant platforms present several drawbacks: time-consuming in generating stable transgenic lines, non homogeneous protein production in different tissues, impact of pests and diseases even in controlled conditions (greenhouses) and, more importantly, growth in non-sterile conditions. This last point may affect the good manufacturing practices (GMP) necessary for the production of pharmaceuticals. Transient expression or in vitro culture have emerged as alternative platforms to circumvent some of these drawbacks. Food and Drug Administration (FDA) has recently approved the first plant-made drug for human use, an enzyme produced in genetically engineered carrot cells for treating type 1 Gaucher’s disease. Plant production of candidate prophylactic and therapeutic HPV vaccines is proven, with evidence of efficacy in animals. There are data showing that an adjuvant-like effect was obtained in immunizations with crude tobacco plant extracts containing the E7 protein of HPV 16 ,74. The DNA vaccines have been used in the clinical arena to elicit antigen-specific immune responses. Although nucleic acid vaccines do not appear to induce as vigorous immune responses as live viral vaccine vectors, they have several advantages, mainly naked DNA is relatively safe, stable, cost efficient, and able to sustain reasonable levels of antigen expression within cells. DNA-based plasmid vectors remain stable in a wide range of conditions over great lengths of time (longer periods than RNA vaccines), and they can be delivered with little risk to individuals who are immunosuppressed. In addition, since DNA vaccines do not elicit neutralizing antibodies in the vaccinated patient, they can be repeatedly administered with similar efficacy. Many strategies have been employed to produce an efficient delivery of targeted antigen-to-antigen presenting cells (APC) such as dendritic cells (DCs), an enhancement of antigen processing and presentation in DCs, and an augmentation of DC and T cell interaction . RecentlA dose-escalation trial of plasmid DNA encoding a transgene that produced E7 linked to Hsp70 showed limited efficacy at the highest dose, with low induction of responses in the IFN-gamma ELISPOT assay and a resolution rate of 33% .A plasmid DNA encoding a 13-amino acid sequence of E7 encapsulated in biodegradable poly  micro-particles was utilized to develop the ZYC101 vaccine expressing a HPV 16 E7 HLA-A2 restricted peptide. Two different phase I clinical trials examining the potential treatment of patients with anal dysplasia or with high-grade CIN, respectively, demonstrated a high number of immunological response i.e. circulating HPV-specific T cells and histological regression/improvement in 1/3 of the patients ,83. The VGX-3100, a DNA vaccine incorporating plasmids targeting HPV 16 and 18 E6 and E7 proteins was utilized in clinical trials employing electroporation technology. This technology was demonstrated particularly effective in animal models where the candidate vaccine is delivered via intramuscular injection followed by electroporation using various devices to deliver a small electrical charge. In a phase I clinical trial, 78% of the VGX-3100 vaccinated high-grade CIN subjects showed T cell and antibody responses . On the Other DNA vaccines have also been associated with other adjuvating treatments. In particular, the TLR7 agonist, Imiquimod promoting the activation of antigen-presenting cells and, in turn, leading to the production of cytokines IFN-alpha, IL-6, and TNF alpha  was showFinally, the use of RNA replicons is a potentially interesting strategy for HPV vaccination. RNA replicons are naked RNA molecules derived from alpha-viruses, such as Sindbis virus ,90, SemlThis ‘suicidal DNA’ is transcribed into RNA within the transfected cell and provides a stable and efficient way to express tumor antigen but the cells may undergo apoptosis. Another replicon system is derived from the flavivirus Kunjin (KUN) . The newThe newest mRNA-based vaccines have also been developed by Novartis as a non-viral delivery system for self-amplifying RNA that has been successfully tested in preclinical models. Another mRNA-based vaccine is the RNActive® vaccine platform from CureVac  that is based on a more stable modified mRNA sequence with increased immunogenicity by complexation with protamine. This mRNA vaccine exploits both the antigenic and the adjuvant properties of mRNAs to activate the adaptive and innate immune system. Two ongoing clinical trials on patients with prostate and non-small cell lung cancer show that RNActive® vaccines are safe and effective in inducing long lasting, humoral and cellular immune responses. No information is available on the possible utilization on HPV-associated cancers.Bacteria, such as Listeria monocytogenes (LM) ,99, LactAmong viral vectors employed for the expression of HPV antigens, like adenoviruses ,106, alpTo date many VV vaccines have been employed in clinical trials to deliver genes and antigens of interest efficiently. Phase I/II clinical trials in patients with vulvar or vaginal and early or late -stage cervical cancer were conducted with a vaccinia vector encoding HPV 16 and HPV 18 E6 and E7 antigens (TA-HPV) recombinant VV ,115-117.In another study, a recombinant VV expressing E6 and E7 antigen together with IL-2 (TG4001/R3484) was administered to CIN 2/3 patients, with very promising clinical results. Ten patients (48%) were evaluated as clinical responders at month 6. At month 12, 7 out of 8 patients without conization reported neither suspicion of CIN 2/3 relapse nor HPV 16 infection . InterimFinally, in alternative to viral vectors, synthetic viral vector like virus like particle (VLP) can be utilized because they are easy to manufacture and have the capacity for compacting DNA, and targeting specific cell receptors. Thus, the same technology used for producing anti-HPV prophylactic vaccines was employed for producing chimeric VLPs. An L1–E7 fusion protein has been shown to self-assemble into chimeric VLPs (CVLP) that can induce E7-specific cellular immunity in mice . A randoThe immune response to initial stages of infection causes inflammatory responses that trigger innate effector cells, such as NK and NKT cells. This inflammatory response, driving the innate immunity, is initiated through pathogen-associated molecular pattern (PAMP) sensors including TLRs 1–9. These receptors in response to specific bacterial or viral components activate APCs via the transcription factor nuclear factor KB (NF-KB). In addition, infection may alter the local metabolic and cellular microenvironment activating danger-associated molecular pattern (DAMP) sensors, particularly nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs), as components of multiprotein complexes termed the inflammasomes, inducing maturation and releasing members of the IL-1 family. In particular, the produced IL-1b and IL-18 mediate repair responses such as angiogenesis and, via upregulation of cytokines and chemokines, induce the recruitment of inflammatory cells to the site of infection. The observed slow clearance of HPV infection and weak immune responses to viral proteins is a likely consequence of the nonlytic nature of HPV infection and a consequent delay in induction of PAMP- and DAMP-induced inflammatory responses through TLRs and the inflammasomes. Thus, in the absence of inflammation a number of events take place  inducing negative regulatory signals that in turn can change the state of the APC by altering costimulatory molecule expression, thus inhibiting induction of cytotoxic effector T cells. Therefore a therapy aimed to reactivate these APCs could be a valid tool for clinical intervention.Among specialized APCs the most potent are DCs, because they express high levels of MHC and co-stimulatory molecules. Therefore, DCs were the research focus of many investigators and a variety of methods for generating DCs, loading them with tumor antigens, and administering them to patients. The success of Provenge, a DC vaccine incorporating prostatic acid phosphatase, in patients with advanced prostate cancer has generated great interest for DC-based vaccines ,126. AltGiven the importance of local microenvironment in the persistence of HPV lesions, strategies aiming to alter local immunity have shown some positive results, therefore therapeutic HPV vaccine strategies have shifted toward combinatorial approaches with radiotherapy and chemotherapy. Low-dose radiation in combination with HPV vaccination was effective in the treatment of tumors in preclinical models . RadiatiA chemotherapeutic agent in combination with DNA-based vaccines was proven an effective HPV therapy in preclinical models -136. LowA randomized study was carried out in 110 recurrent/refractory cervical cancer patients with cisplatin and different doses of HPV bacterial vector-based vaccine ADXS11-001, and preliminary results showed efficacy and manageable toxicity .In addition, other compounds affecting the immunological environment like COX-2 inhibitors, through the prevention of the production of prostaglandin E2 or antibodies to IL-6  or IL-10In particular, Imiquimod is already in clinical use against warts stimulating local innate immunity and potentiating adaptive immune response by activating tissue antigen presenting cells. A number of studies with topical Imiquimod have been reported with favorable results mainly in vulvar intraepithelial neoplasia (VIN) lesions ,140 and Cytokines-based therapies in combination with HPV therapeutic vaccine showed promising results in preclinical models. Treatment with IL-12 gene, administered as gene therapy, viral gene therapy, by adenovirus, and in combination with E6-E7 oncogenes, determined tumor growth suppression ,142.Recently, programmed death-1 receptor (PD-1) is emerging as a pivotal target for combination therapy by affecting immune suppression.Indeed, PD-1 is expressed on T cells following T-cell receptor (TCR) activation and binding of this receptor to its cognate ligands, programmed death ligand (PDL)-1 and PDL-2, down-regulates TCR signals, promoting T-cell anergy and apoptosis, thus leading to immune suppression.An anti-PD-1 antibody (CT-011) with Treg-cell depletion by low-dose cyclophosphamide (CPM), combined with HPV 16 E7 peptide vaccine, produced synergistic antigen-specific immune responses inducing complete regression of established tumors in a significant percentage of treated animals, with prolonging survival .Expanded phase I clinical studies with anti-PD-1 (BMS-936558) and anti-PDL -1 (BMS-936559) showed objective clinical responses in renal cell carcinoma, melanoma, and non–small cell lung cancer, and a relationship between tumor cell surface PD-L1 expression and objective responses to anti-PD1 therapy ,145. In The development of cervical cancer depends not only on efficient negative regulation of cell-cycle control supporting the accumulation of genetic damage, but also on a sophisticated viral mechanism of immune evasion -22. DespSeveral clinical studies on these precursors lesions have so far been completed with promising effects, even if these trials were not powered to detect small effects in the vaccine versus the placebo groups. Indeed, there was no follow-up study with the same strategy, perhaps as a result that these trials were investigator-initiated or sponsored by small biotech companies.Large pharmaceutical corporations that could have ensured continuation of the individual program and guarantee the prosecution in more powered phase II/III trials were completely lacking. Even the partnership announced in 2007 between a large pharmaceutical firm (Roche) and a biotech company (Transgene) on a promising vaccine TG4001 was recently discontinued.In conclusion immune escape and immune suppression are active in cancer and to some extent also in premalignant lesions and, therefore one might consider targeting persistent infections before they become clinically apparent. This ‘paradigm shift’ in vaccination of cancer patients is a quite logical step in HPV-related cervical cancer. Treating this early condition with post-exposure prophylaxis might be more successful than therapy at more advanced stages. Several well-defined cohorts of women with persistent infections do exist, and the design and execution of clinical trials aiming to clear of HPV DNA should be straightforward. However, it is important to take into consideration that patients with HSIL are generally asymptomatic and can be treated by minor surgical procedures. A clinical trial on long overlapping peptide vaccine in CIN 2/3 patients was stopped prematurely because motivational problems of the patients and the local/systemic side effects of the vaccine. Thus, motivational problems must be taken into account when considering studies in patients with premalignant lesions for whom an effective treatment is available . Future The development of prophylactic and therapeutic HPV vaccines must be continued in order to come close to the eradication of HPV-associated malignancies even in other localizations ,149, getThe authors declare that they have no competing interests.The outline was conceived by AV. All authors contributed to initial drafts, edited version, and the final version. All authors read and approved the final manuscript.


Oral cancer is one of the most common cancers and it constitutes a major health problem particularly in developing countries. Oral squamous cell carcinoma (OSCC) represents the most frequent of all oral neoplasms. Several risk factors have been well characterized to be associated with OSCC with substantial evidences. The etiology of OSCC is complex and involves many factors. The most clearly defined potential factors are smoking and alcohol, which substantially increase the risk of OSCC. However, despite this clear association, a substantial proportion of patients develop OSCC without exposure to them, emphasizing the role of other risk factors such as genetic susceptibility and oncogenic viruses. Some viruses are strongly associated with OSCC while the association of others is less frequent and may depend on cofactors for their carcinogenic effects. Therefore, the exact role of viruses must be evaluated with care in order to improve the diagnosis and treatment of OSCC. Although a viral association within a subset of OSCC has been shown, the molecular and histopathological characteristics of these tumors have yet to be clearly defined. The significant role of viruses in cancer was acknowledged finally in the second half of the past century after various rodent tumorigenic viruses were discovered, and evidence had accumulated supporting an association between viruses and human cancer. Indeed, the Nobel Prize was awarded to Rous in 1966 in recognition of his seminal discovery of tumor-inducing viruses. In addition, almost at the same time, a Special Virus Cancer Program (VCP) was launched by the US Congress in 1964 providing enormous funds for intensive research into the supposed role of viruses in human cancer. This program, criticized by some investigators as being a political moonshot-style plan, failed to identify candidate human cancer-causing viruses yet generated fundamental information about the molecular biology and mechanisms underlying, in particular, virus-related animal cancer and cancer in general [The strong cohort effect that accounted for the increased incidence of head and neck cancers after 1915 indicates that oral cancer is a disease largely attributable to behaviors that expose an individual to environmental carcinogens. The majority of oral cancers in individuals above and below the age of 45 can be attributed to the combined effects of alcohol and tobacco smoking. Other risk factors for oral cancers include diet, Body Mass Index, oral hygiene, and viral infections . The mosOf these, HPV and herpes have been the most thoroughly studied and are now considered to be the most likely “synergistic viruses” involved in human oral cancer. The herpes viruses most often linked to oral cancer are the Epstein-Barr virus (EBV), human herpes virus- (HHV-) 8, and cytomegalovirus (CMV) .This review will attempt to focus on the approaches taken to discover human cancer viruses and promising methods for detecting new viruses. We also discuss how causal association is established and possible cofactors that influence development of virus-associated cancers.Even though human oncogenic viruses belong to different virus families and utilize diverse strategies to contribute to cancer development, they share many common features. One key feature is their ability to infect but not kill their host cell. In contrast to many other viruses that cause disease, oncogenic viruses have the tendency to establish long-term persistent infections . ConsequEvans and Mueller Guidelines .Epidemiologic GuidelinesGeographic distribution of viral infection corresponds with that of tumor, adjusting for the presence of known cofactors.Viral markers are higher in case subjects than in matched control subjects.Viral markers precede tumor development, with a higher incidence of tumors in persons with markers than those without.Tumor incidence is decreased by viral infection prevention.Virologic Guidelinesin vitro.Virus can transform cells Viral genome is present in tumor cells but not in normal cells.Virus induces the tumor in an experimental animal.Hill Criteria for Causality , 15Strength of association (how often is the virus associated with the tumor?).Consistency (has the association been observed repeatedly?).Specificity of association (is the virus uniquely associated with the tumor?).Temporal relationship (does virus infection precede tumorigenesis?).Biologic gradient .Biologic plausibility .Coherence (does the association make sense with what is known about the tumor?).Experimental evidence (is there supporting laboratory data?).The human papillomavirus (HPV) family consists of more than 200 genotypes, classified in accordance with the ability to infect and transform epithelial cells. HPVs are DNA viruses that specifically target the basal cells of the epithelial mucosa . GenotypHPV belongs to the family Papovaviridae. These are small nonenveloped icosahedral viruses with an 8 kbp long double-stranded circular DNA genome. The papilloma virus genome comprises early and late genes that encode early proteins E1–E7 and late proteins L1-L2. The early proteins are nonstructural proteins involved in replication and transcription of the genome (E1–E5) or in host cell tumoral transformation (E6 and E7), whereas L1 and L2 are the structural capsid proteins of the virion. The late genes (L1-L2) code for the structural proteins of viral capsid and are activated during the final stages of the viral cycle. Up to six early genes and two late genes can be detected in HPV –23 Tabl.The ability of HPV to transform epithelial cells is divided into high-risk and low-risk types. Low-risk types are associated with development of benign lesions such as warts, while infections with high-risk types may progress to malignant lesions .Both high-risk and low-risk types of HPV can cause the growth of abnormal cells, but only the high-risk types lead to cancer because only the E7 protein encoded by high-risk HPVs can immortalize human epithelial cells. Sexually transmitted, high-risk HPVs include types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, and 73 . It is ithe strongly established etiological role of HPV in cervical SCC , 27;the epithelial tropism of HPV;the similarity between oral and genital epithelia ;the detection of HPV genotypes in samples of OSCC –33.The HPV involvement in oral carcinogenesis was supported on the basis of the following evidences:Several studies examined the incidence of second cancers after an initial diagnosis of anogenital cancers , 35 and A recent study has shown that besides the classical horizontal transmission during the sexual life, a vertical transmission occurs in approximately 20% of case HPV-positive people. In these individuals, HPV-DNA is detected in amniotic fluid, foetal membranes, blood, and placental trophoblastic cells, all suggesting HPV infection in utero, that is, prenatal transmission .E6 and E7 oncoproteins can inactivate the genetic mechanisms that control both the cell cycle and apoptosis. The hallmark of high-risk HPV E6 oncogenic activity is degradation of the p53 tumour-suppressor gene , 40. TheIn addition to these properties, E6 oncoprotein of high-risk HPV types can also mediate cell proliferation through the PDZ-ligand domain. The PDZ domain is located at areas of cell-to-cell contact, such as tight junctions of epithelial cells, and is associated with signal transduction pathways. The binding of high-risk HPV E6 oncoprotein to the PDZ family of proteins may result in degradation of the PDZ domain leading to dysregulation of organization, differentiation, and the chromosomal integrity of HPV infected epithelial cells . This maHPV-induced activation of telomerase prevents the shortening of telomeres resulting in prolongation of the life-span of HPV-infected cells. High-risk HPV E7 oncoprotein has the capacity to initiate DNA synthesis in differentiated epithelial cells mainly by binding and inactivating the Rb apoptosis/tumour suppressor gene. The Rb family of proteins plays an essential role in controlling the cell cycle by governing the checkpoint between the G1 and the S phase. Hypophosphorylated Rb binds to E2F transcription factor forming a Rb-E2F complex, making E2F unavailable for transcription of genes associated with DNA synthesis. Upon phosphorylation of Rb by cyclin-CDK complexes, E2F is released from the Rb-E2F transcription repressor complex, and it induces transcription of the S-phase genes. E7 oncoprotein of high-risk HPV types functionally inactivates the Rb family of proteins resulting in overexpression of E2F transcription factor with upregulation of cell cycle genes resulting in DNA replication, in the transition of the cell from the G1 to the S phase, and in increased cell proliferation –45.E7 oncoprotein can also interact with other cellular factors that control the cell cycle including histone deacetylases, AP-1 transcription complex, and CDK inhibitors, p21 and p27. Furthermore, E7 of high-risk HPV 16 and HPV 18 can decrease the expression of major histocompatibility complex (MHC) class I molecules, thus interfering with MHC class I antigen presentation, resulting in downregulation of cellular immune responses, allowing HPV to persist in infected epithelial cells .In addition to these properties, high-risk HPV E7 oncoprotein can induce chromosome duplication errors leading to dysregulation of mitotic spindle formation and function, contributing to the genomic instability of the cell. The separate pathological effects of high-risk HPV E6 and E7 on the cell cycle complement each other, and together E6 and E7 mediate the HPV-associated epithelial cell transformation and promote cellular genomic instability that predisposes the infected cells to full malignant transformation. High-risk HPV E7 activates the DNA synthesis and cell replication mechanisms that are normally inactive in matured epithelial cells, thus initiating pathological cell growth. By inducing cell survival and delayed apoptosis of cells with DNA damage, E6 allows E7 to exert and sustain its pathological effect .Typically, infected epithelial cells of HPV-associated benign lesions harbor low-risk HPV episomally in the nuclei. In HPV-associated malignancies, high-risk HPV DNA may either be integrated within the cellular genome or it may be maintained as an episome in the nuclei of the malignant cells. It is unclear how the HPV genome, whether episomal within the nucleus or integrated into the nuclear cellular genome, brings about the same end result of malignancy. The integration of HPV DNA favours the inactivation of tumour suppressor genes, p53 and Rb, contributing to increased cellular chromosomal instability and prolonging the life-span of the cell, which are essential steps in the multistep process of HPV-associated carcinogenesis. It is probable that following the initial HPV-induced cellular transformation, additional interactions with chemical carcinogens will provide the necessary additional impetus for the development of frank malignancy  Figure .The HPV involvement in oral and oropharyngeal carcinogenesis was first proposed in 1983 by Syrjänen et al.  Table 2Table 2. Furthermore, other studies have proved the existence of a synergistic effect between HPV and alcohol. The risk of head and neck cancer was statistically significantly increased in heavy alcohol users detected with the virus, compared to that of HPV-negative cancer drinkers. Therefore, it has been proposed that alcohol can biologically modify mucosal tissue, by increasing its permeability to viral infection or by influencing the immune response to HPV . It is bSlightly modifying Koch's postulates, in order to establish a relationship between a causative virus and a disease, four criteria are needed: (1) viral genome ought to be present in tumor lesions or in tumor cells; (2) virus must be isolated from a pathologic lesion and grown in culture; (3) cultured virus should cause disease when inoculated into a healthy organism; and (4) virus must be reisolated from the inoculated host and identified as being identical to the original specific causative factor. However, the use of Koch's postulates to establish disease causation does not fully apply to these phenomena, since the etiology of cancer is multifactorial . On the In situ hybridization and in situ oncogenic protein staining techniques have also increased sensitivity and specificity and are used for HPV testing. These techniques have allowed for not only the detection of HPV in cytological smears or histopathological immunesections but also the determination of the topographic site of the infection [HPV testing is critical for the estimation of HPV prevalence in various oral diseases. HPV testing is usually based on PCR method. General or consensus primers targeting L1 gene are most frequently used for HPV detection because they are able to identify several HPV genotypes at the same time. Sampling techniques together with widely divergent PCR methods in different studies explain most of the variability in HPV prevalence among OSCC and control samples . In situnfection . Accordinfection . Other anfection , 137. HoThe immune response to HPV infection involves both the cell-mediated and humoral responses. However, serological evidence is circumstantial since it provides only data on prior exposure to HPV. Since not all patients with HPV-associated cancers have detectable HPV antibodies, serum antibody determination may be a limited biomarker for HPV infection and carcinogenesis. Serum antibodies to HPV capsid proteins (virus-like particles) are thought to be a marker of lifetime HPV infection , 139. AnHSV is a double-stranded DNA virus that is enveloped. When it infects cells the series of events that takes place is very complex but can be analyzed logically since it follows a coordinated series of steps. The virus exists in two closely related forms, known as HSV 1 and HSV 2. HSV 1 causes mainly oral and ocular infections, while HSV 2 causes mainly genital infections. However, the overall behavior and structure of the two viruses are very similar. Each virus causes a severe primary infection followed by a latent infection that may be followed by recurrent infections. Cells that are infected by HSV are killed. Gene maps of HSV 1 and HSV 2 have been developed and show that the various functions of the two viruses are encoded by identical regions of the genome of each virus .The only viral function that is not in a related location between the two types is the ability to transform cells, and the reason is unknown. The transforming region of HSV 1  is located in the left third of the genome, while the equivalent two functions of HSV 2 (mtr-2 and mtr-3) are close to the center of the genome .Induction of cellular proteins has been studied as a possible mechanism for transformation by HSV. It is known that infection by HSV 1 induces the expression of “stress” or “heat shock” proteins. The mechanism of induction is not known but does depend on the expression of the immediate-early family of HSV proteins. Since HSV might transform cells by stimulating the expression of cellular proteins, some workers have started to study such proteins by isolating cDNA from cells that were transformed by HSV 2 –144.However, the function of these cloned fragments is still not known. Host cell shutoff is recognized as being an important event during infection of cells by HSV. The infected cell ceases to synthesize cellular proteins, and cell RNA is degraded very quickly. Recently, it was found that the gene of HSV that mediates shutoff is located in the same region of the genome as the mtr-2 region of HSV 2, which mediates cell transformation . This haThe first phase of host cell shutoff is due to some activity by the virus particle itself. It happens very soon after infection and occurs even if virus gene expression is prevented by Actinomycin D or by irradiation of the virus .The second phase of the host cell shutoff does require the expression of virus genes and eliminates any remaining host protein synthesis. It seems likely that these phenomena will be studied quite closely in the future to find which of them is most likely to be involved in cell transformation. Other activities of HSV that might be related to cell transformation include the fact that the virus can stimulate the replication of other viruses. This can also occur when cells are exposed to chemical carcinogens .Infection by HSV induces the expression of stress or heat shock proteins. According to Steele and Shillitoe, the exact mechanism is not known but it depends on the expression of the immediate early family of HSV proteins. It was, therefore, concluded that HSV might transform cells by stimulating the expression of cellular proteins .Infected cell ceases to synthesize cellular proteins and cell RNA would be very quickly degraded. A mediating gene was located in the same region of the genome as the mtr-2 region of HSV 2 that mediates cell transformation . Steele This comes from the idea that at least in cervical carcinogenesis, HSV and HPV may act as cocarcinogens, with HSV as an initiator and HPV as a promoter. In oral lesions, however, Scully et al. in 1993 showed that very few premalignant or carcinoma specimens appeared to have both HPV 16 and HSV 1 DNA sequences. They, therefore, concluded that there is no evidence that these potentially oncogenic DNA viruses do play a synergistic role in oral cancer development, but the possibility cannot be discounted .Stich et al. , MinchevEl Sissy  attempteJalouli et al. , using PDelavarian et al.  investigThe herpes simplex viruses (HSVs), types 1 and 2, have been investigated in the past for possible associations with human cancers. Levels of antibody to HSV 1 have been reported to be higher in patients with oral cancer than in control subjects . These ain vitro [in vivo [The explanation for the association between oral cancer and HSV could well be that of a confounding variable, except for the fact that HSV can transform some animal cells to a malignant phenotype in vitro . Indeed [in vivo . Unfortu[in vivo . Instead[in vivo . This re[in vivo . Neither[in vivo . Recent EBV was named after Tony Epstein and Yvonne Barr who were the two scientists who first isolated and described the virus in 1964 from lymphoma samples collected by Denis Burkitt. The EBV genome is a double-stranded DNA molecular of approximately 172,000 base pairs; as in other herpes viruses, the molecule is divided into unique, internal repeat, and terminal repeat domains . The genAll phases of the EBV life cycle are associated with human disease. In immunocompromised individuals, infected cells increase in number and eventually B cell growth control pathways are activated, inducing transformation and leading to malignancies such as nasopharyngeal carcinoma (NPC), Burkitt's lymphoma (BL), posttransplant lymphomas, and gastric carcinomas . EBV encLMP1 also upregulates the expression of numerous antiapoptotic and adhesion genes and activates the expression of IRF-7 , matrix in situ hybridization for detecting the presence of EBV DNA and EBV encoded small messenger RNA. Nearly 60% of SCC was EBV genome positive, but none of papilloma demonstrated EBV genome. On the other hand, oral hairy leukoplakia lesion seen in patients with AIDS has been proved to be EBV associated. Horiuchi et al. concluded that EBV virus infection of oral squamous epithelium may be carcinogenic or, alternatively, the virus may merely exist in epithelial cells of squamous cell carcinoma, carcinoma in situ, and leukoplakia.Horiuchi et al.  carried in situ hybridization and PCR , and latent membrane protein 1 (LMP-1) regions). Sequence analysis of the PCR products was also carried out. In Okinawa, 25 patients were found to be infected with EBV type A by analyzing the 3-sequence divergence of the EBNA2 genes. Six patients were positive for EBV type B and 8 for both types A and B. Therefore, type A virus infection was demonstrated in 33 of 54 patients and type B in 14 of 54. In total, 39 of 54 patients were infected with EBV. Authors concluded that, in Okinawa, EBV infection was frequently demonstrated in oral squamous cell carcinoma (P < 0.001). However, in mainland Japan, there was no significant correlation between EBV and oral squamous cell carcinoma [González-Moles et al.  showed aarcinoma .A study by Li et al.  to detecIn a study conducted by Sand et al.  examininJiang et al.  isolatedThe worldwide prevalence of hepatitis C virus (HCV) is estimated to be around 3%, representing approximately 170 million infected individuals. Morbidity associated with HCV infection is due to not only the sequelae of chronic liver disease but also a variety of extra hepatic manifestations, including that involving the oral cavity .Hepatitis C virus (HCV) is an enveloped, single-stranded positive-sense RNA virus that was isolated in 1989 from a chimpanzee chronically infected by contamination with a human factor VIII concentrate  .There are six HCV genotypes and more than 100 subtypes. Its envelope contains two glycoproteins, E1 and E2, which form heterodimers at the surface of the virion. The genomic RNA is translated into a viral polyprotein which is cleaved by cellular proteases to generate the capsid protein (C), the two glycoproteins E1 and E2, a small protein the role of which is unclear (p7), viral proteases NS2 and NS3, and nonstructural proteins NS4A and 4B and NS5A and 5B, which are required for viral RNA replication .Although details of HCV replication are not known, it is thought to take place in the cytoplasm, where, among other products, negative-sense viral RNA (vRNA), replicative double-stranded forms, and nonstructural proteins are synthesized. Their presence can thus be used as evidence that virus multiplication, as opposed to passive transportation, is occurring  . FurtherJohnson et al. suggested a possible involvement of HCV in diseases outside the liver and concluded that since oral cavity is frequently exposed to HCV viruses, this in turn increases the risk of genetic instability in these cells . Nagao eOral cancer is an important cause of morbidity and mortality, especially in developing countries, and its prevalence may rise in the foreseeable future. The studies of virus-associated head and neck cancers have provided many critical insights into key mechanisms of carcinogenesis. The human tumor virus oncogenes play central roles in viral life cycles and their oncogenic potential is a manifestation of these activities.Some viruses, most notably the high-risk HPVs, play essential roles in the initiation as well as progression of cancers and continued expression of their viral transforming activities is necessary for the maintenance of the transformed phenotype. Recent reviews establish the increased evidence of HPV-related oral cavity cancer in man.Standardization of the methods for sample collection and analysis is mandatory to obtain reliable data and to compare the results obtained in different studies in the presence of HPV in variable proportions in oral squamous cell carcinoma tissues. Some tumors are associated with papillomaviruses and some with viruses of the herpes family; however, the exact role of these viruses must still be evaluated carefully. These viruses may provide targets for therapy and for diagnostic tests and may widen our understanding of the mechanisms by which the tumors develop.Vaccines are designed strictly for prevention of such viral infections which may be involved in cancers of the head and neck. Few studies found sufficient evidence to support a global immunization program against HPV, irrespective of gender and geography, to help to achieve a reduction in HPV-related malignant diseases in the future. Clearly, large, prospective randomized trials are needed to document the clinical usefulness of these vaccines against oral cancer.


Estimates of Human Papillomavirus (HPV) prevalence in a population prior to and after HPV vaccine introduction are essential to evaluate the short-term impact of vaccination.Between 2010 and 2012 we conducted a population-based cross-sectional study in Germany to determine HPV prevalence, genotype distribution and risk factors for HPV-infection in women aged 20-25 years. Women were recruited by a two-step cluster sampling approach. A home-based self-collection of cervicovaginal lavages was used. Specimens were analysed using a general primer GP5+/GP6+-based polymerase chain reaction and genotyped for 18 high-risk and 6 low-risk HPV- strains by Luminex-based multiplexed genotyping.Among 787 included women, 512 were not vaccinated against HPV. In the non-vaccinated population, HPV prevalence of any type was 38.1%, with HPV 16 (19.5%) being the most prevalent genotype. Prevalence of any high-risk type was 34.4%, and in 17.4% of all women, more than one genotype was identified. A higher number of lifetime sexual partners and low educational status were independently associated with HPV-infection. In 223 vaccinated women, prevalence of HPV 16/18 was significantly lower compared to non-vaccinated women . When stratifying by age groups, this difference was only significant in women aged 20-21 years, who at time of vaccination were on average younger and had less previous sexual contacts than women aged 22-25 years.We demonstrate a high prevalence of high-risk HPV genotypes in non-vaccinated women living in Germany that can be potentially prevented by vaccination. Probable first vaccination effects on the HPV prevalence were observed in women who were vaccinated at younger age. This finding reinforces the recommendation to vaccinate girls in early adolescence. Persistent high-risk (hr) Human Papillomavirus (HPV) infections are essential for development of cervical precancer and cancer. Since 2006, two highly effective HPV vaccines have been available to prevent infections with hr-HPV genotypes 16 and 18. Many industrialised countries have adopted routine HPV vaccination into their national immunisation programmes between first intercourse and first dose of vaccination” was calculated by subtracting age at vaccination (first dose) from age at first intercourse among all vaccinated women with evaluable information (n = 173).We expected an all-type HPV prevalence of 45% based on data from our pilot study conducted in 2010 and from a large Danish population-based study,10. We ct-test for numerical variables. We conducted univariate analyses for all binary or categorical exposure variables and calculated crude odds ratios (OR) and 95% CI. Variables with a p-value <0.2 in univariate analysis were included in an initial step for multivariate modelling, followed by stepwise backward removal of all variables with a p-value >0.05 to obtain the final model. The statistical software package STATA®, version 11  was used.We performed descriptive analyses of HPV prevalence and genotype distribution both in non-vaccinated and vaccinated women, and reported results as percentages with 95% confidence intervals (CI). Risk factors for HPV infection of any or of specific HPV types or groups (i.e. hr or lr) were assessed. We performed chi-square tests and Fisher’s exact test for categorical, and Student’s The study was approved by the local ethics committee  and registered at German Clinical Trials Register (DRKS00000599).In total, 787 11.9%) women agreed to participate in the study and returned an adequate cervicovaginal lavage specimen. Of those, 223 (28.3%) reported to be vaccinated against HPV, 512 (65.0%) were not vaccinated and 52 (6.7) missed to report their HPV-vaccination status  participants reported of having received three doses, 14 (6.3%) two doses and 11 (4.9%) one dose, respectively. The quadrivalent vaccine was administered in 194 of 223 individuals (87%).Key characteristics of the non-vaccinated n = 512) and the vaccinated (n = 223) study population and the general female population of the same age group are presented in Table 2 and theThe most commonly detected HPV genotypes in the non-vaccinated population are presented in Table In univariate analysis, a higher number of lifetime sexual partners significantly increased the probability of HPV infection by any type and by hr-types. Furthermore, women with 2 or more sexual partners during the past 12 months had a significantly increased risk of HPV infection compared to those who had no partners during this time interval. Also current smokers had an increased risk of being HPV positive. In contrast, preference for condom use during one-night stands and a higher education significantly reduced the chance of being HPV positive Table . Four ouThe final multivariate model indicated that an increasing number of lifetime sexual partners and lower educational status were independently associated with the presence of HPV infection. Adjusting for age did not substantially affect the model.Three hundred seventy-six women (73.4% (95% CI: 69.4-77.2)) rated the home-based self-sampling method as “very easy” or “easy”  and less than 1%  as “difficult” or “very difficult”). Two hundred forty-one women  stated that they would prefer self-sampling over sampling by a clinician in the future, whereas 187 women (36.5% 95% CI: 32.3-40.9) would prefer sampling by a physician. 14.3% of the remaining participants did not report a preferred method (“do not know”) and 2.2% did not answer. Women who preferred physician sampling were more likely to have prior participated in the cervical cancer screening programme than those who preferred self-sampling at home (p < 0.01).st HPV vaccination prior to sexual debut only 5 (9.3%) were positive for either HPV-type 16 or 18.The prevalence of HPV 16/18 in women who reported to be vaccinated (≥1 doses) was significantly lower than in non-vaccinated women . When stratifying by age groups, this difference was only significant in the age group 20-21 years (p = 0.016), but not in the other age groups Table . AverageSome key characteristics, which are potentially associated with an increased risk for HPV detection, were more frequently found in the population of the non-vaccinated compared to vaccinated women, namely a higher mean number of sexual partners , smoking  and an immigrant background  have been more favourable for HPV than for other vaccines . NeverthWe found a high HPV prevalence in young non-vaccinated women aged 20-25 years in Germany, with the number of lifetime sexual partners and low educational status being risk factors for present infection. A significantly lower prevalence of HPV 16/18 was observed in vaccinated women suggesting first effects of vaccination. The finding that HPV 16/18 prevalence was lowest in the youngest women, 40% of whom had received their first vaccination dose before starting sexual activities, reinforce the recommendation to vaccinate girls in early adolescence and before sexual debut.These data provide a useful baseline HPV prevalence estimate shortly after the introduction of prophylactic HPV vaccination in Germany and are essential to evaluate the impact of HPV vaccination on HPV prevalence and type distribution in forthcoming years. For this purpose, prevalence studies should be repeated in the near future with the same study design and HPV-tests to allow for a direct comparison with these baseline data. Self-sampling at home via cervicovaginal lavage provides a method for monitoring that is highly accepted by study participants and allows easy inclusion of women from all parts of the country even if not attending the screening programme.CI: Confidence interval; DEGS: German health interview and examination survey for adults; DRKS: German clinical trials register; GEDA: German health update survey; HPV: Human papillomavirus; hr: high risk; lr: low risk; OR: Odds ratio; RKI: Robert Koch Institute.The study was financed by internal funds of the Robert Koch Institute. Andreas Kaufmann (AMK) has contributed as expert and member to advisory committees for GlaxoSmithKline (GSK) and Gen-probe. He has received speaker’s honoraria and travel support to conferences from Sanofi Pasteur MSD, GSK and Roche. The remaining authors declare no competing interests.YD was responsible for the overall study design, study performance, data collection and interpretation and manuscript writing. CR performed data collection, statistical analysis, interpretation and manuscript writing. JL carried out HPV testing. MS was responsible for recruitment and trial coordination. MF performed statistical analysis, AS and OW gave important intellectual content and revised the manuscript critically. AMK carried out HPV testing and data collection. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/14/87/prepub


This suggests that they may have specific functions in different organs. In the last years most of the effort has been centred on the study of the function of the p38α isoform, which is widely referred to as p38 in the literature. However, the role that other p38 isoforms play in cellular functions and their implication in some of the pathological conditions have not been precisely defined so far. In this paper we highlight recent advances made in defining the functions of the two less studied alternative p38MAPKs, p38γ and p38δ. We describe that these p38MAPKs show similarities to the classical p38α isoform, although they may play central and distinct role in certain physiological and pathological processes.The mammalian p38 mitogen-activated protein kinases (MAPKs) family is composed of four members (p38 To preserve the homeostasis and health of the organism cells are constantly responding to changes in the physical and chemical properties of the environment by altering many of their cellular functions. The activation of Mitogen Activated Protein Kinases (MAPKs) is involved in the transduction of most extracellular signals, and it is one of the major signal transduction mechanism by which the cell adapts to changes in the surrounding medium. In mammalian cells there are four well-characterised MAPK families: ERK1/2, ERK5, JNK, and p38MAPK, which are serine/threonine kinases that catalyze the reversible phosphorylation of proteins.α, p38β, p38γ, and p38δ. The isoform p38α was identified in 1994 by four groups as a 38 kDa polypeptide that is activated in response to endotoxin treatment, cell stress, or cytokines [β [γ [δ [α and p38β are 75% identical, whereas p38γ and p38δ are 62% and 61% identical to p38α, respectively. In turn, p38γ, and p38δ are ~70% identical to each other. The four p38MAPK isoforms are widely expressed, although p38β, p38γ and p38δ expression appear to be higher in specific tissues; for example, p38β is abundant in brain, p38γ in skeletal muscle, and p38δ in endocrine glands [α and p38β in one group and p38γ and p38δ in the other. In this paper, we provide an overview of the less known p38MAPK isoforms, the p38γ and p38δ MAPK pathways, which are strongly activated by stress, but also play important roles in tissue regeneration, differentiation, metabolic diseases, and cancer.The p38MAPK family comprises four members, p38kines [β –4, p38γ es [β [γ , 6, and [β [γ [δ , 8. Thesγ and p38δ isoforms are serine/threonine protein kinase of 367 and 365 amino acids with a predicted molecular mass of 42–45 kDa and are encoded by different genes located on chromosomes 22q13.3 and 6p21.31, respectively [γ is also known as ERK6, SAPK3, and MAPK12. It was first described by three independent studies as either a MAPK involved in myoblast differentiation [δ, also known as SAPK4 and MAPK13, was cloned as the fourth member of the p38MAPK family by two different groups [Human p38ectively . p38γ isntiation . p38δ, aγ in complex with an ATP analog has been determined by X-ray crystallography [γ is similar to other enzymes of the MAPK family and is characterized by two domains separated by a deep channel where potential substrates might bind. The dually phosphorylated p38MAPK goes through characteristic global conformational changes that alters the alignment of the two kinase halves  of the folded protein and enhances access to substrate. In addition, the interaction between MAPKs and their upstream activators seems to work allosterically to make the MAPKs activation loop available for processing by kinases and phosphatases, which further increases enzymatic activity [γ activation loop is almost identical to that observed in the structure of activated ERK2, contrary to ERK2, the crystal structure of activated p38γ exists as a monomer, suggesting that not all activated MAPKs form dimers [γ unique among other MAPKs is its short C-terminal sequence-KETXL, an amino acid sequence which docks directly to PDZ domains of proteins, such as α1-syntrophin, SAP (synapse-associated protein) 90/PSD (postsynaptic density) 95 and SAP97/hDlg (human disc large), and phosphorylation of these proteins by p38γ is dependent on its binding to the PDZ domains [The structure of doubly phosphorylated, active p38lography . The gloactivity –14. Althm dimers . A featu domains –17.γ and p38δ biological role is limited compared to the extensive knowledge of p38α and p38β functions. This is at least in part due to the lack of specific inhibitors for p38γ and p38δ. In vitro and in vivo assays demonstrated that only p38α and p38β are inhibited by certain compounds, such as SB203580 and other pyridinyl imidazoles, whereas p38γ and p38δ are completely unaffected by these drugs [γ and p38δ compared to p38α and p38β, in the amino acid sequence of the ATP-binding pocket, the site where most protein kinase inhibitors bind and directly compete with ATP [The information about p38se drugs , 18, 19.β-activated kinase 1), and some members of the MEKK (MAPK/ERK kinase kinase) family [The canonical activation of p38MAPKs occurs via dual phosphorylation of tyrosine and threonine residues in a conserved TGY motif, located in the activation loop of kinase subdomain VIII. MAPK phosphatases reverse this phosphorylation and return the p38MAPK to their inactive state. Phosphorylation of p38MAPKs is catalysed by the dual specificity kinases (MKK or MAP2Ks), MKK3 and MKK6, which are in turn activated upon phosphorylation of serine/threonine residues by phosphorylation by a MAPK kinase kinase (MAP3K) . The MAP) family . The div) family .in vitro, it has been suggested that the pattern of downstream p38MAPK activation in the particular response may be determined by the level of MKK6 activity triggered by a given stimulus [α activators but, in some circumstances, such as ultraviolet radiation, MKK4, an activator of JNK, may contribute to p38α activation [in vitro experiments MKK4 also phosphorylates and activates p38γ and p38δ [γ activation induced by environmental stresses, whereas MKK6 is the major p38γ activator in response to the cytokine tumour necrosis factor-α (TNFα). On the other hand, MKK3 is the major kinase responsible for p38δ activation by ultraviolet radiation, hyperosmotic shock, TNFα or by the protein synthesis inhibitor anisomycin  [γ and p38δ dephosphorylation has not been extensively studied, and therefore very little is known about physiological p38γ and p38δ protein phosphatases. Recently, it has been shown in one study that p38γ interacts through its C-terminal binding PDZ motif with the single PDZ domain of the protein tyrosine phosphatase PTPH1. Moreover, PTPH1 can dephosphorylate p38γ, but not p38α, in vitro and in overexpression experiments in cells. This specificity seems to be determined by both p38γ C-terminal PDZ-binding sequence and the conserved TGY motif within the kinase subdomain [The magnitude and duration of p38MAPK signal transduction are critical determinants of its biological effects. Termination of p38 kinase catalytic activity involves the activity of several phosphatases that target the activation loop threonine and tyrosine residues. In mammalian cells there are good es (PPs) , 29, proes (PPs) , and duaes (PPs) . Howeverubdomain .α and p38β than p38γ and p38δ or vice versa. For example, MAPK-activated protein kinase 2 (MK2) and MK3 are very good substrates for p38α and p38β, but cannot be phosphorylated by other p38MAPK isoforms [p38MAPK family members have overlapping substrate specificities, and the genetic ablation of specific p38MAPK family members has also demonstrated the existence of functional redundancy . Howeverisoforms .γ and/or p38δ has slowed down the identification of their in vivo substrates and the elucidation of their biological roles. Nonetheless, this problem can be partly solved by the use of p38 knockout mouse models. p38γ and p38δ and double p38γ/p38δ knock-out mice have been generated, which are viable and fertile [α and p38β, but also inhibits p38γ and p38δ at higher concentrations in cell-based assays providing a new tool for identifying physiological roles of these two p38MAPK isoforms [The lack of specific inhibitors for p38 fertile . Moreoveisoforms , 33.γ MAPK isoform have been described in the past years  Tat protein. This peptide blocks the phosphorylation of PDZ domain-containing proteins by p38γ in intact cells by preventing the association of the kinase with the PDZ domain of the substrate [γ provides a mechanism of dissociating it from the cytoskeleton [Drosophila tumour suppressor Dlg, a scaffold protein that forms multiprotein complexes with a variety of proteins and is targeted to the cytoskeleton by its association with guanylate kinase-associated protein (GKAP). The p38γ-catalysed phosphorylation of SAP97/hDlg triggers its dissociation from GKAP and therefore releases it from the cytoskeleton  protein-associated splicing factor (PSF) and p54us RNAs, . PSF andd repair . p38γ re of hDlg . p38γ acc stress , but notγ regulation of the SAP97/hDlg-PSF complex is independent of its kinase activity. This has been shown using cells from knockin mice expressing an endogenous inactive p38γ mutant in combination with cells from mice lacking p38γ [γ in K-Ras transformation, although the precise mechanism for this regulation remains unknown [α- and yeast p38MAPK-related proteins such as Spc1 or Hog1 may also have kinase-independent roles , which is phosphorylated in filamentous Tau from Alzheimer's disease brain. It seems that Tau-T50 phosphorylation is an early event after p38δ activation. Surprisingly, this phosphorilation causes an increase in the ability of Tau to promote microtubule assembly and help to the adaptive response of neurons to osmotic shock, whereas subsequent Tau phosphorylation at additional sites by p38δ or/and by other protein kinase(s) may then instead induce the detachment of Tau from the microtubule and destabilize the microtubule network [Some p38ed cells –42. Tau assembly , 40. Usi network .δ phosphorylates and inactivates the eukaryotic elongation factor 2 (eEF2) kinase and the protein kinase D1 (PDK1) [δ plays a role in the regulation of insulin secretion [Finally, it has been shown that p381 (PDK1) –45. PDK1γ- and p38δ-functions in vivo. Contrary to p38α, whose constitutive deletion causes death during embryonic development [γ and p38δ deficient mice are viable and have not apparent phenotype [γ- and p38δ-deficient mice. Nonetheless, there are recent reports showing the implication of p38γ and p38δ in tissue regeneration, cancer, and metabolic diseases, further strengthening the interest of these pathways for the development of new therapeutics strategies. Thus, p38δ seems to be a regulator of processes related to the pathogenesis of diabetes, such as insulin secretion and β cells death. p38δ-deficient mice have improved glucose tolerance as a result of enhanced insulin exocytosis by pancreatic β cells. Correlating with this, p38δ-deficient mice show higher levels of active PKD1, which is known to positively regulate secretion in neuroendocrine cells, as a result of the lack of p38δ-mediated inhibitory phosphorylation. In addition, p38δ has been suggested as a potential therapeutic target for human diabetes, since p38δ-deficient mice are protected against the insulin resistance induced by a high-fat diet and the oxidative stress-mediated β-cell failure [Evidence from a number of studies carried out during the past few years suggests that many physiological functions of the p38MAPK isoforms may overlap but may not necessarily be redundant and/or identical . Thus, delopment –48, p38γhenotype . Functio failure .γ and p38δ pathway could be involved in the modulation of some processes implicated in cellular malignant transformation, such as proliferation, cell cycle progression, or apoptosis [δ promotes the malignant phenotype of squamous cell carcinoma by regulating cell proliferation and invasion [γ RNA and protein expression increases during Ras-induced transformation [γ knock-down in IEC blocks the Ras transformation activity and results in the significant diminution of the oncogenic characteristics of breast cancer cells [γ mediates Ras-induced senescence at least partly by stimulating the transcriptional activity of p53 through direct phosphorylation; in contrast p38α appears to regulate senescence in a p53-independent manner [γ gene expression is required for Ras oncogene activity but the mechanism by which p38γ may promote Ras transformation is not clear. Interestingly, p38δ was recently shown to mediate 12-O-tetradecanoylphorbol-13-acetate- (TPA-) induced epidermal cell proliferation in mice, and mice lacking p38δ show reduced susceptibility to the development of TPA-induced skin carcinomas [γ and p38δ. Contrary, there is one study that shows pieces of evidence indicating that p38γ and p38δ have a role in the suppression of tumor development using mouse embryonic fibroblasts derived from mice lacking p38γ or p38δ [γ or p38δ increases cell migration and metalloproteinase-2 secretion, whereas only p38δ deficiency impairs cell contact inhibition. In addition, lack of p38γ in K-Ras-transformed fibroblasts leads to increased cell proliferation as well as tumorigenesis both in vitro and in vivo [α [Using mainly ectopic expression and knock-down model cell lines it has been shown that p38poptosis , 49, 50 invasion . In rat ormation , 52. p38er cells –55. Addit manner . These rrcinomas . All the or p38δ . Lack of in vivo . These d vivo [α .δ has been suggested to play an important role in inducing keratinocyte differentiation by regulating the expression of involucrin, which is a protein expressed during keratinocyte differentiation [δ expression is detected in mouse and human epidermis [δ by differentiation-inducing agents correlates with increased involucrin promoter activity in keratinocytes [α/β-independent manner, and what is more, p38γ is poorly expressed in keratinocytes [δ may play a role in keratinocyte differentiation come from a study carried out in lesional psoriasis skin. Psoriasis is a chronic inflammatory skin disorder characterised by keratinocytes hyperproliferation and differentiation. It has been shown that the activity of p38α, p38β, and p38δ is augmented in lesional psoriasis skin compared with nonlesional psoriasis skin [δ may have a dual role in keratinocytes contributing not only to the differentiation process, but also to their apoptosis in a PKCδ-dependent manner, though the exact mechanisms by which p38δ may regulate keratinocyte differentiation or apoptosis are still unknown [δ in regulating keratinocyte differentiation or apoptosis are based in overexpression experiments and require verification using other tools to both inhibit the activity or the expression of different p38MAPKs.p38ntiation . p38δ expidermis , 61. It inocytes . This ocinocytes . More dasis skin . Additio unknown , 66. It δ and p38γ role in primary human erythroid cells differentiation has been suggested. Analysis of the mRNA expression pattern of each p38 isoform during erythroid differentiation of primary human erythroid progenitors shows that p38α and p38γ are expressed in early and late stages, whereas p38δ mRNA is expressed only at terminal stages of differentiation, indicating a possible role of p38γ in hematopoiesis and of p38δ during the terminal phase of differentiation [A possible p38ntiation .γ expression is very high in skeletal muscle in comparison to other tissues, it is not surprising that it may play a fundamental role in skeletal muscle differentiation. Thus, endogenous p38γ protein level increases when myoblast differentiates into myotubes [γ in skeletal muscle cells leads to differentiation from myoblast to myotubes and that a dominant-negative mutant of p38γ prevented this differentiation process [γ null mice reported that p38γ plays a cardinal role in blocking the premature differentiation of skeletal muscle stem cells, the satellite cells that participate in adult muscle regeneration. p38γ phosphorylates the transcription factor MyoD and promotes MyoD association to the histone methyltransferase KMT1A. This complex acts repressing transcription and the premature expression of myogenin [α in muscle differentiation [γ is involved in muscle-specific exercise-induced skeletal muscle adaptation, and it seems to be required for the upregulation of PGC-1α (peroxisome proliferator-activated receptor-γ (PPARγ) coactivator-1α) in mitochondrial biogenesis and angiogenesis in response to exercise and nerve stimulation in mice [Since p38myotubes , 69. Mor process . Recentlntiation . Moreove in mice .α isoform and report the implication of this p38MAPK isoform in numerous biological and physiological processes. However, the in vivo functions of other alternative p38 isoforms or the molecular mechanism by which these kinases regulate particular cell processes remain largely unknown, and several important questions remain to be answered to address why a variety of p38MAPK isoforms is needed in mammalian cells are: for example, (i) how the p38MAPK isoforms are differentially activated by certain stimuli to mediate specific nonredundant signals, (ii) the identification of specific physiological substrates and how they are modulated by each p38 isoform, and (iii) the elucidation of new in vivo roles. The use of a combination of genetically modified mice, such as mouse lacking one or more p38 isoforms, tissue-specific knock-out mice, and knock-in mice expressing inactive p38MAPK will be a powerful tool to elucidate in vivo functions. Furthermore, high throughput genomic and proteomic technologies will also help to answer these questions and to generate enough knowledge that hopefully could be translated in therapeutics strategies by targeting the alternative, p38γ and p38δ MAPK isoforms.Most of the studies to date have focused on the role of the p38


The burden of cervical cancer is disproportionately high in low-resource settings. With limited implementation of human papillomavirus (HPV) vaccines on the horizon in the developing world, reliable data on the epidemiology of high-risk HPV (HR-HPV) infection in distinct geographic populations is essential to planners of vaccination programs. The purpose of this study was to determine whether urban patterns of HR-HPV occurrence can be generalized to rural areas of the same developing country, using data from Mali, West Africa, as an example.Urban and rural women in Mali participated in a structured interview and clinician exam, with collection of cervical samples for HPV DNA testing, to determine HR-HPV prevalence and correlates of infection. Correlates were assessed using bivariate analysis and logistic regression.A total of 414 women  were recruited across both settings. The prevalence of HR-HPV infection in rural women was nearly twice that observed in urban women (23% v. 12%). Earlier age of sexual debut and fewer pregnancies were associated with HR-HPV infection among urban women, but not rural women. Twenty-six percent of urban women who had sexual intercourse by age 14 had an HR-HPV infection, compared to only 9% of those who had later sexual debut (p<0.01). Overall, age, income, and polygamy did not appear to have a relationship with HR-HPV infection.Compared to urban women, rural women were significantly more likely to be infected with high-risk HPV. The patterns and risk factors of HR-HPV infection may be different between geographic areas, even within the same developing country. The high prevalence in both groups suggests that nearly all rural women and most urban women in Mali will be infected with HR-HPV during their lifetime, so the effects of risk factors may not be statistically apparent. To control HPV and cervical cancer in West Africa and the rest of the developing world, planners should prioritize vaccination in high-burden areas. The developing world bears a disproportionately high burden of cervical cancer . ParticuHuman papillomavirus (HPV) is a common sexually-transmitted pathogen, of which several high-risk types are associated with nearly all cases of cervical cancer worldwide . The higHPV vaccination of adolescent girls may be on the horizon in the developing world. However, the drug and delivery costs of vaccination may prohibit widespread implementation in low-resource countries in the immediate future . DonatioPrevious studies have examined correlates of HPV infection  and invaThe current study sought to determine whether the patterns of high-risk HPV (HR-HPV) infection are different between urban and rural areas of the same developing country, using pooled data from two cross-sectional studies in Mali as an example. In doing so, we measured the prevalence of infection in both an urban and rural area of Mali, and estimated the degree to which certain risk factors were correlated with infection in each setting.et al. 2004 [et al. 2011 [The current study combined data from two cross-sectional studies of HR-HPV in Malian women. Study I recruited urban-dwelling Malian women. The urban study was auxiliary to a study to evaluate the accuracy of visual screening techniques in low-resource settings. A description of that parent study was previously published . Anotheal. 2011 ). Study Two samples of women were recruited separately: an urban sample from Bamako in March 2007, and a rural sample from the village of Naréna, approximately 100 km (60 mi) southwest of Bamako, in August 2008. The urban sample was population-based, recruited from an age-stratified random sample of women who were selected from a census registry sampling frame. In the rural area, where no census registry existed, women were recruited from the catchment population of a community health center via word-of-mouth advertisement. Participating women in both settings were required to be 15 years or older and provide informed consent. Women under the age of 18 were also required to have verbal consent from a parent or guardian. Women were excluded if they self-reported: (i) history of hysterectomy with removal of the cervix; or (ii) history of anogenital, breast, oral, esophagus, lung, bladder, liver, or cervical cancer.By local custom, community meetings were held prior to recruitment in both the urban and rural setting. In the urban setting, community recruiters visited the homes of each of the randomly-selected women to invite each to participate in the study. Women who agreed were then assigned appointments at the research clinic, where study personnel explained the study to the women first in groups and then in greater detail individually. An interviewer obtained informed consent in the privacy of an interview room. A printed consent form was translated by a trained investigator into the local language and read to the study participant, and the participant’s signature, index fingerprint, or witnessed verbal affirmation was obtained as indication of consent to participate.In the rural setting, the study days and times were advertised throughout the community via word-of-mouth. On the study days, potential participants arrived at a community health center, where they underwent an eligibility review and consent process similar to that in the urban setting. The interview was then administered in a semi-private area. Subsequently, the woman entered a private examination room, where a gynecologist performed the pelvic examination and specimen collection.A standardized interview was used to obtain detailed information on sociodemographic factors and sexual and reproductive history. The self-reported value for age should be considered an estimate, as many women, particularly in the rural setting, may not know their exact age. The questionnaire was derived from a more extensive interview used by the International Agency for Research on Cancer (IARC) during an international multisite international study of the epidemiology of cervical cancer . Each inClinical examinations and specimen collection were performed according to standardized procedures ,17. A clHPV DNA testing for samples collected from the urban women was performed in the US using Digene Hybrid Capture II  according to the manufacturer’s protocol. The HC2 is a pooled-probe, nucleic acid hybridization assay with signal amplification that uses microplate chemiluminescent detection. Each sample was determined to be positive or negative for one of 13 high-risk types of HPV, defined as HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68 [Interview and clinical examination data were collected using standardized, scannable paper forms, generated by the Teleform™ software system .The variables obtained from the interview were defined as continuous, categorical, or count variables. Average monthly income was reported in West African CFA and was treated as an ordinal variable, with values 1–4 corresponding to increasing income categories.The urban and rural groups were first compared along sociodemographic and behavioral factors, using Student’s t-tests , Pearson’s chi-square tests , Fisher’s exact tests , or Poisson regression (counts). We then compared women who were infected (HR-HPV+) to those who were not infected (HR-HPV-), using the same tests. This analysis was then repeated within each geographic setting, with reference categories designated for categorical variables.To test whether the effects of different variables on HR-HPV infection were different between urban and rural women, we constructed a series of logistic regression models, with HR-HPV status as the outcome variable. Each independent variable was entered into a separate model, along with geographic setting  and a geography×variable interaction term. The significance of the interaction term  implied how different the effect of the variable was on infection status between the urban and rural women. For categorical variables, this approach produced conclusions that were meaningfully equivalent to those produced by a Breslow-Day test for statistical interaction.A p-value of <0.05 was considered statistically significant. All analyses were performed using STATA 11 .The two studies initially recruited a total of 436 participants. HPV testing was not completed in 22 of these women , and these participants were excluded from the analysis. The final analysis sample included 202 urban and 212 rural women , and reported a greater number of pregnancies .The rural women appeared to engage in slightly more risky sexual behavior than the urban women. The rural women had first sexual intercourse at a younger mean age than the urban women , and had slightly more casual sexual partners and partners before age 20 . Few women in either setting used condoms or had ever undergone a Papanicolaou (Pap) test, and nearly all women in both settings were circumcisedTwenty-four 12%) of the urban women had an HR-HPV infection, compared to 49 (23%) rural women , but no significant difference was seen among rural women Table . InfecteThe study demonstrated that the occurrence and patterns of HPV infection may be different between urban and rural settings within the same developing country, using data from Mali as an example. Our sample of rural women had nearly twice the prevalence of HR-HPV as did an urban sample. Early sexual intercourse and fewer lifetime pregnancies predicted HR-HPV infection, but only in urban women.The much higher prevalence of HR-HPV in rural compared to urban women is not easily explained by sampling techniques, despite differences in recruiting methods that were used in the urban and rural settings of this study. In this and previous studies in these settings, we have encountered a population that is generally very receptive of medical research studies and is eager to participate, regardless of current or previous medical conditions or experiences. The sampling techniques produced samples that were comparable on many sociodemographic characteristics, providing evidence that the women who decided to participate were similar across the settings. The baseline differences that were apparent-for example, the large number of 35–44 year-old rural women, which was likely due to the estimation of ages in the rural setting-did not appear to bias the overall prevalence figures within settings, as demonstrated by subgroup prevalence figures. We therefore do not expect that differences in recruiting techniques, alone, could explain the wide disparity in prevalence between the two settings, nor could it solely explain the most apparent differences in HPV patterns.The difference in prevalence between settings is also unlikely to be caused solely by behavioral or sociodemographic predispositions. The idea that the high rural prevalence is driven by that group’s riskier sexual behavior-a somewhat lower mean age at first intercourse and slightly higher number of partners before age 20-is refuted by the lack of a convincing association between either of those risk factors and HR-HPV status in the rural setting. Several possible explanations remain regarding difference in prevalence between settings; for example, an historic founder effect could contribute to the results. The prospect also exists that sexual and social network construction is inherently different in the rural setting, perhaps due cultural factors such as the occurrence of polygamy, in such a way that facilitates HPV transmission to nearly all women quickly upon sexual debut.et al. study-a hospital-based case-control study of cervical cancer from the mid-1990s in Bamako [et al. study, women with six or more pregnancies had at least three times the odds of invasive cervical cancer compared to those with five or fewer pregnancies. Historically, studies in the developing world-but not in developed countries-have found a positive relationship between parity and HPV or cervical cancer [Contrary to the Bayo n Bamako -this stul cancer ,12,18,19l cancer , as it wEarly sexual debut was a risk factor of HR-HPV infection in our urban sample, a result that has been found elsewhere in the developing world ,4, but cFor some factors, such as female circumcision, lack of screening, and lack of screening knowledge, the sample was too homogenous to detect any meaningful association with HR-HPV status.et al. study, 60% of women tested positive for having ever been infected with the three most common HR-HPV types, and nearly all women had a previous infection with any HPV type [The high baseline prevalence of HPV in this population may obscure the effect of risk factors measured through a cross-sectional study. Beyond the detectable group of HPV-infected women was a presumably much larger but undetectable (by this study) group of women who had recovered from an HPV infection. The HPV prevalence rates seen here were comparable to those measured elsewhere in West Africa . In areaHPV type . BecauseBecause HR-HPV infection may be nearly inevitable for many women in Mali and other parts of the developing world, even in the absence of high-risk behaviors, the focus of vaccination efforts in such resource-limited settings should be on the general female adolescent populations of highly-affected areas, like our rural setting. Based on our results, behavioral intervention may be unlikely to stem the course of HPV infection and cervical cancer. Furthermore, seeking out potential high-risk members (based on projected future behavior or sociodemographic factors other than age) within populations may add an unnecessary layer of difficulty and cost to vaccine implementation. Vaccination in general populations will likely be effective when implemented; a recent mathematical modeling simulation of the Malian population projected that the HPV vaccine could reduce the burden of cervical cancer by approximately the same proportion as vaccine coverage rates .This study used different laboratory testing methods to detect HR-HPV infection in the urban and rural settings-HC2 alone was used in the urban setting, whereas the rural setting saw HC2, LA, and Amplicor. However, any overestimation in HPV prevalence in the rural setting due to the use of parallel tests is expected to be modest. Previous work in a cohort of women with cervical abnormalities in Australia has shown generally high agreement between the three tests, with interassay agreement ranging of about 84% comparing either Amplicor or LA to HC2, and 98% comparing Amplicor to LA . AgreemeInterpretation of these findings should be careful to consider the number of simultaneous statistical comparisons being made. Also, the statistical power of the study to identify risk factors was limited both by the high prevalence of HR-HPV, especially in the rural group, and the number of people in each setting.As with all studies using self-reporting of variables, especially variables of a sensitive sexual nature, the quality of our data is contingent on accurate reporting. Misclassification of sexual behavior variables likely would have made it more difficult for us to find significant differences between urban and rural or infected and uninfected women. The main research variables-HR-HPV status and geographic setting-were not prone to misclassification due to self-report.The finding that HPV occurs according to different patterns in urban and rural settings could have implications that reach far beyond West Africa. Because associations that appear obvious in one area may not be present in others, planners of vaccination campaigns must do exploratory work in every potential setting to measure the burden of disease and uncover patterns of HPV and cervical cancer occurrence.As HPV vaccine implementation unfolds in the developing world, public health planners within countries must identify the subpopulations with the greatest need, so that they can prioritize the allocation of limited resources. This study demonstrated that data from urban areas-where most cervical cancer and HPV surveillance has been done-cannot be used to predict HPV occurrence in rural areas. We tested more than 200 women for prevalent HR-HPV infection in each an urban and rural setting in Mali, West Africa. HR-HPV infection was twice as prevalent among our rural sample of women compared to our urban sample. Earlier sexual debut and fewer pregnancies predicted HR-HPV infection among urban but not rural women. In areas with very high prevalence of HPV, the practice of identifying risk factors can be troublesome from statistical and practical perspectives. The ubiquity of HPV in such a setting may erode the apparent effect of risk factors other than geography, meaning that to vaccination planners, nearly all adolescents in high-burden areas can be considered at high risk for contracting HPV and developing cervical cancer.HPV: Human papillomavirus; HR-HPV: High-risk human papillomavirus; HC2: Hybrid Capture II; LA: Linear Array; PCR: Polymerase chain reactionThe authors declare that they have no competing interests.KT and NS led the writing of the manuscript and the data analysis. In addition, KT conceptualized the study design and methods and secured funding for these studies. SS led the organization and implementation of the study in Mali. CT, KB, RD, and FS helped to design the recruitment and data collection procedures. PG was involved with the conception of the study design and methods and performed HPV testing of rural samples. All authors had access to the data, and have approved of the data analysis and the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6874/13/4/prepubMali Interview-English. Description: English version of the questionnaire administered to both urban and rural participants.Click here for file