From an epidemiological perspective, the practice of universal vaccination of girls and young women in order to prevent human papilloma virus (HPV) infection and potential development of cervical cancer is widely accepted even though it may lead to the neglect of other preventive strategies against cervical cancer.It is argued that removing the deterrent effect – the fear of developing cancer – could encourage teenage sex. This paper reflects on the ethical legitimacy of the universal vaccination of girls and young women against HPV infection, especially regarding safety issues, the need to vaccinate people who have opted to abstain from sex, the presumption of early onset of sexual relations, the commercial interests of the companies that manufacture the vaccine, and the recommendation of universal vaccination in males.Based on the aforementioned information, we believe that the universal vaccination against HPV in young women is acceptable from an ethical point of view, given the medical advantages it presents. Human papilloma virus (HPV) is routinely found in humans. There are more than 100 different types of HPV, but not all are health threatening. Some cause common condylomas and others plantar warts, but there is a group of HPVs that mainly infects the mucous epithelium of the anogenital tract and has an oncogenic nature [Around 40 types of HPV are transmitted primarily through sex, causing a mucous infection, especially in the anogenital region . CondomsIn the reproductive stage of its life cycle, HPV multiplies inside the infected cell, causing cell alterations that lead to morphological changes in the epithelial tissue and other types of lesions .Genital infections due to HPV are very common. Most occur in young women although 70% disappear spontaneously within one year and 90% within two years . When thOf the more than 100 different variants of HPV,  types 16Worldwide, HPV infection is responsible for more than half a million cases of cancer and more than 250,000 deaths every year ,16. The The most effective measure for preventing cervical cancer in the last century was the introduction of cervical screening programmes, especially the Papanicolaou test, for the early detection of precancerous lesions in women that were already infected (secondary prevention). This test has high specificity, but low sensitivity and so erroneous results are not uncommon . FurtherEven though an intervention on the modifiable risk factors  of the HPV vaccine in preventing moderate and severe precancerous cervical lesions associated with HPV types 16 and 18 in women not previously infected by these types -35 as weFor the reasons previously stated, it appears that, from a medical and sociological point of view, recommending universal vaccination of women, especially girls and young women, is an appropriate health measure, and it was introduced into vaccination schedules following pharmaco-economic and opportunity analyses. However, recommending the universal vaccination of girls and young women may entail ethical problems that may make it difficult to implement these campaigns. Assessing these ethical considerations will constitute the main part of our paper.Some of the potential ethical issues that HPV vaccination may give rise to are as follows:The side effects reported after administration of the vaccine have been mainly local reactions, while signs or symptoms of greater importance have been reported in very rare cases ,39,41. SFurthermore, taking into account that the vaccine has an efficacy of practically 100% for preventing precancerous lesions caused by the viral genotypes included in the vaccine -35 and tAdolescents who have decided to maintain sexual abstinence, either due to education or personal choice would not technically be susceptible to HPV infection, as they are not exposed to at-risk relationships, so it would not be necessary to vaccinate them. However, absolute sexual abstinence is not an easy option to maintain and can vary throughout life, especially if we take into account that the age at which the vaccination is recommended is between 9 and 14 years old, an age at which it is unlikely that girls will have adopted a sexual behaviour that will remain unaltered throughout their life.Furthermore, even if they remain true to that decision, this is unlikely to be maintained after marriage. Even though sexual intercourse takes place within the couple itself and is based on the fidelity and exclusivity of the spouses, it may be that one or both spouses has not maintained an attitude of sexual abstinence during their entire premarital life and so there may be the possibility that one of them is infected with HPV and could infect the healthy partner. In order to prevent this hypothetical transmission, it would have to be ensured that neither spouse was infected with HPV before the marriage, but this practice seems difficult to accomplish socially. Vaccination prior to marriage could be recommended, although, as has been mentioned, the effectiveness of the vaccine is highest if administered between the ages of 9 and 14 years because, at that age, a higher antibody titre is generated than at later ages . It appeThe recommendation for the universal HPV vaccination of girls and young women is based, on the one hand, on the possible early onset of sexual relations,  and, on Be that as it may, sometimes universal HPV vaccination may be part of a family or public strategy that seeks to facilitate the onset of sexual relations in young people, sparing them greater risks, especially the danger of developing cervical cancer. In relation to this, even if vaccination were part of that strategy, the vaccination in itself would not be unethical; it is the strategy that would be unethical, i.e. the ethical problem would have to be shifted to the strategy that forms the context of the vaccination and not the vaccination itself, because obviously the instrumentalisation (using something that is not your own for a purpose) of vaccination programmes does have an ethical content, or because it deliberately encourages a good thing to mislead and promote something bad (“safe sex”), or because it prevents a good thing so that it cannot be exploited for something bad (“safe sex”). Both choices are unethical and are not justified by the possible good intention.When making an ethical reflection on the advisability of a universal vaccination policy for girls and young women, the interests of the commercial firms that manufacture the vaccines, as well as the intermediary agents, and to what extent the economic benefits for both could influence decision-making should be taken fully into account. In fact, it is essential that, in order to ensure the correct choice of vaccine and the programmes to be implemented, the administration gives priority to the economic efficiency of the potential vaccines to be used – this would guarantee the suitability of the vaccination programmes chosen.It should also be taken into account that the research processes required to develop viable vaccines are very costly. At present, the main economic investment for the development of new vaccines is made by the commercial firms and so, in order to maintain the investment, the vaccines obtained must be marketed effectively. It is obvious that if they do not achieve high use of the vaccines, they cannot continue investing in research. The information on HPV infection given to the general public should be improved, which is why many companies carry out information campaigns on HPV and its vaccine together with their vaccination campaigns . InformaThe massive vaccination of males might be one measure that could reinforce the results of vaccine protection ,48. AlthMoreover, previous studies have assessed the inclusion of males in the vaccination programmes and have found a cost-benefit relationship that is sufficient to opt for inclusion, ,50 althoWhen making an ethical assessment on whether to promote universal campaigns in favour of the vaccination of girls and young women with the HPV vaccine, in our opinion, the first question that arises is whether it can be considered ethical to remove the deterrent effect that is the fear of acquiring a serious and, in fact, fatal illness, as a result of the universal use of the HPV vaccine, and whether the disappearance of this deterrent effect could promote sexual relations among adolescents and young people. In relation to this, we believe that the first question that should be asked is: “Is it ethically acceptable to base the proper use of human sexuality on the fear of contracting a serious disease  because of the possibility that said medical action might reduce the hypothetical fear of developing the disease, which could indirectly contribute to encouraging sexual relations in young people when the deterrent effect represented by the possibility of infection and development of this disease disappears?” In other words: “Can we stop using a useful medical measure to prevent a disease – a measure that in itself does not have any ethical difficulty – amid fears that it could indirectly encourage sexual promiscuity in young people?” This is a question that, specifically in relation to the HPV vaccine, could be asked as follows: “Is it ethically acceptable not to promote universal HPV vaccination in girls and young women for fear that this could reduce the deterrent effect – i.e. the fear of developing cervical cancer – and that this could encourage sexual relations in young people?”Human sexuality is far from being solely a physiological mechanism for reproduction, undoubtedly an extremely important end in itself, but it is also an expression of physical and spiritual love between a man and a woman. It is not only an instrument to generate pleasure, but is also an expression of human love, hence its greatness, which goes well beyond a mere reproductive mechanism.Due to the physical and spiritual nature of sexuality, it follows that its exercise cannot be evaluated exclusively from a biological aspect, but should also be assessed from an ethical point of view. To that end, the ethical aspect of the exercise of human sexuality forces us to consider this when evaluating actions that affect it in some way – this is true in the case of the HPV vaccine. Therefore, any action or act related to human sexuality requires an ethical evaluation.We are of the opinion that the ethical evaluation on the use of the HPV vaccine, in principle, could be classified within the so-called “double effect” actions. “Double effect” actions are those that simultaneously cause two effects, one of which is morally positive and the other negative.Traditionally, it has been postulated  that to In our opinion, we believe that the vaccination cannot be classified within the moral judgement merited by “double effect” actions, since the effect sought – i.e. to prevent an infection that may cause a serious or even fatal disease, something that can be achieved with the vaccination – is not accompanied by any negative side effects that could morally invalidate the positive primary effect sought. However, in certain circles, it is argued that proposing universal HPV vaccination, especially in adolescents, could encourage sexual relations between adolescents by removing the deterrent effect of the fear of contracting a serious disease, something that, in our opinion, cannot be directly inferred from the very fact of the vaccination, especially if we take into account that by proposing the vaccination at early ages, this is removed in time from the possible onset of sexual relations, and so conflating vaccination and sexual activity does not appear to be justified.Furthermore, a recent study conducted in the United States  assessedLikewise, there is an added circumstance that we believe may invalidate the negative ethical judgement that vaccination might merit when considering, as already indicated, that it could promote sexual promiscuity by removing the deterrent effect for preventing sexual relations between adolescents. This circumstance is that, with the use of the HPV vaccine, development of a serious disease  that is acquired sexually can be reduced or prevented, but it does not prevent the spread of other sexually transmitted diseases, among them some as serious as HIV; in other words, it does not seem that, with the potential to be able to prevent one disease with the HPV vaccination but not to eliminate the risk of spread of other sexually transmitted infections, we can continue using the argument that by removing the deterrent effect, which is reducing the risk of developing cervical cancer, by eliminating a single possibility of infection, one might think that we are going to contribute to an increase in sexual relations among adolescents, if the risk of contracting other equally serious sexually transmitted diseases through these relations remains.In addition to this, and with respect to infection with HIV and other sexually transmitted diseases, it could be argued that together with vaccination, it could promote the use of condoms and so would achieve so-called “safe sex”, i.e. adolescents could have sexual relations without the possibility of being infected, especially with HIV. However, this seems misleading, since it is well known that using condoms does not completely prevent the possibility of being infected with the virus in a heterologous couple, i.e. with one healthy partner and the other HIV-positive, as using a condom for this purpose has a failure rate of between 5% and 10% -60.One aspect that – regardless of the advisability or not of the universal vaccination of girls and young women – must be left quite certain, is the inalienable right of parents to decide about the sex education of their children, and, therefore, the parents must be provided with all the information relative to the use of the HPV vaccine and the consequences that it could have in relation to their sexual activity. It is particularly important that parents, in the context of a universal vaccination programme for adolescents, can refuse to have their daughters vaccinated if they consider it appropriate – a right that should be made explicit in the vaccination campaign. In order to ensure this parental autonomy, even if the vaccine is promoted or recommended by the public health authorities, the final decision on its use should always remain in their hands -63.In summary, we believe that proposing a universal HPV vaccination campaign in girls and young women does not entail any particular ethical difficulties, providing that the aforementioned premises are taken into account, so we do not see any major drawbacks in proposing it.Díez-Domingo, was principal investigator in clinical trials sponsored by GlaxoSmithKlein, Merk and Sanofi Pasteur MSD. The rest of authors don't have any conflict of interests.This article has not been funded.PN-I made substantial contributions to the conception and design, acquisition of data and analysis and interpretation of data. JA made substantial contributions to the ethical debate. JD-D was involved in drafting the manuscript and revising it critically for important intellectual content and has given final approval of the version to be published. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6939/15/29/prepub


We introduce a new method for generating optimal split-plot designs. These designs are optimal in the sense that they are efficient for estimating the fixed effects of the statistical model that is appropriate given the split-plot design structure. One advantage of the method is that it does not require the prior specification of a candidate set. This makes the production of split-plot designs computationally feasible in situations where the candidate set is too large to be tractable. The method allows for flexible choice of the sample size and supports inclusion of both continuous and categorical factors. The model can be any linear regression model and may include arbitrary polynomial terms in the continuous factors and interaction terms of any order. We demonstrate the usefulness of this flexibility with a 100-run polypropylene experiment involving 11 factors where we found a design that is substantially more efficient than designs that are produced by using other approaches. Split-plot designs arise in experimental studies when a completely randomized run order is structurally impossible, expensive or inconvenient. So, a statistician's recommendation to randomize the order of the runs is often ignored in practice. Instead the experimenter rearranges the runs of the design so that hard-to-change factors only need to be changed a few times over the course of the study. This rearrangement, though seemingly innocuous, creates a split-plot structure, i.e. the experiment is performed in groups of runs where the hard-to-change factors stay constant within each group.whole plot. A factor whose levels vary within each plot is called a subplot factor. In industrial application the hard-to-change factors are whole-plot factors and the groups of runs are the whole plots. A subplot factor is one that is easy to reset from run to run.The terminology for split-plot designs comes from their original application in agricultural experiments where a factor that only varies between separate plots of land is called a whole-plot factor. This is because the researcher applies only one of its possible levels to the D-efficiency.Though the primary motivation for doing split-plot experiments is economic or logistic necessity, there are also statistical reasons to prefer a split-plot arrangement over a completely randomized design in some cases. Varying a hard-to-change factor can cause a substantial disruption in the process. This results in a random change in the mean of the process response from one group of runs to the next. If the process is completely reset between each run, this random effect of varying the hard-to-change factors gets added to the usual process variance. This makes detection of the effect of a change in any factor more difficult. By contrast, in a split-plot design, only the whole-plot effects experience this extra variability. Support for this argument in favour of using split-plot experiments has been provided by The design of industrial split-plot experiments received attention in Huang A recent line of references focuses on the arrangement of standard response surface designs like central composite and Box–Behnken designs in a split-plot format so that ordinary least squares estimation provides the same estimates as generalized least squares estimation see , 2006.a priori model. D-optimality criterion to select simultaneously the combinations of the hard-to-change and the easy-to-change factors and to arrange the selected combinations, or runs, in whole plots.The previously referenced literature on the design of split-plot experiments mainly addresses the question of how to superimpose a split-plot structure safely on well-known designs. Another line of research focuses on the algorithmic construction of split-plot designs to allow more flexibility in the choice of sample size, whole-plot size and D-optimality criterion is maximized. This approach, which became traditional in the literature on the algorithmic construction of optimal designs after the publication of the first point exchange algorithm by A difficulty with the algorithms of The purpose of this paper is to present an algorithmic approach to constructing tailor-made split-plot experiments without having to specify a candidate set. After the introduction of the statistical model, the candidate-set-free algorithm is outlined in Section 3 and compared with previous algorithms in Section 4. The good performance of the candidate-set-free algorithm is first demonstrated by using a proof-of-concept example. Next, we apply the new algorithm to a polypropylene experiment and compare the resulting design with the experiment that was actually conducted and a sequential approach that uses the algorithm of n and b whole plots, the model can be written as X represents the n×p model matrix containing the settings of both the whole-plot factors w and the subplot factors s and their model expansions, β is a p-dimensional vector containing the p fixed effects in the model, Z is an n×b matrix of 0s and 1s assigning the n runs to the b whole plots, γ is the b-dimensional vector containing the random effects of the b whole plots and ɛ is the n-dimensional vector containing the random errors. It is assumed that For a split-plot experiment with sample size y), is Under these assumptions, the covariance matrix of the reponses, var,the number η of the whole-plot to the error variance,the ratio a priori model andthe t to consider.the number of starting designs or tries M|=|X′V−1X|. The two parts are performed t times. Each time the value of Given this information, the body of the algorithm has two parts. The first part is the creation of a starting design. The second is the iterative improvement of this design until no further improvement is possible. Improvements are measured by increases in the objective function b random numbers are chosen. All the rows in a given whole plot have the same value. This procedure gives the starting design the desired split-plot structure.The starting design is formed column by column. For subplot factor columns, the values for each row are chosen randomly. For whole-plot factor columns, Improvements are made to the starting design by considering changes in the design element by element. This is inspired by For an element in a subplot factor column, the objective function is evaluated over a discrete number of values spanning the range of that factor. If the maximal value of the objective function is larger than the current maximum, then the current maximum is replaced and the current element in the design is replaced by the factor setting corresponding to the maximal value.For an element in a whole-plot factor column the procedure is more involved. If this element changes then all the elements in the same whole plot for that column must also change. A discrete number of values for that whole-plot value are considered. Again, if the maximal value of the objective function is larger than the current maximum, then the current maximum is replaced and all the rows in that whole plot are replaced by the factor setting corresponding to the maximal value.This element-by-element procedure continues until a complete cycle through the entire design has been completed. Then, another complete cycle through the design is performed noting whether any element changes in the current pass. This continues until no changes are made in a whole pass or until a specified maximum number of passes have been executed.D-optimal designs. A comparison is made in terms of ease of use, quality of the design produced and computing time.The purpose of this section is to compare the candidate-set-free algorithm with alternative methods for constructing The main advantage of the candidate-set-free algorithm that was presented in Section 3 is that it does not require the user to specify a candidate set. For simple design problems, such as problems involving unconstrained continuous factors only and a cuboidal experimental region, good candidate sets for first- and second-order models are given by the points of two- or three-level factorial designs respectively. For other design problems, involving constrained continuous and/or mixture variables, constructing a good candidate set may be difficult and time consuming. The construction of good candidate sets for such problems requires experience and is, for spherical design regions, even a matter of on-going research see . Since rThe fact that the construction of a good candidate set often takes more time than running a classical algorithm for computing an optimal design is not captured by the timing study that is reported in Section 4.3. Obviating the need for the construction of a candidate set, however, is a major contribution of the candidate-set-free algorithm.D-optimal designs for various design problems, including the examples in D-optimal split-plot designs do not always lead to highly efficient designs, even when the designs for the whole-plot factors and the subplot factors are both constructed by using the D-optimality criterion. This is illustrated in Section 6.4.2, where the sequential approaches yield designs that are more than 10% less efficient than the design that is produced by the candidate-set-free algorithm. When the goal is to construct D-optimal split-plot designs, we would therefore not recommend a sequential approach in general.We have computed D-optimality than the algorithm of The candidate-set-free algorithm and the algorithm of The fact that the algorithm of Consider a problem with two whole-plot factors and five subplot factors. Suppose that there are resources for 24 runs to be performed in eight whole plots of three runs each. All the factors have two levels.If the number of runs and the number of runs per whole plot are both powers of 2, then The difficulty with the example under consideration is that the number of runs per whole plot is 3 (not a power of 2). As a result, within each whole plot it is impossible to balance the settings of the subplot factors while maintaining a two-level design. A 24-run Plackett–Burman design with seven columns might serve as a template for solving this problem. One could sort such a design by any two columns to create the desired split-plot structure. However, the resulting covariance matrix of the coefficients will not be diagonal in general. So, the estimates of the coefficients will be correlated unnecessarily.2 factorial design. Within each whole plot the sum of the values of each subplot factor is always either −1 or 1; moreover, the inner product of any pair of columns is 0. So, the design is as balanced as it possibly can be given that each whole plot is only three runs instead of four.η) and η and The information matrix of the design, assuming that the ratio of the whole-plot variance to the error variance , Europlasma (a developer of gas plasma systems), Structuplas  and Techni-Coat International  ran a large experiment to investigate the effect of several additives and a gas plasma surface treatment on the adhesive properties of polypropylene. The experiment was of great interest to car manufacturers who are increasingly using polypropylene because it is inexpensive and light, and because it can be recycled. The experiment was therefore financially supported by Flanders’ Drive, a technological platform that stimulates innovation in the automotive industry in Flanders (the northern part of Belgium) and that itself is supported by the Flemish Government.An undesirable property of polypropylene is that glues and coatings do not adhere well to its surface unless it undergoes a surface treatment, like a gas plasma treatment. The goal of the experiment was to search for economical plasma treatments that lead to good adhesion. For that, several experimental factors related to the plasma treatment: the gas flow rate, the power, the reaction time and the type of gas that was used. Three types of gas were utilized in the experiment: one etching gas and two activation gases. After some pilot tests, the engineer who was in charge of the plasma treatment decided to study gas flow rates between 1000 and 2000 sccm, powers ranging from 500 to 2000 W and reaction times between 2 and 15 min.On the basis of a literature study and the engineers’ experience with applying coatings and glues to various kinds of plastics and with gas plasma treatments, a slack variable statistical model was selected. This choice seemed justified because polypropylene is inactive with respect to adhesion. Also, three of the seven additives have very small ranges so a Scheffé mixture model would force the analyst to deal with severe collinearity. the main effects of the seven additives,the six two-factor interactions involving EPDM and each of the other additives,the main effects of the gas type, the flow rate, the power and the reaction time,all two-factor interactions of these four factors,the quadratic effects of the flow rate, the power and the reaction time, andall two-factor interactions between the seven additives, on the one hand, and the four plasma treatment factors, on the other hand.p, equalled 66.As a result, the number of fixed parameters to be estimated, PolyPropylene Compounds produce 20 different batches or formulations of polypropylene and to use the material in those batches to test the effects of 100 different gas plasma treatments on the adhesion of glues and coatings.For estimating the model, the grant for Europlasma, Structuplas and Techni-Coat International allowed for 100 runs. However, it was impossible for Domo PolyPropylene Compounds to prepare 100 different polypropylene formulations as this was very labour intensive and the minimum batch size was quite large. A reasonable compromise was to have Domo w1,…,w7 in s1,…,s4 in Since each formulation or batch was used for multiple experimental runs, the result is a split-plot design. The seven additives are fixed for each batch, so they are ‘hard-to-change’ or whole-plot factors. They are labelled D-optimality criterion as there were many practical considerations, each of which rendered the use of standard design methods impossible:All the design approaches that are described below use the the presence of the multicomponent constraint,a categorical subplot factor at three levels,a whole-plot size of 20,the interest in all the two-factor interactions involving EPDM,the overall sample size of 100 andthe need to estimate quadratic effects.The experimental design that was actually conducted by the four companies was constructed in a sequential fashion to satisfy the constraint that only 20 different batches of polypropylene could be used. This was done using the SAS procedure.D-optimal completely randomized design with 20 runs for a model involving the main effects of the seven additives and all two-factor interactions involving EPDM, taking into account the multicomponent constraint for the additives talc and mica. The resulting design is given in First, 20 polypropylene formulations were selected by computing a D-optimal design for estimating the full 66-parameter model in the additives and the gas plasma factors. The resulting design is displayed in ki, are displayed in the last column of The 20 polypropylene formulations were combined with an 81-point candidate set for the gas plasma factors to construct a 1620-element candidate set for computing a D-optimal split-plot design with whole-plot sizes equal to 5.The approach that was used in the original experiment might be improved in several ways. First, the design construction method did not allow the researchers to force the whole-plot sizes to be equal. Second, the selection and the arrangement of the design points were not done by taking into account the split-plot correlation structure of the experiment. One way to circumvent these problems is to use the sequential approach that was presented by The approach that was advocated by The sequential approach has several advantages. First, the resulting designs do not depend on prior specification of the variance components of the split-plot model. Second, the construction method does not require the specification of a large candidate set of whole-plot and subplot factor level combinations. This makes the approach attractive for designing the polypropylene experiment which involves seven whole-plot factors and four subplot factors.D-optimality criterion selected the 20 formulations that are displayed in As explained in Section 6.3, the D-optimal 100-point design for the three continuous subplot factors and the categorical factor. The design that was produced by the algorithm of Trinca and Gilmour is displayed in η-value of 1, the relative efficiency of this design relative to the original design in The second building-block that is needed as an input to the algorithm is a 100-run design for the four subplot factors. We selected a Another alternative is to use the candidate-set-free algorithm that was outlined in Section 3. Sequential approaches restrict the solution space, thus making it impossible to find locally optimal designs in general. But, unlike the sequential approaches that were described above, the candidate-set-free algorithm solves the optimization problem for the whole-plot and subplot factors simultaneously. This makes it more likely to find a locally optimal design.D-optimal design that was produced by the candidate-set-free algorithm in the JMP6 software for η=1 is displayed in The η-value of 1, the relative efficiency of the original design in D-optimal design is 91.5%. The relative efficiency of the design that was created by using the D-optimality criterion as well. One could say that, compared with the D-optimal design, using the designs in Using an In addition to the factors that are related to the plasma treatment, the effect of several additives in the polypropylene was studied as well. This was because polypropylene is often compounded with additives such as stabilizers against ultraviolet (UV) light, lubricants, talc, mica and/or colour pigments to tailor the plastic to a specific end use. The engineers from the four companies strongly believed that some of these additives had an effect on the adhesive properties. This was because additives like UV stabilizer affect the surface of the polypropylene, which is exactly where the adhesion should take place. After a long debate, seven additives were included in the experiment: ethylene propylene diene monomer (EPDM) rubber, ethylene copolymer content of the rubber, talc, mica, lubricant, UV stabilizer and ethylene vinyl acetate. The ranges that were utilized for each of those factors are shown in One multicomponent constraint had to be taken into account when designing the experiment: the additives talc and mica were not allowed to have strictly positive levels simultaneously. The reason for this is that talc and mica have similar effects on the properties of the polypropylene and are almost never used together when producing plastics.D-optimal split-plot designs. The algorithm runs in polynomial time with respect to the number of observations and number of factors. This is in contrast with algorithms that are based on a candidate set approach which run in exponential time in the number of factors.We have described a flexible algorithm for finding We have also demonstrated the power of this tool through finding a previously unpublished optimal design. There are two ways in which the tool proposed can save practitioners money. First, the candidate-set-free algorithm provides an opportunity to reduce the sample size compared with the use of fractional factorial designs. Second, it allows the computation of more efficient solutions for challenging practical problems than can be found by using available methods, as in the polypropylene example.I-optimal designs and to multistratum designs is one possibility. Another straightforward extension would be to support Bayesian D-optimal designs as described in a priori model. Finally the need to discretize the range of each co-ordinate could be avoided by using an efficient one-dimensional non-linear optimization technique (see, for example, There are a couple of opportunities for extending this work. An extension of the methodology to


To determine prevalence of genital human papillomavirus (HPV) infection among men in rural China, we analyzed genital swab specimens. Among 2,236 male residents of rural Henan Province, HPV infection prevalence was 17.5%. The most common oncogenic and nononcogenic types were HPV-16 and HPV-3, respectively. Infection was associated with younger age and multiple sex partners. Human papillomavirus (HPV) is an etiologic agent of cervical cancer. Among men, genital HPV infection plays a role in the development of anogenital cancer . We excluded from the study men who were older and reported less risky sexual behavior than those who were included .Of the 40 HPV types we tested for, we detected 36, including 13 oncogenic types. Overall prevalence of HPV infection was 17.5% (95% CI 16%–19%). Oncogenic HPV was detected in 140  specimens, and nononcogenic HPV was detected in 251  specimens. Among 15 HPV-positive specimens that had ambiguous direct sequencing signals and were further cloned and resequenced for genotyping, 3 had a second type and minor types were ignored. Among these infections , the mosPrevalence of infection of any or nononcogenic HPV types decreased significantly with participant’s increasing age . Risk foThis study of genital HPV in a large sample of adult men from rural China addresses the paucity of data on male genital HPV infection in Asia. Prevalence rates for any type of HPV and oncogenic HPV were lower among these men in China than among heterosexual men elsewhere   and 936 older men (because of specimen inadequacy) must be noted. This nonparticipation of younger men might have neutralized the age-related association to some extent. Although circumcision is extremely rare in rural China, the lack of accurate data for this variable is another study limitation, which rendered subgroup analysis by circumcision status impossible.As reported in other studies (Characteristics of 3,172 men from rural Henan Province, by human papillomavirus β-globin status of genital specimens, 2007–2009.


Cervical cancer is a major public health problem in Latin America and the Caribbean (LA&C), showing some of the highest incidence and mortality rates worldwide. Information on HPV type distribution in high-grade cervical lesions (HSIL) and invasive cervical cancer (ICC) is crucial to predict the future impact of HPV16/18 vaccines and screening programmes, and to establish an appropriate post-vaccinal virologic surveillance. The aim was to assess the prevalence of HPV types in HSIL and ICC in studies in LA&C.We performed a systematic review, following the MOOSE guidelines for systematic reviews of observational studies, and the PRISMA statement for reporting systematic reviews and meta-analyses. Inclusion criteria were at least ten cases of HSIL/ICC, and HPV-type elicitation. The search, without language restrictions, was performed in MEDLINE, Cochrane Library, EMBASE, LILACS from inception date to December 2009, proceedings, reference lists and consulting experts. A meta-analysis was performed using arc-sine transformations to stabilize the variance of simple proportions. Seventy-nine studies from 18 countries were identified, including 2446 cases of HSIL and 5540 of ICC. Overall, 46.5% of HSIL cases harbored HPV 16 and 8.9% HPV18; in ICC, 53.2% of cases harbored HPV 16 and13.2% HPV 18. The next five most common types, in decreasing frequency, were HPV 31, 58, 33, 45, and 52.Study's limitations comprise the cross-sectional design of most included studies and their inherent risk of bias, the lack of representativeness, and variations in the HPV type-specific sensitivity of different PCR protocols.This study is the broadest summary of HPV type distribution in HSIL and ICC in LA&C to date. These data are essential for local decision makers regarding HPV screening and vaccination policies. Continued HPV surveillance would be useful, to assess the potential for changing type-specific HPV prevalence in the post-vaccination era in Latin America. Human papillomavirus infection (HPV) is one of the most common sexually transmitted diseases worldwide It is now recognized that virtually all cervical cancers  and their precursor lesions are causally related to cervical infections through at least 14 oncogenic HPV genotypes  We performed a systematic review, following the Meta-analysis Of Observational Studies in Epidemiology (MOOSE) guidelines A search, without language restrictions, was performed on the main international and regional literature databases MEDLINE; EMBASE; CINAHL; NHS R&D Health Technology Assessment Program; ClinicalTrials.gov; LILACS; Cab International Global Health; Pascal Biomed; generic and academic internet search and meta-search engines; and the specialized register of the Cochrane Gynecological Cancer Group from its inception date to December 2009. Databases containing regional proceedings or congress's annals, doctoral theses and experts were also consulted.Appendix S1. An exhaustive strategy module was developed to localize studies from LA&C. According to pre-specified criteria, pairs of authors independently examined the title, abstract, and descriptors of the articles in order to identify potentially relevant studies for full review. The reference lists of the articles finally included were hand-searched for additional information. If data or data subsets of the same population were published in more than one article, only the publication with the largest sample size was selected, after consulting the principal investigator. Discrepancies were resolved by consensus or, finally, by a third author. The full texts of relevant articles retrieved were examined using a pre-designed form.The Medline, LILACS, and EMBASE search strategy is available at the Any descriptive epidemiological study with individual-level data was considered. Participant subjects were women from LA&C countries, in studies of cervical cancer/HSIL associated with HPV. The inclusion criteria were a) to inform at least ten cases of HSIL or ICC, b) confirmed by biopsy, and c) HPV-type elicitation. We excluded those papers that undoubtedly failed to meet the aforementioned inclusion criteria. Studies using both polymerase chain reaction (PCR)-amplified and non-amplified genotyping methods were included. There were no restrictions on PCR primers' utilization. HPV DNA tissue sources included fixed or fresh biopsies and/or exfoliated cells. Outcome measures included global and type-specific HPV prevalence. Two attempts of email contact with the author were made in order to recover missing data.Appendix S2)Two reviewers assessed the methodological quality of studies independently. Discrepancies were solved by consensus of the whole team. Observational studies or control arms of randomized controlled trials were assessed by a checklist of essential items stated in STROBE Pairs of reviewers independently abstracted the following key information: country where the samples were drawn, setting, population, sample size, study design, age, study year, distribution of cases by histological type, type of cervical specimen and PCR primers, type-specific and overall prevalence of HPV infection, reported duration of HPV infection, and quality score. Data on HPV-specific prevalence was extracted independently for squamous cell carcinoma (SCC) and for adeno- and adenosquamous carcinoma. Each study, or regional components of a study, was classified by the following criteria: 1) geographical region  2) income level as defined by the Gross Nation Income (GNI) World Bank Classification , 3) tissue source , and 4) genotyping method , PCR 1 (PCR MY09/11 or Consensus primers), PCR 2  and PCR 3 (PCR MY and GP performed together).HPV prevalence data was expressed as a percentage of all cases tested for HPV. Multiple infections were separated into constituent types, thus type-specific prevalence represents both single and multiple infections. For HPV type-specific prevalence, only studies testing for a particular HPV type contribute to the analysis for that type, and therefore sample size varied between the type-specific analyses. In order to perform a meta-analysis with prevalence data, we first transformed proportions into a quantity (the Freeman-Tukey variant of the arcsine square root transformed proportion) 2 statistic quantifies the heterogeneity between studies. This statistic describes the percentage of the variability in effect estimates that is due to heterogeneity rather than sampling error (chance). One must consider that each HPV type proportion is a pooled estimate of only those studies reporting the particular HPV type. Hence, each proportion has its own denominator and must be considered regardless of the other types. Thus, cumulative point estimates do not sum to 100%. DerSimonian-Laird weights for the random effects model We hypothesized the following possible sources of heterogeneity: age, risk factors of HPV and/or HSIL/cervical cancer, country, geographical region, income level by the Gross National Income (GNI) World Bank Classification, type of cervical lesion, type of tissue source and type of genotyping method used. With the available data we could perform pre-designed subgroup analyses considering the country where the study was carried out, the geographical region, the income level of the country according the Gross National Income (GNI) World Bank Classification, the type of genotyping method and the tissue source. Additionally, we applied a meta-regression analysis in order to further study the possible sources of heterogeneity and to get the adjusted prevalence. Publication bias was unlikely as assessed by funnel plots although this type of bias is unlikely to occur in prevalence studies (data not shown). No ethical approval was required for this study.Checklist S1 and Diagram S1).The present Systematic Review and Meta-analysis met the PRISMA statement requirements  and nine countries from South America (68.2%). One country, was a high-income nation (0.3% of women), six countries were middle-income (72.3%), and eleven countries were low-income (27.4%).Overall, 1452 citations were retrieved from the search strategy. After the assessment Figure 1Appendix S3).We considered 114 sub-studies for the analysis, including seven country-level sub-studies from Bosch 1995 HSIL/ICC cases came mainly from Brazil (23.7%), Argentina (19.0%), and Mexico (17.9%). The HSIL and ICC prevalence, and ICC∶HSIL prevalence ratio by type are presented in The comparison of HPV type-specific prevalence cancer and HSIL cases is illustrated by In the 52 sub-studies included in the HSIL systematic review, 16 were performed in Mexico or Central America and 36 in South America. Overall, a total of 2446 patients' samples were analyzed with a median of 47.5 specimens in each sub-study (range 6 to 130). Most data came from cross-sectional studies (n = 39) while seven came from case-control studies, four from cohort studies/prospective follow up, one from a nested case-control study, one from a before-after study and one from a randomized controlled trial. Mean age of women was 40.4±7.6 years old.2 = 86.4%) of samples, while prevalence of HPV16 was 46.5%  and prevalence of HPV18 was 8.9%   of the analyzed samples.Any HPV in HSIL was found in a pooled proportion of 82.5%  , , 4. M2 = 85.8%). In Brazil (13 studies) the pooled prevalence of HPV16 in HSIL samples was 52.7% . In Mexico (9 studies), the pooled prevalence of HPV16 in HSIL samples was 48.5% .2 81.2%) in Argentina, 9.0%  in Brazil, and 6%  in Mexico. HPV prevalence according to subgroups of geographic region and by GNI World Bank Classification are shown in We found a pooled prevalence of HPV18 in HSIL of 16.9% . Most data came from cross-sectional studies (n = 52) while 10 came from case-control studies, and one nested case-control study. Mean age of women was 41.1±7.0 years old.2 = 94.0%) of the samples, while the prevalence of HPV16 was 53.2%  and the prevalence of HPV18 was 13.2%   of the samples.Any HPV in cervical cancer was found in a pooled proportion of 89.0%  , , 6. M2 = 68.2%), in Brazil (13 studies) 53.2% , and in Mexico (14 studies) 54.9% .2 = 65.7%) in Argentina, 15.8%  in Brazil, and 12.8%  in Mexico. HPV prevalence by the geographic region and by GNI World Bank Classification are shown in  and When we analyzed the prevalence of HPV18 in ICC samples, we found a pooled prevalence of 17.6%  and when MY and GP performed together were used compared to Hybridization techniques and for cancer when the tissue source were Polymerase Chain Reaction SPF, GP5/6, E6, E7 and others compared to Hybridization techniques. However the adjusted prevalence, by the means of each variable and considering the SE of the meta-analysis, remained stable: HPV16 in HSIL women 45.7% (CI 95% 42.9–48.5%) HPV18 in HSIL 8.7% (7.2–10.3%); HPV16 in ICC 55.3% (52.5–58.1%); and HPV18 in ICC 13.4 (11.5–15.3).Funnel plots showed no evidence of publication bias (data not shown).Data on the geographic distribution of HPV type in HSIL and ICC are crucial for estimating the impact of HPV vaccines on cervical cancer and cervical screening programs. This review brings representative estimations of HPV type distribution from the LA&C region. Since multiple HPV genotyping techniques have been included, varying sensitivities of the techniques considered might impact the HPV type-specific prevalence reported In 2003, Smith et al. Data on ICC has greatly enriched previous reports; we increased the number of Latin American cases included from 3,010 considered by the last published meta-analysis Some intra-regional variations of the most common HPV types have been observed, although these apparent differences may happen simply by random fluctuation and/or a lack of sample representativeness of certain countries. For ICC, Mexico, Central America and the Caribbean showed a slightly lower HPV16/18 prevalence than South America (64.2% vs. 67.3% respectively). Particularly, Argentina shows the highest prevalence rate for HPV16/18 in both HSIL (65.4%) and ICC (77.1%). It is interesting to point out that the 12 Argentine studies incorporated samples from women of different provinces of the country, including aboriginal communities ; these cases most likely represent the failed detection of known types  rather than infections of yet-undiscovered types.In this review, multiple-type HPV infections were detected in 16.8% of HSIL and 12.6% of ICC, although the frequency of multiple infections depends largely on the number of HPV types tested for within a given study. The attribution of ICC etiology to HPV types is increasingly complicated by the rising prevalence of multiple co-existing types. It was suggested that infections with multiple HPV types seem to act synergistically in cervical carcinogenesis HPV18 and 16 had the highest ICC∶HSIL prevalence ratio in our studies, as found in Smith et al. meta-analysis As more data is accumulated, it is supportive to observe that HPV16/18 accounts for two-thirds of ICC in LA&C. The proportion of ICC cases potentially averted by the current approved vaccines may be even higher than the aforementioned one if cross-protection against non-vaccine high-risk HPV types (like HPV31 and 45) is found to be clinically effective in reducing the incidence of ICC and HSIL caused by these genotypes. The information given by this work would be also useful in LA&C for the evaluation of polyvalent vaccines (currently in development) for the prevention of ICC associated to more than eight or nine high-risk HPV types.Limitations of our meta-analysis include the cross-sectional design of the included studies and their inherent risk of bias, lack of representativeness, the HPV type-specific prevalence variation and HPV type-specific sensitivity of different PCR protocols Although information on the histological type of ICC was collected, its discrimination was not always clear and the data came mostly from SCC. For this reason we presented only global data of ICC.This study is the broadest summary of HPV type distribution in HSIL and ICC in LA&C to date, and it has included the majority of American countries which have the highest cervical cancer burdens in the region and worldwide. The presented information may be of importance for local decision makers to consider the cervical cancer prevention as a whole, taking into account the relevance of vaccination and updating screening strategies using type-specific high-risk HPV-DNA-based tests. This work comes available at the time some Latin American and Caribbean countries are evaluating the HPV vaccine introduction in their National Vaccination Schedules, in the frame of the Pan American Health Organization purchase using revolving fund, which makes vaccines affordable. Continued surveillance of HPV types in HSIL and ICC as HPV vaccines are introduced would be useful, to assess the potential for changing type-specific HPV prevalence in the post-vaccination era in Latin America.Appendix S1Search Strategy.(DOC)Click here for additional data file.Appendix S2Methodological Quality Assessment.(DOC)Click here for additional data file.Appendix S3Study characteristics and HPV-specific prevalence by study and country.(DOC)Click here for additional data file.Checklist S1PRISMA checklist for reporting systematic reviews and meta-analyses.(DOC)Click here for additional data file.Diagram S1PRISMA study flow diagram for reporting systematic reviews and meta-analyse.(DOC)Click here for additional data file.


Human papillomaviruses (HPVs) are the cause of cervical cancer and possibly a subset of squamous cell carcinomas (SCCs) in other sites. However, the prevalence and distribution of HPV subtypes remain unclear. In the present study, we collected and analyzed 511 paraffin sections of non-cervical SCC from patients in Qingdao, China, for the presence of HPV using polymerase chain reaction (PCR). We identified that 55.77% (285/511) of the samples were positive for HPV infection. There was a significant association between HPV type and the different sites of SCC. An association between HPV-positive cases and tobacco, alcohol, age and tumor differentiation was demonstrated. The information provided by this study may be important for further investigation into the association between HPV and SCC. High-risk HPV subtypes were associated with the malignant degree of SCC. This study provided a theoretical basis for the preventative treatment of non-cervical SCC using HPV vaccines. Human papillomavirus (HPV) infection has been globally reported as being involved in tumors in several types of cancer, including genital mutilation cancer, penis cancer, lung cancer, head and neck tumors, gastric cancer, breast cancer, colon cancer, skin cancer and esophageal cancer. Numerous studies have shown that HPV infection is closely related to squamous cell carcinoma (SCC) and it has been determined as an important factor in the induction of cervical SCC. Preliminary studies have been carried out to determine whether HPV exists in SCCs in non-cervical sites . StudiesThis study aimed to explore the correlation between the different subtypes of HPV and the different sites of non- cervical SCCs in 511 patients from Qingdao, China, using a polymerase chain reaction (PCR) detection method. The high prevalence of HPV6/16 and the lack of HPV18 in esophageal SCC and lung SCC may point to specific virus-tissue interactions.Surgical resection specimens (n=511) were retrieved from Qingdao Hiser Medical Group and Qingdao Center Hospital between 2006 and 2011. All patients belonged to low socioeconomic strata and the majority of these were agricultural workers. None of the cases had been treated with radio- or chemotherapy prior to surgery. The specimens consisted of 27 tongue SCC, 79 nasopharyngeal SCC, 196 lung SCC, 185 esophageal SCC and 24 rectal SCC cases. The specimens for the control group, which were cut from the margins of the tissues in the same cases, were confirmed as non-tumor tissue by pathology. There were 397 specimens from males, while 114 were from females. According to Broders’ classification, the histopathological staging of all SCC cases were as follows: 238 stage I, 234 stage II, 30 stage III and 9 stage IV cases. Their ages ranged from 26–81 years . None of the cases had distant metastasis. All specimens underwent a regular dewaxing process and were cut continuously into 4-μm-thick sections for HPV testing. The histology from all cases was reviewed by two pathologists who confirmed the diagnosis. Discrepant cases were resolved by histological evaluation by a third pathologist.in vitro amplification combined with PCR reverse dot blotting.DNA was extracted from each of the 511 paraffin-embedded specimens using the HPV genotyping test kit according to the manufacturer’s instructions . The kit applied DNA-chip technology based on The kit used special primers to obtain 23 types of HPV amplification products by PCR. It was hybridized with probes, which had 5 low-risk genotypes and 17 high-risk genotypes fixed in the membrane. It determined the HPV genotype by its hybridization signal.Exfoliated SCC cells were collected and added to 50 μl lysate and centrifuged at 13,000 rpm for 10 min after a boiling water bath for 10 min. The supernatant was reserved for template DNA.Template DNA (5 μl) was added to the PCR mix. PCR conditions were as follows: 50°C for 15 min, denaturation at 95°C for 10 min followed by 40 cycles of 94°C for 30 sec, 42°C for 1 min and 30 sec, 72°C for 30 sec and 72°C for 6 min.The PCR product and membrane were incubated for ≥1 h and 30 min in a hybridization incubator and then kept in a boiling water bath for 10 min in 5 ml solution A. The DNA became purified after this step.The membranes were washed in a hybridization incubator for 5 min in solution B at 51°C to remove proteins and other contaminants.2O2. The color liquid was removed and deionized water was added. The HPV genotype was determined by its hybridization signal.After washing the membrane, it was incubated for 30 min in solution A  mixed with POD (Solution A:POD = 2000:1). It was then colored for at least 30 min in color liquid, which was added to 19 ml solution C (1M 100 ml sodium citrate), 1 ml TMB and 10 μl 3% HThe film articles were placed on the reading instrument scanner and the results were saved. The wet film article was placed into an airtight seal in a hermetic sealing bag and stored at 2–8°C in a refrigerator for preservation.Statistical analysis was performed using Fisher’s exact test and Student’s t-test. P<0.05 was considered to indicate a statistically significant difference.Overall, HPV was detected in 285 of 511 (55.77%) SCC tissues compared with 55 of the 511 (10.76%) normal tissues. The HPV-positive distribution for the tissue from each SSC site was higher than for the normal tissue at its corresponding site. The HPV-positive distribution in 137 of 185 (74.05%) esophageal SCCs and 114 of 196 (58.16%) lung SCCs was higher than for the other SCC sites .Five HPV genotypes were identified, including HPV6, 16, 18, 53 and 58. High-risk HPV was composed of HPV16, 18, 53 and 58, whereas low-risk HPV was HPV6 only. Overall, 147 of 285 (51.58%) cases were positive for high-risk HPV, of which 137 (48.07%) were associated with HPV16, 6 (2.11%) with HPV18, 2 (0.70%) with HPV53 and 2 (0.70%) with HPV58. The number of cases with low-risk HPV positivity  was higher compared with other HPV types and the majority of cases had esophageal cancer. HPV6 and HPV16 comprised a large proportion of the HPV-infected cases .Follow-up information was available for 285 HPV-positive patients who were diagnosed with SCC stages I-IV , includiThere were 137 HPV16-infected cases, of which 33 (24.09%) were in stage I, 54 (39.42%) in stage II, 39 (28.47%) in stage III and 11 (8.03%) in stage IV. For HPV6-infected cases, there were 94 (68.12%) in stage I, 31 (22.46%) in stage II, 13 (9.42%) in stage III and 0 (0%) in stage IV. There was 1 (16.67%) case in stage I, 4 (66.67%) in stage II and 1 (16.67%) in stage III for HPV18-infected cases. There were only two cases infected by HPV53, 1 of which was in stage I and the second was in stage II. Only two cases were infected by HPV58 and these were in stage II .Cancer incidence in the Qingdao area has increased rapidly, particularly tongue, nasopharyngeal, lung, esophageal and colorectal cancer. These five types cancers are in the top ten most commonly diagnosed tumors. Of the top ten, squamous cell carcinomas are the most commonly observed types of tumor. Studies investigating the correlation between non-cervical SCC and different subtypes of HPV infection remain limited, despite their increasing incidence in the last 20 years. Studies have reported that HPV-positive tumors have a better response to therapy –5. Howevin situ hybridization. In the noncervical SCC samples, 19.30% (285/511) were positive for HPV detection, while 10.76% (55/285) of the control samples were positive. The presence of HPV infection was significant in noncervical SCC cases (P<0.05). HPVs have been categorized by their genotype into low- and high-risk types, according to the risk of the virus causing SCC of the uterine cervix . The prevalence of HPV6 (75/137) and HPV16 (58/137) is high in esophageal SCC, while for HPV18 (2/137) it is low. The prevalence of HPV6 (48/114) and HPV16 (61/114) is also high in lung SCC, while HPV18 (4/114) is also low. The two subtypes of HPV infection in esophageal and lung SCC had significant differences when compared with other sites of SCC (P<0.05). The high prevalence of HPV6/16 and the lack of HPV18 in esophageal and lung SCC may point to specific virus-tissue interactions.This study demonstrated that the high-risk HPV subtype was the most important factor associated with the malignant degree of SCC. The study provided a theoretical basis for the preventative treatment of non-cervical SCC using HPV vaccines. Further study is required to determine the effect of HPV on survival in patients when observed in combination with other prognostic factors.


CIP1/WAF1tumor suppressor gene, and identify a p21 locus and TSG101 domains that mediate this interaction. TSG101 deficiency in Saos-2 human osteosarcoma cells was accompanied by an increased abundance of p21 mRNA and protein and the retardation of cell proliferation. A cis-acting element in the p21 promoter that interacts with TSG101 and is required for promoter repression was located using chromatin immunoprecipitation (ChIP) analysis and p21-driven luciferase reporter gene expression, respectively. Additional analysis of TSG101 deletion mutants lacking specific domains established the role of the central TSG101 domains in binding to the p21 promoter and demonstrated the additional essentiality of the TSG101 C-terminal steadiness box (SB) in the repression of p21 promoter activity. Neither binding of TSG101 to the p21 promoter nor repression of this promoter required the TSG101 N-terminal UEV domain, which mediates the ubiquitin-recognition functions of TSG101 and its actions as a member of ESCRT endocytic trafficking complexes, indicating that regulation of the p21 promoter by TSG101 is independent of its role in such trafficking.TSG101 (tumor susceptibility gene 101) is a multi-domain protein known to act in the cell nucleus, cytoplasm, and periplasmic membrane. Remarkably, TSG101, whose location within cells varies with the stage of the cell cycle, affects biological events as diverse as cell growth and proliferation, gene expression, cytokinesis, and endosomal trafficking. The functions of TSG101 additionally are recruited for viral and microvesicle budding and for intracellular survival of invading bacteria. Here we report that the TSG101 protein also interacts with and down-regulates the promoter of the p21 TSG101 (Tumor susceptibility gene 101) was identified initially in mouse cells in a genetic screen for loci that modulate neoplastic transformation of NIH3T3 fibroblasts and tumor formation in nude mice TSG101 expression and adventitious overexpression of the gene were both found to produce a neoplastic phenotype TSG101 is lethal during early stages of embryogenesis More than 15 years of study has revealed that TSG101 affects multiple cellular functions beyond growth and proliferation; these include gene expression, cytokinesis, and endosomal trafficking The 46 KDa TSG101 protein contains a domain that is characteristic of ubiquitin conjugases but lacks a site necessary for it to function as an agent of ubiquitination; additional distinct domains in the protein are the proline-rich (PR), coiled-coil, and steadiness box (SB) regions TSG101 also functions as a transcriptional regulator to suppress hormone-receptor-mediated transactivation of targeted genes, which requires interaction of nuclear hormone receptors with the centrally-located PR and coiled-coil domains of TSG101 TSG101 to alter cellular functions TSG101 expression at the pre-translational level.An elevated cellular abundance of TSG101 protein normally is prevented post-translationally, at least in part through autoregulation by the C-terminal SB domain CIP1/WAF1, which negatively regulates cell cycling At least some of the effects of TSG101 on cell proliferation have been attributed its actions on the cyclin-dependent kinase inhibitor p21 TSG101 open reading frame from pLLEXP-Tsg*-Flag A DNA fragment spanning the human p21 promoter from -3227 to the transcription start site +1 was PCR-amplified and cloned into pGL2 (Promega) to create the (-3227/+1) p21-Luc reporter. (-2325/+1) p21-Luc was similarly generated and described previously -/--/-) supplemented with 10% fetal bovine serum. Transfections were carried out using Lipofectamine 2000 (Invitrogen) as per the manufacturer.Saos-2 and U2OS cells were obtained from the American Type Culture Collection. HCT116 and the isogenic cell line HCT116 p21Stable TSG101 knockdown cell lines were established in Saos-2 cells constitutively expressing TSG101 shRNA. A construct with shRNA targeting the 413-433 coding sequence of the gene and a pGFP-IRES-Puro plasmid were introduced into Saos-2 cells by co-transformation and colonies exhibiting both puromycin resistance and GFP green fluorescence were isolated, cloned, and assayed for TSG101 expression. Two clones showed decreased TSG101 abundance to ∼50% of the level observed in parental cells.Western blot analysis was performed as described T (ΔΔCT) program. PCR primer set (p21 qPCR-5′ and p21 qPCR-3′) was used for p21, and (β-actin qPCR-5′ and β-actin qPCR-3′) was used for β-actin, which served as an internal control. Oligonucleotide sequences are listed in As described 2, 1 mM DTT, 0.1% NP-40 and protease inhibitor cocktail). Cell extracts (60 µl) were then mixed with 250 µl luciferase assay buffer , and 100 µl of 0.5 mM luciferin (Promega). Luciferase activity was measured using an AutoLumat LB953 luminometer (Berthold) and normalized to β-galactosidase activity. Statistical analysis of data was performed by two-tailed unpaired Student's t test. A P value of <0.05 was considered significant.Saos-2-2 cells were co-transfected with p21 reporter constructs and empty pcDNA-SRα/HA vector or the TSG101 expression construct pcDNA-SRα/HA-TSG101. A plasmid expressing β-galactosidase  was included as an internal control. 24 hr post transfection, cells were lysed using the freeze-thawing method in 100 µl of lysis buffer  p21-Luc and empty vector or a TSG101 expression construct, and then treated with 0.5 µM TSA or 100 µM RG108. TSA and RG108 stock solutions were dissolved in ethanol.st, IP buffer; 2nd, IP buffer containing 500 mM NaCl; 3rd, 10 mM Tris-HCl, 0.25 M LiCl, 0.5% NP-40, 1 mM EDTA and 0.5% sodium deoxycholate; and 4th, 1x TE buffer. Precipitated DNA fragments were eluted from beads after RNase treatment, followed by proteinase K digestion in the presence of SDS (0.25%) and de-crosslinking by incubation at 65°C. Following phenol/chloroform extraction, DNA was resuspended in 50 µl TE buffer. 5 µl of the DNA solution was PCR-amplified using p21 promoter-specific primer sets  at a ratio of 100 mg cells per 800 µl buffer. The sample was incubated on ice for 5 min to lyse cells after addition of Triton X-100 (Cf = 0.1%). Following centrifugation, the nuclear pellet was collected and the supernatant saved as the cytoplasmic fraction. The nuclear pellet was further rinsed in ice-cold PBS, re-suspended in RIPA buffer supplemented with 1 mM Na3VO4, 1 mM DTT, 1 mM PMSF and a protease inhibitor cocktail (Sigma), and sonicated. After centrifugation , the supernatant was saved as the nuclear fraction.Subcellular fractionation was performed as described -/- cells were transfected with siRNA using LipofectAMINE 2000. To inhibit TSG101expression, 60 nM of TSG101-specific siRNA (5′-CCUCCAGUCUUCUCUCGUCTT-3′ and 5′-GACGAGAGAAGACUGGAGGTT-3′) synthesized by DHARMACON was used. Transfection of an equivalent amount of annealed double-stranded oligonucleotides (5′-UUCUCCGAACGUGUCACGUTT-3′ and 5′-ACGUGACACGUUCGGAGAATT-3′), which do not target any gene, served as a negative control.HCT116 and HCT116 p21-/-cells were seeded at a density of 5×104 in triplicate in 6-well plates. Cells were collected, stained with trypan blue, and counted by a hemocytometer every 24 hr for 3 days. Saos-2 cells and the TSG101-knockdown line Saos-2-2 were transfected with either pcDNA-SRα/HA vector or pcDNA-SRα/HA-TSG101. 36 hours later, cells were seeded at a density of 8×104. Cell counting was performed as described above for 4 days. Statistical significance of the results was analyzed by Student's t test.Following transfection with TSG101-specific or control siRNA, HCT116 or HCT116 p21TSG101 mRNA reduced the abundance of TSG101 protein by approximately 50% as determined by Western blotting (data not shown). During microarray analysis of the effects of such reduction on global gene expression  in which shRNA directed against pression , we obsepression  showed apression . LuciferTo identify the p21 promoter region required for TSG101-mediated regulation, a series of deletions in the p21 promoter was constructed, and luciferase expression from these constructs was measured in Saos-2-2 cells that overexpressed TSG101 from pcDNA-SRα/HA-TSG101. As shown in TSG101-/- mouse embryos and tumor cells treated with siRNA directed against TSG101 have been reported to show reduction of the rate of cell growth -/- cells). A population of HCT116 cells transiently transfected with siRNA directed against TSG101 showed a reduced rate of cell growth relative to control HCT116 cells, concurrent with an increase in p21 expression as assayed by Western blot analysis. In contrast, HCT116 p21-/- cells lacking the p21 gene showed no effects of TSG101 knockdown on cell proliferation (Fibroblasts isolated from feration . ConsistChromatin immunoprecipitation (ChIP) assays showed that sequences of the p21 promoter region interact directly with the TSG101 protein and identified the TSG101-binding sequence in Saos-2 as the region amplified by primer set “d” . TSG101 The possibility that TSG101 repression of the p21 promoter is affected by DNA methylation or histone acetylation, both of which can modulate the functions of a TSG101-co-repressor DMAP1 in a cell cycle dependent manner was tested by evaluating TSG101 mediated repression of the p21 promoter in the presence of the DNA methyltransferase inhibitor RG108 TSG01 transcripts lacking segments present in full-length TSG101 mRNA have been reported to accumulate in multiple types of human cancer TSG101 transcripts on p21-regulated cell proliferation, we constructed a collection of truncated variants of TSG101 (Variant f TSG101 . ChIP asExtension of the ΔUEV deletion through the proline-rich domain (CC.SB) did not affect binding  and PROMO .Multiple transcription activator/repressor proteins can modulate p21 gene expression; sites in the p21 promoter that interact with these proteins have been extensively characterized -/-, we have validated that at least the p21 gene is required for the defect of cell growth by TSG101 deficiency. In the stable line Saos-2-2, cell apoptosis does not substantially increase compared to its parental cells  are direct regulators of cell cycle progression and G1/S transition al cells ; therefoWhile multiple TSG101-deficient clones were obtained during our isolation of the Saos-2-2 stable line, none of these clones showed a reduction in TSG101 greater than 60%, suggesting that a baseline level of TSG101 is needed for cell growth and/or survival. This finding is consistent with earlier evidence that total elimination of TSG101 function is a lethal event TSG101 transcript variations have been detected in multiple types of human cancer, including carcinomas of the liver, lung, breast, cervix, and ovary Aberrant splicing and production of +/+ and isogenic HCT116 p21-/- cells, we’ve shown that proliferation defects associated with TSG101-deficiency are mediated at least in part by the modulation of p21 gene expression. Our findings thus indicate that TSG101 regulation of p21 is an important factor in the cellular function of TSG101, as has been suggested also by results showing that altered TSG101 expression in ovarian cancer may affect disease prognosis by modulating p21 expression Our results demonstrate an inverse correlation between TSG101 and p21 protein levels. Using HCT116 p21Figure S1Luciferase reporter activity in p21 promoter-less construct is negligible and such low activity is not modulated by cellular TSG101 levels. pGL2-basic, a reporter plasmid lacking the p21 promoter sequence, was transfected into Saos-2 and Saos-2-2 cells for luciferase reporter assay. pGL2-p21 (-3227/+1), in which the firefly luciferase gene is driven by the p21 promoter, was included as a positive control. Following β-gal normalization, the reporter activity of each sample is illustrated as Relative Luminescence Unit (RLU). The data presented are the average of three independent experiments .(TIF)Click here for additional data file.Figure S2p21 repression by TSG101 is maintained in the presence of Trichostatin A or RG108. (A) p21 promoter activity in Saos-2-2 cells with or without ectopic TSG101 expression was assessed in the presence or absence of the histone deacetylase inhibitor Trichostatin A (TSA). p21 promoter activity of the mock sample transfected with the empty vector was set as 1. Relative promoter activity of the other samples is shown. (B) Relative p21 promoter activity was examined as described in (A), except the DNA methyltransferase inhibitor RG108 was used. The data presented are the average of three independent experiments .(TIF)Click here for additional data file.Figure S3Saos-2-2 does not show a substantial increase of cell apoptosis compared to its parental cells. (A) Saos-2 and Saos-2-2 were analyzed for cell apoptosis by TUNEL assay. Saos-2 treated with Adriamycin , a DNA-damaging reagent, also were included as a positive control. Scale bars, 100 µm. (B) TSG101, Caspase-3, and Poly ADP-ribose polymerase (PARP) were examined in indicated cells by Western blot analyses. Activated Caspase-3 (17 KDa) and the cleavage form of PARP (85 KDa) both serve as index for the activation of apoptotic signal pathways.(TIF)Click here for additional data file.Table S1Oligonucleotide primers used in Chromatin Immunoprecipitation (ChIP) and quantitative RT-PCR (qPCR).(PDF)Click here for additional data file.Table S2List of genes that are statistically significant up-regulated or down-regulated in Saos-2-2 compared with its parental cells. Microarray data were deposited in GEO with an accession number GSE50808 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE50808), and analyzed by a software package “Limma” at http://bioconductor.org/packages/2.12/bioc/html/limma. html. Genes up-regulated or down-regulated more than 4-fold  are shown.(PDF)Click here for additional data file.


Squamous cell carcinoma of conjunctiva has increased tenfold in the era of HIV/AIDS. The disease pattern has also changed in Africa, affecting young persons, with peak age-specific incidence of 30-39 years, similar to that of Kaposi sarcoma, a well known HIV/AIDS defining neoplasm. In addition, the disease has assumed more aggressive clinical course. The contributing role of exposure to high risk HPV in the development of SCCC is still emerging.The present study aimed to investigate if immunohistochemical expressions of EGFR, pEGFR and p16, could predict infection with high risk HPV in HIV-related SCCC.FFPE tissue blocks of fifty-eight cases diagnosed on hematoxylin and eosin with SCCC between 2005-2011, and subsequently confirmed from medical records to be HIV positive at the department of human pathology, UoN/KNH, were used for the study. Immunohistochemistry was performed to assess the expressions of p16INK4A, EGFR and pEGFR. This was followed with semi-nested PCR based detection and sequencing of HPV genotypes. The sequences were compared with the GenBank database, and data analyzed for significant statistical correlations using SPSS 16.0. Ethical approval to conduct the study was obtained from KNH-ERC.Out of the fifty-eight cases of SCCC analyzed, twenty-nine (50%) had well differentiated (grade 1), twenty one (36.2%) moderately differentiated (grade 2) while eight (13.8%) had poorly differentiated (grade 3) tumours. Immunohistochemistry assay was done in all the fifty eight studied cases, of which thirty nine cases (67.2%) were positive for p16INK4A staining, forty eight cases (82.8%) for EGFR and fifty one cases (87.9%) showed positivity for p-EGFR. HPV DNA was detected in 4 out of 40 SCCC cases (10%) in which PCR was performed, with HPV16 being the only HPV sub-type detected. Significant statistical association was found between HPV detection and p16INK4  and EGFR  expressions, but not pEGFR. In addition, the expressions of these biomarkers did not show any significant association with tumor grades.This study points to an association of high risk HPV with over expressions of p16INK4A and EGFR proteins in AIDS-associated SCCC. These range from benign dysplasias, to carcinoma arcinoma ,2. It isarcinoma ,4. The earcinoma .In Africa, however, the incidence of SCCC has risen rapidly in the recent past, with the peak age-specific incidence reported to be 30-39 years, similar to that of HIV/AIDS pandemics in the tropics. The disease is also more aggressive, with a mean history of three months at presentation ,5. An asAlthough the natural history of the SCCC appears to be unique in this region of the world with etiologic mechanism unclear and therapeutic options limited, immunosuppression from HIV has been thought to facilitate the activity of other infective agents that induce the carcinoma. This has been supported by many studies that have documented the presence of high risk HPV genotypes 16 and 18 DNA in a proportion of SCCC . The oncINK4A[INK4A was found to be reduced in HIV-related squamous cell carcinoma of the uterine cervix, a correlation which has also been supported by other studies [Another cell cycle regulatory protein whose increased expression is reported to be predictive of, and an independent prognostic marker in high risk HPV infection is p16INK4A-22. Howe studies ,24.INKA4, EGFR and its phosphorylated form pEGFR have been proposed to reflect infection with high risk HPV in squamous cell carcinoma of conjunctiva. However, HPV detection in conjunctival neoplasm has been largely controversial, and the pathogenic mechanism not well elucidated. Some of these studies have reported a heterogeneous prevalence of high-risk HPV genotypes, suggesting that only a subset of cases can be attributed to these viruses. This variation in the reported HPV infection rates in conjunctival squamous cell carcinoma could result from differences in detection methods and the studied populations [Few authors have also proposed involvement of high risk HPVs in the oncogenesis through altered expression of other key molecules involved in tyrosine kinase pathways, in which inverse expressions of epidermal growth factor receptor (EGFR) and its phosphorylated form (pEGFR) have been reported to contribute to the pathogenesis of HIV/AIDS–associated SCCC, and correlates with poor prognosis -27. Altoulations -31.INK4A, EGFR and pEGFR; and the potential value of use of these biomarkers as indicators of HPV positivity in selected cases of HIV/AIDS-related SCCC.The present study therefore compared PCR-based detection of HPV with immunohistochemical expressions of p16in situ. Tumor site, tumor size and tumor grades were as shown in Table Out of the 58 samples evaluated, the ratio of females to males was 1:1. The age ranged from 23 to 73 years, with a mean of 41.6 years and median of 39 years. Fifty six cases were classified as squamous cell carcinomas and the remaining two as carcinoma INK4A staining, forty eight cases (82.8%) for EGFR and fifty one cases (87.9%) showed positivity for p-EGFR  were positive for p16% were poINK4A, EGFR and pEGFR expressions were semi-quantitatively scored for both intensity and proportion of staining in the cell nucleus, cytoplasm and membrane as reported previously [The patterns of p16eviously ,33. InteFrom the forty cases where DNA was extracted, four cases (10%) showed positivity for HPV 16 genotype, and no other genotypes were detected. Although this is in tandem with other studies that have documented detection ranges from 8-10% of high risk HPV in head and neck squamous cell carcinomas, this low rate of detection suggests that other mechanisms could underlie pathogenesis of HIV/AIDS-related squamous cell carcinoma of conjunctiva independently from the active role of high risk HPV.INK4 expressions, p = 0.000, at 99% confidence interval and EGFR expression, p = 0.028, at 95% C.I, but not with pEGFR.For the cases in which high risk HPV were positive, statistical significant association was found between HPV detection and p16A causal relationship between the epidemic of HIV/AIDS in the Sub-Saharan region and increased incidence of squamous cell carcinoma of conjunctiva (SCCC) has been hypothesized, with the prevalence of the neoplasm reported in Kenya alone to be 8% ,35. Althin-situ hybridization, detection of serum antibodies directed against HPV epitopes, to immunohistochemical detection of surrogate biomarkers such as p16INK4A, a cyclin dependent kinase inhibitor protein [Detection of high risk HPV associated with squamous cell carcinoma has, however, been controversial, with different authors reporting varied percentages of positivity and prevalence -42. This protein . In our INK4A protein over-expression may serve as a surrogate biomarker for biologically and clinically relevant high risk HPV infection in squamous cell carcinoma of the conjunctiva, as well as squamous cell carcinoma and glandular epithelial dysplasia of the uterine cervix [INK4A has also been shown to correlate with the degree and behavioral characteristics of both cervical as well as head and neck neoplasm [A number of studies have therefore demonstrated that p16e cervix ,45. Besineoplasm ,47.INK4A as potential surrogate marker of HPV-derived squamous cell carcinomas, the development of SCCC in HIV positive patients has been documented to involve activation of epidermal growth factor receptor (EGFR) and its phosphorylated form (p-EGFR) [In addition to over expression of p16(p-EGFR) -50. The (p-EGFR) . HoweverINKA, EGFR and p-EGFR IHC as markers of HPV status and utilized HPV PCR in a subset of cases to correlate the results. Immunohistochemistry was performed in all the fifty eight studied cases, and semi-quantitative scores of the bio-markers enumerated. However, 18 cases were excluded from HPV detection by PCR technique, followed with sequencing, because tumor tissue was either inadequate or had extensive DNA degradation due to poor tissue processing. A prevalence of 10% of high risk HPV 16 genotype detection was realized, which is consistent with the findings of other studies. However, the low prevalence of HPV could be associated with poor tissue processing.Several studies have discussed about the optimal method for determining HPV status on FFPE tumor sections , and in the current study, we evaluated immunohistochemical expressions of p16INK4A expression in the epithelium was characterized by variable, weak to strong, diffuse nuclear and cytoplasmic staining. Thirty nine cases (67.2%) were positive for p16INK4A staining and showed significant statistical correlation between HPV positivity and p16INK4A expression ). This was in agreement with similar findings from other authors who demonstrated that p16INK4A expression was significantly associated with the presence of HPV-16 [In this study p16f HPV-16 .The expression of EGFR and its activated form, p-EGFR, were also independently enumerated in all of the fifty-eight cases, from which the results demonstrated that 82.8% of the cases expressed EGFR while 87.9% expressed p-EGFR as brown membranous and/or cytoplasmic immunostaining, similar to the findings from other studies .Once EGFR is activated into p-EGFR, it undergoes internalization, resulting in a marked decrease in the non-activated membrane-bound EGFR . For corINK4A and EGFR but not p-EGFR as clinically useful surrogate biomarkers for high risk HPV infection in HIV/AIDS-associated squamous cell carcinoma of conjunctiva.Therefore, both immunohistochemical assay and PCR-based techniques showed close agreement in the use of p16INK4A and EGFR over expressions in HIV/AIDS-associated squamous cell carcinoma of conjunctiva.Although the exact role played by high risk human papillomavirus in carcinogenesis is not well known, to the best of our knowledge, this is the first study to show an association of high risk HPV infection with p16hematoxylin and eosin to confirm the previous diagnoses and establish the tumor grades according to the WHO guidelines. Ethical approval to conduct the study was obtained from KNH-ERC.Fifty-eight HIV positive SCCC formalin fixed paraffin embedded (FFPE) tissue blocks were retrieved from the archives at pathology department, UON/KNH for analysis. The criteria for inclusion as HIV positive case included information from the clinician in the patient’s histological request form indicating on follow-up at the comprehensive care centre, on HAART treatment, ARV treatment, immunnosuppression, p24 marker reactive, retrovirus disease and HIV positive. Sections (4 μm) from the tissue blocks were stained with et al. [3) (for p16INK4A) or citrate buffer, pH 6.0 (for EGFR and pEGFR) and subsequently heated in a microwave at 750 W for 20 minutes, with 5 minutes intervals. All the slides were then allowed to cool for 20 minutes, and then rinsed with phosphate buffered saline (PBS) wash buffer. The sections were quenched in 3% hydrogen peroxide (H2O2) containing 15 mmol/l sodium azide (NaN3) for 10 minutes to block endogenous peroxidase activity, followed with a rinse in PBS wash buffer. Ten microlitres of ready to use primary monoclonal mouse anti-human antibodies, each of p16INK4A, EGFR , pEGFR  were applied, and incubated for 30 minutes at room temperature, and according to manufacturers’ instructions  for p6INK4A orDNA was extracted from 20 sections (5 μm) of the FFPE tissue blocks using digestion buffer, 50 mM Tris-pH 8.5, 1 mM EDTA, pH 8.0 and 0.5% Tween 20 (Sigma) and proteinase-K (Roche) at a final concentration of 500 μg/ml. The lysates were purified using EZ1 DNA Tissue Kit (Qiagen) and 1 μl of DNA were used in a reaction to verify DNA integrity, using beta globin and as described previously in other studies -59. DNA 2 (25 mM) 1 μl of either DNA or water. The cycling conditions were as follows: 10 minutes at 95°C activation step for Taq DNA polymerase, followed by 40 cycles each for 40 seconds at 95°C, 50 seconds at 55°C and 40 seconds at 72°C. The last cycle was followed by a final extension step of 10 minutes at 72°C. The PCR products were analyzed by electrophoresis on 2% agarose gel stained with ethidium bromide using the protocol described by Gleissner et al. [For the detection of human papillomavirus (HPV) and the genotypes, PCR was performed, followed with DNA sequencing on ABI PRISM 310 Genetic Analyzer. The sequences compared with the GenBank database. In brief, the PCR reaction was performed in a final volume of 50 μl. Each PCR mixture contained 25 μl of AmpliTaq Gold PCR Master Mix 2X , 30 pmol of primers (A1 5'- TTGGATCCATGTTAATWSAGCCWCCAAAATT –3' A2 5'- TTGGATCCTTATCAWATGCCCAYTGTACCAT –3'), 2 μl of MgClr et al. .et al.[Positive samples were typed using cut and purified DNA bands from agarose gel. The DNA were sequenced with an ABI PRISM 310 Genetic Analyzer , with the Big Dye Terminator V1.1 cycle sequencing kit  using primer (A1). Sequences were then compared with the GenBank database as described by Sgueglia et al..p < 0.05 at 95% CI was considered statistically significant.Data was then analysed for significant statistical co-relations using SPSS Version 16. One-tailed The authors declare that they have no competing interests.Contributions: AM, JN, WO, MK, LP and NO, performed the experiments; ER and JN designed the overall study; WM, coordinated the work; RS, ER and LL contributed their expertise in the field of pathology; AM and JN wrote the work. All authors read and approved the final manuscript.