The distribution of cancers of the lung and stomach in the counties of England and Wales in 1968-81 was mapped, and compared to the distribution in the country in 1921-30 described by Stocks. The high risk of stomach cancer in North Wales noted by Stocks was found still to exist in each sex, although its disparity from the rest of the country has diminished. In general the geographic distribution of stomach cancer in both periods has paralleled that of post-neonatal mortality, at the same time and earlier, as an index of general poverty, but postneonatal mortality in North Wales has not been exceptionally high. In 1921-30 the highest risk of lung cancer was in and around London. In the modern data this was still true for older women, but for men and women under 45 years of age, and to a lesser extent for older men, the pattern has changed greatly; the epidemic has moved north, and highest risk is now in Northumberland and Durham. This spread appears to have occurred earlier for men than for women, and for urban than for rural areas, occurring latest of all for women in rural areas. Regional disparity has also increased, especially in males: risks in the northern regions are now over twice those in much of Wales and the South.


A review of Pap smear diagnoses from a reference laboratory in Grand Forks, North Dakota over a 3-year period (07/00 to 10/03) revealed a two-fold higher rate of high grade squamous intraepithelial lesion in a community in northwest Minnesota  than in northeast North Dakota , in spite of both having similar rates of low-grade squamous intraepithelial lesion (1.33% vs.1.30% respectively)To identify the different types of HPV present in patient populations showing high-grade dysplasia in Grand Forks, ND and Roseau, MN.Formaldehyde-fixed paraffin-embedded cervical tissue samples were analyzed using polymerase chain reaction (PCR) to detect the presence of HPV type 16, 18 and 31.Our studies showed that 41 % of samples from Roseau were triply infected with HPV serotypes 16, 18 and 31 in comparison to 12 % from Grand Forks.Due to the small sample size we were unable to prove the study to be statistically significant. However, our results suggest that the presence of HPV 16, 18 and 31 in triply infected samples may be the cause of the higher percentage of high-grade dysplasia in Roseau, MN when compared to Grand Forks, ND. Human Papillomavirus (HPV), a member of the papovavirus family, is a small circular double stranded DNA virus with a genome of approximately 8 Kb. HPV causes the most common sexually transmitted disease (STD) in the U.S. with at least 5.5 million new infections each year and an actively infected population of approximately 20 million people . There aDuring an analysis of the severity of cervical dysplasia in patients attending clinics in Grand Forks, ND and Roseau, MN, we observed that the number of patients with high-grade dysplasia was approximately twice as high in Roseau compared to Grand Forks  in spite of similar rates of low-grade dysplasia [1.304% and 1.332% respectively] in both these areas. Grand Forks, ND and Roseau, MN are geographically related areas separated by approximately 100 miles. Since none of the typical risk factors including age of 18–28, pregnancy, smoking, high school diploma or less, use of oral contraceptive pills, or presence of coexisting STD (including condylomata acuminata) correlated with the increased incidence of high grade dysplasia, we hypothesized that the increased incidence might be a result of differences in the high-risk HPV types responsible for the infections. The aim of this study was to use polymerase chain reaction (PCR) to identify HPV types 16, 18 and 31 present in patient populations showing high-grade dysplasia in Grand Forks, ND and Roseau, MN.Archival paraffin-embedded, formalin-fixed cervical tissue samples from patients diagnosed with high-grade dysplasia were obtained from Altru Clinic, Roseau, MN and Altru Clinic, Grand Forks, ND over a three year period from 07/00 – 10/03. Grand Forks represented the control group, while Roseau, MN represented the experimental group. Statistical significance was analyzed by Chi square test and confirmed by z test using Sigma Stat software.2, 3 μl of 10X PCR Gold Buffer, 200 μM deoxynucleoside triphosphates, 50 pmol of each primer (IDT Oligos) and 0.5 μl AmpliTaq Gold Polymerase . The amplification conditions were set to 1 min of denaturation at 95°C, 2 min of annealing at 40°C and 1.5 min of extension at 72°C for 40 cycles. HeLa and CaSki cells were used as positive controls for HPV 18 and HPV 16 respectively. The presence of an appropriately sized amplification product was monitored by gel electrophoresis and ethidium bromide staining.DNA from formaldehyde-fixed paraffin-embedded tissues was extracted using the thermal cycler deparaffinization method as previously described  with minOut of the total thirty-four high-grade cervical dysplasia tissues analyzed by PCR with the general primers (GP 5+/GP 6+) targeting a 155 base pair fragment of the L1 open reading frame of HPV, twenty-eight tested positive. Samples from four normal patients, which were used as negative controls, did not show any evidence of HPV infection. Since the general primers have previously been reported to be less sensitive than specific primers in screening for certain high-risk HPV types , all of Of the 17 cases studied from Roseau – experimental group, 14 samples (82%) were positive for the general primers  declare that they have no competing interests.LB conducted the HPV analyses and wrote the manuscript. RC helped in the HPV PCR analyses. TW provided the archival tissue samples. MB was instrumental in getting the project started and doing some background research. BM coordinated the research efforts at UND and edited the manuscript. All co-authors read and approved the final manuscript.


Vz did not produce any gross maternal or neonatal toxicity, nor did it reduce the anogenital distance in male pups. We observed no effects of Vz on play behavior on PND22. However, the 12-mg/kg Vz dose significantly increased play behavior in the male offspring on PND34 compared with controls. The most dramatic increases were seen with the nape contact and pounce behavior components of play. The Vz effect was more pronounced in male than in female offspring. As adults, male offspring showed a significant reduction of erections at all dose levels during the ex copula penile reflex tests. The 12-mg/kg dose was also associated with an increase in seminal emissions. These effects demonstrate that perinatal Vz disrupts the development of androgen-mediated behavioral functions at exposure levels that do not produce obvious structural changes or weight reductions in androgen-sensitive reproductive organs.In this study we examined the effects of exposure to the antiandrogenic fungicide vinclozolin (Vz) on the development of two sex-differentiated behaviors that are organized by the perinatal actions of androgens. Pregnant Long-Evans rats were administered a daily oral dose of 0, 1.5, 3, 6, or 12 mg/kg Vz from the 14th day of gestation through postnatal day (PND)3. The social play behavior of juvenile offspring was examined on PND22 and again on PND34 during play sessions with a same-sex littermate. After they reached adulthood, the male offspring were examined with the  Sclerotinis sclerotiorum (white mold) and Botrytis cinerea (gray mold) (Fungicides are applied to many foods to control plant diseases such as ay mold) . After aay mold) . Consumeay mold) , and comay mold) . Fungiciay mold) .The dicarboximide fungicide vinclozolin (Vz) is used in a number of commercial formulations to treat fruits and vegetables such as lettuce, snap beans, canola, and grapes . Vz beloTo date, most investigations have focused on the impact of the environmental anti-androgens on the development of androgen-sensitive male reproductive organs. For example, adult male rats have reduced anogenital distances (AGDs), reduced seminal vesicle and ventral prostate weights, and lower epididymal sperm counts after perinatal exposure to Vz . Vz is nOne of the questions examined in the present study was whether much lower levels of Vz during the perinatal period affect reproduction via a disruption of male copulatory behavior. An androgen-sensitive neuro-muscular system that is critical for normal male copulatory behavior is the levator ani and bulbocavernosus (BC) skeletal muscles and their motor neuron control centers in the lumbar spinal cord [the spinal nucleus of the BC (SNB)]. In rats, contraction of the levator ani and BC muscles, as well as vascular mechanisms, produces penile erections . The sexp,p′-dichlorodiphenyldichloroethylene , have already been shown to reduce erectile functions in rats  were allowed to acclimate to the University of Southern Maine Vivarium quarters for 2 weeks before breeding. All rats were fed standard pellet chow  Groups of three females were housed with stud males, and vaginal smears were examined each morning for the presence of sperm. We regarded a sperm-positive smear as gestation day (GD)0. Pregnant rat dams were placed individually into polycarbonate shoebox cages and assigned to an exposure condition according to a randomized block design. Each block consisted of five groups: 0, 1.5, 3, 6, or 12 mg Vz/kg maternal body weight. Vz  was dissolved in corn oil, and the appropriate volume (~ 0.5–1.5 mL) was administered via gavage from GD14 through PND3 to coincide with the period of sexual differentiation in the rat . Vz was We recorded maternal body weights daily during the gestational period. Cages were inspected each morning and afternoon for the presence of litters. Litter size, sex distribution, pup weights, and AGDs were recorded on PND1 and every 4 days thereafter. Using a randomized procedure, litters were culled to eight offspring on PND4, maintaining equivalent sex distributions when possible. After weaning on PND21, offspring were housed with same-sex littermates in plastic cages with filter bonnets. All animal procedures complied with approved institutional animal care protocols and in accordance with National Institutes of Health guidelines . Animal The exposure and rearing procedure yielded a total of 51 viable litters . From thWe randomly selected two male and two female pups from each litter. Within each same-sex pair, one animal was designated as the “target” and the other animal served as partner. Before data collection, target and partner animals were marked with a nontoxic marker for identification; they were then separated from their littermates. Twenty-four hours later, the target animal and their same-sex partner were placed together for 10 min in a glass aquarium (12 in. wide ×24 in. long × 12 in. high) with clean cage bedding. We filmed their interactions under dim red light with a Canon XL1s digital video camera  interfaced to an iMac computer running iMovie software . All testing was conducted during the middle of the dark phase of the light/dark cycle. No other animals were present in the room during filming.We examined play behavior on PND22 and again on PND34 in the same animals. The assessment ages were chosen in order to examine play immediately after weaning on PND21 and immediately before the decline in same-sex play in male rats, which begins during PND36–40 .A pair of trained observers later viewed the films using QuickTime software. (Apple Computer Inc.) Observers tabulated the frequency and distribution of the following five behaviors: nape attack ; pounce ; pin ; wrestle ; and mount .In rats, reflexive penile erections and movements can be observed if the penile sheath is retracted with light pressure directed at the base of the penis . Penile We conducted all erection tests during the middle of the dark phase. Tests lasted for 20 min after the first response or for 15 min in the absence of responses. During each test, animals were restrained in a supine position with their head and upper torso positioned in a darkened, ventilated tube (8.5 × 5.5 × 20 cm) fastened to a plastic base. The darkened tube is anxiolytic, and rats rapidly habituate to brief periods of restraint. The penile sheath was retracted and held in place . TypicalTrained observers recorded the frequency and time distribution of three gradations of erections: E1, reddening and distension of glans; E2, tumescence of the base and tip of glans; and E3, intense erection accompanied by cupping of the tip of glans . Penile p < 0.05 statistically significant.For the play procedure, the five behaviors were summed and analyzed as total play behaviors per session with repeated-measures analysis of variance (ANOVA). The individual behaviors were also analyzed separately. The litter always served as the statistical unit of analysis, with the exposure level as a between-litter factor and sex and PND as within-litter factors. In cases where there was a significant main effect or interaction involving the exposure factor, Duncan’s probe tests were used to make pairwise comparisons. We considered For the penile reflex procedure, we tested each animal on 2 consecutive days, and the data from the two sessions were averaged before analysis. If an animal was inactive during one of the sessions, that session was dropped. If an animal was inactive during both sessions, that animal was dropped from the analysis. Only 2 of 47 animals were inactive during both sessions. The averaged behavior was analyzed with one-way ANOVA according to exposure level.10) or 1% (ED01) decrement in behavior. BMDS also provided a 95% lower bound that can be divided by a standard uncertainty factor to calculate a reference dose or generate a margin of exposure.Because the penile erection data showed a clear dose–response relationship with evidence that even the lowest dose of Vz disrupted the behavior, we further examined the data with Benchmark Dose Modeling Software . BMDS is a useful alternative to the no observed adverse effect level (NOAEL) approach because it uses the entire dose–response relationship and does not involve extrapolations far below experimental observations. We used the BMDS continuous model to calculate benchmark doses that represented the model-estimated control mean, minus proportional deviations equivalent to a 10% (EDp = 0.367).We found no evidence of gross maternal or neonatal toxicity , nor werF = 7.72, p = 0.01]. We also examined the total number of behaviors separately for PND22 and PND34. There was a significant effect of sex on PND22  and a significant effect of exposure on PND34 . Probe tests revealed that the male 12-mg/kg and 6-mg/kg Vz groups produced significantly more play behaviors than the did controls on PND34  = 5.51, p = 0.03]. We also examined the number of nape contacts separately for PND22 and PND34. As with the total play behavior variable, there was a significant main effect of sex on PND22  and a significant main effect of exposure on PND34 . Probe tests indicated that the male 12-mg/kg Vz group produced significantly more nape contacts than did the 0- and 3-mg/kg groups on PND34  = 6.44, p = 0.02]. Data were averaged across sex and age, and probe tests indicated that the 12-mg/kg group pounced more than did controls  = 4.62, p < 0.01; F = 10.07, p < 0.01] as well as the number of reflex clusters per session [F = 3.23, p = 0.02; F = 7.37, p < 0.01; ex copula procedure.We found a significant exposure-related decline in total erections per session [10 benchmark for erections was 1.23 mg/kg with a 95% lower bound of 0.84 mg/kg  administered to rat pups on PND2 and PND3 reduced play behavior , this stThe play behavior procedure used in the present study was more sensitive to low-dose effects than those used in previous investigations, possibly because of methodologic differences. In the present study we examined nape contact, pounce, pin, and wrestle, as well as mount behaviors, whereas previous studies examined only pin  or pin aIn the present study we also examined play behavior at two different time points, an approach that detected the apparently transient or age-specific effect of perinatal Vz. In rats, the ontogeny of play is characterized by an inverted U-shaped function that peaks between PND32 and PND40 . Male beFinally, it might be the case that we found significant effects at lower doses in the present study because the offspring were exposed during gestation and the neonatal period via maternal dosing with the gavage procedure. Although many play behavior studies have focused on the role of androgens during the neonatal period, perhaps because of the ease of working with newborn versus fetal rats, the available evidence suggests that the critical period for the differentiation of play begins late in gestation and continues through PND10 . Data onA survey of the developmental toxicology literature indicates that the reduction of erections measured in the present study is one of the most sensitive outcomes observed to date in a perinatal Vz study. Earlier work found that reduced AGD in male neonates and nipple retention occurred after exposure to maternal doses as low as 3.125 mg/kg, whereas at least 50 mg/kg was required to affect ventral prostate weight and increase the incidence of hypospadias . AlthougThe Vz-exposed males showed a selective reduction of low-intensity (E1) erections. In this regard, the exposed offspring resemble males castrated as adults, which also show an early reduction of E1 erections . The behAs mentioned above, animal studies indicate that fetal males are far more sensitive to environmental antiandrogens than adults. Results from maternal stress studies shed some light on the likely developmental mechanisms affected by environmental antiandrogens. Maternal stress during the last week of pregnancy lowers the surge of plasma testosterone that is normally present in male rat fetuses during GD18 and GD19 . AttenuaNo previous studies explicitly link Vz to human erectile dysfunction. However, Vz and other antiandrogenic fungicides are used in agriculture, they may be responsible for the recently noted link between pesticide exposure and erectile dysfunction in otherwise healthy men. Specifically, pesticide-exposed men had a significantly higher incidence of complete impotence, showing little to no change from baseline flaccidity on measures of penile rigidity, tumescence, frequency, and duration . Occupat10 benchmark doses associated with altered juvenile play behavior and erectile dysfunction in our animal model. Typically, the U.S. EPA would divide the 95% lower bound by 100 to calculate a reference dose. If this practice were applied to the juvenile play behavior or erectile data, the average daily intake of Vz would exceed the reference doses based on these data by more than two orders of magnitude. It should also be pointed out that humans are exposed to multiple compounds on a chronic basis, whereas this study examined only Vz, which was administered during a limited period of development. The cumulative effects of chronic exposure to multiple compounds and their metabolites are unknown. Lastly, our benchmark doses should be interpreted as conservative estimates because they are based on maternal doses. The actual amount of Vz and/or metabolite that entered our fetal or neonatal subjects is unknown, although the level was certainly lower than the applied maternal dose.It is estimated that children 1–6 years of age are exposed to 0.167 mg Vz/kg body weight/day . Given tIn conclusion, the results of this study demonstrate that the effects of perinatal exposure to an environmental endocrine disruptor can be observed throughout the life span, provided that age-appropriate, sex-specific end points are examined. Low doses of Vz administered during the GD14–PND3 period significantly increased social play behavior in juvenile male rat offspring. These results are interesting in light of recent findings in humans that higher prenatal levels of PCBs have been associated with less masculinized play behavior in Dutch schoolboys and more masculinized play in schoolgirls . Even loThe AGD data for control females were incorrect in


Candidate human papillomavirus (HPV) vaccines have demonstrated almost 90%-100% efficacy in preventing persistent, type-specific HPV infection over 18 mo in clinical trials. If these vaccines go on to demonstrate prevention of precancerous lesions in phase III clinical trials, they will be licensed for public use in the near future. How these vaccines will be used in countries with national cervical cancer screening programmes is an important question.We developed a transmission model of HPV 16 infection and progression to cervical cancer and calibrated it to Finnish HPV 16 seroprevalence over time. The model was used to estimate the transmission probability of the virus, to look at the effect of changes in patterns of sexual behaviour and smoking on age-specific trends in cancer incidence, and to explore the impact of HPV 16 vaccination. We estimated a high per-partnership transmission probability of HPV 16, of 0.6. The modelling analyses showed that changes in sexual behaviour and smoking accounted, in part, for the increase seen in cervical cancer incidence in 35- to 39-y-old women from 1990 to 1999. At both low (10% in opportunistic immunisation) and high  coverage of the adolescent population, vaccinating women and men had little benefit over vaccinating women alone. We estimate that vaccinating 90% of young women before sexual debut has the potential to decrease HPV type-specific  cervical cancer incidence by 91%. If older women are more likely to have persistent infections and progress to cancer, then vaccination with a duration of protection of less than 15 y could result in an older susceptible cohort and no decrease in cancer incidence. While vaccination has the potential to significantly reduce type-specific cancer incidence, its combination with screening further improves cancer prevention.HPV vaccination has the potential to significantly decrease HPV type-specific cervical cancer incidence. High vaccine coverage of women alone, sustained over many decades, with a long duration of vaccine-conferred protection, would have the greatest impact on type-specific cancer incidence. This level of coverage could be achieved through national coordinated programmes, with surveillance to detect cancers caused by nonvaccine oncogenic HPV types. Using a mathematical model of transmission of HPV 16 infection and progression to cervical cancer, Ruanne Barnabas and colleagues show that HPV vaccination could significantly decrease HPV type-specific cervical cancer incidence. Human papillomavirus (HPV) is a common sexually transmitted infection and has been shown in epidemiological and molecular studies to be a necessary aetiologic agent for cervical cancer –3. At lIn Finland, rates of invasive cervical cancer (ICC) incidence in women have changed significantly over the last four decades, with overall rates decreasing from 14.1 cases per 100,000 women in 1960 to 3.8 per 100,000 in 2000 , comprehDespite this overall decline, unfortunately there has been a recent trebling of the rate among Finnish women aged 30–39 y , from 2.In considering the factors responsible for the changes seen in HPV and cervical cancer epidemiology, viral factors (increased virulence and predominance of high-risk types), host factors (changes in behaviour and immunity), and changes in screening and diagnosis were considered. There have been no changes in organised screening or diagnostics as such. Surveys in 1971, 1992, and 1999 showed changes in reported sexual behaviour among women in Finland; between 1971 and 1999 the average lifetime number of sexual partners for women increased from 2.6 to 7.7, and the average age of sexual debut for women decreased from 18.9 to 16.6 y  17]. I. I17]. IMathematical models provide a framework within which we can explore patterns of risk behaviour, HPV infection, and progression to cervical cancer –22. HerThe candidate HPV virus-like particle vaccines are promising. A randomized, double-blind, phase II trial of an HPV 16 vaccine found that, over a median of 17 mo, the vaccine had an estimated efficacy of 100%  and 91%  in preventing persistent and any HPV 16 infection, respectively . A randoA prophylactic vaccine may eventually reduce the incidence of cervical cancer, but in countries with well-established and effective cervical cancer screening programmes, questions about how the vaccine will be used need to be addressed. In this paper we quantify the effects of vaccinating men and women compared to vaccinating women only, vaccination before and after sexual debut, immunisation through national programmes versus opportunistic vaccination, duration of vaccine-conferred protection, and vaccination combined with screening programmes.A compartmental deterministic model of HPV 16 infection and progression to cervical cancer was developed building on a previous model through the addition of type-specific immunity .Figure The model population was stratified according to age (0 y to 84 y) in 5-y cohorts  and sexual activity class , defined according to the rate of sexual partner change . With seA simple screening and treatment scenario was considered that is based on the reported age-specific percentage of eligible women screened through the national Finnish screening programme see. The scrHPV progression and regression rates converted to transition probabilities  were basAn important epidemiological parameter is the risk of transmission when an infectious individual is in contact with a susceptible sexual partner. To estimate the value of this transmission probability (defined here per partnership), we varied its value between its possible limits of 0 and 1 and compared predicted age-specific seroprevalence of HPV 16 with those observed over time . BecauseThe reported sexual partner numbers from the sexual behaviour surveys may be underestimates if those with extreme behaviours are not included in the small sample due to social desirability biases . TherefoThe estimate of seroprevalence as a function of age and time included the effects of observed changes in risk behaviour. Additionally, the changing pattern of incidence of ICC was compared with model predictions. To compare the modelled ICC associated with HPV 16 to the overall rates reported to the Finnish Cancer Registry, we assumed that HPV 16 accounted for 56% of ICC incidence in the data , a valueThe model based on 1992 behaviours (with a doubling of rates of partner change) and current screening programme (every 5 y between 30 and 60 y of age ) was use2 distribution on one degree of freedom when calculating the 95% confidence interval for β. However, this confidence interval is not reliable, because only one parameter in the model was varied, and its estimate relies on other parameters in the model being accurate. Clearly, the uncertainty in estimates of sexual behaviour along with other potential errors in estimating assumed parameter values and model structure result in great uncertainty in our estimate of the transmission probability per partnership. For example, an assumed overestimate of HPV serology sensitivity would lead to an underestimate of the transmission probability, and vice-versa for underestimated sensitivity.Using reported behaviours to estimate the partner change rate, we found that the maximum likelihood value of the transmission probability is its theoretical maximum of 1, which generates a lower prevalence than that observed. Statistically, this estimate is imposed by the constraint and suggests that the estimated prevalence was too high, the rates of sexual partner change was too low, or acquired immunity might not give lifelong protection. Assuming that survey data will underestimate sensitive sexual behaviour, a maximum likelihood transmission probability (β) of 0.6 per sexual partnership was derived, assuming a doubling of sexual partner change rates, and 0.4 if annual partner change rates are equated with lifetime reported partners. The likelihood profiles for these estimates are shown inThe modelled and observed incidence of cervical cancer amongst 35- to 39-y-olds is compared inTo explore the impact of vaccination, we assume an HPV 16 transmission probability per sexual partnership of 0.6, with sexual behaviour based on reports in 1992 and double the reported new sexual partners per year, and sexual debut at 16.6 y for women and at 17.7 y for men. The vaccine was assumed 100% efficacious with a lifelong duration.The equilibrium reduction in HPV 16-associated cancer incidence is derived for a range of ages of vaccination with 90% coverage B. AssumiThe duration of vaccine-conferred protection has an important impact on ICC incidence that depends on what is assumed about the relationship between age and the likelihood of an infection becoming persistent C. If we Hughes and colleagues have previously estimated the per-partnership transmission probability of HPV to be 0.8 . Our estFurther, caution is required because our assumption of lifelong acquired protection is important. If protection is lost, infected individuals would move back into the susceptible class, and the epidemiology of HPV 16 would be very different, with a high observed prevalence and seroprevalence possible with a much lower basic reproductive number and transmission probability. This would greatly reduce the vaccine efficacy and coverage required for elimination with a vaccine that did generate immune protection. It is also important to note that our estimate is per sexual partnership, not per sex act. The transmission probability per partnership is obviously a function of the transmission probability per act and the number of sex acts, but to estimate the transmission probability per act requires either more detailed sexual behaviour or epidemiological data. While we did not explicitly explore the effect of condom use on our estimation for the transmission probability, a meta-analysis of 20 studies suggested that, while condoms may not prevent HPV infection, they may protect against genital warts, cervical intraepithelial neoplasia 2 or 3 and ICC . Also, uWe explored whether the observed increase in cervical cancer incidence among Finnish women aged 35–39 y, from 3.7 cases per 100,000 women in 1985 to 7.7 per 100,000 in 1999 , could bAlso, it is notable in this context that a new HPV type (HPV 45) is present in 19% of the Finnish ICC cases appearing in fertile-aged women, i.e., especially those women who have experienced the increase of ICC incidence since early 1990′s . It is possible that an increase in HPV 45 cancers accounted for some of the increase seen in the overall incidence for cervical cancer among women aged 35–39 y.Including trends in tobacco smoking and an associated increase in progression to ICC improved the model prediction prior to 1979. However, since the percentage of women smoking between 1978 and 1995 was consistently approximately 20%, smoking trends do not explain changing incidence between 1985 and 1999. A more careful exploration of age-specific patterns of tobacco smoking, to look at cohort effects, may be warranted.Oral contraceptive use and parity are aetiological cofactors that may also account for some of the cervical cancer incidence increase , whereasScreening was incorporated into the model by representing the same mass-screening coverage and efficacy rates reported in 1999 over time starting in 1950, because screening coverage has not changed in the last 40 y. In 1999 the Finnish national screening programme invited 80% of 30-y-old women to screening, screened 60% of those invited—that is, only 49% of those eligible . Among oThe impact of vaccination in our model is comparable with findings from previous modelling exercises ,50–52,In developed countries with low cervical cancer incidence, vaccine coverage, in terms of both the target population and oncogenic HPV types, needs to be high in order to sustain the low ICC incidence associated with effective screening. Continued screening programmes have the potential to detect precancerous lesions in those not vaccinated and lesions associated with nonvaccine HPV types. The most effective strategy is vaccination combined with current screening protocols, compared to both screening alone and vaccination alone. However, cytological screening is costly, and in choosing a cervical cancer prevention strategy, health economic assessments of available options are warranted.Dataset S1(189 KB DOC)Click here for additional data file.Figure S1The peak incidence for low-grade squamous intraepithelial lesions (LSIL) in the model occurs at age 25 y for women, and for HSILs at age 40. It is challenging to compare this model output to observed data because screening, as implemented in the model using a simple strategy of preventing progression from HSIL to cervical cancer, does not change the predicted incidence of premalignant lesions. However, the cytological Pap smear diagnostic test, with varying sensitivity and specificity  and low (92 KB DOC)Click here for additional data file.Figure S2The model underestimates HPV 16 seroprevalence, but captures the trend over time.(88 KB DOC)Click here for additional data file.Figure S3HPV 16 ICC incidence is shown for the model prediction before the introduction of screening and with national screening and changes in sexual behaviour and smoking compared to the observed cancer incidence for Finland, 1999–2003 [(88 KB DOC)Click here for additional data file.Figure S4Maximum log likelihood estimation for different patterns of sexual behaviour  is shown(88 KB DOC)Click here for additional data file.Figure S5The observed incidence is compared to the model prediction. In this case, the model changes in sexual behaviour occurred when they were reported (1992), with the increase in model incidence lagging behind the observed increase in incidence. Changes in patterns of behaviour probably occurred before they were detected in the 1992 survey. (89 KB DOC)Click here for additional data file.Protocol S1(145 KB DOC)Click here for additional data file.Table S1(104 KB DOC)Click here for additional data file.Table S2The demographic rates were taken from the Statistics Finland Web site .(93 KB DOC)Click here for additional data file.Table S3(84 KB DOC)Click here for additional data file.Table S4Statistics are from National Public Health Institute, Finland .(79 KB DOC)Click here for additional data file.Human papillomavirus (HPV) is the name of a group of viruses that includes more than 100 different strains or types; more than 30 of these viruses are sexually transmitted. Persistent infection with high-risk types of HPV is the most important risk factor for cervical cancer; the development of cervical cancer always is preceded by infection with one of these viruses, although the opposite is not always true; infection can occur without it leading to cancer. Nonetheless, as the infection is necessary for cancer to develop, vaccination that prevents HPV infection could potentially also prevent cervical cancer. Several such vaccines have already been developed and tested.HPV 16 is the most common high-risk type, accounting for more than half (56%) of all cervical cancers. Trials with vaccines against this virus suggest that they would be very effective—almost 100%—at preventing infection. The authors wanted to look at how vaccination might fit into current programmes against cervical cancer.The authors looked at survey findings in Finland of how common HPV 16 infection was over a ten-year period, and developed a mathematical way of looking at the possible effect of HPV 16 vaccination on the development of cervical cancer. They showed that changes in sexual behaviour and smoking accounted, in part, for the increase seen in cervical cancer incidence in 35- to 39-year-old women from 1990 to 1999. They estimated that vaccinating women and men had little benefit over vaccinating women alone, and that vaccinating 90% of young women before they became sexually active could decrease HPV type-specific  cervical cancer incidence by 91%.Models such as this one will be important for those planning vaccination campaigns with vaccines against HPV. Although the results are specific to the Finnish population they are derived from, the method of modeling has more general applicability and could be used to estimate the effect of vaccination and other factors on the incidence of HPV-associated cervical cancer in other settings.Medline Plus has a page of links to HPV information:http://www.nlm.nih.gov/medlineplus/hpv.htmlMedline Plus also has links to information on cervical cancer:http://www.nlm.nih.gov/medlineplus/cervicalcancer.htmlThe CDC also has information on HPV:http://www.cdc.gov/std/HPV


The loss of perfluorocarbon (PFC) vapour in the expired gases during partial liquid ventilation should be minimized both to prevent perfluorocarbon vapour entering the atmosphere and to re-use the recovered PFC liquid.Using a substantially modified design of our previously described condenser, we aimed to determine how much perfluorocarbon liquid could be recovered from gases containing PFC and water vapour, at concentrations found during partial liquid ventilation, and to determine if the amount recovered differed with background flow rate (at flow rates suitable for use in neonates).The expiratory line of a standard ventilator circuit set-up was mimicked, with the addition of two condensers. Perfluorocarbon (30 mL of FC-77) and water vapour, at concentrations found during partial liquid ventilation, were passed through the circuit at a number of flow rates and the percentage recovery of the liquids measured.-1 . Amounts of perfluorocarbon vapour recovered were 47%, 50%, 81% and 91% at flow rates of 10, 5, 2 and 1 Lmin-1, respectively.From 14.2 mL (47%) to 27.3 mL (91%) of the infused 30 mL of FC-77 was recovered at the flow rates studied. Significantly higher FC-77 recovery was obtained at lower flow rates . As a percentage of the theoretical maximum recovery, 64 to 95% of the FC-77 was recovered. Statistically significantly less FC-77 was recovered at 5 LminUsing two condensers in series 47% to 91% of perfluorocarbon liquid can be recovered, from gases containing perfluorocarbon and water vapour, at concentrations found during partial liquid ventilation. Partial liquid ventilation involves filling the lungs to functional residual capacity with a liquid, in which oxygen and carbon dioxide are soluble, while continuing ventilation with a conventional gas ventilator.Perfluorocarbon (PFC) liquids seem to be physiologically ideal for liquid ventilation, yet they have two major drawbacks. The first is environmental and the second major concern is cost. All PFCs are photochemically stable, have a high global warming potential  and are We have previously shown that up to 74% of PFC can be recovered from saturated vapour using a simple chilled condenser . HoweverUsing a substantially modified design of our previously described condenser, we aimed to see how much PFC liquid could be recovered from gases containing PFC and water vapour, at concentrations found clinically during partial liquid ventilation, and to determine if the amount recovered differed with background flow rate (at flow rates suitable for use in neonates).The experimental apparatus is shown in Figure -1, 2 Lmin-1, 5 Lmin-1 and 10 Lmin-1. For each test 30 mL of FC-77 (vapour pressure = 9.99 kPa at 37°C) [To produce the required gas mixture a humidifier  was set to give a chamber outlet temperature of 39°C and an inspiratory line outlet temperature of 37°C, as is commonly used in clinical practice. A self-filling chamber  and conventional heated inspiratory circuit  were used. A sponge  was placed in the outlet of the humidifier chamber as an FC-77 evaporator. Air was delivered to the chamber at flows of 1 Lmin2.The air, water vapour and FC-77 vapour from the chamber passed through two series-connected condensers. The first condenser was designed to remove water vapour from the gas mixture, in order to prevent the formation of ice in the second condenser. The first condenser consisted of two coaxial tubes . The outer coaxial tube was cooled to 1°C  and the gas mixture passed through the inner tube with a total condensing surface of 0.063 m2.The second condenser was designed to remove PFC from the gas mixture. It consisted of a 1 m long stainless steel tube  with an outer jacket of 20 mm PVC tubing cooled to -30°C  and the gas mixture passed through the inner tube with a condensing surface of 0.041 mAt the conclusion of each experiment the condensers were warmed to ~2°C to ensure any water ice had melted, the volumes of water and FC-77 in each trap were measured and the total volumes calculated. The percentage of theoretical maximum recovery was calculated (see below). The humidifier chamber was visually inspected for the presence of any residual FC-77. The system was flushed with dry air for at least 60 minutes between experiments.-1 at 37°C and 5.2 mgL-1 at 1°C [-1, 2 Lmin-1, 5 Lmin-1 and 10 Lmin-1 respectively.At all flow rates studied the gas stream would be saturated with water vapour which has a density of 44 mgL-1 at 37°C, 0.28 gL-1 at 1°C and 0.04 gL-1 at -30°C [-1, 0.45 gL-1, 0.18 gL-1 and 0.09 gL-1 at 1 Lmin-1, 2 Lmin-1, 5 Lmin-1 and 10 Lmin-1 respectively. The condensers cannot reduce the vapour density below the saturation vapour density at the condenser temperature, dictating the maximum amount of FC-77 that can theoretically be condensed. The maximum possible FC-77 recovery was 28.7 mL, 27.3 mL, 23.3 mL and 16.5 mL at 1 Lmin-1, 2 Lmin-1, 5 Lmin-1 and 10 Lmin-1 respectively. Statistical comparisons were made using ANOVA with Bonferroni's multiple comparison test.The saturated vapour density of FC-77 is 1.6 gLat -30°C . In thesNo unevaporated FC-77 remained in the humidifier chamber at any flow rate. From 14.2 mL (47%) to 27.3 mL (91%) of the infused 30 mL of FC-77 was recovered at the flow rates studied, and from 4 to 26 mL of water was recovered  and significantly higher water recovery was obtained at 1 Lmin-1 (p < 0.01).As a percentage of the theoretical maximum recovery, mean recoveries were 64 to 95% for FC-77 and 88 to 140% for water Figure . Statist-3 and is not miscible with water [For economic and environmental reasons it is desirable to minimise the loss of PFCs during partial and total liquid ventilation. Some of the properties of PFCs that make them ideal for liquid ventilation also make recovery and re-use possible. FC-77 has a density of 1.78 gcmth water  and thusPerfluorooctyl bromide ,9 loss rThe amount of vapour passing into the exhalation line during partial liquid ventilation depends primarily on the surface area of the PFC-air interface and the temperature. Hence, the concentration in the expiratory line depends on the amount of PFC and the gas flow rate. The vapour pressure (perfluorooctyl bromide (1.47 kPa at 37°C)  and FC-7Using a humidified circuit, we studied the recovery of FC-77 with a loss rate of 30 ml/hr, corresponding to the loss rate expected for an approximately 3.5 kg infant . A previTwo series-connected condensers were employed, the first at 1°C to remove water (which would solidify in the second condenser) and the second at -30°C to condense the FC-77.-1 may be attributed to the carry over of a few drops of particulate water, the humidifier over heating slightly at this low flow rate, or inaccuracies in the flowmeter.Water is readily condensed at 1°C and over 88% of the theoretical maximum , was recA small amount of water (up to 3 mL) was found in the second (-30°C) condenser, particularly at higher flow rates. This water forms ice in the second condenser and may block the condenser. In research or clinical practice it would be necessary to have two condensers which could be used alternatively to allow any water ice to melt. Condenser switching would be automated based on an increase in differential pressure across the condenser. Additional safety 'blow-off' valves would also used to prevent excessive circuit pressures in the event of condenser blockage.-1, 86% of the theoretical maximum was recovered. The drop in recovery at 5 and 10 Lmin-1 can be attributed to either reduced efficiency of the second condenser at these high flow rates or a probable change from laminar to turbulent flow. Various strategies could be employed to improve the efficiency of the second condenser at the higher flow rates: increasing the length of the condenser would increase the surface area; packing the condenser with stainless steel wool would cause turbulence in the flow, as well as increasing the surface area for condensation.In absolute terms, 47% to 91% of the evaporated PFC was recovered. Over 65% of the theoretical maximum amount of PFC was recovered at all flow rates studied. At the commonly used flow rate of 10 Lmin-1, compared with a theoretical maximum recovery of 55% of the infused volume with a condenser temperature of -30°C [Further cooling of the second condenser would also increase the recovery. Cooling to dry ice temperature (-78°C) would increase the theoretical maximum recovery of FC-77 to >99% of the infused volume at a flow rate of 10 LminUsing a complicated rebreathing system, Schhrader et al obtained a 90% recovery rate for perfluorooctyl bromide in animal studies utilizing partial liquid ventilation . SpecialThis study represents a substantial advancement in the development of a perfluorocarbon vapour recovery circuit. We have been able to overcome the problems of expired gases not fully saturated with PFC vapour and yet fully saturated with water vapour. We have demonstrated this at flow rates that are used in neonatal ventilation. We have further refined the design of the expiratory circuit with a small surface area, which would not significantly alter conventional ventilator function, that achieves PFC recovery similar to other methods. For the first time, recovery of PFC from expired gases that are not saturated with PFC, and saturated with water, have been demonstrated. Further studies will be necessary to determine the PFC recovery that can be achieved with flow rates and ventilator circuits suitable for paediatric and adult ventilation. Whilst recovery would be less at higher flow rates in bigger patients some recovery would still be possible, and some is better than none. Also the increase in size of the patients would make modifications possible to improve condensation.Using two series connected condensers 47% to 91%, of PFC liquid (FC-77) can be recovered from clinical relevant gas mixtures without altering the function of the expiratory line of a ventilator circuit. Further development of the system could allow re-use of the PFC recovered during partial liquid ventilation; thus curtailing loss of PFC to atmosphere and the potential high costs of partial liquid ventilation.PFC – perfluorocarbonANOVA – analysis of varianceThe author(s) declare that they have no competing interests.KRD conceived the study, constructed the apparatus, carried out the experiments, performed the statistical analysis and drafted the manuscript. MWD conceived the study, participated in its design and coordination, assisted in the statistical analysis and revised the manuscript. JFF assisted in data analysis and interpretation, and revised the manuscript. All authors read and approved the final manuscript.


EHP 115:941–946; Davis et al.][.According to demographic data compiled by the United Nations, an average of 105 boys are born for every 100 girls. The male proportion of births, equal to 0.515, varies slightly between years and populations, but these factors do not fully explain consistently shifting ratios in several industrialized countries over recent decades. A new study examines birth and fetal death sex ratios in Japan and the United States and reveals significant male-to-female shifts in both nations The research team calculated birth and fetal death sex ratios in Japan based on 1949–1999 data from the Japanese Vital Statistics Bureau. The proportion of male births varied yearly before 1970 but declined steadily since then, from 0.5172 to 0.5135. Between 1960 and 1999, the male proportion of fetal deaths increased from 56% to 67.7%. The male fetal death rate is approximately four times higher in Japan than in the United States.For U.S. calculations, the researchers drew 1983–1995 fetal death data and 1970–2002 birth data from the National Center for Health Statistics. The proportion of male births dropped in the United States, from 0.5134 in 1970 to 0.5117 in 2002. There are significant racial differences, however: between 1970 and 2002 the proportion of non-Hispanic white male births fell from 0.5143 to 0.5122, whereas the proportion of black male births rose slightly from 0.5076 to 0.5079. The male proportion of black fetal deaths also increased, rising from 53.5% to 54.5%; among whites, the male proportion of fetal deaths rose by less than 0.5%.Why birth sex ratios differ so much between white and black women is unknown, but hormonal differences due to race and to incidence of obesity may be involved. A possible explanation for the increased ratio among black births may stem from improved prenatal and obstetric care in general, reducing the overall number of fetal deaths.SRY gene on the Y chromosome.The researchers speculate that parental exposures to endocrine-disrupting chemicals, including metalloestrogens such as methylmercury, might be factors undermining the conception and survival of male children. They suggest particular scrutiny of Japanese body burden of mercury and other metalloestrogens to understand this difference. Additionally, future investigations of declining sex ratios should consider the types and timing of prenatal and parental exposures to endocrine-disrupting chemicals. The researchers hypothesize that paternal exposures prior to conception might affect expression of the


Fetal loss is a painful experience. A history of second or early third trimester fetal loss, after painless dilatation of the cervix, prolapse or rupture of the membranes, and expulsion of a live fetus despite minimal uterine activity, is characteristic for cervical insufficiency. In such cases the risk of recurrence is high, and a policy of prophylactic cerclage may be safer than one of serial cervical length measurements followed by cerclage, tocolysis and bed rest in case of cervical shortening or dilatation. In low risk cases, however, prophylactic cerclage is not useful. There is a need for more basic knowledge of cervical ripening, objective assessment of cervical visco-elastic properties, and randomized controlled trials of technical aspects of cervical cerclage (e.g. suturing technique). In the absence of a second- or early third-trimester fetal loss, it is incorrect to use the term cervical insufficiency in connection with a short or traumatized cervix alone. High risk of cervical insufficiency may result from developmental abnormality , previous surgery (amputation or exconization), and laceration by previous transvaginal cerclage or cervical rupture.Classic cervical insufficiency is a diagnosis, based on an obstetric history of activity . In the Observational studies show that in classical cases with a severely traumatized or virtually absent cervix, neonatal survival may be up to 93% after effective cerclage as compared to 27% before the cerclage . That raOne may wonder why no large-scale randomized controlled trials have been performed to definitively prove the effectiveness of cervical cerclage, while there is such an obvious need for those studies. One reason could be that patients at high risk of yet another fetal loss are unwilling to give their consent to randomization after being informed that observational studies have shown approximately 90% infant viability after cerclage and a low rate of procedure related complications . That maThe aim of this report is to determine if, from a clinical-mechanistic point of view, there is reason to question either the concept of cervical insufficiency or the efficacy of cerclage treatment in selected high-risk cases.The physiological changes of cervical tissue remodeling, called ripening, as recently reviewed, are complex and incompletely understood . What isFor obvious reasons, both women and doctors are unwilling to wait till the diagnosis of classic cervical insufficiency has been established by recurrence of fetal loss. In women considered to be at high risk for cervical incompetence, the recurrence risk of fetal loss without cerclage is not exactly known, due to lack of properly designed studies. Uncontrolled studies suggest that infant viability is about 25% without cerclage, whereas it is 75–90% after cerclage . For thaOnce the conclusion has been reached that cervical cerclage is likely to benefit the patient at high or medium-high risk of cervical insufficiency, the question arises how and when to perform it.Figure Not only the position relative to the cervical canal, but also the position relative to the cervical tissue itself might affect the strength of the cerclage. Figure Based on timing, one may differentiate between 1. prophylactic cerclage prior to conception, 2. prophylactic cerclage in pregnancy, 3. urgent cerclage after shortening of the cervix, and 4. emergency cerclage after exposure of the membranes. In high-risk patients, prophylactic cerclage has the advantage that one will not be surprised by yet another fetal loss or sudden shortening or opening of the cervix that requires urgent or emergency cerclage. An additional possible advantage of prophylactic cerclage could be that it may serve as an early warning system, as any serious force on it is likely to induce pain or blood loss, which would allow time to start tocolytic therapy.Prophylactic transvaginal cerclage generally is an easy procedure, and morbidity is limited to hospital admission, mild pyrexia and tocolytic therapy . ProphylEmergency cerclages have traditionally been associated with a high risk of chorioamnionitis up to 37%) and/or rupture of the membranes within 2 weeks of the operation (up to 65%), as a result of cervical shortening and exposure of the membranes to vaginal bacteria 7% and/or,13, withDespite all the controversy with respect to cervical insufficiency and cerclage, for women with a classic or characteristic history a prophylactic cervical cerclage may still be the best option. In contrast, in low risk cases prophylactic cerclage is not useful. Recent studies suggest that emergency cerclage plus antibiotics, tocolysis, and bedrest, has a better chance to increase neonatal survival than previously thought. Further randomized studies are needed to determine the effectiveness of cervical cerclage in women at high risk of fetal loss, but such studies are likely to be hampered by difficulty to obtain informed consent. In addition, there is a need for more basic knowledge of cervical ripening, objective assessment of cervical visco-elastic properties, and randomized controlled trials of technical aspects of cervical cerclage (e.g. suturing technique).The author declares that he has no competing interests.


Merkel cell carcinoma (MCC) is an unusual primary neuroendocrine carcinoma of the skin. MCC is a fatal disease, and patients have a poor chance of survival. Moreover, MCC lacks distinguishing clinical features, and thus by the time the diagnosis is made, the tumour usually have metastasized. MCC mainly affects sun-exposed areas of elderly persons. Half of the tumours are located in the head and neck region.MCC was first described in 1972. Since then, most of the cases reported, have been in small series of patients. Most of the reports concern single cases or epidemiological studies. The present study reviews the world literature on MCC. The purpose of this article is to shed light on this unknown neuroendocrine carcinoma and provide the latest information on prognostic markers and treatment options.The epidemiological studies have revealed that large tumour size, male sex, truncal site, nodal/distant disease at presentation, and duration of disease before presentation, are poor prognostic factors. The recommended initial treatment is extensive local excision. Adjuvant radiation therapy has recently been shown to improve survival. Thus far, no chemotherapy protocol have achieved the same objective.Although rare, the fatality of this malignancy makes is important to understand the etiology and pathophysiology. During the last few years, the research on MCC has produced prognostic markers, which can be translated into clinical patient care. The Merkel cell (MC) was first described by the German histopathologist Friedrich Sigmund Merkel in his classic article published in 1875: Merkel F: "Tastzellen und Tastkörperchen bei den Haustieren und beim Menschen" [MCs are normal constituents of the basal layer of the epidermis and the follicular epithelium  Figure . They arOn light microscopy, MCs are large, oval, amphophilic, clear cells situated in the basal or suprabasal layer of the epidermis. They are not easily distinguished from other nonkeratinocytic epidermal cells, e.g. melanocytes and Langerhans cells, by light microscopic immunohistochemistry. Special techniques such as immunohistochemistry, electron microscopy or transmission electron microscopy are therefore required for their identification.th gestational week [In human development, MCs can be detected in the epidermis in the 8nal week . In the nal week ,5, and inal week . In the nal week . The funnal week .The origin of the MC is still controversial. The cell may derive either from the epithelial cells of the epidermis or from the neural crest migrating to the epidermis during embryogenesis. In the 1980s and 1990s, the epidermal origin from keratinocytes with an aberrant differentiation  was the The Merkel cell carcinoma (MCC) was first described in 1972, when Toker presented the first five cases under the name "trabecular carcinoma of the skin", assuming them to represent an eccrine, sweat gland-derived carcinoma . In elecThe "cell of origin" of MCC is, however, still speculative. There are morphological and biological similarities between the MC and MCC. The common presence of neuroendocrine granules  and the Toker called the first five cases of MCC "trabecular carcinoma of the skin." This name derived from the trabecular architecture of the tumour, which is the most characteristic, but the uncommon, configuration of three histological patterns. Over the years, the name of this tumour has been the subject of lively discussion. Several names and synonyms have been proposed; either based on histological features or derived from the term "Merkel cell". These include derivatives such as "Malignant Merkel-cell tumour" , "MerkelThe histology of MCC is typical of small round blue cell tumours, an entity that includes a wide variety of highly malignant tumours: the Ewing family of tumours, olfactory neuroblastoma (esthesioblastoma), rhabdomyosarcoma, neuroblastoma, lymphoma, desmoplastic small cell tumour, osteosarcoma, small cell lung carcinoma (SCLC), small cell melanoma and mesenchymal chondrosarcoma ,28.Histologically, MCC can be classified into three distinct subtypes -31. In htrabecular subtype, described originally by Toker[The classification to the subtypes is usually reserved for the anatomical studies. The first of these is the  by Toker. This isintermediate subtype is the most frequent histological subtype [small cell type mimics small cell tumours of other sites, e.g. SCLC [The  subtype . It exhi.g. SCLC . The tumDiagnosis is based on typical histology representation on haematoxylin-eosin -stained slides together with the results of immunohistochemistry . A typicThe tumour expresses both epithelial and neuroendocrine markers, and thus exhibits both epithelial and neuroendocrine differentiation.Of the intermediate filaments, MCC expresses low-molecular-weight cytokeratins , the simple epithelial type being the most marked . The mosTissue-specific transcription factors control cell determination and differentiation. Thyroid transcription factor 1 (TTF-1) is a tissue specific transcription factor expressed in epithelial cells of the thyroid and lung, as well as in certain areas of the brain . TTF-1 iCombining TTF-1 with CK-20 provides a sound basis for diagnosis. Most adenocarcinomas from other sites  and neuroendocrine carcinomas such as SCLC are essentially negative for CK-20  while moDue to neuroendocrine differentiation, MCC always stains positively for neuron-specific enolase (NSE), which is a general marker of neuroendocrine tumours -50. CD56et al, demonstrated MAP-2 expression in all MCC samples, even though CK-20 staining was negative [Newer markers of neuroendocrine differentiation introduced for MCC are the microtubule-associated proteins (MAPs). These are the major component of the cytoskeleton family of proteins associated with the microtubule assembly of the central and peripheral nervous system ,63. Specnegative . In genenegative .Different types of chromosomal irregularities have been documented in MCC tumours and cell lines -68. The The clinical presentation of this tumour is rather non-specific. MCC is usually, at least at early stages of the disease, defined as a small painless erythematous intradermal mass, usually with no ulceration ,78. EspeThere is no classification scheme for MCC such as there is for most carcinomas or sarcomas. At present, staging of most malignant tumours is based on the T(umour) N(odes) M(etastasis) classification . Concerning the skin malignancies the TNM classification applies to carcinomas of the skin in general and malignant melanoma. In most published series MCC is not classified according to the TNM system.Yiengpruksawan has, however, proposed a staging system that has been widely recognised in the treatment of MCC , which iThe reported annual incidence of MCC ranges from 0.2 to 0.45 per 100 000 ,80. InciMCC usually occurs in sun-damaged skin. The tumours are often found in close proximity to other lesions of actinically damaged skin, for instance, in cases of Bowen disease, squamous cell carcinoma, basal cell carcinoma, solar keratosis and lentigo maligna. The most common site is the head and neck region . PathogeThe natural history of MCC is variable and dependent on the stage of disease at presentation . TypicalThe typical clinical course of the disease is rapid progression of the primary tumour with early and frequent metastasis to the regional lymph nodes. The sentinel lymph node biopsy (SLNB) technique has provided information on the metastatic dissemination at the presentation. In a meta-analysis of 60 MCC patients, Mehrany and co-workers detected 33% of the patients having metastatic dissemination in the sentinel lymph node at the presentation . Then aget al, at the Memorial Sloan-Kettering Cancer Center's identified 251 patients who had been treated between 1970 and 2002. The median time to recurrence was 9 months, and 102 patients (43%) recurred [Recurrences are frequent. Allen recurred . Primaryrecurred .Although the natural course of MCC is usually defined with metastatic spread, there are some reports in the literature (10 cases) of spontaneous regression ,107. MorMCC has been associated with other skin tumours  and haematological (B-cell) malignancies -112. In The incidence of MCC is abnormally high (8%) among immunosuppressed patients. Such patients are moreover younger, 49% being under the age of 50 ,116. WeaThe prognosis is rather poor. The 2-year survival rate is 30 – 50% ,119, KokLocal recurrences are frequent, occurring in up to 44% of patients ,88,93. TBecause of the rarity of the tumour, there is multitude of treatment protocols. However, the surgical treatment seems to be the corner stone of the different treatment protocols.Early, radical surgery is the recommended procedure for the treatment of primary MCC ,130. MarAccording to the literature on MCC, the sentinel lymphnode biopsy (SLNB) technique was used in approximately 100 cases between 1976 and 2002. Most reports advocate SLNB because morbidity is low, and because it provides an easy and reliable way of locating occult metastasis ,133,134.It is widely accepted that patients with regional node metastases or local or regional recurrence should undergo excision of the primary lesion and lymph node dissection . AdjuvanMohs micrographic surgery is a surgical technique developed by Frederick Mohs in the 1930s at the university of Wisconsin . BrieflyMohs micrographic surgery has been recommended as an advanced technique for local control, especially in areas calling for excellent cosmetic results (i.e. head and neck region) without compromising the principles of cancer surgery ,143. Theet al reported similar results, the rates of response to second-line (n = 33) and third-line (n = 10) chemotherapy being 45% and 20%, respectively [Chemotherapy is generally reserved for stage III (distant metastasis) cases of MCC. The carcinoma has often been shown to respond to chemotherapy but, as in SCLC, remission is brief. Recently, Waldman and co-workers reported a complete remission that lasted for 6 months. The remission was achieved through the use of high-dose polychemotherapy following autologous blood stem cell transplantation . Some moectively . No chemectively .No standard chemotherapy protocol has yet been established for the treatment of MCC. Because of the morphological and immunohistochemical similarity of MCC to SCLC, chemotherapy has been performed with protocols based largely on agents active in SCLC. A wide variety of chemotherapeutic agents, including the cytostatic drugs cyclophosphamide, doxorubicin, epirubicin, vincristine, etoposide, cisplatine, carboplastin, 5-fluorouracil, dacarbazine, mitoxantrone, bleomycine and iphosphamide, have been discussed ,145-147.MCC is a highly radiosensitive tumour, which has been demonstrated in in vitro studies . Many auRecent advances in the research of MCC have revealed features in the natural biology of this malignancy. However, several questions regarding the biology and pathology of the tumour remain to be addressed. The molecular events leading to oncogenic alterations must be identified. Mechanisms of metastasis are still largely concealed. Revealing these biological properties, hopefully, will lead to discovery of new treatments, e.g. the role of anti-angiogenic medication is still uncertain. Of the surgical treatments, the sentinel lymph node biopsy has proven its efficacy in finding the clinically occult metastasis and accrual of patients to adjuvant and further surgical and oncological treatment protocols.Several markers for prognosis have been studied, but none have proven its value like large, ≥ 2 cm, primary tumour size. The wide range of the factors associated to survival may reflect the absence of large institutional studies.The author(s) declare that they have no competing interests.VK: concived the idea, did literature search, wrote the article and edited it for its final content.


Steinernema and their bacterial symbiont Xenorhabdus are very tractable model systems. Previous studies demonstrated (i) a highly specialized relationship between each strain of nematodes and its naturally associated bacterial strain and (ii) that mutualism plays a role in several important life history traits of each partner such as access to insect host resources, dispersal and protection against various biotic and abiotic factors. The goal of the present study was to address the question of the impact of Xenorhabdus symbionts on the progression and outcome of interspecific competition between individuals belonging to different Steinernema species. For this, we monitored experimental interspecific competition between (i) two nematode species: S. carpocapsae and S. scapterisci and (ii) their respective symbionts: X. nematophila and X. innexi within an experimental insect-host . Three conditions of competition between nematodes were tested: (i) infection of insects with aposymbiotic IJs (i.e. without symbiont) of both species (ii) infection of insects with aposymbiotic IJs of both species in presence of variable proportion of their two Xenorhabdus symbionts and (iii) infection of insects with symbiotic IJs  of both species.Symbioses between invertebrates and prokaryotes are biological systems of particular interest in order to study the evolution of mutualism. The symbioses between the entomopathogenic nematodes Xenorhabdus symbionts in the insect-host during competition between Steinernema modified the proportion of each species in the adults and in the global offspring.We found that both the progression and the outcome of interspecific competition between entomopathogenic nematodes were influenced by their bacterial symbionts. Thus, the results obtained with aposymbiotic nematodes were totally opposite to those obtained with symbiotic nematodes. Moreover, the experimental introduction of different ratios of Xenorhabdus symbionts modified the competition between their Steinernema associates. This suggests that Xenorhabdus not only provides Steinernema with access to food sources but also furnishes new abilities to deal with biotic parameters such as competitors.We showed that  Steinernema spp. and the enterobacteriacae Xenorhabdus spp. are associations in which both partners receive benefits from each other [Steinernema spp. was able to associate with a Xenorhabdus spp. genetically distant from its natural one [Xenorhabdus spp. might select special traits in order to enhance their vector's fitness. It is known that Xenorhabdus spp. are beneficial to their nematodes in providing the latter with a better ability to kill the insect and feed on it [Steinernema species (S. carpocapsae and S. scapterisci) have provided us with insights into the association characteristics [S. scapterisci appeared less dependent upon its native symbiont (X. innexi [S. carpocapsae (associated with X. nematophila). Thus, S. scapterisci's symbiont increased the reproductive rate of its naturally associated nematode by a factor of 1.3, whereas X. nematophila increased the reproductive rate of its naturally associated nematode by sevenfold (i.e. S. carpocapsae) [S. scapterisci, transported 700-fold fewer cells of its Xenorhabdus than S. carpocapsae (i.e. ~50 bacteria per nematode for S. carpocapsae and ~0.07 bacteria per nematode for S. scapterisci) [Steinernema species also differed in their ability to deal with non-native Xenorhabdus strains in case of co-infection in an insect [S. scapterisci reproduced in co-infection situations with all the tested Xenorhabdus strains (even if its reproduction was better with its native one than with others), S. carpocapsae could not reproduce at all with most of them in the same situation [Xenorhabdus strains tend to be antagonist against nematodes species which cannot disperse them. One can easily think that this antagonistic effect of Xenorhabdus strains on the fitness of nematodes naturally associated with others Xenorhabdus strains could be selected in case of frequent interspecific competition. In such an evolutionary context, we can postulate that each Xenorhabdus strain should try to provide its own nematode-vector with competitive advantages by producing antagonistic molecules against foreign nematodes. Indeed, a previous study has shown with the association S. carpocapsae-X. nematophila as a model-system that, in insects co-infected by antagonistic Xenorhabdus, X. nematophila partly counteracted their antagonistic effect on the nematodes fitness most probably by the mean of bacteriocins [Symbioses between the entomopathogenic nematodes ch other -3. In thch other . Previouural one ,5,6. As ed on it ,7,8. Preeristics ,5,6,9-11. innexi ) than S.eriocins ,14.Xenorhabdus symbionts on the progression and outcome of interspecific competition between individuals belonging to different Steinernema species. For this, we monitored experimental interspecific competition between (i) two nematode species: S. carpocapsae and S. scapterisci and (ii) their respective symbionts: X. nematophila and X. innexi within an insect-host . In this study, three conditions of competition between nematodes were tested: (i) infection of insects with aposymbiotic IJs (i.e. without symbiont) of both species (ii) infection of insects with aposymbiotic IJs of both species in presence of variable proportion of their two Xenorhabdus symbionts and (iii) infection of insects with symbiotic IJs  of both species.The goal of the present study was to address the question of the impact of X. nematophila was clearly less represented within the hemolymph 72 h post-infection with nematodes when an initial injection of a suspension containing 50% of each bacterium in the insect was performed  the ratio of S. carpocapsae among all females found in the insect 150 h after infection  and (ii) the ratio of S. carpocapsae among all males found in the insect 150 h after infection . Because of the non-significant differences observed in these comparisons, the results obtained in each situation with and without GFP labelled X. nematophila were pooled in further analyses. The ratios of S. carpocapsae among all males found in the insects were highly heterogeneous for the different competition conditions . The Noether test showed that no significant difference in the ratio of S. carpocapsae males occurred under four competition conditions: (i) competition resulting from infection of insects with aposymbiotic IJs of both nematode species without injection of bacterium, (ii) competition resulting from infection with aposymbiotic IJs of both nematode species in insects injected with 100% of X. innexi, (iii) competition resulting from infection with aposymbiotic IJs of both nematode species with 50% of each symbiont injected in the insects and (iiii) competition resulting from infection with aposymbiotic nematodes in insects injected with 70% of X. nematophila and 30% of X. innexi . In these four conditions, the ratios of S. carpocapsae among males were significantly lower than in the three other conditions of competition: (i) competition resulting from infection with aposymbiotic IJs in insects injected with 90% of X. nematophila and 10% of X. innexi, (ii) competition resulting from infection between aposymbiotic IJs in insects injected with 100% of X. nematophila and (iii) competition resulting from infection with symbiotic IJs  . The Noether test showed that the ratios of S. carpocapsae within females were lower in competition resulting from infection with aposymbiotic IJs in insects injected with 100% of X. innexi than (i) in competition resulting from infection with aposymbiotic IJs in insects injected with 100% of X. nematophila and (ii) in competition resulting from infection with symbiotic IJs . In competition resulting from infection with aposymbiotic IJs in insects injected with 100% of X. innexi, no S. carpocapsae females were observed.To know if GFP labelling of S. carpocapsae and S. scapterisci was heterogeneous under the different competition conditions . The Noether test showed that competition resulting from infection with aposymbiotic IJs without injection of bacteria into the insect resulted in the production of significantly fewer IJs than (i) competition resulting from infection with symbiotic IJs , (ii) competition resulting from infection with aposymbiotic IJs in insects injected with 50% of each symbiont , (iii) competition resulting from infection with aposymbiotic IJs in insects injected with 70% of X. nematophila and 30% of X. innexi  and (iiii) competition resulting from infection between aposymbiotic IJs in insects injected with 90% of X. nematophila and 10% of X. innexi .The total number of IJs emerging from insects in the different competition situations for S. carpocapsae IJs within pools of 500 IJs containing respectively 75% and 90% of S. scapterisci were easily detected (i.e. three bands were observed after incubation of the PCR product with Hind III)  50% of each symbiont, (ii) 70% of X. nematophila and 30% of X. innexi and (iii) 90% of X. nematophila and 10% of X. innexi gave offspring in which almost no IJs harboured GFP labelled X. nematophila  Fig. . In all II) Fig. . This reila Fig. . These oila Fig. . The latVibrio which provides its sepiolids host with a better discretion towards its predators [Xenorhabdus symbionts provided Steinernema nematodes with diverse adaptations to their environment: (i) they produce virulence factors in order to kill the insect [Xenorhabdus to modulate the progression and outcome of interspecific competition between Steinernema species. For this, we made experimental competitions between S. carpocapsae and S. scapterisci and their bacterial symbionts. We assessed several parameters of these competitions: (i) the proportion of each symbiont (i.e. X. nematophila and X. innexi) within the hemolymph of the insect after delay of multiplication, (ii) the ratio of S. carpocapsae among the adults found in the insects and (iii) the offspring of each nematode species after competition. The assessment of bacterial multiplication within insect hemolymph showed that X. innexi appeared more competitive than X. nematophila. Moreover, in insects infected with aposymbiotic IJs and in which 50% of each Xenorhabdus strains were initially injected, we found X. innexi highly dominant within the hemolymph after 48 h of multiplication e insect ,4, (iii)e insect -29. The  insects . In compXenorhabdus. In such a situation, we showed that (i) significantly fewer nematodes developed into adults and that (ii) S. scapterisci was highly dominant in both males and females found in the insects. These results showed that S. scapterisci had a better competitive ability without its Xenorhabdus symbiont than did S. carpocapsae  the development of nematodes within the insects and (ii) their ability to reproduce in them. Regarding the first aspect, we showed that the nematode's development into adults differed significantly for (i) competition resulting from infection with aposymbiotic IJs of both nematode species without injection of bacteria and (ii) competition resulting from infection between symbiotic IJs of both nematode species. The competition between aposymbiotic nematodes (without injection of bacteria) allowed evaluation of the intrinsic ability of each nematode species to outcompete the other one without the help of its symbiotic sae Fig. . On the lts Fig. . Experimph. Fig. . The morX. innexi was injected [even at the lowest dose , the association S. carpocapse-X. nematophila totally outcompeted the association S. scapterisci-X. innexi. The outcome of the competition between symbiotic nematodes in favour of S. carpocapsae is quite understandable since this competition occurred in a bacterial environment constituted exclusively of X. nematophila. This dominance of X. nematophila within the insect hemolymph is due to the fact that S. scapterisci is poorly associated with its symbiont compared to S. carpocapsae (700-fold less cell transported) [X. innexi than X. nematophila were released by the worms within the insect hemolymph.Regarding the offspring production of each nematode species, the experimental modification of the bacterial symbiotic environment within the insects led to surprising results. In all insects where injected  but thatpocapsae . In the sported) . Thus, dXenorhabdus modify the progress and outcome of interspecific competitions between their Steinernema associated nematodes. This suggests that Xenorhabdus not only provides Steinernema with access to food sources but also furnishes new abilities to deal with biotic parameters such as competitors. Thus, the evolution of the specific association between Steinernema and Xenorhabdus in environments where interspecific competitions occurred frequently should lead to the selection for an increased number of bacterial cells retained by each nematode in order to outcompete other specific nematodes-bacteria complexes. A previous study has reported that there is dramatically different bacterial retention between some species of Steinernema [S. carpocapsae is a worldwide distributed nematode contrary to S. scapterisci which is only found in South-America [S. carpocapsae is supposed to have a wider insect-host range than S. scapterisci [Steinernema species suggest that S. carpocapsae could have evolved under a higher competition pressure than S. scapterisci. This evolutionary constraint could have induced a selection toward a tighter relationship  in the S. carpocapsae-X. nematophila association than in the S. scapterisci-X. innexi association.This study shows that the bacterial symbionts inernema . This ba-America -32. Morepterisci . These sSteinernema species naturally associated with their Xenorhabdus  were established in the laboratory as soon as they were sampled by successive experimental infections of the last instar of the wax moth Galleria mellonella. The aposymbiotic and symbiotic IJs used for experiments were always freshly emerged from insects. Insect hosts were reared in the dark in aired plastic boxes at 28°C, 65% RH, on a diet of pollen and wax. Aposymbiotic IJs (i.e. without symbiont) of the species S. carpocapsae and S. scapterisci were obtained by disinfecting nematode eggs with a bleach solution as described previously [S. carpocapsae associated with GFP (Green Fluorescent Protein) labelled X. nematophila (strain F1D3) were produced as described previously [The two eviously . The symeviously . The GFPBacterial suspensions were prepared by transferring a single colony of one bacterial strain to 5 ml of Luria-Bertani broth for liquid culture incubated at 28°C for 15 h. 100 μl of this liquid subculture was used to perform a culture to reach an optical density of 0.7 (600 nm wavelength). Before inoculation into the insect, the number of bacterial cells in each culture was counted with a Thoma cell and diluted in order to obtain a suspension of 100 cells/μl. A control of the actual number of bacteria in the injected suspension was measured by plating it onto three NBTA plates . These pX. nematophila and X. innexi) and (iii) competition resulting from infection with symbiotic IJs . In each of these competition experiments, we monitored (i) the proportion of each bacterial symbiont within the hemolymph 72 h post-infection with IJs, (ii) the proportion of males and females from each species within adult nematodes found in the insect and (iii) the proportion of each nematode species within the offspring (IJs). In order to be able to specifically distinguish IJs emerging from the cadaver, we labelled S. carpocapsae with its symbiotic bacteria, X. nematophila, hosting a plasmid expressing GFP. Previous studies showed that 96% of S. carpocapsae IJs were associated with X. nematophila and that S. scapterisci was unable to associate with this bacterial strain [S. carpocapsae's offspring within the total number of IJs emerging from an insect, we made two different sets of insects for each type of competition (except the competition between aposymbiotic nematodes without injection of bacteria): (i) one set of insects was infected with GFP labelled X. nematophila and (ii) one set with wild type of X. nematophila.  competition resulting from infection with aposymbiotic IJs without injection of bacteria into the insects, (ii) competition resulting from infection with aposymbiotic nematodes in insects followed by injection of variable proportions of their respective bacterial symbiont  [X. innexi, (ii) 50% of each symbiont, (iii) 70% of X. nematophila and 30% of X. innexi, (iiii) 90% of X. nematophila and 10% of X. innexi and (iiiii) 100% of X. nematophila  were counted and deposited into 1.5-ml Eppendorf tubes containing a filter paper. A last instar of  innexi) . The difXenorhabdus cells into the insects). For infections made with symbiotic IJs, the same assessment was made 72 h post-infection with symbiotic IJs. To evaluate the proportion of each symbiotic bacterium within the insect, the hemolymph of each Galleria mellonella was collected using a syringe. The sampled hemolymph was diluted by 105 and 100 μl of the obtained suspension was plated out onto three NBTA plates. The plates were then incubated at 28°C during 48 h. After incubation, few cells from each colony were sampled with the help of sterile tooth picks and transferred in lines onto two NBTA plates containing control samples of X. nematophila and X. innexi. One of these plates was incubated at 28°C during 48 h and the other one at 37°C during 48 h. These two different incubation temperatures helped us to discriminate between the two Xenorhabdus strains. Indeed, both of these bacteria grew at 28°C but only X. innexi was also able to grow at 37°C [X. nematophila in the insect was then obtained by subtracting the number of colonies grown at 28°C from the number of colonies grown at 37°C and then dividing the result by the number of colonies grown at 28°C.For infections made with aposymbiotic IJs followed by the injection of variable proportions of each symbiont, the proportion of each symbiont was assessed 72 h post-infection with aposymbiotic IJs  examined the vesicle of 100 IJs by epifluorescence microscopy and (ii) made isolation of bacteria contained in 500 IJs as previously described by Sicard et al., 2003. The first experiment led to assign each IJ to one nematode species. Indeed, if GFP labelled X. nematophila were observed within their vesicles, the IJs were assigned to S. carpocapsae, if not to S. scapterisci. The second experiment led to check if all the clones of X. nematophila isolated from IJs expressed GFP. In case of competition resulting from infection with aposymbiotic IJs without injection of bacteria into the insects, the presence/absence of each nematode species in the offspring was evaluated by performing PCR-RFLP (as described above) with DNA extraction on four pools of 500 IJs coming from four different insects. Preliminary experiments showed that when IJs of S. carpocapsae were mixed with IJs of S. scapterisci three bands were observed after PCR-RFLP. Such an approach led to detect 10% of IJs of S. carpocapsae within pools containing 90% of S. scapterisci. We used this latter experiment as a standard in our experiment  test followed by the pair wise comparison test of Noether .MS designed experiments, carried out some of them, interpreted the data and wrote the first draft of the manuscript. JH carried out main experiments and revised the manuscript, NLB carried out some experiments and revised the manuscript, SP helped to the design of some experiments, NB revised the manuscript and CM participated to the design of the study, the interpretation of the data and revised the manuscript. All authors read and approved the final manuscript.


Objective monitoring of cough may be preferred to subjective reporting of the symptom in clinical and research settings. Therefore, a self-contained, ambulatory cough monitoring system is needed that is non-invasive, usable for children and adults of all ages, inexpensive, and highly accurate with easy to use analysis software.After development of a new device, 15 subjects with frequent coughing were recorded with the novel cough monitor and a simultaneous video recording in order to validate the monitor compared with a gold standard. Two investigators independently analyzed the recordings and counted the number of coughs during the study period from both the cough monitor and the video recording.p < 0.001). In the comparison of video counts, the sample concordance correlation coefficient was 0.997 (p < 0.001). For the comparison of investigator 1's video counts to the corresponding audio counts, the sample concordance correlation coefficient was 0.968 (p = 0.026). For the comparison of investigator 2's video counts to the corresponding counts, the sample concordance correlation coefficient was 0.973 (p = 0.015).When measuring agreement between the two investigators, the sample concordance correlation coefficient for audio counts was 0.998 (We have developed and piloted a new, valid, and reproducible method of objectively recording and analyzing cough. This device appears to be useful for subjects of any age and in clinical and research settings. Cough is one of the most bothersome symptoms of illness, and is the most common cause of outpatient acute care visits in the United States each year . The cauAs such, we developed a self-contained, ambulatory cough monitoring system that was designed to be non-invasive, usable for children and adults of all ages, inexpensive, and highly accurate for the detection of cough with easy to use software for data analysis. Upon its completion, we aimed to pilot its ability to accurately record cough frequency and validate its accuracy in quantitating cough by comparing the auditory recordings of the device with simultaneously performed video recordings serving as the gold-standard.The self-contained monitor consists of several components: 1) an accelerometer, 2) an electronic package , 3) a cable connecting the accelerometer to the electronic package, and 4) a CompactFlash memory card. The accelerometer chosen was the BU-1771 Figure  that is Attachment at the suprasternal notch is advantageous for numerous reasons. First, because it is below the larynx, any speech that causes vibrations is unintelligible on the audio recording. This maintains the privacy for the subject that may be recorded for extended periods in an ambulatory setting. Next, it eliminates any interference from swallowing. Third, it is a relatively comfortable location that does not interfere with typical daily activities. Lastly, this location and its method of placement eliminate the problem of movement artifact or distance from an externally located microphone.® , a graphical user interface (GUI) enables the user to analyze the cough recordings . In the comparison of video counts, the sample concordance correlation coefficient was 0.997 with 95% lower confidence bound of 0.991 (p < 0.001). There was good agreement between the two investigators' audio counts. There was also good agreement between the two investigators' video counts, and experience with objective cough recordings did not impact the findings.When measuring agreement between the two investigators, the sample concordance correlation coefficient for audio counts from the new device was 0.998 with a 95% lower confidence bound of 0.994 (p = 0.026). For the comparison of investigator 2's video counts to the corresponding counts, the sample concordance correlation coefficient was 0.973 with a 95% lower confidence bound of 0.930 (p = 0.015). The agreement level was slightly higher for the more experienced counter; however, this difference was not statistically significant (p = 0.990).Next, the agreement between video counts and audio counts were calculated for both investigators. For the comparison of investigator 1's video counts to the corresponding audio counts, the sample concordance correlation coefficient was 0.968 with a 95% lower confidence bound of 0.918  declare that they have no competing interests.