Each year, nearly 500,000 new cases of cervical cancer are diagnosed around the world, and more than 250,000 women die from the disease. Most of these cases occur in developing countries where there is no routine screening for precancerous lesions. By contrast, in developed countries, national screening programs have greatly reduced the number of women dying from this cancer—between 1955 and 1992 in the US, for example, cervical cancer deaths dropped by 74%.Infection with a sexually transmitted human papillomavirus (HPV) is a precondition for the development of cervical cancer. Of the 35 HPV types that can infect the genital tract, about half have oncogenic potential—the rest cause benign warts. The immune system clears most HPV infections but persistent infection with HPV type 16 accounts for approximately 55% of cervical cancers. Because of the strong association between cervical cancer and HPV infection, several HPV type-specific vaccines are being developed. Early results suggest that these vaccines can prevent almost 100% of persistent infections with the relevant HPV type, raising the possibility of reducing the incidence of cervical cancer by prophylactic vaccination. But what would the impact of such vaccines be in countries that already have cervical cancer screening programs? To find out, Ruanne Barnabas and colleagues have developed a dynamic transmission model of HPV 16 infection and progression to cervical cancer using epidemiological data from Finland. Their analyses indicate that high coverage of women alone over many decades with a vaccine that provides long-term protection would greatly reduce type-specific cancer incidence, a reduction that would be maximized by combining vaccination with routine screening.The researchers' model is represented by a flow chart in which susceptible women acquire an HPV infection that, in most cases, is cleared by their immune system. In some women, persistent infection induces precancerous lesions that can progress to invasive cervical cancer, regress spontaneously, or be screened and treated. HPV infection in men is represented by a simpler flow chart—they simply become infected and then develop immunity. The researchers incorporated values for parameters such as sexual activity, screening protocols, and treatment rates obtained from published Finnish studies in their model and calibrated it using historical data on the proportion of the Finnish population with antibodies to HPV 16.To allow them to model how vaccination will affect the incidence of cervical cancer, Barnabas and colleagues first estimated the transmission probability of HPV in the Finnish population. This probability provides a measure of how easily HPV spreads—if it were 1.0, every sexual partnership a woman had with a man infected with HPV would result in her also becoming infected. The researchers' transmission probability estimate of 0.6 is high, which indicates that universal coverage with a very effective vaccine will be needed to eliminate HPV infection in the population. The researchers put this value (which is subject to great uncertainty) and estimated values for age at sexual debut and the annual number of sexual partners into their model; they also assumed that vaccination takes place before sexual debut, is 100% effective, and gives life-long protection.The model predicts that vaccinating both men and women will be little better than vaccinating women alone, irrespective of whether vaccine coverage is high or low. Furthermore, although vaccinating 90% of young women before sexual debut could decrease HPV type-specific cervical cancer incidence by 91%, delaying vaccination until after sexual debut could decrease the impact of vaccination. The model also predicts that if 90% of women were vaccinated without screening, there would be 0.6 cases of cervical cancer per 100,000 women per year ; vaccination plus screening every five years would reduce this incidence further, by two-thirds. Finally, the researchers investigated how the duration of vaccine-conferred protection might affect invasive cervical cancer rates. The model predicts that—unintuitively—short-lived protection will marginally increase cervical cancer rates compared with no vaccination if, as some people believe, older women are more susceptible to the persistent HPV infections that progress to cervical cancer than are younger women. Booster vaccinations would avoid this potential problem.The researchers conclude that the most effective strategy for the reduction of cervical cancer in developed countries in which incidence is already low is widespread vaccine coverage (both in terms of the HPV type targeted and the fraction of the population vaccinated), combined with current screening protocols. Whether this recommendation is adopted will depend on how vaccines perform in ongoing phase III trials and on a detailed economic assessment of the options available.


Human papillomavirus (HPV) typing and quantitation by polymerase chain reaction was performed on exfoliated cells from 133 women referred for colposcopy because of an abnormal smear. High levels of HPV 16 correctly predicted cervical intraepithelial neoplasia (CIN) grade II-III in 93% of its occurrences, but only 59% of cases of CIN III were associated with high levels of this type. Eighty-four per cent of CIN III lesions contained high levels of at least one of HPV types 16, 18, 31, 33 and 35, but the other types were less specific for CIN III than HPV 16. Overall HPV testing compared favourably with cytology for predicting high-grade CIN lesions, but it would appear that some combination of the two modalities will produce better performance than either alone. In particular, HPV testing appears to be helpful in determining which women with mildly abnormal smears have high-grade underlying lesions in need of immediate referral for colposcopy.


For eight head-and-neck cancer patients, we generated imrt plans with localization uncertainty margins of 0 mm, 2.5 mm, and 5.0 mm. The beam intensities were then applied on repeat computed tomography (ct) scans obtained weekly during treatment, and dose distributions were recalculated.We assessed the effect of geometric uncertainties on target coverage and on dose to the organs at risk (ct scans showed that target coverage was adequate (V100 ≥ 95%) for only 12.5% of the gross tumour volumes, 54.3% of the upper-neck clinical target volumes (ctvs), and 27.4% of the lower-neck ctvs when no margins were added for ptv. The use of 2.5-mm and 5.0-mm margins significantly improved target coverage, but the mean dose to the contralateral parotid increased from 25.9 Gy to 29.2 Gy. Maximum dose to the spinal cord was above limit in 57.7%, 34.6%, and 15.4% of cases when 0-mm, 2.5-mm, and 5.0-mm margins (respectively) were used for prv.The dose–volume histogram analysis for the repeat imrt treatment delivery for head-and-neck cancer. The use of 2.5-mm to 5.0-mm margins for ptv and prv greatly reduces the risk of underdosing targets and of overdosing the spinal cord.Significant deviations from the prescribed dose can occur during  In the treatment of head-and-neck cancer, imrt is used mainly to preserve salivary function after radiotherapy imrt allows a higher dose to be delivered to the macroscopic tumour than to the area at risk for microscopic disease within the same fraction, the imrt technique is ideally suited to the simultaneous delivery of integrated-boost accelerated fractionation schemes Intensity modulated radiotherapy  for targets and planning organ-at-risk volumes (prv) for critical structures, as recommended by the International Commission on Radiation Units and Measurements (icru) ptv are usually chosen to reflect expected setup uncertainties.Accounting for geometric uncertainties is an important issue with imrt is often impractical because of the complex modulation of the beam intensity. Thus, set-up verification for imrt usually consists of acquiring orthogonal images to check the isocentre localization. Verification methods that rely on this kind of two-dimensional imaging may not provide a comprehensive overview of all the geometric variations that can influence dose distribution in an imrt treatment. Slight variations in the patient’s neck and shoulder positioning may not be properly assessed, and some significant changes in shape because of a patient’s weight loss or tumour shrinkage may not be detected. Furthermore, the effects of these changes on the dose distribution are not as intuitive during treatment with imrt techniques. A proper ptv and prv definition for imrt should not only focus on the potential location of the target and the organs at risk during treatment, but should also integrate any geometric variation in the surrounding tissue that may result in significant deviation from the prescribed dose.During treatment, portal imaging can be used to assess the accuracy of field alignment. However, acquiring port films for imrt dose distributions on repeat computed tomography (ct) scans taken during treatment. In addition, we generated imrt plans using 2.5-mm and 5.0-mm localization uncertainty margins to estimate the ptv and prv that would be effective in preventing significant underdosing of the target volume and overdosing of the critical structures.In the present study, we assessed the effect of geometric variations—including changes in patient anatomy and set-up uncertainties—on the target coverage and the dose to organs at risk by recalculating imrt at our institution in this dosimetric study. Primary tumour sites included oropharynx (4 patients), nasopharynx (2 patients), maxillary sinus (1 patient), and nodal metastasis from an unknown primary (1 patient). All patients had locally advanced stage iii–iv(a) disease according to the 2002 American Joint Committee on Cancer classification. In 7 patients, the treatment volume consisted of comprehensive nodal irradiation (upper and lower neck nodes), and 1 patient received treatment to the primary tumour site and upper neck region only. Two patients also received concurrent chemotherapy with cisplatin.We included 8 head-and-neck cancer patients consecutively treated with ct simulation in the treatment position. To ensure proper immobilization and treatment reproducibility, custom-made thermoplastic face masks and shoulders pulls were used. The slice thickness of the ct scan was 5 mm through the entire head and neck region. Each patient then underwent repeat ct scans at weekly intervals during treatment, with the goal of obtaining at least four ct scans per patient. However, because of severe weight loss (3 patients) or significant shrinkage of a large neck mass (1 patient), some patients required a new mask during the course of treatment. In each of these cases, a new imrt plan was generated for the remainder of the treatment. As a result, only the repeat ct scans that were made with the initial mask were used for the study. Thus, 4 patients had four repeat ct scans as planned, 2 patients had three ct scans, and 2 patients had two ct scans, for a total of 26 repeat ct scans. All the repeat ct scans were acquired using the same ct protocol that was used in the planning ct scans and radio-opaque markers were placed on the thermoplastic masks to indicate the treatment isocentre.Two weeks before the start of treatment, all patients first underwent gtv), clinical target volume (ctv), and organs at risk (oars) were contoured on the initial planning ct scan according to icru definitions. The primary tumour and each positive node were delineated as separate volumes, except when they were immediately adjacent. The ctv was defined as any potential area of microscopic disease and usually included a margin of at least 1 cm around the gtv to account for direct tumour invasion (except when it was adjacent to structures known not to be involved), plus the lymph-node regions at risk. For dose prescription and dvh analysis, the upper and lower nodal neck regions were contoured separately. The normal structures that were defined were the spinal cord and brain stem, the ipsilateral and contralateral parotids, the mandible, and the larynx. The target volumes (gtv and ctv) and oars were then redrawn on all the repeat ct scans by a single radiation oncologist. An anatomy-matching fusion algorithm semiautomatically matched the original with the repeat CT scans so that the original targets were superimposed on the repeat CT scans. The target contours were then modified with respect to changes in patient anatomy.The gross tumour volume , and imrt plans were generated using 7–9 equally spaced 6-MV photon beams. The prescribed dose was 66–70 Gy to the gtvs, 54–60 Gy to the upper neck ctv, and 54 Gy to the lower neck ctv, planned for delivery in a single phase using fractions of 1.6–2 Gy for the ctvs and 2.12–2.4 Gy for the gtvs. The planning goal was to provide adequate target coverage (prescription isodose encompassing at least 95% of each target) while limiting the dose to the oars .The imrt plans were generated: one with no margins for localization uncertainty, and two with margins  around the gtv and ctv (resulting in ptvs) and around the spinal cord and brain stem (prvs). No margins were added around the parotids and the other oars.For each patient, three imrt plans generated from the planning ct scans were then applied on each repeat ct scan. The radio-opaque fiducials placed on the thermoplastic mask were used for isocentre alignment. Dose distributions were recalculated, and dose–volume histograms (dvhs) were generated for the targets and oars. To assess dose coverage, the dosimetric parameters that were analyzed included the percentage of the ptv that received at least 100% and 95% of the prescribed dose . In addition, the lowest dose to 1% of the target volume, which can also be defined as the dose delivered to at least 99% of the volume (D99), was used to monitor for cold spots inside the target volume.The beam configurations and intensity profiles of the initial max) and the highest dose to a volume of 1 cm3 (D1cm3) were analyzed. The mean doses (Dmean) to the ipsilateral and contralateral parotids were used to assess parotid sparing, and the Dmean to the larynx, plus the D1cm3 to the mandible were also recorded.For the spinal cord and brain stem, the maximum dose , resulting in a compromise in target coverage to achieve adequate organ sparing. In addition, the ptvs generated by adding 5.0-mm margins around the nodal-region ctvs frequently extended into the build-up region close to the skin surface, where the calculated dose may be lower.The coverage of the 100, V95, and D99 were significantly lower for the repeat ct scans than for the initial imrt plans. The dose coverage improved for all target volumes when ptvs were generated. Most of the benefit was seen with the use of the 2.5-mm margins; the 5.0-mm margins resulted in modest further improvement. The rate of adequate target coverage, as defined by the proportion of targets with a V100 greater than or equal to 95% also improved with the use of ptvs. When no margins were added for uncertainty, only 12% of the gtvs, 54% of the upper-neck ctvs, and 25% of the lower-neck ctvs had a V100 ≥ 95%. With the use of 2.5-mm and 5.0-mm margins, this proportion increased to 87.5% and 97.5% for the gtvs, and to 97.1% and 100% for the upper-neck ctvs respectively. However, with the 2.5-mm and 5.0-mm margins alike, only 72.7% of the lower-neck ctvs were adequately covered by the prescription isodose. This result reflects significant variation in shoulder position at the time of the repeat ct scan from the position at the initial planning ct scan. As shown in ct scan acquired during week 2 caused a major change in the shape of the external contour at that level, resulting in increased attenuation of the oblique beams. However, the impact of positioning errors on coverage by the 95% isodose was not as pronounced, and when 5.0 mm margins where used, the V95 for the lower-neck ctv was greater than or equal to 95% in all cases.Overall, when no localization uncertainty margins were added at the time of planning, the mean values of Voars for the initial imrt plans and of the recalculated dose distributions from the repeat ct scans. Note that, for the initial imrt plans, the values of Dmax and D1cm3 for the spinal cord and brain stem are for the prvs generated with 0-mm, 2.5-mm, and 5.0-mm localization uncertainty margins; for the repeat ct scans, the values are for the anatomic structures only.max to the spinal cord and brain stem prvs was slightly above the specified limit for the initial imrt plans generated with 2.5-mm and 5.0-mm localization uncertainty margins. However, the dose to the anatomic structures (without margins) was below 45 Gy for the spinal cord and 54 Gy for the brain stem in all plans.The average Dmean to the parotids and larynx, and the D1cm3 to the mandible increased when 2.5-mm and 5.0-mm margins were added around the target volumes to generate ptvs.Unsurprisingly, the Dmax to the spinal cord was greater than 45 Gy in 57% of repeat ct scans when no prv was used, as compared with 35% with 2.5-mm margins and 15.4% with 5.0-mm margins. However, the Dmax to the brain stem was above 54 Gy in only 3.8% of the cases when no prv was generated and in 7.8% of the cases when 2.5-mm and 5.0-mm margins were added. A possible explanation is that the prvs were generated only when similar margins were also added for the ptv. Because of the narrower separation between the target volume and the brain stem when 2.5-mm and 5.0-mm margins were added, the 54 Gy isodose was closer to the brain stem for the plans with ptv and prv than for the plans with no margins. The highest D1cm3 to both the spinal cord and the brain stem were below 45 Gy and 54 Gy, respectively, for all repeat ct scans.The Dmean to the parotids and larynx and the D1cm3 to the mandible were also higher for the recalculated dose distributions than for the imrt plans. The average increase in the Dmean was 1–1.5 Gy for the contralateral parotid, but 2–3 Gy for the ipsilateral parotid. The magnitude of the increase was similar for all plans whether generated with 0-mm, 2.5-mm, or 5.0-mm localization uncertainty margins.The Dct scan imaging for treatment verification provides a more accurate representation of the geometric variations that occur during a course of head-and-neck imrt, including uncertainties in patient position, isocentre localization, organ motion, and changes in external contour because of weight loss or tumour shrinkage. The anatomic information from the ct scan can also be used to generate three-dimensional (3-D) dose distributions to estimate with greater accuracy the dose delivered to the targets and the critical structures during a fractionated course of imrt. However, such evaluation is labour-intensive and not without inconveniences.As compared with conventional portal images, oars may not be identical for the treatment planning ct scan and each of the repeat ct scans acquired during treatment. A recent study by Barker et al. gtvs decreased in size throughout the course of treatment at a median rate of nearly 2% per treatment day. Contouring variations or changes in tumour shape or size may thus influence the dvhs for these structures.First, delineation of target volumes and ct scan and the repeat ct scans. As shown in gtvs and ctvs occurred, except in the case of a few positive nodes that shrank during treatment. Also, the use of dosimetric parameters alone to assess target coverage and organ sparing may not be sufficient for imrt dose distributions. Analyzing dvhs does not give information about whether an area of lower dose is localized in the periphery or in the central region of the target volume, or if an area of higher dose represents a clinically significant hot spot within a critical structure or instead consists of multiple hot foci of negligible size. In addition, it is impossible to tell if areas of dose perturbation are always located at the same place on each of the ct scans.In our study, image-fusion techniques and standardized target volume definitions based on well-defined anatomic landmarks ct imaging together with 3-D dosimetric analysis constitutes an effective method to study the effects of geometric uncertainties on the dose.However, despite these limitations, the use of Adding margins for localization uncertainty at the time of treatment planning can effectively reduce the hazardous effect of these variations on target coverage and dose to critical structures. However, the addition of margins may not compensate completely for dose perturbation caused by a significant change in external contour.ptv cannot compensate for the underdosing in the lower neck region observed when the shoulders were not properly repositioned at the time of the repeat ct scan. The effect of less radical changes in the shape of the external contour, such as those related to weight loss or tumour shrinkage, are probably not as important. A dosimetric study by Kim et al. imrt plan does not result in significant dose perturbation for the tumour and critical structures.In fact, redesign or modification of the ptv required to assure ideal target coverage during treatment may vary, depending on target volume location, size, and shape. In our study, adding 2.5-mm localization uncertainty margins appeared sufficient to provide good target coverage more than 95% of the time (V100 ≥ 95%) for the upper neck ctv; 5.0-mm margins were required to achieve the same level of coverage for gtvs. This finding may be explained by the fact that a given set-up error—a 3-mm misalignment of the isocentre, for example—will have a greater influence on the target coverage (as assessed by the V100) for a positive lymph node measuring 1 cm than for the entire upper-neck region. In addition, set-up accuracy is rarely uniform through the whole treatment volume; the importance of the geometric uncertainty may vary depending on the location of the target.Our results also suggest that the amplitude of the et al. et al. In a study by Gilbeau ptv margins should not only focus on target coverage, but also consider the effect on normal-tissue sparing. Our results showed that the Dmean to the parotids and larynx increases when larger margins are added for ptv at the time of planning. Similar observations have also been made by van Asselen et al. ptv margins reduces parotid sparing and increases the normal-tissue complication probability for xerostomia. Geometric variations between the planning ct scans and the repeat ct scans also seem to influence the mean dose to the parotids in a similar manner, with the greater increase being observed when 5.0-mm localization uncertainty margins are added around the upper-neck ctv. Manning et al. prv for the contralateral parotid to compensate for the detrimental effect of the geometric variation on parotid sparing. Their work suggests that 5.0-mm margins were effective in preventing the increase in the contralateral parotid volume irradiated to 30 Gy when the isocentre was shifted toward that side.Proper et al. prv margins around the parotids may not compensate properly for the overall effect of geometric uncertainties on the Dmean to the parotids. Moreover, in the study by Manning et al. prv margins around the contralateral parotids resulted in decreased coverage of the nodal region adjacent to the parotids. To avoid this problem and to limit planning complexity, we consider that prv should be used only for structures such as the brain stem and spinal cord, for which the consequence of overdosing may be serious.In addition to physical position uncertainties, other geometric variations, such as changes in the volume of the glands during treatment, may also influence the dose delivered to the parotids. In the study by Barker ptv and prv. Because the repeat ct scans were not acquired with the patient lying on the treatment couch, this study did not assess some of the mechanical uncertainties inherent to imrt treatment delivery. Thus, we cannot assume that the variations in set-up between the planning ct and each repeat ct scan are truly representative of those encountered at the treatment machine. Uncertainties in couch and gantry geometry or in alignment of the lasers relative to the treatment machine isocentre may effectively introduce systematic errors between the ct planning and treatment machine set-up ct/linac unit In the present study, the selection of 2.5-mm and 5.0-mm margins was based mainly on the range of the set-up errors reported for head-and-neck cancer patients treated with custom immobilization devices et al. imrt for head-and-neck cancer had a set of orthogonal portal images taken before and after each fraction of radiotherapy for 3 consecutive days. Overall, the magnitude of intrafraction patient motion was minimal , and no persistent intrafraction motion was observed. In another study using an infrared camera monitoring system, Kim et al. imrt. Based on these results, intrafraction patient motion does not seem to differ significantly from interfraction set-up uncertainty.Our study did not address the issue of the intrafractional variations attributable to patient and internal-organ motion. However, in a study by Lee ct scans per patient (median: 3). For that reason, dvhs from the 26 ct scans obtained for the 8 patients were pooled to assess target coverage and dose to oars. This population-based approach may not reflect recurrent systematic deviations in some patients ct scan acquired with the same mask and immobilization system as that used for the initial planning ct scan were included in our analysis, we cannot presume that our findings apply to patients with severe weight loss or tumour shrinkage that produces changes in the surrounding tissue important enough to affect the quality of the immobilization. At our institution, a new mask is made whenever the mask does not fit properly or the isocentre does not align correctly on repeated portal verifications. One of the problems with trying to adjust patient position to fit more tightly within the mask is that these attempts may modify the geometry of the entire neck region relative to the isocentre, thus resulting in misalignment of the beams in the some part of the treated volume despite a somewhat adequate isocentre alignment.Finally, our results are based on a relatively low number of repeat imrt plans were applied on repeat ct scans obtained weekly during treatment to assess the impact of geometric uncertainties on the dose distributions for head-and-neck cancer patients treated with imrt. Significant deviation from the prescribed dose occurred during fractionated imrt treatment because of variations in patient positioning and changes in the surrounding tissue. Adding localization uncertainty margins at the time of planning to generate ptvs and prvs reduces the risk of underdosing targets and overdosing the spinal cord. However, the Dmean to both parotids and to the larynx tends to increase with the size of the margins added for ptv. Based on our findings, use of 5.0-mm ptv margins for the gtv and 2.5-mm ptv margins for the upper neck ctv achieves a satisfactory compromise between target coverage and parotid sparing.In the present investigation, the beam intensities of initial imrt, errors in shoulder repositioning can cause significant dose perturbations in the lower neck region despite the use of 5.0-mm ptv margins. The use of a thermoplastic mask extending down to the level of the shoulder should be considered in these patients to improve set-up reproducibility.Our results also show that, for patients treated with comprehensive nodal ptv and prv will certainly evolve with the use of inverse planning methods for imrt. Integration of the geometric uncertainty in the form of probabilistic algorithms in the optimization process has been proposed to maximize the probability of target coverage and oar sparing Finally, the concept of


An immunohistochemical study has been carried out on fibre optic-biopsy specimens from patients with small cell lung cancer (SCLC) who had either died within 3 months, or who had survived more than 2 years. Long term survivors (LTS) were identified from completed clinical trials at major UK centres and were matched for age and sex within the trial with short term survivors (STS). The panel of immunohistochemical markers included those previously reported to be associated with prognosis, and reagents representative of both neuroendocrine and epithelial differentiation. A preliminary screen of 17 antibodies identified 11 as consistently reactive on paraffin-embedded material using streptavadin-biotin immunoperoxidase. Of 186 identified patients, 110 biopsy samples were retrieved. Of these, 70 gave sufficient material for analysis. All sections were scored by three observers without knowledge of the prognosis. The analysis failed to identify any antigen whose expression was correlated with prognosis. We conclude that, in fibre-optic biopsy specimens, immunohistochemical analysis does not add prognostic information in SCLC.


Background: Epidemiological data suggest that the prevalence of syphilis, gonorrhea and trichomoniasis hasincreased in both urban and rural areas of Mongolia. These data are primarily substantiated by notifications of casesof clinically apparent disease in both rural and urban areas, plus laboratory diagnoses from the AIDS/STD ReferenceCenter,Ulaanbaatar. In the past 5 years, however, there has been a marked decline in the total number of patientsbeing screened for sexually transmitted infections (STIs). An assessment of true prevalence of STIs in a femalepopulation attending an urban sexually transmitted diseases (STD) clinic was therefore commenced.Methods: Consecutivewomen attending an STD clinic in Ulaanbaatar had genital samples collected by the insertionand immediate removal of a tampon, which was then tested for the presence of Neisseria gonorrhoeae ,Chlamydia trachomatis, human papillomavirus (HPV) and Trichomonas vaginalis , using polymerase chain reaction(PCR) amplification.Results: A total of 110 women were studied (mean age 26.7 years). Overall, 58 (53%) patients had one or morepathogens identified; 43 (39%) had a single pathogen, while 15 (14%) had mixed pathogens. C. trachomatis wasfound in 15 (14%), N. gonorrhoeae in 12 (11%), T. vaginalis in nine (8%) and HPV in 39 (36%). Among the 39HPV-positive patients, oncogenic genotypes  were found in 17 (44%) patients.Conclusions: Sexually transmitted infections as defined by PCR were common, and found in 53%of femaleattendees of an urban STD clinic in Mongolia. As infections with conventional STIs increase the risk of humanimmunodeficiency virus (HIV) transmission, it is imperative that strategies be introduced to reduce the prevalenceof STIs. Furthermore, detection of oncogenic HPV was common, indicating that it is vital that a strategy to reducecervical cancer such as a pre-cancer cervical cytology screening program also be introduced.


Hormone level differences are generally accepted as the primary cause for sexual dimorphism in animal and human development. Levels of low molecular weight metabolites also differ between men and women in circulating amino acids, lipids and carbohydrates and within brain tissue. While investigating the metabolism of blue crab tissues using Phosphorus-31 Nuclear Magnetic Resonance, we discovered that only the male blue crab  contained a phosphorus compound with a chemical shift well separated from the expected phosphate compounds. Spectra obtained from male gills were readily differentiated from female gill spectra. Analysis from six years of data from male and female crabs documented that the sex-specificity of this metabolite was normal for this species. Microscopic analysis of male and female gills found no differences in their gill anatomy or the presence of parasites or bacteria that might produce this phosphorus compound. Analysis of a rare gynandromorph blue crab  proved that this sex-specificity was an intrinsic biochemical process and was not caused by any variations in the diet or habitat of male versus female crabs. The existence of a sex-specific metabolite is a previously unrecognized, but potentially significant biochemical phenomenon. An entire enzyme system has been synthesized and activated only in one sex. Unless blue crabs are a unique species, sex-specific metabolites are likely to be present in other animals. Would the presence or absence of a sex-specific metabolite affect an animal's development, anatomy and biochemistry? A metabolite is generally defined as any product of metabolism. For this paper we define a sex-specific metabolite as being synthesized by just one sex and as being a small, non-hormonal compound synthesized by inherent biochemistry. It is not simply a modification of an ingested compound. Using metabolite concentration terminology recommended by the Human Metabolome Project Hormones are trace metabolites occurring at concentrations less than 1 nanomolar. Hormone level differences between men and women are accepted as underlaying human sexual dimorphism and are believed to explain the preponderance of one sex over the other for numerous diseases: cardiomyopathy Most metabolites occur at intermediate concentrations greater than 1 nanomolar but less than 1 micromolar. Metabolite levels in this intermediate concentration can also vary between the sexes. In humans, sex-specific variations occur in circulating lipids Natural product levels in plants can also differ between the sexes, as documented in at least one dioecious plant, that is male and female flowers on separate plants In some reptiles the sex of offspring is determined not by sex chromosomes, but by the ambient temperature during development An excellent and complex H-1 NMR study of metabolites in rat urine employing Principal Component Analysis demonstrated differences between the sexes in metabolite levels, but the study also isolated and identified a novel aromatic metabolite found in males only Expression of rat liver nuclear proteins is significantly different depending upon the sex of the rat The blue crab  is a pugnacious, keystone predator and commercially valuable species that occurs in estuaries and coastal habitats of the Western Atlantic, Gulf of Mexico and Caribbean Sea Blue crabs live in the brackish waters of estuarine systems that they inhabit with salt concentrations varying from fresh water to marine. They undergo approximately 17 molts to reach maturity at about 2 years of age and are omnivores, feeding upon various invertebrates including other blue crabs The blue crab has also been used as a model for various physiological processes including mineralization during molting The term “nuclide” specifies the number of protons and neutrons in an atomic nucleus. Hydrogen, deuterium and tritium are three nuclides of hydrogen having zero, one and two neutrons respectively. Nuclides having an even number of protons plus an even number of neutrons do not have a property called nuclear spin. Nuclides with any other ratio possess nuclear spin. Common nuclei such as Carbon 12 (C-12) and Oxygen 16 (O-16), having an even number of protons and neutrons, cannot be observed using NMR.Many nuclei contain the appropriate number of protons and neutrons to have a spin of 1/2, making their signals narrow and their observation easier. Examples of spin 1/2 nuclides are common hydrogen H-1, carbon C-13, nitrogen N-15, fluorine F-19 and phosphorus P-31. Happily, these nuclides are found in most molecules of biological and medicinal interest and provide extensive information about their structure.When placed in a magnetic field these nuclides can absorb radio frequency (RF) energy, and then release it, thus undergoing resonance. Each nuclide absorbs RF depending upon its intrinsic gyro-magnetic ratio and the strength of the magnetic field. The greater the magnetic field strength the higher the frequency of RF at which it resonates.A measure of magnetic field strength is a “Tesla” which is roughly 20,000 times greater than the Earth's magnetic field. In a magnet with a field strength at 11.7 Tesla , H-1 resonate at 500 MHz, C-13 at 126 MHz, N-15 at 51 MHz, F-19 at 471 MHz and P-31 at 203 MHz. We observe nuclei in a magnet when they resonate, thus was born Nuclear Magnetic Resonance.To observe a nuclide, the NMR spectrometer is adjusted to observe a specific frequency. The nuclide's exact resonance frequency is established by its gyro-magnetic ratio, the magnet's field strength and modified by the electronic environment surrounding the nuclide. As an example, ethanol has six hydrogens. One is bonded to oxygen. Two are in a methylene group and three are in its methyl group (HO-CH2-CH3). Each of these types of hydrogens experience different electronic environments thus produce three different groups of signals with three chemical shifts.Chemical shift is measured in parts per million (ppm) which is a dimensionless unit commonly used to describe where an NMR signal is observed. A signal's ppm value is universal for all spectrometers regardless of their field strength and apply for the spectra they produce. The measured ppm depends on the electronic environment surrounding a nuclide and describes how far the signal is from a standard compound defined to be 0 ppm. I'm using 85% phosphoric acid as the ppm reference compound for P-31 NMR.NMR spectra can also observe the relative amounts of nuclides present in the sample. This is done by comparing the area of each group under specific NMR conditions. NMR can determine the relative amounts of a nuclide within a single molecule and can also measure the relative concentrations of molecules in a mixture.The normal NMR spectrum is a plot of signal amplitudes versus their ppm values. The relative amount of a signal is not set by its amplitude but rather by the area of the signal compared to other signal areas.In a particular magnet, the signal to noise ratio in an NMR experiment depends on the inherent sensitivity of the nuclide and the natural abundance of that nuclide. H-1 is the most sensitive natural nuclide. The inherent sensitivity of C-13 compared to H-1 is 1.6%, for P-31 6.6%, but the natural abundance of C-13 is only 1.1% while P-31 is 100% in natural abundance. For observing the same amount of compound from natural sources, P-31 NMR is over 100 times as sensitive as C-13 NMR. Excellent and more thorough descriptions of NMR are available via web site P-31 NMR spectra of tissues contain far fewer signals than those found in H-1 and C-13 NMR spectra, thus P-31 spectra are more easily interpreted. Although P-31 is less sensitive than H-1, in situ its signals are not so drastically broadened and yields interpretable spectra. A P-31 NMR spectrum can be obtained from whole tissues in less than 20 minutes with signals approximately 30 Hz wide.Earlier P-31 NMR studies of not perfused blue crab muscles were able to differentiate orthophosphate, arginine phosphate, phosphate esters and the phosphates in ATP, thus it seemed feasible to study other blue crab tissues. P-31 NMR provided a quick assessment of the types and relative amounts of small phosphorus metabolites in whole tissues without the need for extraction procedures. Extracts produced much better resolved P-31 spectra, but were not essential for determining the general types and relative quantity of phosphorus compounds present.We expanded the types of tissues examined and saw nothing unexpected until we examined crab gills. For gills there was an obvious difference in the spectra obtained from males and females with spectra from males having a major NMR signal with a chemical shift far down field from the usual phosphate signals. The presence of this Unidentified Phosphorus Compound (UPC) only in males was the impetus for this work.The aim of this research was to determine if UPC was a sex-specific metabolite in blue crabs and to ascertain its structure. The absence of the UPC in females is difficult to prove, because an argument can always be made that it is present at a level below the sensitivity of the analytical technique used. We will provide strong evidence for the existence of a sex-specific metabolite naturally occurring only in male blue crabs.All blue crabs used in this study were handled in accordance with the University of Illinois at Chicago animal protocol guidelines. All crabs had a normal anatomy except for the gynandromorph crab described later. Crabs were purchased from commercial suppliers and arrived via ice cooled, overnight air delivery. Several crabs could be analyzed in a single day. Remaining crabs were kept in a cold room at approximately 12 degrees C in individual containers and were given a diet of fish twice weekly until sacrificed for this study.The crabs were ice chilled until lethargic, the carapace was opened and the following tissues were excised: gill, hepatopancreas, heart, claw muscle, leg muscle, eye stalks, hemolymph and ovaries or vas deferens plus testes. The initial surveys of whole tissues from blue crabs showed that an abundant Unidentified Phosphorus Compound (UPC) was highly concentrated in the male's gill with much smaller concentrations found in the hepatopancreas and muscle tissues. UPC was not observed in female tissues. After the correlation of UPC and the sex of the gill was initially noted, efforts focused on comparing male versus female gill tissues. Gills were excised, separated into their primary lamellae for insertion into a 5 mm NMR tube and the remaining primary lamellae frozen for later analysis. Normally gills were analyzed by P-31 NMR immediately after being excised, but frozen then thawed gills produced similar results. P-31 NMR results from male and female gill analyses are presented in The gill tissue was analyzed using a Bruker AVANCE 360 MHz NMR spectrometer having a QNP 5 mm probe producing the H-1, C-13 and P-31 spectra at 360.13, 90.55 and 145.78 MHz respectively. P-31 NMR employed broad banded H-1 decoupling at room temperature without a lock solvent such as deuterium oxide being added to the NMR tube. Normally analyses took 20 minutes. To identify the UPC, P-31 NMR was used to determine the types of phosphorus compounds present at each step of the isolation. The UPC extraction procedure from gills used approximately 2 g of tissue vigorously mixed in a laboratory blender with 60 ml of organic solution  for at least 5 minutes. The slurry was filtered through Whatman #1 filter paper under vacuum to hold the caked solid and pass the lipid extract that was flash evaporated, dissolved in 1 ml , analyzed by NMR and called the lipid fraction (LF)The caked solid plus filter was vigorously mixed in a laboratory blender with 40 ml de-ionized water for at least 5 minutes. The slurry was filtered through glass wool on top of Whatman #1 filter paper under vacuum to capture the remaining residue on the glass wool. The filtrate was flash evaporated 3 times from water then dissolved in 1 ml D2O and labeled as the aqueous low MW fraction (LOMWF).The remaining residue was digested using concentrated hydrochloric acid for 24 hours at 104 degree C to create a black slurry which was filtered, yielding the high molecular weight fraction (HIMWF). This procedure hydrolyzed all phosphorus anhydride bonds and almost all phosphate ester compounds to orthophosphate, but does not break the carbon phosphorus bond.For male gills, P-31 NMR analysis showed that the LF yielded several phospholipid signals. The vast majority of UPC was found in the LOMWF with trace amounts in the LF. The HIMWF had just orthophosphate and small amounts of phosphate esters. The UPC in the low molecular weight fraction was further purified. Any solutions containing particles were filtered through 0.22 um filters from Millipore corporation or were centrifuged at 13,000 rpm for 8 minutes before NMR analysis. The purification results were followed by P-31 and H-1 NMR analysis.The LOMWF containing the UPC was passed through a Accubond II AMINO form solid phase extraction cartridge (100 mg) (Agilent Technologies) which captured virtually all phosphate compounds, but allowed the UPC through the cartridge. The filtrate still contained many non-phosphorus compounds, as evidenced by H-1 NMR analysis. In order to isolate the UPC, a 0.5 ml aliquot of the solution was applied to 2 ml of dry silica gel (Whatman bulk microparticle media 70–230 mesh) contained in a 12 ml Bio-Rad poly-prep chromatography column. The UPC loaded silica was dried by drawing air through the column using a water aspirator. The silica was washed with 10 ml of analytical grade methanol with the UPC remaining on the silica. The methanol eluted almost all non-phosphorus compounds. Finally the UPC was eluted by washing with 10 ml water, concentrated by flash evaporation, flashed 2 times with water to reduce residual methanol and dissolved in 1 ml of D2O.The purified UPC was analyzed using 1D and 2D P-31, C-13 and H-1 NMR techniques and determined to be 2-aminoethyl phosphonate (AEP), which is an uncommon, but well documented compound. Authentic AEP was added to the purified UPC to verify the assignment using P-31, C-13 and H-1 NMR. Small quantities of a modified AEP were also extracted and were most likely mono-methyl additions to the amino group in AEP. The 2-aminoethyl phosphonate (AEP), also known as ciliatine, is an analog of 2-aminoethylphosphate, a normal metabolite which is incorporated into many phospholipids. All reagents were analytical grade and all water was de-ionized.To determine if the sex specific presence of AEP was an anomaly and limited to the initial group evaluated, blue crabs were surveyed in six different years using both commercial and scientific suppliers. To determine if geographic location affected this phenomenon, crabs were surveyed from both the Chesapeake Bay and later, the Gulf of Mexico coast of Florida. All the analyses produced similar results to those of the initial group sampled, confirming that the presence of AEP in males and its absence in females is the norm for blue crabs.In this paper only spectra from intermolt blue crabs were presented, but data obtained from pre-molt and post-molt blue crabs produced comparable results. In whole gill surveys of females, the vast majority contained no observable AEP, but a few females exhibited less than 1% of AEP, which was attributable to cannibalism by the females upon juvenile males The fractionation procedure yielding lipid, low molecular weight aqueous and high molecular weight aqueous fractions was used for a female gill showing less than 1% levels of AEP and yielded the expected low levels of AEP. This demonstrated that there was nothing in female tissue masking the appearance of AEP in P-31 NMR spectra from female gill tissue.In the LF from both sexes orthophosphate esters in phospholipids were present. In the HIMW fractions of both male and female gills, orthophosphate was the only major compound along with small amounts of unhydrolyzed phosphate esters.AEP was only abundant in the LOMW fraction of males. In order to eliminate the possibility that some factor extrinsic from gill metabolism was producing the AEP signals, gills from 2 males and 2 females were microscopically analyzed at the Shedd Aquarium, Chicago, Illinois for anatomical factors or disease conditions that could differentiate males from females, but none were found. See supporting information Surveys of blue crab habitats, migration routes and diets indicate that male and female crabs have similar diets and habitats A rare gynandromorph blue crab, with an anatomy symmetrically and bilaterally divided between male and female, was captured in the Rappahannock River, a tributary of Chesapeake Bay. After the crab died, it was frozen. Gills were removed from the male and female side of this crab. The P-31 NMR analysis of aqueous extracts from the male and female side gills showed that the percentage of AEP found in the male side gill was over seven times greater than that found in the female side gill. Male side gills averaged 21% AEP. The female side gills averaged just 3%. See The sexually asymmetric metabolism in the gynandromphic blue crab exactly mirrors its external anatomy. The high internal AEP level found in the male side gills reflect the external male anatomy. The low AEP levels found in the female side gills are consistent with a female incorporating AEP from a male. For an anterior view of this crab see The statistical analysis of the AEP was first identified in 1959 by Horiguchi Previously, labor-intensive chromatographic methods were needed to separate phosphonate compounds from their analogous phosphate compounds. The carbon-phosphorus bond of AEP is extremely stable to acid hydrolysis and most hydrolyzing enzyme systems. Phosphorus of this type has been classically determined indirectly, as the difference between total phosphorus and that released as orthophosphate upon prolonged acid hydrolysis. Early investigations needed to use phosphonate's stability to quantify its presence, by oxidative ashing to determine total phosphorus, followed by extensive acid hydrolysis of a second aliquot to measure the orthophosphate produced. The difference between the total phosphorus minus the acid-hydrolyzed phosphorus represented the amount of phosphonate Whole invertebrate tissues can be studied by NMR because they do not rapidly deteriorate. P-31 NMR is an efficient method for analyzing these tissues and can readily distinguish phosphonate from phosphate signals Some invertebrates synthesize AEP from orthophosphate Our findings document that there is a sex-specific metabolite in blue crabs, indicating that an entire synthesizing system is being produced and activated only in males. Only because AEP was a readily discernible metabolite, was its sexual specificity obvious. If other species have sex specific metabolites, then their presence could go unrecognized in the mass of variable data obtained from H-1 and C-13 NMR spectra and from Mass Spectrometry. That AEP is specific to males was surprising. That it was found primarily in a non-sexual organ was extraordinary.Data supporting AEP being sex-specific was obtained from P-31 NMR analysis of intact gills over several years, from fractionation and analysis of specific male and female gills and finally from a comparison of AEP results of the gynandromorph's male and female side gills. Because conservation of biological systems is common across species, it is likely that sex-specific metabolites occur in other species.The synthetic pathway for and the function of AEP in blue crabs still need to be investigated. AEP may be a pheromone, endocrine or paracrine agent. Raising juvenile blue crabs segregated by sex then analyzing female gills for AEP, should determine if the AEP found in the two AEP containing females, was obtained through cannibalism of male crabs. Plans for testing AEP as a pheromone are ongoing.Major questions raised by this study are: how many other species exhibit sexually specific metabolites and how would the presence or absence of these metabolites affect animal development, anatomy and biochemistry?Figure S1Shedd Aquarium-unpublished results(0.19 MB TIF)Click here for additional data file.


Rodents are known to display fear-related responses when exposed to the odor of natural predators, such as cats, even when they are totally naïve to these stimuli. Based on that, a behavioral test in which rats are exposed to cat odor has been developed and proposed to model some forms of anxiety. The objective of the present study was thus to compare the LEW (Lewis) and SHR (spontaneously hypertensive rats) inbred rat strains, which display genetic differences in other classical models of anxiety, in the cat odor test. As expected, cat odor produced an increase in fear-related behaviors. However, no clear differences were found between the two strains tested. These results suggest that the type of stress experienced by LEW and SHR strains exposed to cat odor is different from that elicited by exposure to classical models of anxiety such as the elevated plus-maze, black/white box and open-field tests. Many studies have shown that laboratory rodents that have never been exposed to a live cat (or any cat vestiges), demonstrate strong fear responses when exposed to cat odor -13. PharThe inbred rat strains Lewis (LEW) and Spontaneously Hypertensive Rats (SHR), when compared with each other, display high and low basal indices of anxiety-related behaviors, respectively, when tested in classical anxiety models. These strains, however, do not differ in their activity levels in either novel or familiar environments -25 but pIn spite of showing consistent differences in a variety of anxiety models, LEW and SHR rats do not differ in the social interaction test of anxiety  and in tad libitum under a 12-h light/dark cycle (lights on at 07:00 h) at 21 ± 2°C.LEW and SHR rats (16/strain/sex) coming from our own colonies were used . All aniThe apparatus was made of wood covered with formica and consisted of a rectangular box divided in two compartments: one smaller sheltered area covered with a ceiling (25 × 25 × 30 cm height) and one larger open area (40 × 25 × 30 cm height) where a collar, worn (cat odor) or not (control) by a cat during 3 weeks was hung in the opposite end in relation to the entrance (6 × 6 cm) of the shelter. The light inside the open area was at 7 lux. Each rat was placed in the center of the open area, facing the collar, and the following measures were registered for 5 min: the time spent with all four paws outside the shelter, the time spent in direct physical contact with the collar, the number of approaches towards the collar and the number of transitions between the sheltered and the open compartments. One transition was considered as each time that the animal left the shelter, went to the open area and came back to the shelter with all four paws. After experimental sessions the apparatus was cleaned with 70% ethanol. To minimize odor contamination, cat odor and control groups were tested in different days. Males and females were also tested in alternate days. For each sex, a total of four separate days were used. The animals were tested between 14:00 and 18:00 h.The results were analyzed by a three-way ANOVA for the factors strain, sex and odor condition (cat odor vs. control). Newman-Keuls test was used for post-hoc comparisons. The accepted level of significance for all tests was p < 0.05. = 191.2; p < 0.0001) and in contact with the collar  = 172.8; p < 0.0001), spending more time inside the shelter  = 70.1; p < 0.0001), approaching less frequently the collar  = 135.4; p < 0.0001) and making less transitions  = 146.1; p < 0.0001) between compartments than the animals exposed to the apparatus containing the collar alone (control). Moreover, there was an overall effect of strain for the time spent inside the shelter  = 4.5; p < 0.0375), with LEW rats spending more time inside this compartment than SHRs. Furthermore, an overall effect of sex  = 15.8; p < 0.0002) and an interaction between sex and odor  = 5.8; p < 0.0189) were detected for the number of transitions. The post-hoc comparisons indicated that males made less transitions under the control condition than females (p < 0.0002).The results of the behavioral responses showed by SHR and LEW rats of both sexes exposed to a collar with or without cat odor are illustrated in Figure In agreement with the concept that emotionality is a multidimensional trait, studies applying multiple behavioral tests on groups of animals with well defined genotypes can be useful to investigate whether or not different experimental paradigms (or different testing conditions) assess the same psychological phenomenon . In thisCompared to SHRs, LEW rats of both sexes display lower levels of approach towards the aversive, less-protected areas of the open-field, elevated plus-maze, and black/white box and show increased startle reflex, which suggests that they are more anxious-like than SHRs -25,28. LThe present results agreed with Hogg and File  who repoZangrossi and File -13 have In conclusion, the expected behavioral differences between the LEW and SHR strains, which differ in some classical models of generalized anxiety, were not found in the COT. Further evaluation of these rats, with and without pharmacological treatment, in tests with predator odors as well as in other behavioral tests, should improve our understanding of their psychological profile, which will represent an important step towards the investigation of new neurobiological/genetic mechanisms underlying anxiety-related traits.The author(s) declare that they have no competing interests.GRB carried out the data collection, helped to design the study and wrote the manuscript. LFV participated in the data analyses, interpretation of data and elaboration of the manuscript. AR designed the study and the data analysis strategy, and participated in the interpretation of data and elaboration of the manuscript. All authors read and approved the final manuscript.


In a study of factors related to cervical carcinogenesis, a cohort of 1,107 cervical dysplasia along with 1,077 controls matched for age and parity were followed up prospectively. During the follow up 75 dysplasia cases progressed to carcinoma in situ. The overall rate of progression of dysplasia to malignancy was observed to be 15.7% at the end of 108 months of follow-up. The analysis of progression rates in relation to various factors revealed significantly higher progression rates for initially higher grade of dysplastic lesions, and early age at consummation of marriage (ACM). The other factors, such as religion, literacy status of the patient, number of pregnancies, presence of cervical erosion, history of fetal loss and positivity to HSV-II antibodies, did not reveal statistical significance. The case-control comparison for detection of HPV 16/18 by in situ hybridisation revealed the presence of HPV 16/18 sequences in 67.3% of the dysplasia subjects progressed to carcinoma in situ while 27.3% of precancerous cases regressed to normalcy. The difference was found to be statistically significant (P less than 0.001).


Biomarkers were measured in a subset. Most (71%) of these women were located within eight blocks of the WTC at 0900 hr on 11 September, and 12 women were in one of the two WTC towers. Daily EIs were determined to be highest immediately after 11 September and became much lower but remained highly variable over the next 4 weeks. The weekly summary EI was associated strongly with women’s perception of air quality from week 2 to week 4 after the collapse (p < 0.0001). The highest levels of polycyclic aromatic hydrocarbon–deoxyribonucleic acid (PAH-DNA) adducts were seen among women whose blood was collected sooner after 11 September, but levels showed no significant associations with EI or other potential WTC exposure sources. Lead and cobalt in urine were weakly correlated with ∑EI, but not among samples collected closest to 11 September. Plasma OC levels were low. The median polychlorinated biphenyl level  was 84 ng/g lipid and had a nonsignificant positive association with ∑EI (p > 0.05). 1,2,3,4,6,7,8-Heptachlorodibenzodioxin levels  were similar to levels reported in WTC-exposed firefighters but were not associated with EI. This report indicates intense bystander exposure after the WTC collapse and provides information about nonoccupational exposures among a vulnerable population of pregnant women.We have characterized environmental exposures among 187 women who were pregnant, were at or near the World Trade Center (WTC) on or soon after 11 September 2001, and are enrolled in a prospective cohort study of health effects. Exposures were assessed by estimating time spent in five zones around the WTC and by developing an exposure index (EI) based on plume reconstruction modeling. The daily reconstructed dust levels were correlated with levels of particulate matter ≤ 2.5 μm in aerodynamic diameter (PM Depending on the World Trade Center (WTC) source characteristics and the date after 11 September 2001, emissions from the WTC site during September 2001 through early 2002 affected lower Manhattan as well as immediately adjacent areas in Brooklyn and New Jersey. Environmental and personal air monitoring, dust analyses, and biomonitoring have identified that dust and air particulates and fibers, alkaline dust, and polycyclic aromatic hydrocarbons (PAHs) were greatly elevated in the first days after 11 September, with some contaminants remaining above background for 1–3 months . Higher We have characterized and estimated WTC-related exposures among 187 women who were pregnant and were at or near the WTC on 11 September or within the subsequent 3 weeks. A preliminary report on birth outcomes in this group has been published .n = 170) on that day or within the next 3 weeks (n = 17). The five exposure zones were a) the area including and surrounding the WTC site bordered by Murray Street (north), Nassau Street (east), the Battery (south), and the Hudson River (west); b) the area of Manhattan south of Chambers Street excluding zone 1; c) south of Canal Street and north of Chambers Street; d) Brooklyn Heights; and e) the easternmost part of New Jersey across the Hudson River from the WTC ; evacuation on 11 September through the dust and debris; perception of air quality (PAQ) after 11 September; and presence of WTC dust in the home. The time–activity log recorded all the time spent indoors and outdoors at any distinct street address in one of five zones around the WTC for 4 weeks after 11 September . These zBeginning in February 2002, we obtained blood and urine specimens from 178 women, 55 of whom were in the third trimester of pregnancy (the remainder had delivered), to determine levels of PAH-DNA adducts and other biomarkers of exposure. For 160 samples, DNA was successfully obtained from mononuclear cells by standard RNase and proteinase K treatment and phenol/chloroform extraction. Mononuclear cells have higher adducts than do granulocytes , and thuIn a randomly selected subset of 100 women, the Centers for Disease Control and Prevention (CDC) determined that these women had metals in urine and blood and organochlorines  changed very little whether LOD values were assigned a censored value (LOD/√2) or the actual value was reported. However, the measures of central tendency for concentrations of ∑PCB4/total PCBs were influenced by choice of surrogate value for the LOD. Few samples had ∑PCB4 congeners below the LOD, but many samples had values below the LOD for other PCBs. As a result, the ∑PCB4: total PCB ratio decreased artificially when a surrogate value (LOD/√2) was used. The mean ∑PCB4: total PCB ratio increased by 40% and the median by 50% when using LOD/√2 rather than zero or the actual value below the LOD. Therefore, we used the actual values  or the lowest positive value (to avoid zeroes) of congeners for the sum of PCBs (see discussion and citations in A daily dust exposure index (EI) was estimated for each woman. The EI each day was derived by reconstruction of the post-11 September WTC plume. The Computational Chemodynamics Laboratory  developed simulations of the plume that was generated by the collapse of the WTC buildings and the subsequent fires at the site .The plume reconstruction was obtained by generating a set of simulations that were the result of employing the Regional Atmospheric Modeling System (RAMS)/Hybrid Particle and Concentration Transport (HYPACT) model, which is employed as part of the Modeling Environment for Total Risk (MENTOR) developed by the Environmental and Occupational Health Sciences Institute. The RAMS/HYPACT model was used to reconstruct the atmospheric dispersion of “generic” particulate matter (PM) emitted from the location of the WTC. This simulation employed a triple-nested modeling domain of 4 × 4 km (grid 1), 1 × 1 km (grid 2), and 250 × 250 m (grid 3) resolutions. The RAMS/HYPACT results were averaged over time to produce 8-hr concentrations for five zones across lower Manhattan, Brooklyn, and New Jersey. A source decay factor was incorporated into each 8-hr concentration average estimate, corresponding to a sigmoidal weakening function. This was done to simulate the decrease in the intensity of the fires and other emission sources during the days after 11 September.The RAMS/HYPACT model accounts for advection, convection, and dispersion of emissions after the collapse of the towers, but is not constructed to model the initial wave of dust and debris. A geographic information system (GIS)-based database developed by the U.S. Environmental Protection Agency EPA;  from oveReflecting the RAMS/HYPACT model start time of 0900 Eastern Daylight Savings Time, only two 8-hr averages exist for 11 September. Therefore, the total number of hours per person was set to be ≤16 for 11 September. Because the time–activity diary for an individual does not include the specific hours spent in a particular zone in a particular day, the data sets cannot be used to directly estimate exposure based on the time of day. As a result, the daily average plume density in each zone was combined with the time–activity diary entries  to develop an average EI.The EI accounts for time spent both indoors and outdoors. Various indoor/outdoor (I/O) concentration penetration factors have been reported and used for microenvironmental models. These values typically range from 0.05 to 0.9 . To accoThe EI was calculated for 187 women for 11 September and each of the 27 days immediately thereafter. Because the exact emission amount resulting from the WTC collapse is unknown, a hypothetical unit pollutant release rate was used in the RAMS/HYPACT simulation. The simulation with a unit release provides the relative “normalized” concentration of airborne contaminants at different locations. These calculations result in a dimensionless EI. Because the values are relative, identical EIs on different days may not represent exactly equivalent daily exposures. However, the potential misclassification was small enough to justify their summation. This was shown by the correlation of the daily median EI with EPA PM monitoring data at four sites (see Results). In addition, for most women, their exposure sums were dominated by the value on 11 September or on a few other days with very high readings, because of the logarithmic nature of the EI distribution. Therefore, for the purposes of estimating total exposure, the indoor and outdoor EIs have been summed over certain time periods, with ∑EI being the 28-day (4-week) sum. If additional monitoring data become available from ongoing data analyses, the current EIs can be analyzed to determine if there is reason to improve the estimates and more accurately calibrate the EIs.rS) were used to examine associations between continuous variables. In addition, we used multivariate analyses to predict biomarker levels of those analytes that demonstrated any association with ∑EI . For these models, the log-transformed biomarker level was the dependent variable, and the independent variables were EI (dichotomized below or above the median ∑EI) and factors that might potentially affect selected biomarkers . We used the general linear models program (PROC GLM) in SAS with the LSMEANS option to determine whether the biomarker level differed significantly by ∑EI (below vs. above the median).Statistical analyses were conducted using Microsoft  Excel and SAS PC . The EIs were explored as continuous variables  and as quantiles (dichotomized at the median). The significance of associations among categorical variables was assessed by chi-square analysis, using the Fisher exact test and the Mantel-Haenszel test for trend where appropriate. Because the exposure variables were not normally distributed, nonparametric methods  were used to test for differences in the medians between groups. Spearman correlations . Therefore, although we do not have data on average time spent in these zones before 11 September, it is likely to be much higher than the durations reported in the 4 weeks after 11 September. As shown in 10), r = 0.91; PM ≤2.5 μm in aerodynamic diameter (PM2.5), r = 0.93; 290 West Broadway: PM10, r = 0.73; West Broadway–Park Place: PM2.5, r = 0.68; n = 7–15 per site]. Using the models of reported PM data tested for performance against daily EI .In contrast to the increasing time spent in zones 1–3, the relative dust concentrations within the five zones declined rapidly during the 28 days after 11 September . Howeverdaily EI , we estin = 33) had a higher ∑EI than did women who only worked (n = 113), only lived (n = 38), or spent time for other reasons in one of the zones (n = 3) (n = 40) than among those who stayed for 1–39 min  or not at all . Women who wore dust masks during cleaning also had higher EIs, although the reported presence of dust in the home was not associated with EI.Sociodemographic factors did not differ by the ∑EI  among participants . The coh (n = 3) . Answers (n = 3) . Notablyn = 39, 92, 127, and 143, respectively).The pattern of EI versus time as shown in p < 0.0001; PAH-DNA adducts were measured in 160 women. Most were nondetectable ; the median of detectable values was 60 apmn. Eleven women had levels higher than 100 apmn, and nine of these women had blood drawn in February or March 2002 , a time rS = 0.20 for Co and 0.21 for Pb; p < 0.05), were not significant among the February–March biospecimens . The median values of four urine metal concentrations in rS = 0.29, p = 0.01), but the comparison with ∑EI above and below the median was not significant for any of these . No significant associations of blood metals with exposure were seen among the February–March biospecimens.Thirteen urinary metals as well as Pb, cadmium, and Hg in blood were measured in a subset of 100 women. After comparing the percentage detected and the median values of the urinary levels in our data with data from earlier reports , in TablSelected organohalogen compounds  were measured in blood plasma in the same randomly selected subset of 100 women as were the metals. Data on specific PCBs, PCDFs PCDDs, and PBDEs presented in We performed multivariate analyses to evaluate the association of analytes that showed some pattern of association with ∑EI, by computing the geometric means of the biomarker (dichotomized above vs. below the ∑EI median) adjusted for age, nonwhite race, being currently pregnant, breast-feeding, body mass index, and smoking. We also adjusted models for PCBs and Hg for fish intake. We found no significant differences in the adjusted geometric means with regard to dichotomized ∑EI for PCBs, OCDDs, PBDEs, Pb (blood and urine), Cd, or Hg among all 100 women tested or among the 44 women who donated blood samples in February–March 2002.10 and PM2.5 as close as six blocks northeast of the site (2.5 levels measured five blocks east of the site from 14 September through 16 October 2001 were 15–60 μg/m3 (2.5 were > 60 μg/m3 (one to two blocks away). At Chambers Street (six blocks north-northwest), the median PM10 level was 43 μg/m3 and the median PM2.5 level was 10 μg/m3 in October 2001. At 290 Broadway (six blocks northeast), the median PM2.5 level was 22 μg/m3 in late September (n = 4 days) and 15 μg/m3 in October, whereas the median PM10 levels were 32 and 30 μg/m3, respectively, during those time periods. However, daily excursions of PM10 and PM2.5 levels near or higher than 100 μg/m3 occurred periodically, depending on site operations, weather, and traffic . At Public School 64 (about 50 blocks northeast), PM2.5 levels were 11.2 μg/m3 in September and 13.8 μg/m3 in October 2001 (median), which is similar to annual data at Public School 64 . Women in our cohort in zone 1 were probably exposed to > 100 μg/m3 of particulates on 11 through 12 September, based on our extrapolation of levels of PM2.5 and PM10 from the daily EI correlations with the air monitoring data. The U.S. EPA 24-hr standard is 65 μg/m3 for PM2.5 and 150 μg/m3 for PM10 (The predominant air pollutant at the WTC was dust composed of large-size particles (> 10 μm) . Air monthe site . Daily P60 μg/m3 . At the  traffic . Some difor PM10 .As the dust exposures in lower Manhattan gradually diminished in weeks 1–4 after 11 September, women gradually returned to that area for longer time periods. Therefore, fluctuation in the daily EIs  reflectsUncertainties exist in our exposure estimates, including the recall data and the plume reconstruction. We used recall information to derive the GIS time–activity variables and to obtain data on PAQ from the exposed women. Plume reconstruction was used to calculate the GIS dust exposure levels. There were no air monitoring data during the first 24 hr after the events of 11 September, and the plume dust level estimates for the GIS were evaluated for precision during that time using plume density derived from satellite photographs.3 in the PM2.5 fraction soon after the incident compared with < 1 in normal urban air and in the plume later on .In summary, we were able to successfully incorporate results of a WTC plume reconstruction model into predicted exposures and doses of airborne emissions among these women who were near the WTC immediately after 11 September. The results provided daily estimates of exposure by using both geographic dust concentrations and individual activity patterns to predict individual EIs among 187 women for 28 days. Women experienced very high dust exposures; as a result of being close to the WTC in September 2001, the PM exposures were likely > 100 μg/mThe values listed in “Materials and Methods” for the sample  were incorrect in the online version. Also, in


Despite the high incidence of cervical cancer reported from India, large scale population based studies on the HPV prevalence and genotype distribution are very few from this region. In view of the clinical trials for HPV vaccine taking place in India, it is of utmost importance to understand the prevalence of HPV genotypes in various geographical regions of India. We investigated the genotype distribution of high-risk HPV types in squamous cell carcinomas and the prevalence of high-risk HPV in cervicovaginal samples in the southern state of Andhra Pradesh (AP), India.HPV genotyping was done in cervical cancer specimens (n = 41) obtained from women attending a regional cancer hospital in Hyderabad. HPV-DNA testing was also done in cervicovaginal samples (n = 185) collected from women enrolled in the cervical cancer screening pilot study conducted in the rural community, of Medchal Mandal, twenty kilometers away from Hyderabad.High-risk HPV types were found in 87.8% (n = 36/41) of the squamous cell carcinomas using a PCR-based line blot assay. Among the HPV positive cancers, the overall type distribution of the major high-risk HPV types was as follows: HPV 16 (66.7%), HPV 18 (19.4%), HPV 33 (5.6%), HPV 35 (5.6%), HPV 45 (5.6%), HPV 52 (2.8%), HPV 58(2.8%), HPV 59(2.8%) and HPV 73 (2.8%). Women participating in the community screening programme provided both a self-collected vaginal swab and a clinician-collected cervical swab for HPV DNA testing. Primary screening for high risk HPV was performed using the Digene Hybrid Capture 2 (hc2) assay. All hc2 positive samples by any one method of collection were further analyzed using the Roche PCR-based line blot for genotype determination. The prevalence of high risk HPV infection in this community-based screening population was 10.3% (19/185) using the clinician-collected and 7.0% (13/185) using the self-collected samples. The overall agreement between self-collected and clinician-collected samples was 92%; however among HPV-positive specimens, the HPV agreement was only moderate (39.1%). The most frequently detected HPV types in the Medchal community are HPV 52 and 16.Our results suggest that the HPV type distribution in both cervical cancer tissues and in a general screening population from Andhra Pradesh is similar to that reported in India and other parts of the world. We also conclude that an effective vaccine targeting HPV 16 will reduce the cervical cancer burden in AP. Cervical cancer is one of the most common malignancies and is the major cause of cancer mortality among Indian women. ,2. CytolIn the past decade, a strong etiologic association between infection with high-risk HPV types and development of cervical cancer has been established, and vaccines targeting HPV 16 and 18 have been shown to prevent persistent HPV infections in clinical trials ,8. Mass Most studies in India have only assessed the prevalence of HPV 16 and 18 in cervical cancer tissues. In spite of the fact that cervical cancer burden in India is high, there are very few large scale studies from India describing either HPV prevalence or type distribution in the general population and in invasive cervical cancer . Because of subtle regional cultural differences that exist in various states in India, it is important to describe the distribution of HPV genotypes in cancer cases and community samples from multiple representative populations before these data can be generalized for application in national cancer prevention strategies. The present study reports the HPV type distribution in a rural community (20 Km from Hyderabad city) and invasive cancer samples from women attending the regional cancer centre in Hyderabad.For the cancer study conducted at the MNJ regional cancer hospital (between 2002–2003), women were consecutively recruited at the time of their visit to the cancer clinic. A tissue biopsy was collected from women clinically diagnosed with cervical cancer and who gave consent (n = 45). Following the cervical punch biopsy, a small piece of tissue was sent for histopathology and the rest of the specimen was snap frozen in liquid nitrogen and stored at -70°C. Out of the total 45 women, DNA was extracted from forty-two histopathologically proven cases of squamous cell carcinomas. Histopathology was not available from the remaining 3 participants. The study protocol was approved by all participating institutional bioethical committees.A community-based sample of women aged 30 years and above was recruited from a single village in Medchal Mandal during the period July–October 2003. These women were participating in a cervical cancer screening pilot study entitled Community Access to Cervical Health, or CATCH. A total of 657 age-eligible women were recruited to participate. Consent was obtained from women participating in the study who had not undergone hysterectomy and were not pregnant. Of the 489 women who were eligible, 190 (38.9%) consented to participate and were enrolled. Of the 190 women, HPV analysis was done for 185 women. Both self-collected vaginal samples and clinician-collected cervical samples were collected using a Digene sampling brush which was placed into Digene STM collection medium. Samples were stored at 4°C for no more than 24 hours. Before aliquoting, the STM tubes are vortexed to dislodge cells and then aliquoted without removing the brush. The aliquoted samples were stored at -80°C.At the MNJ cancer hospital the attending nurse explained the procedure and written consent obtained. For the Medchal community study, the consent was read aloud to the participants as a group. Each eligible participant was then asked privately if they had any questions regarding study participation and written consent was obtained.The samples collected in the STM tubes were vortexed to dislodge from the brush and 200 μl aliquots from both the self and clinician collected samples were transported to Centre for DNA Fingerprinting and Diagnostics (CDFD) for HPV testing. Aliquotting was done prior to denaturation of the sample for the hc2 test in order to preserve the integrity of the DNA for future PCR analyses. Each sample was tested for the presence of one or more high risk HPV types using probe B of the Hybrid Capture 2 (hc2) assay  according to the manufacturer's instructions. The hc2 test is based on the hybridization of HPV DNA with RNA specific probes and they hybrid detected by a chemiluminescent assay. The values obtained are recorded as relative light unit (RLU) per positive control (PC). The sample is considered positive for HPV-DNA when the RLU/PC ratio is greater than or equal to one.HPV genotyping was performed on all cervical cancer tissues and on cervicovaginal samples which were hc2 positive by either collection method. The PCR and line blot reagents were kindly provided by Roche Molecular Systems, Inc. The cervical punch biopsy (approximately 50 mg) was pulverized using liquid nitrogen, suspended in 1000 μl of digestion buffer , extracted using standard phenol:chloroform methods , and resAgreement between self- and clinician-collected samples was calculated using kappa statistics to provide estimates beyond chance agreement. To test for differences in HPV prevalence by age, we calculated Pearson's chi-square. Results were considered statistically significant at p < 0.05. All frequencies, kappa estimates, and chi-square tests were computed using Stata/SE version 9.0 .A total of 42 specimens were tested. The age range of the women was 30 to 65 years with a median age of 55 years. The majority of women attending MNJ for cancer diagnosis and treatment are of low socioeconomic status (average income 1000–1500 Rs per month) based on data from hospital records. However, specific details regarding the socioeconomoic or demographic status were not collected from individual women participating in the invasive cancer study. HPV genotyping of the samples was done using the line blot assay and of the total 42 samples tested, one was negative for beta-globin and was excluded from further analyses. Of the 41 tissues with satisfactory β-globin amplification, 36 (87%) were HPV positive. Among the 36 HPV positive tissues, HPV 16 was the most prevalent type detected , followed by HPV 18 (7/36 19.4%). Three of the HPV16 positive specimens were co-infected with other HPV genotypes  consented to participate and were enrolled. Women aged 30–45 were twice as likely to participate (60.4%) relative to the next age group . Of the 190 women, HPV analysis was done for 185 women. The median household income reported by the women in the community study was 1500 Rs, which is similar to the general characteristics of the population served at the regional cancer hospital.Of the 190 women enrolled, 185 had samples collected for HPV testing. HPV genotyping was done in women who tested hc2 positive in either the self- or the clinician-collected sample. Approximately 10.3% (19/185) of clinician-collected cervical samples tested positive for high-risk HPV types by the hc2 assay. The HPV prevalence was similar across age strata  of the self-collected vaginal swabs were positive for high-risk HPV using the hc2 assay. When tested by PCR, two of the vaginal swabs failed to amplify the β-globin control, leaving a total of 11 hc2-positive self-collected samples available for analysis. Of these, 7 (64%) were confirmed as high-risk HPV positive samples, two of which were positive for multiple HPV genotypes resulting in total of 11 infections. HPV 16 and 52 were each present in 2 of the 11 infections (18%), and HPVs 18, 31, 39, 58, 62, 81, and 84 detected once.Of the five hc2-positive samples in the clinician-collected set that were not confirmed as positive by PCR, three had RLU/PC value ratios between 1.38 and 2.26, while the remaining two had somewhat higher RLU/PC ratios of 4.5 and 14.38. All four hc2-positive self-collected samples that were PCR negative had RLU/cutoff ratios ranging from 1.17–3.91. One of the self-collected samples although negative by the hc2 assay showed the presence of oncogenic HPV type 52 on line blot analysis.The overall agreement of the hc2 results between the two sampling methods was good  collected from women attending a cancer clinic at the regional cancer hospital in Hyderabad, Andhra Pradesh. High risk HPV prevalence was approximately 87.8% in our sample of invasive squamous cell carcinomas. In the present study, low risk HPV types were not detected in the carcinomas. A case-control study undertaken in Chennai, southern India, reported HPV prevalence as high as 99.4% in their invasive cancer samples . Both stThe most prevalent HPV types found in the invasive cervical cancers in Andhra Pradesh were HPV 16 (66.7%) followed by HPV 18 (19.4%). The distribution of HPV types found in our study is quite similar to a recent large-scale study reported from India and is also consistent with the most common types found in South-East Asia -14,21,22While the incidence of cervical cancer is high in the state of Andhra Pradesh, with an age adjusted rate of 10.3 per 10,000 women  few dataOur HPV prevalence (10.4%) is very similar to two large population-based studies of largely cytologically normal women. Sankarnarayanan, et al. reported 10.3% high risk HPV prevalence as detected using hc2 testing in Osmanabad District in West India , and somWe evaluated HPV-DNA testing using both clinician- and self-collected samples from each woman. In this pilot study, the initial screening for high risk HPV DNA was done by Hybrid Capture 2 with positive samples tested for HPV genotype using a PCR-based line blot assay. There was a considerable variation in the RLU/PC values for the self and clinician collected samples, however more than half of the discordant samples had viral loads <10 RLU/PC. Similarly, most hc2-positive samples that were not confirmed by PCR had low viral load suggesting sampling error as a potential source of variability when sample viral loads are near the assay sensitivity threshold. Further, it is possible that the discrepancy between hybrid capture positive and PCR negative samples in samples with RLU/CO less than 5 is because of non-specific binding in the hc2 assay leading to false positive results.Clinician collected samples (10.3%) showed a slightly higher HPV prevalence relative to the self collected samples (7%) in the pilot study. The good overall agreement is reflective of a large number of HPV negative samples; continued evaluation of HPV testing in this community with a larger sample size will be required to determine the relative performance of self- vs. clinician-collected samples for HPV testing. Self-sampling as a means to monitor HPV infection post-vaccination may prove to be a valuable tool in post-immunization surveillance in India.The most prevalent HPV genotypes in the general population of Medchal is HPV 52 followed by HPV 16, which differs slightly from that of rural community in Chennai where the major types were HPV 16 and 56. However, screening of larger samples size in Medchal will give a better picture on the distribution of the HPV types.Our efforts are therefore continuing in the characterization of HPV genotypes prevalent in the rural areas of Andhra Pradesh. The successful completion of our ongoing studies will help in (a) understanding the distribution and prevalence of HPV types in Medchal Mandal community of Andhra Pradesh, (b) feasibility of self collection methods for HPV-DNA testing in India as an alternate to clinician based sampling, and (c) comparing the three different screening modalities . Furthermore, combining our results with the ongoing IARC study conducted in the Mahabub Nagar district of Andhra Pradesh and the recently completed IARC assisted studies will help in predicting the most cost-effective method of cancer cervix screening programmes in India . A compaOur results confirm the earlier studies on the role of high risk HPV infection as a major risk factor development of cancer cervix. Further, the distribution of high risk HPV types in Hyderabad is similar to those reported from a recent study conducted in Southern India. The prevalence of HPV-16 in the cancer samples suggests that effective vaccination against HPV 16 can considerably bring down the cancer burden in the southern states of India.Keerti Shah and Patti Gravitt were responsible for the overall planning and coordination of the Medchal community based study. Usha Rani Poli, Radha Rama Devi, B.N.Rao helped in providing the invasive cancer specimens for the study. Pavani and Padma were involved in the cancer cervix sample collection. Pavani was responsible for HPV-DNA testing on all the samples using line blot and Hybrid capture-II assay. Meenakshi Jain collected all the cervicovaginal samples from women enrolled in the CATCH programme. Manik Das was responsible for biospecimen processing and inventory. Patti Gravitt provided all the expertise involved with the HPV Line blot assay system. Gayatri Ramakrishna was involved in the coordination and setting up of the HPV-DNA testing system at CDFD and together with Patti Gravitt compiled and finalized the manuscript.The author(s) declare that they have no competing interests. We have received reagents from Roche Molecular Systems. These are the same reagents used in this manuscript.The pre-publication history for this paper can be accessed here:


Calcineurin (CaN) is an important serine-threonine phosphatase (PP2B), which plays a crucial role in calcium-calmodulin mediated signal transduction events. Calcineurin has been implicated in pathogenesis of various diseases cardiac hypertrophy, diabetic neuropathy and Alzheimer's, however its role in neoplasia remains unclear.In view of this we evaluated the calcineurin activity in serum and biopsy samples collected from women diagnosed with invasive squamous cell carcinoma of cervix. A significant reduction was observed in the calcineurin activity in cancer cervix patients compared to the control group. However the calcineurin activity remained unaltered in the cervical scrapes obtained from patients diagnosed with low-grade squamous intra epithelial lesions (LSIL). Interestingly the downregulation of calcineurin activity in squamous cell carcinomas was not accompanied by any significant change in DNA-binding affinity of the transcriptional factor NFAT (Nuclear Factor of Activated T-cells). All the squamous cell carcinoma samples used in the present study were positive for high-risk human papillomavirus (HPV) types.The present study demonstrates the downregulation of calcineurin activity in squamous cell carcinoma of cervix with high risk HPV infection. We conclude that perturbations in calcineurin-mediated pathway may be involved in development of cervical neoplasia. Cervical cancer is one of the most prevalent neoplastic diseases affecting women world wide, especially in the developing countries ,2. In InThere is a plethora of information existing on the involvement of various kinases such MAPK , PI3K 99, JNK 1, cdk4 11,12, cdc0/G1 checkpoint element  from both the normal and cancer patients. Blood was allowed to clot and serum was separated by centrifugation at 3000 rpm for 10 min at room temperature. The serum samples were stored at -20°C till further use.Cervical scrapes were also collected using Ayer's spatula from women attending the cervical screening outpatient clinic (N = 30). Four of these women were found to have low-grade intraepithelial lesion (LSIL) and the rest with chronic inflammation only on cytology. The cervical scrape was collected in chilled phosphate buffer saline (pH 7.2) and the cells were collected by centrifugation at 13000 rpm for 10 min at 4°C. The supernatant was discarded and the cells were immediately frozen in liquid nitrogen till further use.2, 10% glycerol, 10 μg/ml PMSF and 10 μg/ml aprotinin for 3 min. The homogenates were cleared of the debris by centrifugation at 13,000 rpm for 10 min at 4°C. The supernatants were aliquoted and stored at -70°C till further use.Tissues (100 mg) and cervical scrapes were homogenized in 500 μl or 250 μl of buffer A respectively, containing 25 mM HEPES (pH 7.2), 150 mM NaCl, 1% NP-40, 10 mM MgCl2, 1 mM CaCl2) and 5 μl of the cell/tissue homogenate. The samples were incubated with 2X-assay buffer for 10 min at 30°C. The total volume was adjusted to 50 μl by the addition of water. Reactions were initiated by adding RII phosphopeptide [5 μM] and incubated for a further period of 10 min at 30°C. Reactions were terminated by addition of 100 μl of Malachite green reagent . The color was allowed to develop for 30 min and the absorbance read at 660 nm. The calcineurin activity was calculated as nmoles of inorganic phosphate released/min/mg total protein in case of tissues and for serum samples as nmoles of inorganic phosphate released/min/dl of serum.An aliquot of the homogenized sample (100 μl) was passed through sephadex G-25 column (Amersham Pharmacia) to eliminate the free phosphate and further used for calcineurin activity. The protein estimation was done using the BCA Kit [Pierce company] as per the manufacturers protocol. Calcineurin activity was assayed in a total volume of 50 μl containing 25 μl of 2X assay buffer  and the labeled oligo for 30 min at room temperature. The reactions were terminated using 25 μl of the loading dye and loaded onto a 7% gel retardation assay gel. The run was done at 250 V for 3 hrs at 4°C. At the end of the run, the gel was removed, dried and exposed in a phosphoimager overnight at room temperature.Electrophoretic mobility shift assay (EMSA) was performed using oligonucleotide with consensus binding site for NFATc (5'CGCCCAAAGAAAATTTGTTTCATA3'). Briefly, biopsy tissues were homogenized and the nuclear pellet was collected by centrifugation . The nuclear lysates (100 μg) were prepared and incubated in 25 μl of reaction mixture containing polydI/dC, 10X binding buffer (50 μM ZnCl The DNA isolated from the squamous cell carcinoma samples were tested for presence of HPV by a PCR based reverse line blot assay as described earlier (40)t-test for paired data.Comparisons between the normal and the cancer samples were done using the Student's Calcineurin activity was assayed in both sera and tissue lysate by measuring the dephosphorylation of RII peptide, a specific substrate of calcineurin. The biopsy samples collected from the cancer patients were classified as moderately differentiated large cell non-keratinising squamous cell carcinomas of the cervix. We observed a significant reduction in the calcineurin activity both in the cervical tissues p = 0.0001) and sera (p = 0.0037) of patients with invasive cervical cancer in comparison to the control group  by cytology and the rest were cytologically normal but with chronic inflammation. We didn't observe any significant change in the calcineurin activity between the LSIL and the controls in the cervical scrapes Fig. .In view of our observation on downregulation of calcineurin activity in cervical carcinoma, we wanted to investigate if there is any alteration in the DNA-binding activity of NFAT, one of the major substrates of calcineurin. We analyzed the nuclear extracts from 5 squamous cell carcinoma and 5 controls by incubating it with the radiolabelled oligonucleotide probes specific for NFAT binding. Interestingly we didn't find any significant change in the DNA binding affinity of NFAT between cancer and the control samples  was used to check the presence of HPV types in the squamous cell carcinomas. We could detect oncogenic HPV-types  in all of the cervical cancer but none in the control samples, however we could not correlate the presence of HPV-types with any variation in calcineurin activity (data not shown).The present study shows downregulation of calcineurin activity in invasive cervical cancer. High calcineurin activity can lead to apoptosis , thereby2+-Zn2+ binuclear center in its active site is oxidized to Fe3+-Zn2+ by superoxide and hydrogen peroxide ions [The basis for the decrease in calcineurin activity observed in cervical neoplasia in absence of a change in Calcineurin A content needs to be identified and we speculate that a change in redox state of the cancer cell due to oxidative stress contributes to the alteration in the activity of calcineurin without altering its content. Calcineurin by virtue of its Feide ions . Superoxide ions ,44. Low ide ions ,46, whicide ions  and it cide ions . Anotheride ions ,49. The ide ions , which iThe natural history of invasive cervical cancer involves sequential progression of persistent HPV infection to reversible LSIL (CIN1) and then to high-grade intraepithelial lesions (HGIL/CIN2). In the presence of other precipitating factors HSIL is a direct precursor of invasive cancer . To evalThe regulation of gene expression in response to calcium stimuli is one of the most explored functions of calcineurin. Importantly, the critical target of calcineurin is the NFAT family of transcription factors. Calcineurin dephosphorylates NFAT allowing translocation of this protein into the nucleus . StudiesIn conclusion, our pilot study is indicative of a possible role of calcineurin-mediated pathway in pathogenesis of cervical neoplasia. While NFAT is a major target of calcineurin, our study rules out the involvement of this transcriptional factor indicating presence of a NFAT independent calcineurin pathway atleast in cervical cancer. The present investigation has opened up new avenues to evaluate further the role of calcineurin in cervical cancer progression.The author(s) declare that they have no competing interests.SP carried out the calcineurin assays and data compilation. HPV testing and typing in cancer cervix samples was carried out by APS. Both SP and APS performed NFAT assay. The samples were provided by URP, MJ and BNR. The study was conceived and coordinated by GR. The manuscript was finalized by GR and approved by all the investigators.