Identifying syntenic regions, i.e., blocks of genes or other markers with evolutionary conserved order, and quantifying evolutionary relatedness between genomes in terms of chromosomal rearrangements is one of the central goals in comparative genomics. However, the analysis of synteny and the resulting assessment of genome rearrangements are sensitive to the choice of a number of arbitrary parameters that affect the detection of synteny blocks. In particular, the choice of a set of markers and the effect of different aggregation strategies, which enable coarse graining of synteny blocks and exclusion of micro-rearrangements, need to be assessed. Therefore, existing tools and resources that facilitate identification, visualization and analysis of synteny need to be further improved to provide a flexible platform for such analysis, especially in the context of multiple genomes..We present a new tool, Cinteny, for fast identification and analysis of synteny with different sets of markers and various levels of coarse graining of syntenic blocks. Using Hannenhalli-Pevzner approach and its extensions, Cinteny also enables interactive determination of evolutionary relationships between genomes in terms of the number of rearrangements . In particular, Cinteny provides: i) integration of synteny browsing with assessment of evolutionary distances for multiple genomes; ii) flexibility to adjust the parameters and re-compute the results on-the-fly; iii) ability to work with user provided data, such as orthologous genes, sequence tags or other conserved markers. In addition, Cinteny provides many annotated mammalian, invertebrate and fungal genomes that are pre-loaded and available for analysis at Cinteny allows one to automatically compare multiple genomes and perform sensitivity analysis for synteny block detection and for the subsequent computation of reversal distances. Cinteny can also be used to interactively browse syntenic blocks conserved in multiple genomes, to facilitate genome annotation and validation of assemblies for newly sequenced genomes, and to construct and assess phylogenomic trees. The increasing number of newly sequenced genomes is stimulating interest in comparative genomics and highlighting the need for the development of efficient tools that enable assessing their evolutionary relatedness. One problem of central importance is the identification of blocks of genes (or other markers) with evolutionary conserved order. These synteny blocks help in tracing back the evolution of genomes in terms of rearrangement events, such as reversals, translocation, fusion, fission etc. Consequently, genome evolution and phylogenetic (or phylogenomic) trees can be reconstructed from the analysis of synteny -5. FurthThe evolutionary distance between two genomes can be expressed in terms of the minimum number of reversals to transform one genome into another, which is also referred to as the reversal distance -5. In thA number of browsers, including Ensembl SyntenyView ,10 and NThanks to remarkable algorithmic developments, the reversal distance (RD) can be efficiently computed in linear time ,8. UtiliIn order to efficiently represent the linear order of markers on a genome, Cinteny uses a tailored data-structure, which is implemented as a straightforward extension of ternary search trees (TST) . In partMouse Srm → Mouse E2f2 → Mouse Wnt4. The strandedness (orientation) of a gene is stored in the node using an additional variable, and thus the whole genome may be formally represented as a signed permutation. The latter is required in order to apply the Hannenhalli-Pevzner theorem and based on it efficient algorithmic solutions [Figure Nlog(N) where N is the number of markers), which makes it is suitable for large genomes. As result, a typical query, involving computation of the reversal distance for a pair of mammalian genomes, takes only a few CPU second on a typical desktop PC.There are several advantages of this hybrid data-structure: i) a gene may be searched in logarithmic time as the complexity of TST is similar to a binary search tree; ii) starting with a gene, one can directly enumerate all orthologs; iii) starting with a gene, one can visit the genes upstream and downstream by traversing up or down the walk. These factors facilitate the identification of conserved blocks and their representation in the form of a signed numeric permutation in linear time. Consequently, the reversal distance can be computed in linear time overall, using the approach by Hannenhalli and Pevzner , with moInitially, we define the synteny blocks by identifying blocks of conserved markers without any disruption of order and all the genes or other markers having the same relative orientation, i.e., two signed permutations representing these blocks being identical up to one reversal operation. These initial blocks with perfectly conserved order are subsequently extended (aggregated) into larger (non-overlapping) synteny blocks by ignoring smaller blocks, such as those resulting from micro-rearrangements. The aggregation strategy used here is based on that of Peng and colleagues  and invoThese parameters effectively define the extent to which small divergences are tolerated within extended synteny blocks. We note also that many divergences in gene (marker) order may involve markers only observed in some genomes; these are thus automatically filtered out when multiple genomes are used, as described in the next section. The resulting coarse-graining leads to further computational simplifications, making Cinteny suitable for large scale comparisons of multiple genomes and a comprehensive sensitivity analysis of the results.Finding synteny blocks and the reversal distance typically involves identifying markers  that are shared by two species of interest. For example, the synteny between human and mouse can be analyzed using 15,645 orthologs, as identified by HomoloGene . Using CCinteny enables analyses of genome rearrangements at three different levels: genome-wide, chromosome or around individual genes. The graphical layer for synteny browsing and analysis is built using PHP and enables intuitive interpretation of the results. All graphical representations are cross-linked and can be browsed interactively. For example, one can easily jump from a genome level view to a chromosome level view, or further magnify a synteny block for a detailed study. The graphical elements are also linked to external resources, such as NCBI. Public domain packages, including GBrowse  and SynVIn this section, we demonstrate examples of interrogating evolutionary relatedness of genomes using the Cinteny server. Different types of queries and different setup of parameters are illustrated. In particular, we show how the Cinteny web server can be used to identify synteny blocks and compute the reversal distance (RD) between whole genomes as well as between any two chromosomes of two genomes.In order to perform sensitivity analysis for the computation of RD and identification of synteny blocks, Cinteny allows the user to interactively adjust several parameters, including: i) minimum length of synteny blocks (denoted as min_len); ii) maximum gap between adjacent blocks for aggregation (denoted as max_gap); iii) minimum number of markers in a block (denoted as min_num). Aggregation refers here to combining smaller synteny blocks to form larger blocks, wherever feasible. For example, by increasing the length between adjacent blocks (max_gap), one may effectively join segments which are otherwise far apart and obtain longer synteny blocks. Thus, such aggregation provides an effective coarse-graining of synteny blocks and affects the resulting RDs.Another problem in applying algorithms for synteny block identification is posed by degenerate markers, such as paralogs . In particular, the Hannenhalli-Pevzner theorem assumes that markers are unique . In ordeFinding synteny and the RD between whole genomes is discussed here as an example of a typical application in comparative genomics. Figure The figure was generated using a particular level of coarse-graining (aggregation), as defined by min_len = 300 Kb, max_gap = 1 Mb and min_num = 3. The number of synteny blocks found with these parameters and using the set of 15,645 human-mouse orthologs, as identified by HomoloGene 46.1 , is 359 Cinteny can also be used to identify synteny blocks and reversal distance between two chromosomes. In this case we use common markers located on the chromosomes of interest. In Figure In addition to queries illustrated above, one may use Cinteny for visualization and analysis of the synteny around a specific marker or gene. For example, starting with human and mouse genomes and default aggregation parameters, we find that the human BRCA1 gene is present in a conserved region of size 7.2 Mb in human chromosome 17, whereas its mouse ortholog (Brca1) is present in a conserved region of size 6.1 Mb on mouse chromosome 11  when using the 2-way approach, which can be easily verified using the Cinteny server. It also interesting to note that in relative terms  the reversal distances appear to be quite constant for a range of aggregation parameters, especially when using the multiple genome approach (data not shown). This may suggest that RDs can be used to indicate evolutionary relatedness in relative terms, as long as proper parametrization of the problem is used. This is the subject of a future work.To further illustrate the usefulness of multi-way approach, we performed a 5-way analysis for the X chromosome of mouse and rat without any aggregation, yielding the RD of 14 and 19 synteny blocks. Comparison with Figure The identification of synteny blocks, and the subsequent calculation of the reversal distance, is highly sensitive to the choice of parameters. Therefore, sensitivity analyses and careful assessments of the choice of critical parameters are important for drawing meaningful conclusions from inter-genomic comparisons. We present a new tool which offers a flexible platform for such analysis, enabling customization of synteny block detection and sensitivity analysis for the resulting estimates of evolutionary relatedness and plausible scenarios of genome rearrangements from ancestral genomes.Cinteny performs all computations on the fly, allowing the parameters to be adjusted and the results recomputed interactively. Since the computation of the reversal distance for a pair of mammalian genomes takes only about a few seconds, one can easily assess the effects of various approximations and different levels of coarse-graining. For example, blocks larger than 300 kb are typically used for analysis of mammalian genomes. However, a different threshold may be more appropriate for smaller genomes, e.g., fungal genomes. In particular, a natural aggregation of synteny blocks can be achieved using multiple genomes. In addition, the effect of paralogs may be assessed by choosing a range of options, from removing all paralogs to using the ones that are contained within the largest conserved blocks.While some well-annotated genomes like human, mouse, rat, dog, chimpanzee, nematode, drosophila, fungal genomes, etc. are pre-loaded, Cinteny also allows users to upload their own data. Hence, it can readily be used for the analysis of newly sequenced genomes, with user provided set of markers. Automated identification of conserved sequenced tags for multiple, unannotated  genomes is being implemented as an extension of the current work.• Project Name: Cinteny• Project home page: • Operating System(s): Platform independent (Web-based)• Programming languages: C++ and PHP• Other Requirements: None• License: GNU GPL• Restriction to use by non-academics: license needed.RD – Reversal DistanceJM conceived of the study. AUS and JM designed the software and AUS implemented it. AUS and JM drafted the manuscript. All authors read and approved the final manuscript.


Viruses including Epstein–Barr virus (EBV), a human equivalent of murine mammary tumour virus (MMTV) and human papillomavirus (HPV) have been implicated in the aetiology of human breast cancer. We report the presence of HPV DNA sequences in areolar tissue and tumour tissue samples from female patients with breast carcinoma. The presence of virus in the areolar–nipple complex suggests to us a potential pathogenic mechanism.In situ hybridisation supported the PCR findings and localised the virus in nipple, areolar and tumour tissue.Polymerase chain reaction (PCR) was undertaken to amplify HPV types in areolar and tumour tissue from breast cancer cases. Papillomavirus DNA was present in 25 of 29 samples of breast carcinoma and in 20 of 29 samples from the corresponding mamilla. The most prevalent type in both carcinomas and nipples was HPV 11, followed by HPV 6. Other types detected were HPV 16, 23, 27 and 57 (nipples and carcinomas), HPV 20, 21, 32, 37, 38, 66 and GA3-1 (nipples only) and HPV 3, 15, 24, 87 and DL473 (carcinomas only). Multiple types were demonstrated in seven carcinomas and ten nipple samples.The data demonstrate the occurrence of HPV in nipple and areolar tissues in patients with breast carcinoma. The authors postulate a retrograde ductular pattern of viral spread that may have pathogenic significance. Breast cancer is the most frequently diagnosed cancer among women in the USA, and the incidence of breast carcinoma has increased by more than 40% over 25 years . The aetViral infection as an aetiological factor has been addressed in other studies. The data obtained from studies investigating the presence of viral sequences in breast cancer biopsies and cell lines have been controversial. A role for herpes viruses, and specifically Epstein–Barr virus (EBV), in the aetiology of this cancer has been questioned ,10. A huSamples from breast tumour tissue for the HPV analysis were supplied by two of the authors (RES and CEB). The 29 cases used for the retrospective study and demonstration of papillomavirus sequences were selected from routine mastectomy specimens undertaken in a course of treatment for carcinoma of the breast. Case selection was based on the availability of adequate nipple and tumour tissue from the same patient and the presence of histological features suggestive of HPV infection in nipple and areolar sections. The patients, all female, ranged in age from 30 to 88 years, with an average age of 61.3 years. Twelve tumours arose in the right breast and 17 in the left. Five tumours were subareolar and 24 were located at a distance from the nipple and areola. Tumours ranged in size from 6 mm to 15 cm. One patient received cytoxan chemotherapy before surgery. In all other cases primary treatment was surgical as represented by the specimen. One patient had undergone contralateral mastectomy ten years earlier for a localised breast cancer, which was judged clinically and pathologically to represent a separate primary tumour. Among the 29 patients there were three with remote histories of malignancy without evidence of recurrence . One patient had undergone hysterectomy for high-grade cervical dysplasia. There were no patients with documented cervical carcinoma. No patients were immune-impaired or immunosuppressed.n = 29) and the carcinoma of the breast (n = 29) from the same patient were sectioned, and the total DNA was extracted from each sample. Great care was taken during sectioning to avoid any contamination between samples. Two different individuals sectioned the blocks in several small random groups at different time intervals over a period of one week. The microtome was cleaned thoroughly and exposed to ultraviolet between samples; in addition, new blades were used for each sample. Deparaffinisation was performed by rotation overnight in 1 ml of xylene, followed by centrifugation and subsequent removal of the supernatant. This step was repeated twice (each for one hour) with fresh xylene. The xylene was in turn removed by rotation in 1 ml of 100% ethanol for one hour followed by centrifugation and subsequent replacement of the supernatant with 90% ethanol for 45 min, and repeated by steps of 80% ethanol for 45 min and 70% ethanol for 45 min. Samples were freeze-dried, and total cellular DNA was extracted as described previously [Formalin-fixed paraffin-embedded samples from both the nipple n =  and the +/GP6+ primers [2 using 40 cycles with an annealing temperature of 40°C. The expected size of the amplicon was 140–150 bp. The expected size with the FAP primers is 480 bp after 45 cycles in 3.5 mM MgCl2 and an annealing temperature of 50°C. The nested amplification approach with the CP primers results in an amplicon size of 370–400 bp. The initial amplification was performed in 2 mM MgCl2 and 40 cycles at 50°C annealing temperature, after which an aliquot was re-amplified with nested primers but with the same PCR conditions. All amplicons were eluted after gel electrophoresis, purified and cloned. At least ten fragments of each amplicon were sequenced. Sequencing was performed with either the Sequenase 2.0 DNA Sequencing Kit  or an ABI Model Sequencer with Big Dye Terminator chemistry . All precautions were taken to avoid contamination before or during extraction of the DNA from the embedded formalin-fixed samples. After initial results had been obtained, a second sectioning was performed on the same embedded samples and DNA was again extracted to ensure that no contamination had taken place during the first round of experiments. This second round of analyses was performed in different rooms and by different individuals.DNA (50–100 μg) of each sample was amplified by polymerase chain reaction (PCR). The quality of the DNA obtained from the fixed samples was controlled by amplification with primers to detect the β-actin gene . The pri primers , the CP  primers  and the  primers . All thrSequences were compared with all available data banks with the aid of the Husar software package (Deutsches Krebsforschungszentrum).In situ hybridisation was performed as described by Kawase and colleagues [in situ hybridisation. Reading of the samples was blinded and performed by one of us (HzH).lleagues . Papillolleagues . This wain situ was identified in lactiferous ducts in four samples and small benign papillomas were present in three samples. Most invasive carcinoma samples (18 of 29) displayed a duct cell carcinoma pattern  for histological features consistent with human papillomavirus infections. Features identified included epithelial hyperplasia, hypergranulosis, parakeratosis, and distorted nuclear shape associated with nuclear clearing and pyknosis suggestive of koilocytosis. In addition, squamous metaplasia of lactiferous ducts and shedding of metaplastic cellular elements into lactiferous ducts and sinuses were noted Fig. . In addi samples 8 of 29 dNormal breast and areolar tissue was not available for use as control samples. The described methods of testing have been used routinely in our laboratory over several years in the analyses of tissues from a large variety of organs. We have analysed large numbers of samples from benign and malignant tumours of the oesophagus, head and neck and skin, as well as normal skin tissue for the presence of papillomavirus DNA with identical methods. HPV positivity varied depending on the type of tumour and normal tissue ,36-38 E. Other HIn situ hybridisation was performed on all samples that had previously been positive by PCR amplification for HPV 6, HPV 11 or HPV 16. Positive signals were clearly distinguishable from the surrounding negative tissue  amplify an overlapping region within the L1 open reading frame. By using all three primer combinations to amplify the DNA of a single sample we are able to detect all known HPV types and also putative new HPV types. In addition, the cloning of the amplified product, in combination with sequencing of the cloned inserts, allows us to distinguish individual HPV types present in one sample, including multiple infections in one sample. Studies applying the techniques described here have demonstrated a larger spectrum of papillomavirus types associated with different types of benign and malignant tumours and argue that the historical grouping into 'mucosal' and 'cutaneous' HPV types can no longer be upheld . We are in vitro conditions [in vivo situation.Other investigators have analysed nipple and areolar specimens for the presence of HPV DNA. HPV 6/11 was detected in one of 20 papillomas of the nipple, whereas ten nipple duct adenomas were negative  and sevenditions -18. It iThe present study detected papillomavirus sequences in 86% (25/29) of the breast carcinoma samples and 69% (20 of 29) of the nipple samples. HPV 6 and HPV 11 infections were most prevalent in our collection of samples. Either of the two HPV types was present in 20 of 29 (69%) of the breast carcinoma biopsies and in 12 of 29 (41%) of the samples of the mamilla. We detected the simultaneous presence of more than one HPV type in about one-third of the samples. HPV types associated with mucosal lesions, and also several associated with skin lesions, were detected. HPV 27 and HPV 57 are closely related HPV types. HPV 27 is very frequently found in cutaneous warts, whereas HPV 57 has been demonstrated in mucosal and cutaneous lesions . Severalin situ hybridisation in our hands lends substantive support to the conjecture that viral sequences are present and not an artefact in the tissues we have examined. The absence of HPV DNA in several samples, as well as the detection of different HPV types in the carcinoma versus the nipple of the same patient, might be attributed to the viral load at the time of sampling in combination with the sensitivity of the tests performed. The tissue sections also harboured unaffected tissue, leading to the dilution of virus-containing cells. In addition, the amplification of viral DNA by PCR with degenerate or consensus primers will detect replicating viral sequences and is not sensitive enough to detect all individual HPV types with equal sensitivity in a mixed population of cells. The in situ hybridisation technique is also less sensitive and will not detect a single viral genome copy per cell.The confirmation of PCR-derived evidence of HPV sequences in nipple, areolar and tumour tissue by in vitro [HPV 6 and HPV 11 are regarded as low-risk HPV types because they have only rarely been demonstrated in premalignant or malignant tissue, and the early genes E6 and E7 do not immortalise primary keratinocytes in vitro . HPV 6 Din vitro ,52 and gin vitro ,54. The in vitro , minimalin vitro ,59 and iin vitro  of HPV 6in vitro studies investigating the influence of hormones, smoke adducts and genetic factors on the interaction of the HPV 6 and 11 proteins with cellular proteins should provide valuable information on a possible role for HPV 6 and 11 in the pathogenesis of breast carcinoma. The data presented in this study also indicate a strong need for epidemiological studies correlating HPV, cigarette smoking, hormone use and family history to substantiate in vitro findings.The mere presence of the low-risk HPV 6 and 11 in most of the mamillae and in the concomitant breast carcinoma samples does not prove its role in the aetiology of the disease, although it merits further investigation. Unfortunately, because of the previous fixation of our samples, RNA from these tumours was not available for the detection of viral transcripts as additional support of our results. Relatively few studies have been performed in an attempt to unravel the intracellular mechanisms through which HPV 6 or 11 may immortalise cells, and information is still fractional in comparison with data on the high-risk HPV types. Introduction of the HPV 6 E6 into normal mammary cells leads to immortalisation and reduced levels of the p53 protein . BindingThis study suggests that human papillomavirus may infect the epithelium of the nipple and areola. Human papillomavirus infection may be identified with recognisable histological features comparable to human papillomavirus infection at other sites. Furthermore, this study is consistent with a pathogenic mechanism involving transfer in a retrograde fashion via the nipple, areola, lactiferous ducts and sinuses of the human papillomavirus. If confirmed, the pathogenic mechanism proposed may have significant implications for the prevention and treatment of breast carcinoma and the identification of individuals at risk for carcinoma development. Examination of cellular samples of nipple and areola inclusive of cytological examination and methods for identifying the presence of human papillomavirus might aid early diagnosis and perhaps therapy.The data demonstrate the occurrence of HPV in nipple and areolar tissues in patients with breast carcinoma. The presence of papillomavirus DNA in most mamillae and concomitant breast carcinoma samples merits further investigation.The author(s) declare that they have no competing interests.EBV = Epstein–Barr virus; HPV = human papillomavirus; MMTV = murine mammary tumour virus; PCR = polymerase chain reaction.


Objective: To determine the rate of positive group B streptococcus (GBS) cultures at 35–37 weeks gestationin women who have first trimester asymptomatic GBS bacteriuria.Methods: Pregnant women with asymptomatic first trimester GBS bacteriuria had genital cultures for GBSperformed at 35–37 weeks gestational age. Serotyping was performed by the standard Lancefield capillaryprecipitin method.Results:  Fifty-three women with positive urine cultures had genital cultures performed at 35–37 weeks. Sixteenof the 53  third trimester vaginal cultures were positive for GBS.Five of eight (63%) of the women with typable urine serotypes had the same typable serotype in the thirdtrimester genital culture.Conclusion: Genital tract cultures at 35–37 weeks for GBS correlate poorly with first trimester asymptomatic GBSbacteriuria. Recommendations for GBS prophylaxis in labor in women who have first trimester asymptomaticGBS bacteriuria should be investigated further and reconsidered.


The aim of the present study was to analyse in invasive carcinomas of the uterine cervix, the anionic glutathione S transferase (GST pi) gene, possibly implicated in the drug resistance of human cancers. Total RNA preparations obtained from invasive cervical cancers (106 specimens), carcinomas in situ (CIS) (three specimens) and normal cervical epitheliums (24 specimens) were analysed by Northern and slot blot hybridisation. A 0.7 kb GST pi transcript band was detected in all the cervical specimens. GST pi mRNA levels were lower in normal cervix (mean: 0.7 +/- 0.1 arbitrary units) than in invasive carcinomas (mean: 2.5 +/- 1.5 units) (Student test P less than 10(-4)). However no significant difference was observed between invasive cancers of advanced stages (III and IV) and those of early stages (I and II). The presence of human papillomavirus in cancers and in normal cervices did not influence significantly the GST pi mRNA level. Neither amplification nor gross rearrangement of GST pi gene could be observed after Southern blot analysis of genomic DNA. In conclusion, our data indicate that the presence of high levels of GST pi transcripts in invasive cancers may be a consequence of the multiple biochemical changes which accompany cervical carcinogenesis.


Parameters characterising the progression of cervical neoplasia were estimated from population-based cancer and mortality statistics in Sweden for 1958-1981 by means of a dynamic computer model. Proceeding from that model and these data, the incidence and prevalence curves were constructed, the effects of the extensive cytological screening measures introduced during the 1960s were assessed, and future gains due to the measures already undertaken up to 1981 could be simulated. About 4,000 cases of cancer in situ were diagnosed annually in Sweden after the end of the 1960s, most of them in women born later than 1919. The maximum reduction in the number of invasive cancers up to 1981 was 42% for women born in 1919-1923, but increased progressively for later birth cohorts and reached 69% for those born in 1934-1938. The corresponding reduction in mortality rates was of the same magnitude. The screening measures up to 1981 will ultimately result in a reduction of invasive cancer by about 12,500 cases and of the number of deaths due to this disease by about 4,100. Only a part of the total gain in the number of lives saved had been revealed at the end of the study period in 1981.


Data from the screening programme in British Columbia are used to test hypotheses about the natural history of cervical cancer, especially about progression and regression of preclinical lesions (dysplasia and carcinoma in situ). Three models are considered. A model without regression does not give an adequate fit of the data (P less than 0.001), and results in an implausible estimate of 33 years for the mean duration of pre-invasive lesions. A model with an equal regression rate at all ages still does not result in a good reproduction of the data. A good fit is achieved for a model with different regression rates in lesions that develop under and over age 34. Under age 34, 84% of the new lesions will regress spontaneously, with a 95% confidence interval of 76-92% regression. Over age 34, we estimate that 40% of the new lesions will regress. The average duration of dysplasia + CIS is 11.8 years, and the sensitivity of the Pap-smear is 80%. It is concluded that a considerable proportion of pre-invasive lesions in young women do not progress. The findings about progression and duration of pre-invasive lesions do not support the still prevailing tendency of frequently making Pap smears in young women.


Schistosomiasis affects the reproductive health of women. Described sequelae are ectopic pregnancy, infertility, abortion, and cervical lesions and symptoms mimicking cervical cancer and STIs. There are indications that cervical schistosomiasis lesions could become co-factors for viral infection such as HIV and HPV.In a retrospective descriptive histopathological study clinical specimens sent between 1999 and 2005 to the pathology department of a consultant hospital in Tanzania were reviewed to analyse the occurrence and features of schistosomiasis in female genital organs.During the study period, schistosomiasis was histopathologically diagnosed in 423 specimens from different organs , out of those 40% were specimens from female and male organs. The specimens were sent from 24 hospitals in 13 regions of mainland Tanzania. Female genital schistosomiasis was diagnosed in 125 specimens from 111 patients. The main symptoms reported were bleeding disorders (48%), ulcer (17%), tumor (20%), lower abdominal pain (11%) and infertility (7%). The majority of cases with genital schistosomiasis were diagnosed in cervical tissue (71 cases). The confirmation of cervical cancer was specifically requested for 53 women, but the diagnosis could only be verified for 13 patients (25%), in 40 cases only severe cervical schistosomiasis was diagnosed. Vulval/labial schistosomiasis was seen in specimens from young women. Infertility was reported in four patients with schistosomiasis of the Fallopian tubes.Genital schistosomiasis adds to the disease burden of women in all age groups. Pathological consequences due to the involvement of different genital organs can be damaging for the affected women. Clinical unawareness of genital schistosomiasis can lead to misdiagnosis and therefore false and ineffective therapy. In endemic areas cervical schistosomiasis should be considered as differential diagnosis of cancer. Ill-health due to reproductive health problems and sexually transmitted infections (STIs) is mostly both preventable and remediable to its nature. These illnesses, notwithstanding, account for almost one fifth of the health burden in women . Many goAlthough both health professionals and the public are aware of the dismal statistics for STIs, few seem to realize that there are other infectious agents than bacteria and virus that can havoc and complicate reproductive health. Symptoms and signs associated with a STI are not uncommon in schistosomiasis, a parasitic helminth disease afflicting around 250 million people, again in the poorest countries where services are limited , but stiSystematic histopathology studies made by pathologists and gynecologists in schistosomiasis endemic areas in the 1970s and 1980s have confirmed the presence of schistosoma eggs and adult worms in upper and lower female genital organs . The relTanzania belongs to the poorest countries in the world . Its heaIn this article we present evidence of female genital schistosomiasis in referred specimens to a pathology department in Northern Tanzania.The Kilimanjaro Christian Medical Centre (KCMC) is the consultant hospital for over 12 to 15 million inhabitants of Northern Tanzania. It is also the Medical College of the Tumaini University. The Department of Pathology receives biopsies and surgical specimens from over 50 hospitals all over the country, which means that they are responsible for about 35 percent of this type of examinations made in Tanzania. Around 5,000 specmines of all kinds are processed yearly.The specimens are routinely fixed and preserved in 10 percent formaldehyde for transportation. At KCMC the tissues were paraffin-embedded. Five to 7 μm thick sections are cut on a microtome and mounted onto microscope slides. The sections are stained with Hematoxylin and Eosin for the microscopical examination. Two slides are prepared and analysed from each paraffin-embedded block.All surgical specimens sent to or taken at KCMC between 1994 and 2005 in which the histopathological diagnosis of schistosomiasis was established were reviewed. Age, provisional diagnosis and symptoms, when available, as well as all specific histopathology information were noted for the female patients where the examination had shown the presence of schistosomiasis.This study is part of a larger project "Sustainable Prevention of Endemic Schistosomiasis" (SPES), a research collaboration between KCMC and the Skaraborg Institute for Research and Development, Skövde, Sweden. Ethical clearance was given by the Research and Ethical Committee of the KCMC, Tumaini University, Tanzania. Research data were processed and stored anonymously.During the study period (12 years) 423 organ specimens examined at Department of Pathology  had a histopathologically confirmed schistosomiasis diagnosis. In 172 cases (40.7%) schistosomiasis was diagnosed in bladder and/or urethra and/or urether specimens, in 15 cases (3.5%) in liver specimens, in 45 cases (10.6%) in intestine specimens  and in 8 cases (1.9%) in mesentery/omentum. Genital schistosomiasis was diagnosed in 176 specimens from genital organs . Totally, genital specimens from 111 women were examined. The age of the patients ranged from five to 61 years, with a median age of 34 years. The specimens were sent from 24 hospitals in 13 regions of mainland Tanzania. Organs represented were peritoneum, uterus, cervix, ovary, Fallopian tubes, vagina/vulva, perineum and labia. The specimens consisted of biopsies from in situ and removed organs. The provisional diagnosis on the referral form was mostly a suspicion of malignancy. The main symptoms reported were bleeding disorders (48%), ulcer (17%), tumor (20%), lower abdominal pain (11%), and infertility (7%).Eleven percent of the specimens came from women less than 20 years old. Table Schistosomiasis was seen in simultaneously in two sites in 13 patients and in three sites in two patients with the following combinations, cervix and uterus; uterus and peritoneum; ovary and Fallopian tube; ovary and peritoneum; uterus, cervix and ovary. In five specimens schistosome eggs as well as adults worms were detected .Of all the 71 cervical specimens 14 (20%) showed signs of malignancy; epithelial dysplasia (4), carcinoma in situ (2), squamous cell carcinoma (6), adenocarcinoma (1) and lymphangiosis carcinoma (1). In the rest schistostosomias associated with chronic inflammation was diagnosed.For the 62 patients in this group symptoms reported were intermenstrual bleeding, post-coital bleeding, irregular menstruation, contact bleeding, and post-menopausal bleeding, with a median duration of symptoms given as four months (range 2 – 96 months). The clinical findings were described in 31 patients as suspicious appearance of the cervix (61%), ulcer/erosion (35%), growth/polyp (16%), tumor (13%), and friable/hard/nodular cervix (13%).The median age of the women with or without cervical malignancy was 39 years.The verification of a provisional carcinoma diagnosis was explicitly requested for 53 patients Table . The carThree of the ten uterine specimens showed only severe schistosomiasis. Schistosomiasis was associated with leiomyoma in four specimens, and in one specimen each with adenomyosis, adenomyosis/leiomyoma, and endometritis. Seven patients had undergone total hysterectomia, in one case with right salpingo-oophorectomia. One patient (28 years) had a hysterectomia performed due to a tumor of the cervix, however, the histopathology diagnosis of this case was severe schistosomiasis of the cervix and uterus. In one patient a squamous epithelial carcinoma associated with schistosomias was diagnosed in the bladder, the right and left ureter, and the uterus. In seven specimens of the endometrium, schistosomiasis was diagnosed; in one of these cases concomitant tuberculosis could not be ruled out.This group consisted of 14 samples from ovarian tissue and five from the Fallopian tubes. Severe scarring due to schistosomiasis was seen in one case and in two cases there was a specific inflammatory reaction. In three cases schistosomiasis of the peritoneum, in one schistosomiasis of pelvic tissue, and in one schistosomiasis of a lymphnode was also noted. Infertility was reported in four patients where tubal schistosomiasis was found . A provisional diagnosis of cancer was confirmed in two out of seven women , perineum (2 cases: 5 and 6 years old) and labia . Labial tumors were reported in the request form in four cases, in two cases associated with ulcera and verrucous skin.Schistosomiasis is an important and highly prevalent helminthic infection related to water contact and poverty and is affecting approximately 250 million people living in the tropical and sub-tropical parts of Sub-Saharan Africa, Asia and South America  have notRetrospective histopathology studies carried out in Egypt, Malawi, Mozambique, South Africa, Tanzania and Zimbabwe have shown that genital involvement mainly caused by Schistosoma haematobium most often occur in the vagina and cervix, less frequently in the ovary, tube and uterus. Gelfand et al.  found inOut of the 423 cases with a histopathological confirmed diagnosis of schistosomiasis the genital and urinary involvement occurred to the same degree (41% and 42%). In no case a provisional diagnosis of female genital schistosomiasis was mentioned on the request forms. Whereas six percent of the cases were due to multi-focal infections in Malawi , we founGenital schistosomiasis is associated with bleeding disorders, lower abdominal pain but also with infertility. A simultaneous affection of the ovaries and Fallopian tubes and/or peritoneum, as was found in our material, has been reported previously -31. A hiCervical schistosomiasis as a chronic disease persisting for years causes damages of the cervical epithelium and can theoretically render the local immune environment favourable for viral infections such as caused by HIV and human papilloma virus (HPV) ,6. ImpaiThe possible association between cervical cancer and cervical schistosomiasis have been studied retrospectively by histopathology analysis. Genital schistosomiasis was diagnosed in 1.7, 1.9 and 3.0 percent of cases with cervical cancer in three studies from Malawi and Tanzania ,13,14. IThe retrospective histopathological data presented give an overview of the occurrence of schistosomiasis in genital organs in women living in a schistosomiasis endemic area. Conclusions on the prevalence of female genital schistosomiasis in the different genital organs can not be drawn from this type of study. However, the results indicate that genital schistosomiasis has to be considered as differential diagnosis in a schistosomiasis endemic areas. Genital schistosomiasis misdiagnosed as cancer can be detrimental and devastating, if surgery is done. Furthermore, cervical genital schistosomiasis misdiagnosed as an STI can lead to ineffective treatment. Schistosome-, HIV- and HPV-infections are highly prevalent in sub-Saharan Africa, indications that cervical schistosomiasis can influence the course of STIs are at hand.The author(s) declare that they have no competing interests.BS participated in the collection of data, interpretation fo findings and the drafting of the manuscript. GP participated in the analysis, the interpretation, the presentation of findings and the preparation of the manuscript. SM participated in the interpretation of the results. IK participated in the interpretation of the findings and the preparation of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


A phylogenetic approach to anxiety is proposed. The different facets of human anxiety and their presence at different levels of the phylum are examined. All organisms, including unicellular such as protozoan, can display a specific reaction to danger. The mechanisms enabling the appraisal of harmful stimuli are fully present in insects. In higher invertebrates, fear is associated with a specific physiological response. In mammals, anxiety is accompanied by specific cognitive responses. The expression of emotions diversifies in higher vertebrates, only primates displaying facial expressions. Finally, autonoetic consciousness, a feature essential for human anxiety, appears only in great apes. This evolutive feature parallels the progress in the complexity of the logistic systems supporting it . The ability to assess one's coping potential, the diversification of the anxiety responses, and autonoetic consciousness seem relevant markers in a phylogenetic perspective. In human, anxiety is present in most psychopathological conditions . The regTo achieve this goal, we used a comparative approach, which consisted in assessing in animal species the presence of the process described in psychology and thus designed for humans. Such rationale provides operational criteria for the study of emotions in animals and may be a heuristic framework for interspecies comparison, which may be used also for emotions other than fear and anxiety. This approach is necessarily theoretical and requires to review many findings obtained in animals research, trying to analyze data obtained within other frames. To this aim, the present paper first describes the different elements constituting human anxiety and examines their presence along the phylum. Then, it reviews the different neurological and physiological systems of the organism supporting the anxiety responses along the phylum. Finally, the different conjunctions in a given species of the elements constituting anxiety will be examined.Fear, anxiety, and panic are three related concepts that need to be differentiated. Fear is considered by most emotion theorists as a basic emotion in humans e.g., , 6). As . As 6]).In human psychology, anxiety is often thought of as a secondary emotion, this is, as an emotion in response to a primary emotional reaction , 7. AnxiFear and anxiety are complex phenomena that articulate different components. For example, when confronted with a danger, a subject may display a specific response that includes a behavioural component , a physiological one , and an expressive one . As an emotion, anxiety supposedly orients the organism toward a specific type of interaction with its environment  and thusIn this section, we will present the different facets that constitute an emotion, focussing on fear, in the perspective that anxiety is the fear of an emotional state.Emotions have been conceived as action tendencies  resultinWhen a relevant stimulus is identified, physiological, motor, and expressive response systems are activated, which constitutes the action tendency. This concept refers to the inner dispositions (or their absence) of performing certain actions or achieving certain relational changes with the environment. In other words, an action tendency is the activation of a behavioural plan aiming at changing the individual-environment relation. Impulses of “moving towards,” “moving away,” and “moving against” are examples of action tendencies . The varAplysia californica, a gastropod mollusc, is able to react to a threatening stimulus by escaping locomotion [Aplysia displays a reduction of nondefensive behaviour such as feeding [Such actions tendencies can be found in a ubiquitous manner across the phylum. For example, avoidance of danger and flight has been observed in protozoan such as paramecia , 16, whicomotion . Furthercomotion . Finally feeding . All thepreparation of the organism to react in a certain way, but the actual reaction would depend upon a sufficient activation of the action tendency. Thus, some species would benefit from a buffer between the activation of a response mode and its actual enactment. This is found in many species, including invertebrates. For example, it has been shown that environmental disturbances such as light, a drop of water, or a pebble dropped in the aquarium induce a modification of the ventilatory rate and the heart rate in crustaceans such as crayfish. These modifications occur before the animal would undergo behavioural activity. Further, in case the intensity of the fearful stimulus is low, the animal will not display any behavioural modification. These physiological modifications have been interpreted as indicative of an animal's intention for body movement before physical movement occurs [It is to be noted that, in humans, action tendencies are not necessarily immediately enacted . They wot occurs .  Regarding the appraisal or evaluation component, Scherer  proposesIn human, specific emotions would be brought into play by the operations of a series of five stimulus evaluation checks (SECs). These checks are performed rapidly by mechanisms that continually scan the objects in the perceptual field, with different patterns or outcomes of the check process seen as giving rise to different emotions. Based on logical, phylogenic, and ontological arguments, Scherer  postulatCaenorhabditis elegans (which is much studied by neurobiologists because it has a fully mapped nervous system comprising exactly 302 neurons) [Aplysia [The first SEC, “novelty check,” looks for potential changes in the pattern of the situation. The orientation reflex is one of its consequences. Scherer , inverteneurons) , insectsneurons) , or moll[Aplysia , and ver[Aplysia , rats[26[Aplysia .Caenorhabditis elegans is able to display preferences for some stimuli over others [The second SEC is the “intrinsic pleasantness check.” On the basis of innate feature detectors or of learned associations, this second SEC evaluates the pleasantness of the stimulus or situation, hence determining approach or avoidance . Schererr others , to avoir others , and to r others . Insectsr others . In factr others  argued tr others , 34). Thr others , page 7 Goals and needs of the organism come into play in the third SEC, the “goal/need conductiveness check.” It examines the extent to which the introduction of the detected stimulus or event will advance or hinder the attainment of a specific goal or the satisfaction of a need. The goal/need conductiveness check is divided into three subchecks: the relevance subcheck that examines the relevance of the stimulus or event for important goals/needs of the organism, the expectation subcheck that determines the stimulus consistency with the state expected at this point in the goals/needs sequence, and the conductiveness subcheck that determines if the stimulus is conducive or obstructive to the respective goals or needs. This check can also be entirely genetically determined.Euplotes are able to change their morphology [If a given animal is able to display specific behaviour to escape stimuli that are incompatible with its survival such as predators or high temperatures, one can consider that it has this capacity. This can be seen in almost all invertebrate species. For example, nonsegmented worms such as nematodes escape when exposed to temperature above 33°C (for a review on nematodes see ). Other rphology  and beharphology  in resporphology . Of courSo, this third SEC has not been altered significantly through evolution, as it is described in invertebrates, and even protozoan such as ciliates as well as higher vertebrates. This is probably related to the fact that it is essential to the survival of the different species. One should note that, at the methodological level, the distinction between the second  and third  SEC might not be possible to operate in lower-order species. Beyond this methodological limitation, an alternative hypothesis should be considered: this distinction might not be relevant. In species low on the phylogenetic scale, these two SECs might not be differentiated. Their distinction would only appear in higher-order species.These three first checks have also been studied in an extensive and systematic way in some mammals, such as for example lambs , 44. TheThe fourth SEC, the “coping potential check,” determines the cause of the event, and the capacity of the organism to control it or to confront it, or to adjust to the final outcome. If a species is able to react in a different way in function of the predictability/controllability of a signal, one may claim that it has this ability. To our knowledge, no study has been published addressing the presence of such processes in ancestral worms or protozoan. Ancestral worms such as nematodes possess the ability to assess the rhythmicity of some events; this is necessary but probably not sufficient to possess the ability to react in function of the uncontrollability of an event. Such changes of behaviour in function of the controllability of a stimulus have been described in mammals such as dogs by Overmier and Seligman . Indeed,Macaca fuscata), a species displaying acquisition of innovative behaviours, such as potato and wheat-washing, first displayed by a young female and then transmitted to social partners as well as to successive generations [Pan troglodytes), behavioural variants (traditions) have been described in different communities, such as differences in tool usage, grooming and courtship behaviours [Finally, the last SEC, the “norm/self compatibility check,” evaluates the congruence of the event with the social and individual norms and standards such as mental prescriptions, self-concept, and self-ideal. This check needs the presence of cultural transmission. The presence of culture in animals such as nonhuman primates is still debated. Some authors claim that “proto-cultures” or “traditions”  can be erations . In chimhaviours . Howeverhaviours , humans haviours . Similarhaviours .The pattern of the outcome of the different SECs determines a particular emotional meaning and directly activates the corresponding action tendency. In human anxiety, the central features are that aspects of the situation are evaluated as intrinsically negative (intrinsic pleasantness check), as threatening important goals of the organism  , and as unpredictable or uncontrollable . Thus, to experience full-blown anxiety, a species would need to have the capacity for the first four SECs defined by Scherer's theory. As previously shown, all these four checks seem to be present in an ubiquitous manner in the different phyla, from invertebrates such as insects to lower vertebrates (fish) and mammals and even, for some of them, in unicellular organisms such as protozoan. Therefore, according to this theoretical frame, some rudimentary form of anxiety may be present from invertebrates to humans. However, as we will see, the level of sophistication, as well as of awareness of these evaluations and of the resulting experience vary tremendously from species to species, according to their cognitive capabilities.As action tendencies, emotion and anxiety recruit all the logistic capacities of the organism. The physiological systems are activated in order to support the actions and transactions with the environment called for by the emotional situation. In humans, many physiological and endocrine responses have been observed in emotion and in anxiety in particular. There is still a debate regarding whether specific emotions (and anxiety can be considered as such) have unique physiological characteristics. Despite a century long tradition of physiological research in human emotion, no definite conclusion has been reached yet . PhysiolRegarding fear and anxiety, meta-analyses of the literature have documented marked changes in most peripheral responses: cardiovascular changes, respiratory changes, muscles tonicity changes, or skin temperature changes when compared to neutral states . These cMost of these reactions are present in rodents such as rats, and they can vary as a function of the behavioural response that the subject may display. For example, a flight response can occur in response to threat that is associated with increased blood pressure and tachycardia, enhanced cardiac output and respiration, increased cerebral perfusion and redistribution of blood flow to increase limb circulation –72. SomeEven if not possessing an autonomic nervous system similar to the one enabling the physiological response to danger seen in vertebrates, invertebrates need the same rapid cardiovascular and respiratory regulation to be primed for the defensive behaviours they exhibit toward threatening stimuli. Indeed, such modifications provide the organism with the metabolic/energetic resources that will be necessary to deal with environmental challenges. Are such physiological responses observed in invertebrates when confronted with danger? Are they associated with the behavioural response? In crustaceans, perception of changes in the surroundings of the animal can induce modifications of some physiological variables such as heart rate and ventilatory rate . This isThus, it is possible that the representation of the body changes occurring during danger may be very different depending on the species: mammals may perceive environmental-induced changes driven by the autonomic nervous system in their body and including modifications in heart and ventilatory rate, in skin temperature, and mydriasis, lower vertebrates  and some invertebrates  may exhibit modified heart and ventilatory rate without changes in temperature or mydriasis.Emotions are not only inner states. They are also communicated to the environment, as they convey the behavioural intend of the individual. In human, the expressive component has certainly been the most studied, at least for facial expression. A series of studies has demonstrated innate and cross-cultural aspects of emotional facial expressions in humans. However, these innate facial displays are modulated by a set of cultural and display rules , 78. TheWhile much work has been devoted to the facial display of fear, the literature in human is almost silent regarding a facial expression that would be specific to anxiety. Most scholars do not distinguish facial expression between these two states , 79. SimAn interesting phenomenon for emotion regulation, known as facial feedback, has been documented in humans , 79. A wIn humans, some studies have documented that different emotions were expressed by different postures . FurtheIn animals too, emotional state can be communicated to the environment by specific signals, including facial, postural, vocal, or chemical ones. Further, other kind of expressive components are also documented, including more specific ones such as camouflaging.Modification of facial expression in relationship to emotions can be seen only in species having a well-developed facial musculature. Facial musculature is highly conserved across primates , the onePostural changes have been extensively described in higher vertebrates confronted with danger. For example, rodent may display a posture characterized by immobility, flattening of the ears, piloerection, and marked mydriasis. Indeed, specific postures have been repeatedly seen in vertebrates in emotional situations: they have been nicely illustrated by Darwin .Specific vocalizations to threat have also been documented across the phylum. For example, vervet monkeys emit specific alarm calls to different predators such as leopards, eagles, or pythons . VariatiThe use of pheromones to alert conspecifics of the presence of a danger is common in many animal species. For example, in the presence of an intruder, several species of social hymenoptera secrete pheromones that cause defensive behaviour among conspecifics . Such reOctopus cyanea), which forage on coral reefs, produce colour patterns capable of instantaneous matching to backgrounds from sand and reef rubble, through to spiked corals and seaweeds. More rarely, this kind of defence strategy can also be seen in mammals. For example, in the rock pocket mice Chaetodipus intermedius and in the deer mouse Peromyscus maniculatus, variation in coat colour, as a function of the colour of rock substrate, has been documented. This strategy is adaptive, providing the mice cryptic protection against predators [Camouflaging can be considered as a form of behaviour intermediate between emotional expression and coping with the situation. Indeed, it often appears when a species is confronted with a danger such as a predator. The most common form of it involves the modification of the visual appearance, but calls, songs, and scents can also be changed. Different strategies of camouflaging have been described, such as crypsis, aposematism, Müllerian mimicry, and Batesian mimicry. Crypsis enables to minimize the signal to noise ratio, thus rendering the detection of the subject very difficult for a predator. It generally consists in matching colours and patterns between an animal and its background –106. It redators . The othredators . BatesiaIn human psychology, extensive research has documented that emotion in general, and anxiety in particular, are accompanied by specific cognitive response. Threat and anxiety have been shown to powerfully affect attention allocation. Laboratory studies have documented that threatening stimuli automatically attract attention, even during subliminal exposure (very rapid presentation that cannot be consciously perceived) . In peoAttention bias toward anxiogenic stimuli has rarely been examined as such in nonhuman animal species. However, different phenomena have been described in animals that can be interpreted within this frame, including fear-potentiated startle, increased cognitive performance in stressful situations, anxiety-induced increased attention toward negative stimuli and a bias for threat cues in anxious mice.Fear-potentiated startle corresponds to an increase of the amplitude of the acoustic startle response in the presence of a cue previously paired with a shock. It has been described in rhesus monkeys  but alsoβ-CCT, a selective benzodiazepine receptor antagonist, blocks the antianxiety but not the amnesic action of benzodiazepines in mice [Another phenomenon that has been widely documented is the increased mnesic performance observed in anxiogenic situations: this is generally attributed to the fact that anxiogenic situations increase attention, thus increasing mnesic encoding. This facilitation has been repeatedly observed in rodents such as mice but also in birds. The processes used to increase anxiety include pharmacological manipulations, lesions studies, maternal separation in pups, genetic invalidation, and strain variations. For example, a principal component analysis showed that, in mice, higher emotional memory performance is related to heightened state anxiety . Further in mice , suggest in mice , suggest in mice . This phMost of these studies suggesting an attentional bias toward threat in anxious animals have been conducted in mammals, specially rodents, the sole exception being the pharmacological studies that were also conducted in birds. Even if the absence of such studies does by no ways mean that such processes do not exist in lower vertebrates, it suggests that it is at least difficult to assess in fish, amphibians, or reptiles. A reason for that could be that this facilitation does not occur in that species, but this remains to be confirmed by experimental studies.In the human literature, an important component of emotion is of phenomenological nature: the subjective feeling state. It reflects the notion that, when emotional, the individuals feel in a different state that colours their perception of the world and of themselves. Most authors agree that the subjective feeling component results from the global perception by the individual of the changes operating in the different emotion facets . There iReflexive emotional awareness is particularly relevant for emotion regulation in general and anxiety in particular. This capacity enables humans, not only to be reflexively aware of their on-going experiences, but also to reactivate past experiences, or to imagine future ones . The capThe capacity for autonoetic consciousness is one of the last cognitive features to develop in the human ontogeny. Its first manifestation in terms of reflexive capacities to one's own experience appears around 4 years of age and it is believed to be only fully developed around 14 years of age . To dateAs autonoetic consciousness is a key feature of episodic memory , the devIt should however be noticed here that the assumption that great apes are able of self-recognition of their image in a mirror has been questioned by some authors, and is still matter of controversy. Indeed, according to some authors , the beIn humans, the anxiety response is supported by several biological systems, including neurotransmitters such as biogenic amines, stress hormones, activity driven by the autonomic nervous system, and changes within specific brain areas. Are these different features present at all levels of the phylum?Fear triggers the release of various biogenic amines, including the catecholamines adrenaline, noradrenalin, octopamine, and dopamine and the indolamine serotonin. Adrenaline, noradrenalin, and dopamine have been described in all vertebrates, with some variations that have been suggested to be related to an evolutive trend . Indeed,Dendrobaena subrubicunda, the insect Periplaneta americana, and the mollusc Planorbarius corneus  axis, including a release of several stress hormones such as corticotropic-releasing hormone (CRH), adrenocorticotropic hormone (ACTH), and glucocorticoids. Stress hormones seem also highly conserved across the animal kingdom. Indeed, CRH has been described in various mammals but also in birds such as pigeons and quails, frogs, and several fish species . Such molecules are not only found in vertebrates. Indeed, CRH-like molecules have been reported in some invertebrates including in the nervous system of the annelid iew, see ). ACTH riew, see ). Therefiew, see ). So, agThe phylogeny of the autonomic nervous system has been extensively studied by Nilsson , 144. ItAnother important system supporting the human anxiety response is the facial musculature, enabling the facial expression of emotions. Such musculature is not present in invertebrates having an external skeleton, such as insects or bivalves. In nonmammalian vertebrates, this musculature is very rudimentary, enabling only opening and closing of the apertures such as mouth, eyes, and nostrils , 147. GrSeveral functional neuroimaging studies have investigated the brain structure whose activity is modified during fearful experience. For example, activation of the amygdala has been observed during acquisition of conditioned fear . This inIs such a pattern of activation also observed in other species? How does the anatomy of these brain areas evolve across the phylum? We will answer these questions mainly focusing on vertebrates, as the nervous system is organized in a different manner in invertebrates making a comparative approach difficult.We will first consider the phylogeny of the hypothalamus, the amygdale, and the hippocampus. The hypothalamus is a very old area and unlike most other brain structures, it has been conserved throughout phylogeny and exists in all vertebrates, including fishes. Amygdala and hippocampus have not been described as such in fishes; however, on the basis of anatomical and developmental data, it has been suggested that the fish medial and lateral regions of the telencephalic pallia might be the homologous neural structure to the mammalian amygdala and hippocampus, respectively –159. FurWe now consider the phylogeny of the neocortical areas  involved in the human anxiety. The classical view concerning the origins of the mammalian neocortex considers that it may be inexistent in nonmammalian vertebrates such as birds or reptiles. In fact, a three-layered cortex has been described in reptiles , 182 andIn rats, the frontal cortex is subdivided into three topologically different regions: the medial prefrontal cortex (that includes the anterior cingulate), the orbital prefrontal cortex, and the agranular insular cortex . Rats haad hoc structures for more functional diversity and efficiency. This view is in line with a Lamarckian perspective on the phylogeny of anxiety.At first sight, Another remarkable point that can be seen in Further, Different aspects of the literature reviewed above clearly suggest that anxiety as a conscious anticipation of danger only appears in great apes. This capacity, that implies autonoetic awareness, is directly related to the development of the neocortex and its connections with the limbic system and with the thalamus. This suggests that the capacity to represent oneself and one's reactions to hypothetical situations depends upon the capacity to strategically activate emotion networks or representations of emotional states. This reflexive capacity would be shared only by great apes and humans. Thus it might be that only great apes experience anxiety as humans, with its apprehension component. This does not mean that other species  may not have the aptitude to experiment anxiety with its anticipation dimension. However, in the case of lower mammals, this anticipation may not be conscious and may not be related to the ability to activate a representation of the situation with its possible consequences.Finally, To come back to our initial question, whether there is a qualitative difference between human and animal anxiety, In conclusion, the present review proposes a general frame for discussing anxiety in the context of phylogeny. In many cases, the data necessary to assess the presence of a given process are not available and additional empirical work may be necessary to clarify this question. Still, as testified by the points highlighted in the general discussion, this approach proves to be heuristic, both for our understanding on how a phenomenon such as anxiety varies across the phylogeny, and for our understanding of the processes and logistic systems underlying anxiety.


Human papillomaviruses (HPV) are the most common sexually transmitted viruses. Infection of the cervical epithelium by HPVs can lead to the development of cervical cancer. Recent advances in vaccine research have shown that immunization with papillomavirus-like particles (VLPs) containing the major structural viral protein, L1 from HPV 16 can provide protection from the establishment of a chronic HPV 16 infection and related cervical intraepithelial neoplasia (CIN) in baseline HPV 16 naïve women.To better understand the quantitative and qualitative effects of aluminum adjuvant on the immunogenic properties of an HPV 6, 11, 16 and 18L1 VLP vaccine, we used an HPV-specific, antibody isotyping assay and a competitive immunoassay that measures antibodies to neutralizing epitopes to profile sera from rhesus macaques immunized with the HPV L1 VLP vaccine formulated with or without aluminum adjuvant.Immunization with VLPs formulated with the aluminum adjuvant elicited a significantly stronger immune response with higher peak antibody titers both at four weeks post vaccination (12.7 to 41.9-fold higher) as well as in the persistent phase at week 52 (4.3 to 26.7-fold higher) than that of VLPs alone. Furthermore, the aluminum adjuvant formulated HPV VLP vaccine elicited a predominantly T helper type 2 response, with high levels of IgG1 and IgG4 and low levels of IgG2. The vaccine also elicited high levels of serum IgA, which may be important in providing mucosal immunity to impart protection in the anogenital tract.These results show that the HPV 6, 11, 16 and 18 L1-VLP vaccine formulated with Merck aluminum adjuvant elicits a robust and durable immune response and holds promise as a vaccine for preventing cervical cancer. Cervical cancer remains a leading cause of cancer-related deaths in women. HPV infection is the obligate first step in the development of cervical cancer . Nearly Human papillomaviruses are small double-stranded DNA viruses. Infection with HPV is the most common viral sexually transmitted diseases worldwide . HPV infRecently, we reported a proof-of-concept efficacy study for a prophylactic vaccine composed of HPV 16 L1 virus-like particles (VLP) formulated on Merck Aluminum Adjuvant (MAA) . The douCurrently, we are investigating a quadrivalent vaccine, composed of HPV 6, 11, 16, and 18 L1 VLPs formulated with MAA. This vaccine has also been shown to be effective in preventing persistent infection and HPV 6, 11, 16 and 18 related cervical dysplasia . To bettSaccharomyces cerevisiae expressing the L1 genes of HPV 6, 11, 16 and 18, respectively [Virus-like particles were prepared as previously described (with modifications) from individual lysates of types- ectively . Equal cectively .Groups of male and female Rhesus macaque (n = 5) were immunized at weeks 0, 8 and 24 with the two experimental vaccines described above. Serum was collected at weeks 0, 2, 4, 8, 10, 12, 16, 20, 24, 26, 28 and 52. The animals were maintained in accordance with the Institutional Animal Care and Use Committees of Merck Research Laboratories .The monoclonal antibodies used in the HPV cLIA included H6.M48  for HPV A multiplexed antibody isotyping Luminex assay was developed to characterize the qualitative aspects of the HPV-specific humoral immune response. The multiplexed, isotyping assay was used to classify HPV-specific antibodies as IgM, IgA, IgE, IgG1, IgG2, IgG4, or total IgG. IgG3 was not evaluated since rhesus macaques do not make the IgG3 subclass of antibodies ,19. A pa® membrane filter plate . The plate contents were washed 3 times with 200 μl of PBST and resuspended in PBST. HPV type-specific antibodies bound to the VLPs were incubated for 2 hours with biotinylated, isotype-specific secondary detection antibodies at the concentrations shown in Table For the multiplexed, HPV-specific antibody isotyping assay, HPV 6, 11, 16, and 18 VLPs were covalently conjugated to four distinct Luminex microspheres identified as microsphere 6, 11, 16 and 18, respectively. Test sera from vaccinated rhesus macaques were serially diluted 5-fold starting from a dilution of 1:10 with ADHS and incubated overnight with the VLPs conjugated to microspheres  in a total volume of 100 μl in a 1.2 μm hydrophilic, low protein binding, Durapore8 virions in the athymic mouse xenograft assay [An optimized and previously validated HPV competitive Luminex Immunoassay (cLIA) first described by Opalka et al.  was usedft assay . The titft assay . The lowAntibody titers obtained from the HPV cLIA were log-transformed and analyzed using a one-tailed, non-paired, Student's t-Test. P-values were obtained by comparing the average log-transformed titers of each of the 5 monkeys from week 2 to 52 within the two groups. The existence of a statistically significant difference between antibody titers from rhesus macaques vaccinated with VLP + MAA versus VLP alone was shown by a P-value of less than 0.05. Error bars represent the standard error of log-transformed titers.The purpose of this study was to characterize the quantitative and qualitative effects of including MAA to the HPV 6, 11, 16 and 18 VLP vaccine. First we wanted to measure antibody titers to neutralizing epitopes on HPV L1 induced by a quadrivalent vaccine formulated with or without aluminum adjuvant using the HPV cLIA. Antibody titers for all four HPV types in both vaccine groups developed after the first vaccination, declined, and then increased again following each boost Fig. . StudentWe also wanted to measure the total HPV-specific IgG levels to determine whether the formulation with MAA increased the total HPV-specific Ab titers. The total HPV-specific IgG peak titers, and titers seven months post dose three were consistently higher in the VLP+MAA group compared to the VLP group alone Fig. . An anamTo characterize the antibody isotypes elicited by VLPs formulated with MAA or VLPs, alone we modified a multiplexed Ab isotyping assay first developed to measure Ab isotypes in humans . These cWe first examined the HPV-specific IgM responses because IgM antibodies are the first to be produced following vaccination and would indicate a primary immune response to the vaccine. As expected, an IgM response for all four types was measured after the first immunization at week 0 and is shown for the representative type HPV 16 Fig. . InteresSince HPVs are sexually transmitted viruses that cross the mucosal barrier in the anogenital tract, we next examined whether the VLPs induced an IgA response and what effect formulation with MAA would have on an IgA response. In theory, the induction of IgA would greatly enhance the effectiveness of an HPV vaccine. Both vaccine groups demonstrated type-specific IgA responses to the VLPs that were maintained through one year Fig. . Each vaWe also examined whether there was an IgE response induced following vaccination. The geometric mean titers (GMTs) of IgE responses for the two vaccine groups were not above background, however one animal in the VLP group had a detectable IgE response at week 4 for HPV 6 and 16, and at week 24 for HPV 16 (data not shown). This response was just barely above the limit of detection and was not detected following the booster doses given at weeks 8 and 24.We next measured the IgG subtypes to determine whether formulation with Merck aluminum adjuvant affected the Ig isotype profile. Characterizing the IgG isotype profile would also provide insight into whether formulation with MAA altered the TH1 or TH2 profile of the immune response. The main IgG subclass observed in both vaccination groups was IgG1 Fig. . SimilarThis is the first study to directly examine the effects of the Merck aluminum adjuvant on immune responses to a quadrivalent HPV 6, 11, 16 and 18 L1 vaccine. The results here were generated using a novel HPV-type specific antibody isotyping assay to characterize the immunoglobulin subclasses to HPV 6, 11, 16 and 18 in rhesus macaques, and a competitive Luminex immunoassay (cLIA) that measures HPV-type specific antibodies to known neutralizing epitopes. The quadrivalent HPV L1 VLP vaccine formulated with MAA induced higher titers for total IgG Fig. , as wellResults from the HPV cLIA showed that the vaccine induced antibodies that competed with MAbs to known neutralizing epitopes, and are, thus, theoretically able to neutralize HPV. It is worth noting that we have previously shown that a competitive radioimmunoassay (cRIA)  and the + or CD4+ cells [Previously, we reported that the Merck HPV 16 L1 VLP vaccine induced a predominantly TH2 immune response with IL-4-producing T-helper cells, high levels of neutralizing Abs and low levels of IFN-γ secreting CD84+ cells . Given t4+ cells . Using t+ and CD4+ T-cells [Predictably, abundant amounts of IgG antibody isotype were found, and of all the IgG subclasses, IgG1 was most readily detected. The abundant presence of IgG1 elicited in both vaccination groups affirms that the vaccines did induce immune responses through a TH2 pathway. It is also interesting to note that the next abundant IgG subclass detected, primarily detected in the VLP+MAA group, was IgG4 Fig. . IgG4 is T-cells , suggest T-cells . Isotype T-cells . Detecti T-cells ,19.We also measured high levels of serum IgA observed in vaccinated animals. IgA, the secretory immunoglobulin, is thought to be very important in blocking viral entry into mucosal tissues . These fIn these isotyping studies, we used polyclonal goat anti-monkey IgM, IgA, and total IgG; because monkey-specific reagents are not available for the IgG subtypes and IgE, we used anti-human mouse MAbs to measure IgG1, IgG2, IgG4, and IgE responses. The polyclonal anti-IgA, IgM and total IgG reagents were selected for their specificity and their ability to broadly detect African Green monkey, rhesus macaque and human antibody isotypes. The various differences between human, rhesus macaque and African green monkey sera could account for changes in the sensitivity and specificity of the antibody-isotyping reagents and so the appropriate antibody reagent concentrations used in the HPV type-specific antibody isotyping assay were optimized for use with rhesus macaque serum.Due to the unavailability of purified rhesus Igs, we used purified human Igs to determine the specificities of the type-specific antibody isotyping reagents. There was some observed reactivity of the anti- human IgG2 antibody to purified human IgG1, and vice versa, but these data were obtained using purified human Igs and the differences between human and rhesus antibodies may account for why we saw a strong IgG1 response with low IgG2 titers Fig. . HoweverIn some cases, the test animals had low Ab titers to the VLPs prior to vaccination. This was not unexpected since more than 100 papillomaviruses have been identified  and manyCurrently, the only FDA approved adjuvants for use with prophylactic vaccines are aluminum based . The purThe differences observed in the immune responses elicited by the vaccine reported by Harro et al. and the Merck quadrivalent HPV vaccine may be attributed to several factors. First, would be the differences in the formulation and purification processes of the vaccines. The vaccine reported by Harro et al. was produced in insect cells as opposed to yeast for the Merck vaccine. Second, the differences in vaccine recipients, humans and rhesus macaques may respond differently to adjuvanted HPV VLP vaccines. Lastly, differences in formulation and composition of the two aluminum adjuvants used could have profound impacts on immunogenicity. Different aluminum salt adjuvants were used in these two studies; our study adsorbed VLPs to a proprietary formulation of aluminum hydroxyphosphate sulfate (MAA), while the Harro et al. study used aluminum potassium sulfate. Without a head to head comparison of the two vaccines, it is difficult to establish why the results are different. However, the magnitude and durability of antibody responses found in the quadrivalent VLP+MAA vaccinated rhesus macaques reported in this study are consistent with those found in human clinical trials of the HPV 11 or HPV 16 monovalent vaccines .The isotyping and cLIA assays described in this study are useful in characterizing the immune responses to HPV virions after natural infection or following vaccination. The novel cLIA utilized in this study is a robust and sensitive method for detecting HPV-specific antibodies in sera to known neutralizing epitopes on HPV virions and has proven to be a valuable tool for monitoring HPV immune responses in human clinical trials of an HPV 6, 11, 16 and 18L1-VLP vaccine. These assays have potential use in future epidemiology studies and other vaccine clinical trials.The results presented here show that an HPV 6, 11, 16 and 18 L1-VLP vaccine formulated with Merck aluminum adjuvant has increased immunogenicity without affecting the isotype profile. The VLPs formulated with Merck aluminum adjuvant elicited a robust and durable immune response that lasted up to 52 weeks. This vaccine holds promise as a vaccine for preventing cervical cancer.MAb, monoclonal antibody; PBS, phosphate-buffered saline; cLIA, competitive Luminex immunoassay; ADHS, antibody depleted human serum; VLP, virus-like particles; HPV, human papillomavirus; GMT, geometric mean titers; Ig, immunoglobulin; Ab, antibody.W. Ruiz, W. McClements, K. Jansen and M. Esser were all employees of Merck Research Laboratories, a division of Merck & Co., Inc. when this study was performed and potentially own stock and/or hold stock options in the Company. Merck is developing a quadrivalent HPV vaccine. Merck also funded this study in its entirety.W. McClements and K. Jansen designed the immunization study. M. Esser and W. Ruiz developed the competitive Luminex immunoassay and the new isotyping assays. W. Ruiz performed all the laboratory assays. W. Ruiz and M. Esser wrote the manuscript and all authors read and approved the final manuscript.


Although colorectal cancer mortality rates in the general U.S. population declined slightly from 1992 to 2000, the rates for Hispanic men and women did not. Disparity in colorectal cancer screening among Hispanics may be an important factor in the unchanged mortality trends. This study examined rates of colorectal cancer test use among Hispanic and non-Hispanic adults in the United States.Using sampling weights and logistic regression, we analyzed colorectal cancer test use among 5680 Hispanic and 104,733 non-Hispanic adults aged 50 years and older who participated in the 2002 Behavioral Risk Factor Surveillance System. We estimated the percentages and adjusted odds ratios (ORs) of the respondents' reported test use by sociodemographic characteristics, health care access, and state or territory of residence.Hispanic respondents aged 50 and older reported having had either a fecal occult blood test within the past year or a lower endoscopy (sigmoidoscopy or colonoscopy) within 10 years less frequently (41.9%) than non-Hispanic respondents (55.2%). Rates of test use were lower for respondents who reported less education, lower income, no health insurance, and no usual source of health care, regardless of Hispanic ethnicity. After adjusting for differences in education, income, insurance, and having a usual source of health care, Hispanic respondents remained less likely than non-Hispanic respondents to report colorectal cancer testing . Greater disparity in screening rates between Hispanics and non-Hispanics was observed in Colorado, California, and Texas than in other states.A disparity exists between Hispanic and non-Hispanic U.S. adults in colorectal cancer test use. This disparity varies among the states, highlighting the diverse health care experience of Hispanic adults in the United States. Colorectal cancer is a leading cause of cancer-related death in the United States . AlthougRecommendations for colorectal cancer screening are that men and women of average risk begin regular screening at age 50 with one of the following: 1) an annual fecal occult blood test (FOBT), 2) a flexible sigmoidoscopy every 5 years, 3) a combination of FOBT annually with flexible sigmoidoscopy every 5 years, 4) a colonoscopy every 10 years, or 5) a double-contrast barium enema every 5 years -11. SuppIdentifying disparities in colorectal cancer testing among Hispanics could be useful in focusing interventions to reduce cancer mortality. The U.S. Hispanic population, representing 12.5% of the U.S. population, is heterogeneous, with each state having a unique composition of Hispanic subgroups and other demographic characteristics . PreviouWe analyzed data from the 2002 BRFSS to estimate Hispanic-specific rates for receiving colorectal cancer tests. We determined national percentages and odds ratios (ORs) of Hispanic and non-Hispanic adults who reported having had colorectal cancer testing within recommended screening periods. We then examined state-specific test use rates among Hispanic adults in Puerto Rico and nine states that administered the survey in Spanish and had an adequate number of Hispanic respondents. Finally, to explore ethnic disparities within these states, we examined differences in the percentage of reported colorectal cancer test use between Hispanic and non-Hispanic respondents.The BRFSS is a national standardized, continuous random-digit–dial telephone survey begun in 1984 to monitor the behavioral risk factors associated with mortality and morbidity ,31. The Respondents aged 50 years and older were asked questions about the use of tests that screen for colorectal cancer. Background knowledge of whether the respondent "ever heard of" the specific test was not assessed before asking about test use. The use of an FOBT was determined by asking, "A blood stool test is a test that may use a special kit at home to determine whether the stool contains blood. Have you ever had this test using a home kit?" If the respondent answered yes, the question was followed by, "How long has it been since you had your last blood test using a home kit?" Similarly, for sigmoidoscopy and colonoscopy, respondents were asked, "Sigmoidoscopy and colonoscopy are exams in which a tube is inserted in the rectum to view the bowel for signs of cancer or other health problems. Have you ever had either of these exams?" and "How long has it been since you had your last sigmoidoscopy or colonoscopy?"lower endoscopy. BRFSS questions do not differentiate between sigmoidoscopy and colonoscopy. The recommended screening interval is every 10 years for colonoscopy and every 5 years for sigmoidoscopy; we defined reported use of lower endoscopy within the past 10 years as "within the recommended interval." Although the FOBT and lower endoscopy are tests that are commonly recommended for colorectal cancer screening  to view the rectum and colon for abnormalities. Colonoscopy examines the entire colon, whereas sigmoidoscopy focuses on the lower third of the colon. In this report, we refer to both procedures collectively as creening , the BRFHispanic individuals are defined by the U.S. Census Bureau as individuals who indicate their origin as Mexican, Puerto Rican, Cuban, Central or South American, or other Hispanic origin . For theThe total number of 2002 BRFSS respondents aged 50 years and older in all states, territories, and the District of Columbia was 111,036. Of these respondents, 5680 identified themselves as Hispanic. Respondents who answered "Don't know/Not sure" or "Refused" to ever having had an FOBT (1.6%) or lower endoscopy (2.1%) were excluded. In addition, 1.2% of respondents were excluded because they were unsure or refused to answer questions about Hispanic ethnicity, education, insurance status, and usual source of care.Statistical estimates were produced using SAS 8.02  and SUDAAN 7.5.3 . All data were weighted by age, sex, and race to reflect the population of each state. Percentages and 95% confidence intervals (CIs) were age-adjusted using 5-year age groups to standardize to the 2000 U.S. census. Using logistic regression, we calculated crude ORs and CIs for receiving recommended colorectal cancer testing, then adjusted the ORs for age, sex, race, Hispanic ethnicity, education, insurance, income, and having a usual source of care. The respondents who chose not to give information on income were grouped into an "unknown" category to be included in the logistic regression.The state-specific analysis was limited to states and territories where the survey was administered in both Spanish and English and where there were at least 100 Hispanic respondents aged 50 or older. These states and territories were Arizona, California, Colorado, Florida,  Massachusetts, New Jersey, New Mexico, New York, Puerto Rico, and Texas. Of the 5680 respondents who reported Hispanic ethnicity, 3808 (67%) lived in one of the included states. It was not possible to determine from national BRFSS survey data which language was used during the interview . TherefoAmong both Hispanic and non-Hispanic respondents, a higher percentage of respondents reported having had a lower endoscopy test than having had an FOBT ever and within the recommended screening intervals . The estA higher percentage of non-Hispanic men than women reported having received colorectal cancer testing, whereas there was no difference by sex observed for Hispanic respondents . For nonBoth non-Hispanic and Hispanic respondents who reported more education and a higher household income reported higher percentages of having had either an FOBT within 1 year or lower endoscopy within 10 years. Of note, the difference in the percentages of non-Hispanic and Hispanic respondents who reported lower endoscopy within 10 years was smallest among respondents in the highest income category. Respondents without insurance or without a usual source of care had the lowest percentage of reported colorectal cancer test use, regardless of ethnicity. Because of the small number of Hispanic respondents who were uninsured, the percentage for FOBT use within 1 year among this group could not be reported. The small number of respondents would lead to an unstable estimate. Among non-Hispanic respondents who reported no insurance and no usual source of care, the percentage of use of either colorectal cancer test within recommended intervals was 34.7% (data not shown).The percentage of Hispanic respondents aged 50 and older who reported an FOBT within the past year ranged from 8.5% in Texas to 24.1% in Massachusetts, and the percentage of Hispanic respondents who reported a lower endoscopy within the past 10 years ranged from 28.9% in Colorado to 47.1% in Massachusetts . For recThe The results of this study confirm that, regardless of ethnicity, colorectal cancer test use among men and women aged 50 years and older is low compared with other well-known cancer screening tests -6,34. WeThis study also shows that Hispanic colorectal cancer test use differs among states. Colorado, California, and Texas had low percentages of Hispanic respondents reporting recommended test use and a greater disparity in colorectal cancer testing between Hispanic and non-Hispanic respondents than other states. In Massachusetts and New Jersey, test use among Hispanic men and women was higher than other states, and the disparity between Hispanic and non-Hispanic respondents was less. For states that had too few Hispanic BRFSS respondents for this analysis, colorectal test use and disparities are unknown. Often, Hispanic populations are analyzed as a single group, and these state-specific differences are not apparent. In addition to variation among states, there may be variation within each state as well. For example, Coughlin et al suggested that Hispanic women living in counties along the U.S.–Mexico border, regardless of their state of residence, were less likely to receive breast and cervical cancer screening than Hispanic women in nonborder counties .A factor that may account for state differences is the unique composition and culture of Hispanic subgroups in each state. In general, the Western states have an overall higher Hispanic proportion of the population and, in the Western United States, Hispanic individuals are more likely to be of Mexican descent than they are in the Northeast, which has Puerto Rican, South American, and Central American populations . HispaniUnderstanding the individual influences of ethnicity, socioeconomic factors, and health care access on colorectal cancer test use is challenging. Previous studies cite the lack of a usual source of health care as the most consistent reason that Hispanic individuals are not being screened for colorectal and other cancers -40 and aThe BRFSS survey did not ask whether Hispanic respondents were born outside of the United States and, if so, time since immigration to the United States. Therefore, we were not able to assess the role of acculturation and language of BRFSS respondents in this study. Analyses of other national health behaviors surveys have shown acculturation to be associated with reduced colorectal cancer rates . HoweverThere were several limitations to this study. Actual compliance with screening guidelines may be lower than percentages reported in this article. In our study, to fully capture the use of colonoscopy, having a lower endoscopy (which refers to both sigmoidoscopy and colonoscopy) "within 10 years" is considered to be within recommended screening intervals. With this definition, individuals who received sigmoidoscopy outside the recommended 5-year screening interval but within 10 years were considered compliant with screening guidelines. In addition, there was no way to differentiate between tests performed for screening purposes and tests performed for diagnostic purposes. Also, the use of a double-contrast barium enema, an acceptable but less often recommended choice , was notTelephone survey data are limited by several factors. The response rate to the 2002 BRFSS was 58%. Low response rates may bias results by selecting a unique population that differs from the general population in health care access, use, and beliefs. Despite this low response rate, BRFSS data have been shown to be valid and reliable when compared with other national surveys, and bias in demographic characteristics of respondents in BRFSS data was not associated with response rate . AdditioResponses were self-reported. The BRFSS questions did not ascertain whether the respondents had "ever heard of" the colorectal cancer test of interest. Despite previous studies, which have shown moderate and good concordance between self-reported colorectal cancer testing when validated with medical records ,48, cognColorectal cancer test use is estimated to be lower among Hispanic than non-Hispanic adults. Differences in test use cannot be fully explained by education, income, and health care access because, after adjusting for these factors, Hispanic men and women remain less likely to report having had colorectal cancer testing at recommended intervals. Certain Western states had a large disparity between the percentages of Hispanic and non-Hispanic test use, whereas Northeastern states had similar percentages of use. Regardless of the differing degrees of disparities, increasing awareness of and access to colorectal cancer screening among Hispanics is needed. The differences in colorectal cancer test use among states call attention to the diverse health care experience of Hispanic adults in the United States. Future studies that explore the reasons for differences in test use among Hispanic communities may highlight effective programs and practices that encourage increased screening.