Serologic tests for HIV infection in infants less than 18 months do not differentiate exposure and infection since maternally acquired IgG antibodies may be detected in infants. Thus, the gold standard for diagnosis of HIV-1 infection in infants under the age of 2 years is DNA or reverse transcriptase polymerase chain reaction. There is an urgent need to evaluate alternative and cost effective laboratory methods for early diagnosis of infant HIV-1 infection as well as identifying infected infants who may benefit from cotrimoxazole prophylaxis and/or initiation of highly active antiretroviral therapy.Whole blood was collected in EDTA from 137 infants aged 0 to 18 months. DNA polymerase chain reaction was used as the reference standard for diagnosis of HIV-1 infection. T-cell subset profiles were determined by flow cytometry.Seventy-six infants were DNA PCR positive while 61 were negative. The median CD4 counts of PCR negative infants were significantly higher than those of the PCR positive infants, p < 0.001. The median CD4/CD8 ratio and the %CD4 of the PCR positive infants were both significantly lower than those of the negative infants, p < 0.001. The CD4/CD8 ratio had a >98% sensitivity for diagnosis of HIV-1 infection and a specificity of >98%.The CD4/CD8 ratio appears useful in identifying HIV-infected infants. The development of lower cost and more robust flow cytometric methods that provide both CD4/CD8 ratio and %CD4 may be cost-effective for HIV-1 diagnosis and identification of infants for cotrimoxazole prophylaxis and/or highly active antiretroviral therapy. Endemic HIV infection, in sub-Saharan Africa, where in many countries more than 20% of pregnant women are HIV seropositive leads to a diagnostic problem in the evaluation of their infants. Without intervention, more than 25% of infants born to seropositive women will acquire HIV infection in the first year of life. HIV testing with enzyme immunoassay-based rapid tests have expanded capacity to identify seropositive women and provide interventions, but even with single dose Nevirapine and other antiretrovirals, infection of infants still exceeds 10% in the first year of life. Serologic tests for HIV do not accurately identify those infants who have acquired infection within the first 18 months of life because of transplacentally acquired maternal IgG antibodies. As antimicrobial and antiviral interventions are developed to reduce morbidity and mortality, among infants born to seropositive women, the early diagnosis of infection is increasingly important. The gold standard for diagnosis of HIV-1 infection in infants under the age of 2 years is DNA polymerase chain reaction (PCR) or reverse transcriptase (RT)-PCR . With thThe World Health Organization (WHO) recommends initiation of highly active antiretroviral therapy (HAART) in HIV-seropositive infants under the age of 18 months, who have WHO Pediatric Stage III disease and CD4 percentage (%CD4) <20% in resource-poor countries where %CD4 are available but virologic tests (DNA PCR, RT-PCR or immune-complex dissociated p24 antigen) for confirmation of HIV infection are not available . Thus, i+ cells, a transient increase in CD8+ cells, total lymphocytes and inversion of the CD4/CD8 ratio [+ cells decline, while the CD8+ cells which may remain at high levels for long periods, eventually decrease but not to baseline levels. Since in healthy children the CD4+ and CD8+ cells account for 60% and 30% of the T lymphocytes respectively, a normal CD4/CD8 ratio should always be >1.0. Thus, in HIV-1 infection where there is a decrease in CD4+ cells and an increase in CD8+ cells, the reversal of the CD4/CD8 to <1.0 should in theory be useful for diagnosis of HIV-1 infection.Immunological changes in HIV-1 infection include a decrease in CD4D8 ratio ,7. As HI+ cell counts, their percentages as well as the CD4/CD8 ratio. The use of the ratio in combination with %CD4, may lead to a timely identification of infected infants, who meet the WHO criteria for initiation of CTX prophylaxis and/or ARV therapy. While the infrastructure needed to conduct flow-cytometric analyses of HIV infection are still largely confined to a few centers, an increasing number of point of care diagnostic testing systems, inexpensive methods to measure CD4 cells are currently in development. These include the development and evaluation of simplified volumetric flow cytometric methods using a low cost flow cytometer that can be powered from a car battery or by solar panels (Cyflow SL, Partec, Munster, Germany) by Cassens and colleagues [Certain flow cytometers when used in tandem with a haematological analyzer can provide absolute CD4lleagues  and modilleagues . Furtherlleagues ,11.The main objective of the current study was to evaluate the CD4/CD8 ratio for diagnosis of subtype C HIV-1 infection in infants under the age of 2 years among infants where DNA PCR was performed to diagnose HIV infection.The infant specimens used in this study were obtained from two independent prospective studies; the short course zidovudine (AZT) and Pediatric AIDS Clinical definition (PACD). The Medical Research Council and the Institutional Ethics Committees approved both studies and present study. The infants included in the study were under the age of 2 years. All the infants were breastfeeding at the time of specimen collection.Infants who were followed in the short course AZT study aimed at preventing MTCT were enrolled between May 2001 and June 2002. In the AZT study, mothers received short course AZT starting at 36 weeks gestation and throughout labor. Their infants received AZT for 7 days.In the PACD study, hospitalized children aged between 2 months and 18 months were prospectively enrolled into the study, between July 2002 and July 2003, following informed consent from their mothers. The mothers of these children did not receive antiretroviral therapy for prevention of MTCT nor for HIV disease. The PACD study is a hospital based analytical cross-sectional survey. Laboratory specimens were obtained once from each subject. Children presenting in the moribund state, requiring immediate resuscitation or those with known HIV status or those whose mothers/guardians refused to sign the informed consent were excluded from the study. The objective of the PACD study is to identify clinical symptoms, associated with HIV-1 infection in infants under the age of 18 months, which may be used in the absence of laboratory tests, for HIV-1 diagnosis.A total volume of 2 ml whole blood was collected in ethylenediamine tetraacetic acid from each of the 137 infants in the short course AZT and the PACD studies. The whole blood was then aliquoted into two tubes (500 microlitres in each) for determination of T cell subset profiles, and the second for DNA PCR and the rest centrifuged at 200 g to obtain plasma, which was stored at -80°C.The laboratory tests described below were conducted in a blind fashion.+ and CD8+ cell counts, CD4/CD8 ratios, as well as the %CD4 and %CD8 values among lymphocytes were automatically calculated.T cell subset profiles were determined by flow cytometry using a Coulter Epics XL equipped with System II software (Beckman Coulter, Miami, Florida, USA) within 4 hours of blood collection. This flow cytometer was run, in a double platform setting where the absolute counts for both white blood cells and lymphocytes were obtained on a Celldyn 3500R haematological analyzer (Abbott, GmbH, Germany). Then from the combined results, the absolute CD4DNA PCR Roche amplification assay version 1.5 (Roche Diagnostics, Branchburg, NJ, USA) was employed as the reference standard for diagnosis of infant HIV-1 infection status. DNA extraction, amplification and detection were performed and results interpreted following the manufacturer's instructions (Roche Diagnostics, Branchburg, NJ, USA) as we previously described ,13.Reference ranges of T cell subset profiles for infants and children are usually stratified by age as <12 months, 1 to 5 years and 6–12 years. In the current study, in addition to overall evaluation of samples obtained from all the infants aged 0 to 18 months, the evaluated parameters were also compared based on infant age groups 0–11, and 12–18 months.Diagnostic tests , negative predictive value (NPV) and test efficiency (TE) with 95% confidence interval) were used to assess the assay under evaluation with DNA PCR as the reference standard. A MTCT prevalence rate of 30.7% (based on the current rate in Zimbabwe) was used in the calculations of PPV and NPV. Sensitivity was defined as the percentage of reference standard HIV positive samples found reactive by assay under evaluation. Specificity was defined as the percentage of reference standard HIV-negative samples that were negative by the assay under evaluation. TE refers to the overall ability of a test to correctly identify all positives and negatives . It is a combination of the sensitivity and the specificity of an assay and gives an idea of the total effectiveness of the assay. PPV was defined as the probability that a specimen had CD4:CD8 ratio less than 1 when the test was DNA PCR positive. NPV was defined as the probability that a specimen does not have a CD4:CD8 ratio ≥ 1 when the test was DNA PCR negative.P values less than 0.05 were considered statistically significant.Comparisons of T cells between infected and uninfected infants were done using non-parametic equivalent of the T-test . in utero transmission as determined by serial DNA PCR of birth and subsequent samples tested in longitudinal short course AZT MTCT studies .Of the 137 infant specimens tested using DNA PCR, 76 were HIV-1 positive and 61 were HIV-1 negative. The 76 PCR positive infants included 9 infants who had evidence of p = 0.08). As expected, the median CD4+ cell counts of PCR negative infants were significantly higher than those of the PCR positive infants, p < 0.001  and 8.0 months (IQR: 4–14) for HIV infected and uninfected respectively (Seventy-five infants of the 76 PCR positive group (98.7%) had CD4/CD8 ratio <1 while 60 infants of the 61 who were PCR negative had CD4/CD8 ratio ≥ 1. All the 9 infants infected in utero had a CD4:CD8 ratio less than 1. Their median CD4/CD8 ratio was similar to that of the rest of the PCR positive babies , albeit the numbers are too small for statistical significance considerations. The infant (aged 11 months at specimen collection) who was DNA PCR positive with a CD4/CD8 ratio of 1.1 was from the PACD study. The PCR negative infant (aged 4 months at specimen collection) with a CD4/CD8 ratio of 0.3 was also from the PACD cohort.The overall sensitivity and specificity of the CD4/CD8 ratio were 98.7% : 95.1–100) respectively with PPV and NPV of 96.3% and 99.4% respectively and a test efficiency of 98.5% (95% CI: 96.5–100) Table . CompariIn resource-poor countries, the major constraint in the use of DNA PCR for diagnosis of HIV-1 infection in infants under the age of 18 months is the cost of the equipment and the reagents. In addition, highly trained laboratory personnel and stringent quality assurance measures are needed to run this assay for routine diagnosis of HIV infection in infants.In Zimbabwe, enzyme linked immunosorbent assay (ELISA) is routinely performed in both public and private laboratories for diagnosis of various infections including HIV-1. However, because of the transplacental transfer of maternal IgG antibodies, which may persist in infants for up to 18 months, ELISA is not suitable for diagnosis of HIV-1 infection in these infants. Therefore alternative methods are needed for this purpose. In addition, both public and private laboratories have flow cytometers or FACSCount machines for enumeration of T cell subset profile and automated haematological analysers for routine full blood counts with differential. With this equipment available in the country we evaluated the CD4/CD8 ratio as an alternative diagnostic test for infant HIV-1 infection.+ cell counts and both %CD4 and %CD8 values among lymphocytes. The %CD4 value among lymphocytes is the recommended parameter for analyzing pediatric samples, as absolute counts for infants are age sensitive and variable. A simpler single platform system such as the FACScount (Becton-Dickinson Immunocytometry Systems, San Jose, CA, USA), is not fully suited for pediatric work as it provides CD4+, CD8+, CD3+ T lymphocyte counts and CD4/CD8 ratio but %CD4 are not available. Of note, %CD4 expressed among CD3+ T lymphocytes, is a different parameter from the customary %CD4 expressed among lymphocytes. The %CD4 expressed among lymphocytes and not the %CD4 expressed among CD3+ T lymphocytes is recommended for decision making to initiate ARV therapy in children under the age of 18 months [In our investigation we used Epics XL Coulter flow cytometer equipped with System II software (Beckman Coulter, Miami Florida, USA) for the following reasons. With this instrument, CD4/CD8 ratios can be conveniently obtained. In addition, when used in double platform setting tandem with a haematological analyzer, the results also show absolute CD48 months .+ cell count, is less expensive than PCR and may provide additional information of value to the clinician with respect to prognosis, and the need for prophylaxis and treatment. Optimal flow cytometry for determination of T cell subset profile, offers the added advantage that CD4/CD8 ratio will determine the infection status of the infant while the % CD4 will provide information on decision-making for commencement of HAART.In Zimbabwe, CD4Overall, the CD4/CD8 ratio had a ≥ 98% sensitivity for diagnosis of infant HIV-1 infection and a specificity ≥ 98%. Both sensitivity and specificity were 100% for infants in the 12–18 months age group. Interestingly, in parallel studies performed in 250 HIV-1 seropositive adults, 249 had a CD4/CD8 ratio of <1. The CD4/CD8 ratio of the one patient was 1 at enrollment and has remained so for over one year.When interpreting our data, it is important to note that normal T cell subset values among African children differ from those of other populations -16. A st+ cell count rather than a CD4+ cell count was a more specific prognostic marker of disease progression in HIV infected children.A very few studies in Africa have compared T cell subset profiles between HIV-1 infected and uninfected infants under the age of 2 years ,17. MoodEmbree and colleagues, in Kenya, also reported that HIV-1 infected children had lower %CD4 and higher %CD8 by 3 months when compared to uninfected children . The autThe two African studies mentioned above, have documented the clinical value of %CD4, CD4/CD8 ratio and CD8 counts in HIV-1 infection in infants.In summary, the CD4/CD8 ratio may be useful in identifying infected infants while %CD4 will identify infants who may benefit from cotrimoxazole prophylaxis and/or initiation of HAART, and for management of HIV-infected infants in developing countries in general. We thus propose use of flow cytometry, where available, as a point of care diagnostic tool for ill infants admitted to hospitals with clinical symptoms suggestive of HIV infection and/or AIDS.The author(s) declare that they have no competing interests.LSZ designed the study, analyzed the data and drafted the manuscript. DAK, KJN, AB and GJ participated in the design of the study and preparation of the manuscript. SR conducted the statistical analysis and participated in the preparation of the manuscript. OT performed DNA PCR and analysed the data. AB and CG conducted flow cytometry and analysed the data, with the guidance of GJ. MN and PM participated in the collection of infant specimens and demographic data as well as preparation of the manuscript. All authors read and approved the final manuscript.Table 3. Gender-based comparison of T cell subset profiles of HIV infected and uninfected babies.Click here for file


The callipyge mutation is located within an imprinted gene cluster on ovine chromosome 18. The callipyge trait exhibits polar overdominant inheritance due to the fact that only heterozygotes inheriting a mutant paternal allele  have a phenotype of muscle hypertrophy, reduced fat and a more compact skeleton. The mutation is a single A to G transition in an intergenic region that results in the increased expression of several genes within the imprinted cluster without changing their parent-of-origin allele-specific expression.DLK1, PEG11, and MEG8 in the muscles of lambs with the callipyge allele. DLK1 and PEG11 transcript levels were elevated in the hypertrophied muscles of paternal heterozygous animals relative to animals of the other three genotypes. The PEG11 locus produces a single 6.5 kb transcript and two smaller antisense strand transcripts, referred to as PEG11AS, in skeletal muscle. PEG11AS transcripts were detectable over a 5.5 kb region beginning 1.2 kb upstream of the PEG11 start codon and spanning the entire open reading frame. Analysis of PEG11 expression by quantitative PCR shows a 200-fold induction in the hypertrophied muscles of paternal heterozygous animals and a 13-fold induction in homozygous callipyge animals. PEG11 transcripts were 14-fold more abundant than PEG11AS transcripts in the gluteus medius of paternal heterozygous animals. PEG11AS transcripts were expressed at higher levels than PEG11 transcripts in the gluteus medius of animals of the other three genotypes.There was a significant effect of genotype (p < 0.0001) on the transcript abundance of DLK1, GTL2, PEG11 and MEG8 in the hypertrophied skeletal muscles. Transcript abundance of DLK1 and PEG11 was highest in paternal heterozygous animals and exhibited polar overdominant gene expression patterns; therefore, both genes are candidates for causing skeletal muscle hypertrophy. There was unique relationship of PEG11 and PEG11AS transcript abundance in the paternal heterozygous animals that suggests a RNA interference mechanism may have a role in PEG11 gene regulation and polar overdominance in callipyge sheep.The effect of the callipyge mutation has been to alter the expression of  CLPGPat) exhibit the callipyge phenotype. Maternal heterozygotes (CLPGMat/+Pat) and callipyge allele homozygotes (CLPGMat/CLPGPat) have muscling and carcass compositions that are similar to normal sheep .The mutation responsible for the callipyge trait is located within an imprinted gene cluster on the distal end of ovine chromosome 18 . The calDelta-like 1 (DLK1), DLK associated transcript (DAT), gene-trap locus 2 (GTL2), paternal expressed gene 11 (PEG11 / PEG11AS) and maternal expressed gene 8 (MEG8). The same conserved gene order was also found as an imprinted domain on human chromosome 14 and mouse chromosome 12 [DLK1 locus  encodes a transmembrane protein that contains epidermal-growth factor repeats [DLK1 produces the circulating protein fetal antigen-1 [DAT is a short non-coding RNA that has been proposed to be a cleavage product of extended DLK1 transcripts [DLK1and DAT are expressed from the paternal allele [GTL2  and MEG8 genes express non-coding RNA from the maternal allele [PEG11 gene contains an intronless open reading frame of 1333 amino acids in sheep [retrotransposon-like 1 (RTL1/rtl1) encode 1358 and 1745 amino acids, respectively. RNA transcripts were detected from the opposite strand of the same gene, referred to as PEG11AS (formerly known as antiPEG11) [Rtl1 [A physical contig spanning the region containing the callipyge mutation was constructed using overlapping ovine bacterial artificial chromosomes  and 215  repeats -19. Cleantigen-1 . DAT is l allele ,13-15. G1) [Rtl1 .CLPG) in the intergenic region located between DLK1 and GTL2 [Mat/CLPGPat genotype based on haplotype analysis [cis without altering their parent-of-origin-specific expression [The causative mutation for callipyge is a single base transition of A  to G  was different from the other three genotypes. A sense/antisense interaction of PEG11 and PEG11AS, such as an RNA interference mechanism, would be consistent with a trans interaction between reciprocally imprinted genes that has been previously proposed as a mechanism for polar overdominance [In this study, we analyzed the expression of five genes within the callipyge cluster in the muscles of lambs of all four genotypes. Quantitative analysis of gene expression using a series of orthogonal contrasts showed that ominance ,28.PEG11 and PEG11AS expression. Hybridization of longissimus dorsi, semimembranosus and gluteus medius northern blots with a PEG11 probe indicated expression of a 6.5 kb PEG11 transcript in paternal heterozygotes (+Mat/CLPGPat) that was not readily detectable in the other three genotypes  by a probe from the complementary strand. PEG11 and PEG11AS transcripts were not detected in the supraspinatus. The expression of DLK1 was detectable at various levels in each of the four muscles and all four genotypes, although DLK1 appeared to be up-regulated in the loin and pelvic limb muscles of +Mat/CLPGPat and CLPGMat/CLPGPat animals , semimembranosus, and gluteus medius (pelvic limb), and one muscle that does not undergo hypertrophy, the supraspinatus, (thoracic limb). Strand specific probes were used to analyze PEG11 and PEG11AS transcripts to the contig sequence  and from the 3'UTR (C) showed equivalent results to riboprobe P in the gluteus medius  did not detect any PEG11 transcripts, indicating that transcription of the 6.5 kb PEG11 was initiated within 350 bp of the start codon of the open reading frame. Riboprobe F detected PEG11AS transcripts in the gluteus medius and supraspinatus of all four genotypes, with the lowest expression in the paternal heterozygous (+Mat/CLPGPat) animals. The PEG11AS riboprobe G shows three protected fragments, suggesting variable splice junctions or transcription termination sites for PEG11AS transcripts.Two probes from upstream of the DLK1 and PEG11 in gluteus medius was the same  animals had the highest transcript abundance (p < 0.05), followed by CLPGMat/CLPGPat animals, which had significantly greater transcript abundance (p < 0.05) than CLPGMat/+Pat or +/+ animals. The mRNA abundance of DLK1 in the gluteus medius was 6-fold and 2.5-fold greater in +Mat/CLPGPat lambs and CLPGMat/CLPGPat lambs respectively, relative to normal lambs  but was not significantly changed in the other three genotypes  in the CLPGMat/+Pat and CLPGMat/CLPGPat animals relative to the +Mat/CLPGPat and +/+ animals  animals. In this study, both genes showed a polar overdominant expression pattern in that paternal heterozygotes had significantly higher levels of gene expression than the other three genotypes. The major differences between DLK1 and PEG11 were the magnitude and the muscle specificity of the up-regulation. DLK1 was readily detectable in all muscles and genotypes by northern blot, but the up-regulation in paternal heterozygous animals was restricted to muscles of the loin and pelvic limb. The quantitative results showed a 6-fold increase in DLK1 transcript abundance in the gluteus medius and no change of DLK1 abundance in the supraspinatus. This pattern of gene expression is consistent with studies on individual muscle growth that show significant muscle hypertrophy in the gluteus medius but not in the supraspinatus [DLK1 transcripts were significantly increased (2.5-fold) in the gluteus medius of CLPGMat/CLPGPat animals, which do not exhibit muscle hypertrophy. The lack of a phenotype in CLPGMat/CLPGPat animals could be due to a threshold effect that requires more than a 2.5-fold increase in DLK1 transcript abundance to change muscle growth.Due to their paternal allele-specific expression, the spinatus . DLK1 trPEG11 was very low in the gluteus medius and supraspinatus of normal sheep and was induced in both muscles of callipyge lambs. Northern blot analysis, ribonuclease protection assay and quantitative PCR results all show that the expression of PEG11 and PEG11AS transcripts was much lower in the supraspinatus than the other muscles. The high level of expression of PEG11 in the gluteus medius relative to the supraspinatus of callipyge lambs indicates that PEG11 could also be the gene responsible for muscle hypertrophy if there is a threshold level required to change muscle growth. The PEG11 gene has a long intronless open reading frame but it is not known if a protein is produced or what function it may have.The expression of CLPGMat/CLPGPat genotype is one of the more intriguing aspects of the trait and has led to a hypothesis of trans effects by other reciprocally imprinted genes in the callipyge region [PEG11 gene has a direct role in muscle hypertrophy, either alone or in concert with DLK1, then PEG11AS may have a role in a trans effect on PEG11 expression. In +Mat/CLPGPat animals, PEG11 transcripts were 14-fold and 4-fold more abundant than PEG11AS transcripts in the gluteus medius and supraspinatus respectively. PEG11AS transcripts were more abundant than PEG11 transcripts in the other three genotypes for both muscles. Therefore, the relative abundance of PEG11 transcripts to PEG11AS transcripts was unique in paternal heterozygous animals. MicroRNAs are a central component of RNA interference mechanisms. In the mouse, two antisense microRNA have been identified for the orthologous rtl1 locus [PEG11AS RNA and be involved in normal regulation of the locus and in generating overdominance [PEG11AS may normally cause repression of the paternal PEG11 locus since very little PEG11 mRNA was detectable in the muscles of normal animals. In the animals with a paternally inherited callipyge allele, (+Mat/CLPGPat and CLPGMat/CLPGPat), the normal repression of the PEG11 locus has been disrupted by the mutation in a putative long range control element [PEG11 transcripts only accumulate in significant excess of PEG11AS in the paternal heterozygous animals, whereas in the CLPGMat/CLPGPat animals the PEG11AS transcripts remain in excess and may prevent the accumulation of the threshold level of PEG11 mRNA required to produce a muscle hypertrophy phenotype. Although the expression of PEG11AS was not affected by genotype in quantitative PCR, the northern blots and transcript mapping indicate there are multiple transcripts that are likely to undergo different intron splicing. Further analysis of PEG11AS expression will be necessary to determine its role in the PEG11 locus regulation.The overdominant nature of the callipyge phenotype through the lack of muscle hypertrophy in animals with the e region ,27. If tl1 locus . RNA intl1 locus -32 and tl1 locus -35. Microminance . ExpressDLK1, GTL2, PEG11 and MEG8, within the imprinted cluster in skeletal muscles that become hypertrophied. The increase in transcript abundance was consistent with each gene's parental allele-specific expression. The DLK1 and PEG11 genes were both expressed at their highest levels in paternal heterozygous animals and exhibited polar overdominant gene expression patterns. Therefore, both genes are candidates for causing muscle hypertrophy. DLK1 expression was only elevated in muscles that undergo hypertrophy, so its muscle-specific increase was consistent with the callipyge phenotype. PEG11 was 12-fold more abundant in hypertrophied muscle than non-hypertrophied muscle and only paternal heterozygous animals had PEG11 transcript levels in excess of PEG11AS transcript levels. The unique relationship of PEG11 and PEG11AS in paternal heterozygous animals suggests that an RNA interference mechanism may have a role in regulating the PEG11 locus and polar overdominance in callipyge sheep.The effect of the callipyge mutation has been to increase transcript abundance of four genes, free™ reagents (Ambion Inc.) to remove trace genomic DNA prior to use in the ribonuclease protection assay and quantitative PCR.A series of planned matings were conducted to produce the four possible callipyge genotypes. The genotypes of all lambs were verified using the single nucleotide polymorphism ,24 and set al. [32P]-dATP (Amersham-Pharmacia, Piscataway, NJ USA). Unincorporated nucleotides were removed by spin column chromatography . The probes were hybridized to the membranes without denaturation using Ultrahyb™ (Ambion Inc.) at 42°C overnight. After hybridization, the membranes were washed in 2X SSC (0.3 M sodium chloride, 0.03 M sodium citrate)/0.5% SDS followed by 3 washes in 1X SSC/ 0.1% SDS at 65°C for 30 min and a final high stringency wash in 0.1X SSC/ 0.1%SDS at 65°C for 30 min. The northern blots were exposed to Kodak XAR autoradiography film for 18 to 72 h at -80°C. After autoradiography, the probe was degraded and removed from the membranes using Strip-EZ™ reagents (Ambion Inc.).Northern blots were prepared using denaturing formaldehyde gel electrophoresis  of 10 μg of total RNA and transferred to positively charged nylon membranes using standard methods . Primer et al. , and the32P]-UTP (Amersham-Pharmacia). The RPA were conducted using RPA III™ reagents (Ambion Inc.) and standard urea/acrylamide gel electrophoresis methods [Templates for synthesizing strand specific RNA probes for the ribonuclease protection assay (RPA) were generated from PCR products Table  by ligat methods . Dried g8 to 102 molecules. The standards were used to calculate a regression of threshold cycle on molecule copy number to determine a log value of starting abundance for each of the cDNA samples based on their threshold cycle. The PCR reactions were run for 40 cycles in an iCycler Real-Time PCR Detection System (Bio-Rad Inc.). The log value of starting abundance for each gene was analyzed by analysis of variance using the PROC MIXED procedure of SAS [et al. [Complementary DNA was synthesized from 3.2 μg of total RNA using random hexamer priming and MMLV reverse transcriptase reagents (Invitrogen, Carlsbad, CA USA) with RNase inhibitor supplementation (Superase Inhibitor, Ambion Inc). The cDNA samples were diluted with water and aliquoted so that the quantification was based on 213 ng of total RNA for analysis of glyceraldehyde-3-phosphate dehydrogenase, DLK1 and MEG8 transcripts. PEG11 and PEG11AS transcripts were measured using gene-specific priming of cDNA synthesis from 1.6 μg of total RNA. The cDNA was diluted with water and aliquoted for quantification based on 400 ng of total RNA. Each cDNA sample was amplified in triplicate using the SYBR Green Jump Start™ system (Sigma-Aldridge, St. Louis, MO USA). Quantification standards were composed of aliquots of plasmids containing target PCR products in 10-fold serial dilutions ranging from 10e of SAS . The anaCB participated in planning the study and developing the experimental design, conducted the northern blot analysis and ribonuclease protection assays, and wrote the drafts of the manuscript. LK and AP isolated RNA and performed the quantitative PCR assays. TH collected the muscle samples and genotyped the animals used in this study. DM participated in experimental design and performed the statistical analysis. NC participated in planning the study, set up a series of matings to generate all four genotypes and provided the experimental animals. All authors read and approved the final manuscript.


The growing amount of structural, pharmacological and physiological data for these receptors indicates the need for a dedicated and accurate database to provide a means to access this information in a coherent manner.Nicotinic acetylcholine receptors (nAChRs) are pentameric proteins that are important drug targets for a variety of diseases including Alzheimer's, schizophrenia and various forms of epilepsy. One of the most intensively studied nAChR subunits in recent years has been α7. This subunit can form functional homomeric pentamers (α7) is a new relational database of manually curated experimental physiological data associated with the α7 nAChR. It aims to store as much of the pharmacology, physiology and structural data pertaining to the α7 nAChR. The data is accessed via web interface that allows a user to search the data in multiple ways: 1) a simple text query 2) an incremental query builder 3) an interactive query builder and 4) a file-based uploadable query. It currently holds more than 460 separately reported experiments on over 85 mutations.A7DB A7DB will be a useful tool to molecular biologists and bioinformaticians not only working on the α7 receptor family of proteins but also in the more general context of nicotinic receptor modelling. Furthermore it sets a precedent for expansion with the inclusion of all nicotinic receptor families and eventually all cys-loop receptor families. Distinceceptors . All areeceptors ,5. Mutateceptors ,7 and seeceptors . There ird level normalization, the principal information prototype stored in the database is referred to as an "experiment event". An experiment event is a collection of simultaneous measurements and their associated measurement conditions. For example, two drugs tested against the wild type α7 and a mutant under the same experimental conditions would be described by 4 (2 drugs × 2 sequences) experiment events. If the identical measurements were repeated in, say, calcium-free saline, this would yield 8 experiment events. Using this prototype a unique set of experimental conditions is associated with a single set of data. Therefore, the database tables  the positions of these data can be highlighted on a homology model based on the acetylcholine binding protein AChBP . The modFor any database, data integrity will ultimately determine its usefulness. The initial population of the database has been carried out by the authors, but it is desirable and practical in the long term to have a procedure whereby any laboratory can submit data. To help ensure reliable deposition procedure, each depositor will have a unique identifier (thus making the data accountable). A depositor will submit their data which is then tabulated and presented as a form which the depositor is then asked to confirm as being correct. Only when this confirmation is received is the data committed to the database. Although this will reduce some entry-based mistakes, the database will nonetheless need to be curated to maintain integrity.The database does not attempt to duplicate the functionality of other databases. For example we store a local alignment of α7 subunits taken directly from pfam , but we Access to the database is through various routes. The first route is by a very simple search string (e.g. citation = Smith*). The user is then presented with a page informing him of the number of hits and asking what information should be displayed from those hits. The second is a simple incremental search in which the user applies criteria sequentially until he chooses to examine the results. As the query is built, the number of hits returned by the query in its current state is shown. The third search method is also incremental but via the use of a set of tabbed pages which divide the information into intuitive categories such as pharmacology and physiology. Additionally, the user can make choices about alignments used and even upload their own. As the query is built, summary information of its status is also displayed. The fourth method to access the data is provided by a 'fast lane' route which provides more experienced users with a quicker and more direct route to the data of interest. The user formulates and constructs a query offline and then uploads it as a simple ASCII file. A sample query form and details of the format are presented in the supplementary information.The result of a query is presented in tabular form. Only the information requested is actually presented with the option for further or refined searches. In addition to this, the results of any position matches can be displayed via the homologous 3-dimensional structure of the AChBP. This is automatically available if the user selects the alignments available from the server. If the user uploads their own alignment the model will only be produced if AChBP was included. The server streams a Rasmol script or spawns a Chime window depending on browser configuration. In addition to these query routes, the user is also able to browse the contents of the database.The current database has been designed such that it could easily be extended to include other nAChR subunits and then on to other members of the cys-loop ligand-gated ion channel family. We are currently pursuing this aspect. Such expansion will allow trends that link structure to function in this receptor family to be more readily identified.The database is very complementary to some existing resources, in particular the LGICdb ,15. ThisTorpedo marmorata [We have shown here how the collation and careful storage of experimental data pertaining to one sub-family (the α7 nAChR sub-family) of the ligand-gated ion channels can be assembled into a useful resource. However, the real power of the database will be when it is combined with machine-learning technologies to explore complex relationships between sequence, structure and function . We beliarmorata . Its devThe database is freely available at: The project was conceived and technical aspects managed by PCB. The coding and design was done by SB. MSPS and DBS provided critical evaluation of the design and construction process. DBS, LP, AKJ and LB populated the database. All authors have approved the manuscript.Supplementary information. This doc file provides a complete description of the table entries and also provides a description of the uploadable query file. Further information about the online help is also provided.Click here for file


Haemophilus influenzae (HI). OM often occurs to young children, and is mostly treated with antibiotics. Due to concerns over bacterial resistance toward antibiotics, reliable prophylactic treatments such as administrating anti-adhesion agents are now viewed as viable alternatives.One of the primary causes of otitis media (OM), an inflammation of the middle ear, is the bacterium 111- indium-oxine, pre-incubated with various individual phosphoinositides for 15 minutes at 37°C, and incubated with a monolayer of human pharynx carcinoma (DT 562) cells for 20 minutes at 37°C. The result showed that at 0.1 mg/mL dipalmitoylphosphatidylinositol-3,4-diphosphate (PI-3,4-PP) had the highest anti-adhesion activity, followed by phosphatidylinositol-3-phosphate (PI-3-P) and phosphatidylinositol-4-phosphate (PI-4-P). The anti-adhesion activity of PI-3,4-PP was dose-dependent ranging from 0.006 to 0.1 mg/mL. In addition, results from an in vivo study demonstrated that pre-incubation of HI cells with PI-3,4-PP at 1 mg/mL suppressed the growth of HI in nasopharynx of neonatal rats.The present study tested the feasibilty of using phosphoinositides as anti-adhesion agents against HI cells. Cells of non-typeable HI were radiolabeled with These findings suggest that PI-3-P and PI-4-P and more so PI-3,4-PP may serve as prophylactic agents against HI adhesion and colonization. Haemophilus influenzae (HI). Complications of OM include permanent hearing loss and perforation of the tympanic membrane.Otitis media (OM) is an inflammation of the middle ear, often seen in children younger than six year of age. OM is caused by infection of nasopharyngeal cells by the bacterium Generally, OM is treated with antibiotics such as penicillin derivatives. However, in spite of the effectiveness of antibiotic prophylaxis, the increasing bacterial resistance to antibiotics has caused some concerns. This has prompted the development of anti-adhesive agents against HI infection . Today, in vitro effect of various phosphoinositides on the attachment of HI to nasopharyx was evaluated. The activity was defined as the percent of inhibition of HI adhesion to human pharynx carcinoma cells as compared to the control. Results in Table The attachment (adhesion) of bacteria to a mammal's nasopharynx area is believed to be the first stage of the bacterial infection, which can lead to OM and other disorders and diseases caused by HI. For this study, 10 [cfu/mL]) of HI recovered for a total of 10 rat pups for each treatment. In trial 1, at the inoculum dose of approximately 100 cfu/pup, the treated group (PI-3,4-PP) containing 1 mg/mL of PI-3,4-PP showed an 80-fold (1.9 logs) reduction in the number of bacteria recovered 24 hours post-inoculation as compared to the control group (HBSS). In trial 2, the rat pups were exposed to much higher doses (approximately 700 cfu/pup) of bacteria. The treated group (PI-3,4-PP), which was protected by the same level of PI-3,4-PP, showed a 4-fold (0.6 log) reduction in the number of bacteria recovered. The result demonstrates that PI-3,4-PP can inhibit the attachment and thus the growth of nontypeable HI in the nasopharynx of neonatal rats.The inhibitory activity of PI-3,4-PP against the attachment of nontypeable HI was further demonstrated in two trials using a neonatal rat model. Figure myo-inositol hexaphosphate), myo-inositol penta-and tetra-phosphates (data not shown). Taken together, these results suggests that the presence of both a phosphatidyl group and an additional phosphate group is essential for the high anti-adhesion activity of PI-3,4-PP as compared to PI-3-P and PI-4-P.The exact mechanism for the anti-adhesion activity of the phosphoinositides is not known at this time. However, the anti-adhesion activity may be associated with the structures of the phosphoinositides. Molecules containing no phosphatidyl group such as Ins-1,2,6-P, Ins-1,3,4-P and GPI, exerted no effects on adhesion. PI, which contains a phosphatidyl group, also had no activity. In addition to the presence of a phosphatidyl group, phosphorylation of inositol seems to be required for the bioactivity. Since PI-3,4-PP had a higher anti-adhesion activity than PI-3-P and PI-4-P, it is possible that the presence of extra phosphate groups at the positions 3 and 4 of the inositol moiety may exert an even greater effect. Results from a preliminary study  showed the presence of multi-phosphate groups in a molecule (such as phytate, inositol phosphate, and phosvitin) were inhibitory to HI adhesion. On the other hand, results from our study showed that PI-3,4-PP was more active than phytate  and 1-D-myo-inositol-1,3,4-triphosphate sodium salt (Ins-1,3,4-PPP) , class III L-α-phosphatidylinositol ammonium salt (PI), class IV dipalmitoylphosphatidyl-inositol-3-phosphate ammonium salt (PI-3-P), and L-α-phosphatidylinositol-4-monophosphate sodium salt (PI-4-P), and class V dipalmitoylphosphatidylinositol-3,4-diphosphate ammonium salt (PI-3,4-PP)  of phosphoinositides were used for this study Figure . Class I) Figure . Class I) Figure . Ins-1,22 at 37°C until reaching at least 90% confluency. Plates were washed three times with 20 mL of Hanks Balanced Saline Solution (HBSS)  to remove serum proteins.The Detroit 562 human pharynx carcinoma cell line (DT 562) was obtained from the American Culture Type Collection. The DT 562 cells were seeded into Costar 96-well plates (Corning Life Science, Acton, MA) at a density of 20,000 to 25,000 cells per well, and cultured in Dulbeco's modified Eagle Medium (GIBCO, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS) (Hyclone, Logan, UT). The plates were incubated in a humidified atmosphere of 95% air: 5% CO2 for 18 hours. Bacteria were then harvested in phosphate buffered solution (PBS) supplemented with 0.05% bovine serum albumin (BSA) (Miles Inc., Kankakee, Ill.). After centrifugation, the cell pellets were resuspended in a volume of PBS/BSA yielding an optical density of 2.4 at a wavelength of 660 nm. The bacteria were then radiolabelled with 111-Indium-oxine (111- In), a high energy, short-lived tracer. Fifty μCi of 111-In solution was added to 2.5 mL of the bacterial suspension and incubated for 20 minutes at 37°C. The radiolabeled bacteria were then washed two times with 10 mL HBSS and unbound 111- In were removed by centrifugation. The bacteria pellets were then resuspended in 5 mL HBSS supplemented with 30 mM 2-hydroxyethyl-piperazine-N'-2-ethane sulfonic acid buffer .For the adhesion study, an HI nontypeable bacterial strain was used. The HI isolated from the middle ear of an infected child was a gift from Dr. Lauren Bakaletz of The Ohio State University, Columbus, Ohio. HI was streaked onto Chocolate agar plates (Becto Dickinson Diagnostic Instrument System, Sparks, MD) from frozen aliquots of a low passage number. The plates were then incubated at 37°C in a humidified atmosphere of 95% air: 5% CO111- In-labeled bacterial suspension were pre-incubated with 25 μL of the test chemical (containing various phosphoinositides) in a Costar polypropylene 96-well plate for 15 minutes at 37°C to allow binding of the test agent to the HI. For adhesion quantitation, aliquots (25 μL) of the pre-incubated mixture were pipetted into the wells of an assay plate containing the DT 562 human pharynx carcinoma cells. The assay plate was incubated for about 15 to 20 minutes at 37°C to allow adhesion of the bacteria to the cell monolayer. Nonadherent bacteria were removed by washing the plate three times with HBSS. The cell monolayer and the adhering of the HI cells were disrupted by the addition of 100 μL of 0.05 N sodium hydroxide. The contents of each well were placed in Cobra polypropylene tubes and the radioactivity counted on a Cobra Gamma Counter (Packard Instrument Co., Meriden, CT). After calibration of the background, the average radiation count of four replicates (per sample) was calculated. The percents of inhibition of bacterial adhesion, as compared to bacterial attachment in control wells containing no test chemical were then calculated.Prior to adhesion test, aliquots (25 μL) of the in vivo activity of PI-3,4-PP against nontypeable HI. Prior to the test, overnight cultures of nontypeable HI were prepared, washed twice and diluted with HBSS to obtain a bacterial suspension of less than 105 colony-forming-units (cfu) per mL. Three different test chemicals were prepared for this test: (i) HI + PI-3,4-PP, an aliquot (0.5 mL) of PI-3,4-PP (2 mg/mL in HBSS) mixed with 0.5 mL of the diluted bacterial suspension (5 × 104 cfu/mL) and incubated for one hour at 37°C; (ii) HI + HBSS, This bacterial control prepared by mixing and incubating 0.5 mL HBSS with 0.5 mL of the diluted bacterial suspension; and (iii) HBSS, the solvent blank prepared by incubating 1 mL of HBSS under the same conditions. A 10 μL of the test material was used to inoculate 24-hour-old or younger Sprague Dawley rats (Charles River, Portage, MI) by intranasal administration. Twenty-four hours after the inoculation, samples of nasopharyngeal fluid were collected by the slow instillation of 25 μL of HBSS into the left naris, and the initial 10-μL discharge from the right naris was collected for plate count. This procedure insured that the fluid had passed through the nasopharynx. The nasal wash was then spread (or diluted and then spread), onto Chocolate agar plates. The plates were incubated at 37°C overnight and counted for the number of cfu's, an indicator of the number of viable bacteria.A neonatal rat model ,15 was mYSH conceived of the study. JWL screened and identified the phosphoinositides, and drafted the manuscript. SNA carried out cell adhesion assay. JM carried out assay for in vivo activity. SMH and PM participated in experiment coordination.


Deregulation of the Wnt/ β-catenin signal transduction pathway has been implicated in the pathogenesis of tumours in the mammary gland, colon and other tissues. Mutations in components of this pathway result in β-catenin stabilization and accumulation, and the aberrant modulation of β-catenin/TCF target genes. Such alterations in the cellular transcriptional profile are believed to underlie the pathogenesis of these cancers. We have sought to identify novel target genes of this pathway in mouse mammary epithelial cells.Gene expression microarray analysis of mouse mammary epithelial cells inducibly expressing a constitutively active mutant of β-catenin was used to identify target genes of this pathway.Hig2, previously shown to be induced by both hypoxia and glucose deprivation in human cervical carcinoma cells, was strongly repressed upon ΔNβ-catenin induction. The predicted N-terminus of Hig2 contains a putative signal peptide suggesting it might be secreted. Consistent with this, a Hig2-EGFP fusion protein was able to enter the secretory pathway and was detected in conditioned medium. Mutation of critical residues in the putative signal sequence abolished its secretion. The expression of human HIG2 was examined in a panel of human tumours and was found to be significantly downregulated in kidney tumours compared to normal adjacent tissue.The differential expression in response to ΔNβ-catenin for five putative target genes, Autotaxin, Extracellular Matrix Protein 1 (Ecm1), CD14, Hypoxia-inducible gene 2 (Hig2) and Receptor Activity Modifying Protein 3 (RAMP3), was independently validated by northern blotting. Each of these genes encodes either a receptor or a secreted protein, modulation of which may underlie the interactions between Wnt/β-catenin tumour cells and between the tumour and its microenvironment. One of these genes, HIG2 represents a novel non-cell autonomous target of the Wnt pathway which is potentially involved in human cancer. The Wnt/β-catenin signal transduction pathway plays a central role in metazoan development, controlling such diverse processes as cell growth, proliferation and organogenesis . Wnt-1 iAPC was identified as the tumour suppressor gene mutated in the hereditary colorectal cancer syndrome, Familial Adenomatous Polyposis [Axin and β-catenin have also been detected in tumours of the colon and other tissues [Overexpression of Wnt-1 in the mammary glands of transgenic mice leads to extensive hyperplasia and tumorigenesis . APC wasolyposis ,11. Muta tissues . DeregulMany of the Wnt targets thus far identified are cell-cycle regulators ,14 and t2 at 37°C in RPMI 1640, supplemented with 10% Foetal Bovine Serum, 2 mM L-glutamine, 2.5 μg/ml insulin, 5 ng/ml epidermal growth factor and 50 μg/ml gentamycin [lacZ and HC11-ΔNβ-catenin cells were routinely cultured in 2 μg/ml tetracycline to repress transcription of the tetracycline-regulated transgene. HEK293 and MDCK cells were grown in DMEM supplemented with 10% Foetal Bovine Serum.All reagents were purchased from Sigma unless otherwise noted. HC11 mouse mammary epithelial cells were cultured in 5% COntamycin . HC11-lalacZ and HC11-ΔNβ-catenin cell lines were generated by infecting the cells with an ecotropic retrovirus (TRE-tTA) in which the tTA cDNA is under the control of a tetracycline responsive promoter. Consequently, tTA expression is minimal in the presence of tetracycline and, upon tetracycline withdrawal, tTA activates its own transcription in an autoregulatory manner [The HC11-y manner . HC11 ceBosc23 cells were used to produce ecotropic retroviruses . Cells wCell monolayers were washed twice in ice-cold Phosphate Buffered Saline and lysed by addition of Trizol (Invitrogen). Total RNA was isolated according to the manufacturer's instructions. PolyA+ RNA was purified from total RNA using Oligotex (Qiagen) according to the manufacturer's instructions.Autotaxin – 533819; CD14 – 2936787; Ecm1 717050; Hig2 – 367488; Ramp3 – 615797, HIG2 4366895). Following overnight hybridisation with the labelled probe, the membranes were washed twice in 1X SSC, 0.1% (w/v) SDS at room temperature for 20 mins, and twice in 0.2X SSC, 0.1% (w/v) SDS at 68°C for 10 mins and exposed to film at -80°C for 48 hours. Bound probe was quantitated using a phosphorimager (Molecular Dynamics).10 μg of total RNA from each cell line was fractionated on a denaturing formaldehyde agarose gel and transferred to a positively charged nylon membrane (Hybond N+, Amersham Pharmacia Biotech) in 10x SSC. Membranes were prehybridised for four hours in 50% (v/v) formamide, 5X SSPE, 2X Denhardt's reagent, 0.1% (w/v) SDS and 100 μg/ml denatured herring sperm DNA. Radiolabelled probes were prepared from PCR-amplified cDNA clones using the Rediprime II kit (Amersham Pharmacia Biotech) according to the manufacturer's instructions. EST sequences corresponding to the coding sequence of the genes-of-interest were identified by BLAST  and obtaCell monolayers were rinsed twice with ice-cold Phosphate Buffered Saline and total cell lysates were prepared by scraping cells into a minimal volume of 50 mM Tris. HCl pH 7.5, 150 mM NaCl, 0.5% NP40 and Complete protease inhibitor cocktail (Roche). Aliquots containing 80 μg protein from each sample were analysed by SDS-PAGE , and tralacZ cDNA was excised from the CMV-lacZ construct  and sub-cloned into BamHI digested RevTRE to make RevTRE-lacZ. A plasmid containing a myc-tagged ΔNβ-catenin was obtained from Hans Clevers. The myc-tagged ΔNβ-catenin was excised with KpnI and NotI and the ends were blunted, and subcloned into HpaI digested RevTRE to make RevTRE-ΔNβ-catenin. The mouse Hig2 open reading frame was amplified by PCR from I.M.A.G.E. cDNA clone 367488 using the primers TTTACTAGTAGGAGCTGGGCACCGTCGCC and TTTTACCGGTGCCTGCACTCCTCGGGATGGATGG. The PCR product was digested with AgeI and SpeI and subcloned into the AgeI and NheI sites in pEGFP-C1 (Clontech) to make the Hig2-EGFP fusion gene. Site directed mutagenesis was carried out by the method of Sawano and Miyawaki (2000) [A BgIII fragment containing the i (2000) . The pri5 cells were plated in 3.5 cm wells on the day prior to transfection. Each well was transfected with a total of 0.9 μg DNA under serum-free conditions for six hours, after which the cells were washed and incubated for a further 48 hours before assaying expression.Transient transfections were performed using Lipofectamine (Invitrogen) according to the manufacturer's instructions. Briefly, 1.5 × 10lacZ cells for each condition, were cultured in triplicate in 96 well plates for 72 hours, and beta-galactosidase activity was determined as previously described [For the tetracycline dose response curve, 5000 HC11-escribed .Stable cell lines were generated in which either ΔNβ-catenin or β-galactosidase was expressed in a tetracycline dependent manner. These cell lines were established using a novel autoregulatory system in which the expression level of the tetracycline transactivator (tTA) protein is minimised during routine culture and is induced upon withdrawal of tetracycline with concomitant upregulation of the transgene-of-interest . This stlacZ cell line is shown in Figure A dose-response analysis for the HC11-lacZ and HC11-ΔNβ-catenin cells by withdrawal of tetracycline for 72 hours. Total RNA was isolated, from which mRNA was purified. cDNAs were labelled and hybridized to an 8962 element Incyte mouse GEM1 cDNA microarray . These data are provided as supplementary material , Autotaxin, Receptor Activity Modifying Protein 3 (Ramp3), Cd14 and Hypoxia Inducible Gene 2 (Hig2). Each putative target gene was initially subjected to a secondary screen by Northern blotting to confirm the differential expression in response to ΔNβ-catenin  cells by transient transfection   Figure . The levcDNA microarray analysis of the transcriptional changes resulting from overexpression of a constitutively active β-catenin revealed a panel of putative target genes of the Wnt/β-catenin pathway in mouse mammary epithelial cells. This differential expression was confirmed by Northern blotting in five cases.Autotaxin [in vivo [Autotaxin was originally identified as a secreted enzyme with potent motility stimulating activity  and has utotaxin , and pur[in vivo . Autotax[in vivo . Autotax[in vivo ,41.In situ hybridisation showed that Ecm1 was strongly expressed in most newly formed blood vessels and experiments using purified recombinant Ecm1 showed that it could increase the proliferation rate of vascular endothelial cells in vitro and also stimulate angiogenesis in vivo. The ability to induce de novo angiogenesis is an absolute requirement for tumours to grow beyond a size which can be readily perfused by oxygen and nutrients from the interstitial fluid. ECM1 is overexpressed in many epithelial tumours including 73% of breast tumours analyzed [Autotaxin and Ecm1 as genes upregulated by activation of this pathway, together with VEGF [Extracellular Matrix Protein 1 was first identified as a novel 85 KDa protein secreted by a mouse osteogenic stromal cell line . In situanalyzed . Homozyganalyzed . The ideith VEGF  suggestsCD14, which can function as both a receptor and a secreted protein, was downregulated upon ΔNβ-catenin expression. CD14 is a glycosyl-phosphatidylinositol-linked cell surface protein, preferentially expressed in monocytes, where it acts as a receptor for Lipopolysaccharide Binding Protein:Lipopolysaccharide complexes [in vitro and has been detected in human milk where it is postulated to play a role in neonatal immunity [in vivo at the onset of involution [omplexes . Solubleimmunity , and is volution .Receptor Activity Modifying Protein 3 (RAMP3) was downregulated upon ΔNβ-catenin induction and is one of three members of the RAMP family. These proteins are involved in mediating the cellular response to the neuropeptides calcitonin, calcitonin gene related peptide, amylin and adrenomedullin. The RAMP family members function as chaperones for the seven transmembrane domain G-protein coupled receptors for these neuropeptides, shuttling the receptor to the cell surface and altering receptor glycosylation. The ligand binding phenotype of the receptor is dependent on the RAMP family member with which it is associated [sociated . RAMP3-Csociated , and RAMsociated .in vivo [Expression of both CR and CRLR was detected in HC11 by RT-PCR (data not shown) suggesting that functional receptor-RAMP complexes are present in this cell line. Adrenomedullin, the ligand for the CRLR/RAMP3 receptor dimer, functions as a growth factor in several human tumour cell lines , in addiin vivo  via CRLRin vivo ,55.Hypoxia-inducible gene 2 (Hig2) was one of several genes identified in a representational difference analysis screen for genes regulated by hypoxia in a human cervical epithelial cell line. The human gene encodes a 63 amino acid polypeptide of unknown function [Hig2 was downregulated in HC11 cells overexpressing ΔNβ-catenin. The identification of a group of mammalian orthologues revealed a well conserved hydrophobic region in the N-terminus, reminiscent of a signal peptide. A Hig2-EGFP fusion protein entered the secretory pathway and was detected in conditioned medium of transfected cells. The introduction of a pair of charged amino acids into the hydrophobic region abolished secretion, lending support to the hypothesis that this region contains a functional signal peptide. The nature of the large vesicular structures observed in Hig2-EGFP overexpressing cells is as yet unclear. Mammary epithelial cells are known to contain membrane-enclosed lipid droplets, as well as a variety of vesicular compartments involved in the secretion of casein, citrate, lactose and calcium [function . Express calcium , howeverHIG2 using a matched Tumour/Normal tissue cDNA array showed that HIG2 is widely expressed. In most cases, the levels of HIG2 in the tumours and the associated normal tissue controls were similar. HIG2 was, however, strongly and consistently downregulated in the majority of the kidney and stomach tumours analysed. This represents an significant validation of our in vitro findings in human tumours, and suggests that HIG2 may exert a tumour suppressive effect in vivo. Human HIG2 is located on 7q32.2, a commonly deleted region in several tumour types, most prominently leukaemias and lymphomas [Analysis of the expression of ymphomas . Deletioymphomas .The identification of this panel of candidate target genes for this clinically important signal transduction pathway adds to those identified by other workers in a variety of model systems and suggests that, as well as promoting tumour cell proliferation and survival in a cell autonomous manner, this activation of this pathway is likely to have a series of non-cell autonomous effects. Here we have focussed on the identification of Wnt/β-catenin target genes that are either secreted signalling molecules or receptors. It is likely that such targets are involved in mediating autocrine proliferation, promotion of angiogenesis and the mediation of reciprocal communication between Wnt/β-catenin tumours and their microenvironmental milieu.The authors declare that they have no competing interests.PK carried out all of the experimental procedures and drafted the manuscript. PK, TE and AA contributed to the design of the study. All authors read and approved the final version of this manuscript.The pre-publication history for this paper can be accessed here:cDNA microarray dataset: HC11 ΔNβ-catenin v. HC11 lacZ. This file contains the dataset from the Incyte GEM1 cDNA microarray comparison between HC11 cells overexpressing ΔNβ-catenin (Probe 1) and β-galactosidase (Probe 2). This file may be easily imported into MS Excel, Genespring or other microarray analysis software to facilitate further analysis.Click here for file


Lymphoma-associated hemophagocytic syndrome (LAHS) occurs in mostly extra nodal non-Hodgkin's lymphoma. LAHS arising from gastrointestinal lymphoma has never been reported. Here we report a case of gastric T-cell lymphoma-associated hemophagocytic syndrome.A 51-year-old woman presented with pain, redness of breasts, fever and hematemesis. Hematological examination revealed anemia. Gastroscopy revealed small bleeding ulcers in the stomach and the computed tomography scan showed liver tumor. She underwent total gastrectomy for gastrointestinal bleeding and the histopathology revealed gastric T-cell lymphoma. She continued to bleed from the anastomosis and died on the 8th postoperative day. Autopsy revealed it to be a LAHS.If Hemophagocytic syndrome (HPS) occurs in lymphoma of the gastrointestinal tract, bleeding from the primary lesion might be uncontrollable. Early diagnosis and appropriate treatment are needed for long-term survival. Hemophagocytic syndrome (HPS) in adults is characterized by reactive and systemic proliferation of benign histiocytes that phagocytose blood cells . It is o4/mm3, hemoglobin 10.3 g/dl (post transfusion), WBC 4,900/mm3, Platelets 51,000/mm3, serum albumin 1.5 g/dl, total bilirubin 0.6 mg/dl, AST 691 IU/l, ALT 187 IU/l, LDH 2976 IU/l, fibrinogen 134 mg/dl, FDP 10 μg/ml, and AT-III 40%. Bleeding from the stomach continued and did not stop with conservative treatment; therefore, two days later the patient underwent total gastrectomy and a partial liver resection. Histopathology of the resected specimen showed it to be a gastric lymphoma (pleomorphic medium-large cell type, non-Hodgkin's T-cell lymphoma) with liver metastasis . There was no generalized lymphadenopathy. On gastroscopic examination multiple small ulcers were observed in the stomach. An abdominal computed tomographic (CT) scan showed liver tumor and a normal spleen. Hematological and biochemical examination at admission showed the following results: RBC 352 × 10in situ hybridization. The final diagnosis was EBV-related T-cell LAHS.On autopsy, malignant lymphoid cell infiltration and hemophagocytosis were observed in the liver, spleen, heart, small bowel, lung, both breasts, kidney, pancreas, uterus, and gastroduodenal lymph nodes Fig. . The bonet al, [et al, [HPS is a clinicopathological entity characterized by systemic proliferation of benign hemophagocytic histiocytes, fever, cytopenia, liver dysfunction, hepatosplenomegaly, and coagulopathy . This syet al,  are wideet al, ,3,4. On  [et al,  has propIn Japan, T-cell LAHS accounts for 48.5% of all adult LAHS . UncontrThe poor prognosis of LAHS, especially T-LAHS, is well known. The median survival time from the diagnosis is reported to be 143 and 69 days respectively in Japan . For LAHIn this case, the initial presentation was mastalgia and hence it took a considerable amount of time to reach a diagnosis. Furthermore, bleeding from the anastomosis continued leading to a rapidly progressive fatal clinical course.In HPS occurring in lymphoma of the gastrointestinal tract uncontrollable bleeding from the primary lesion might occur. Therefore, an earlier diagnosis of HPS should be made by bone marrow aspirates, and appropriate treatments should be started as soon as possible. Surgery if performed, must be performed with utmost caution.LAHS could also occur from lymphoma of the gastrointestinal tract. For long-term survival; early diagnosis and appropriate treatment are needed. Surgery if performed without a proper diagnosis could prove fatal.The author(s) declare that they have no competing interests.RF, FH, TY, RD, KO and KH were gastrointestinal surgeons.MO referred this patient to us.KK and MS performed pathological examination and the autopsy.KS was a member of the intensive care team.TH, YY, HN gave us helpful comments about the manuscript


Due to the low participation in colorectal cancer screening, public preference for colorectal cancer screening modality was determined.A cross-sectional survey was performed of healthy ambulatory adults in a pediatrics primary care office and neighboring church. Overall preference was ranked for each of four colorectal cancer screening modalities: Faecal Occult Blood, Fiberoptic Sigmoidoscopy, Barium Enema and Colonoscopy. Four additional domains of preference also were ranked: suspected discomfort, embarrassment, inconvenience and danger of each exam.80 surveys were analyzed, 57 of which were received from participants who had experienced none of the screening tests. Fecal Occult Blood Testing is significantly preferred over each other screening modality in overall preference and every domain of preference, among all subjects and those who had experienced none of the tests.Efforts to increase public participation in colorectal cancer screening may be more effective if undertaken in the context of public perceptions of screening choices. Screening for colorectal cancer lessens the risk of dying from that disease . KnowledMost publication concerning colorectal cancer screening relates to the choice of screening modality; discussing accuracy, efficacy and cost, since the most inexpensive technique, faecal occult blood testing, is inaccurate in the detection of colorectal neoplasia, though effective in significantly diminishing disease specific mortality , and theHowever the greatest problem related to screening remains the low level of participation by those for whom it is intended: asymptomatic individuals over the age of 50 years with no specific risk factors for colorectal cancer, i.e., no past history of colorectal polyps, cancer, rectal bleeding, colitis, change in bowel habits, iron deficiency anemia, weight loss or a close family member with colorectal cancer. We agree with Dr. Woolf , that stWe have in this report chosen to focus our survey differently and uniquely; first to inform healthy, ambulatory and younger people, and not ailing patients, concerning only the preparation and conduct of each screening test. Secondly, in order to determine how their perceptions of the conduct of each test might affect their participation, participants were then asked to rank not just their overall preference based upon the preparation and conduct of the tests alone, but four other domains of preference for each screening modality: perceived physical discomfort, inconvenience, embarrassment and danger. Test accuracy was not included in the preamble on test performance, first, because we wanted to isolate perceptions of the physical conduct of the screening test, and second, because test accuracy has been part of many of the previous surveys, often presented with considerable bias. Randomized trials of decision aids have also shown that description of a test's ability to detect colorectal cancer has not been successful in increasing participation in screening -17. LastParticipants were a convenience sample of parents or grandparents of children visiting a general pediatrics office , personnel working in that office, or parishioners attending a church social gathering, all aged 18 and over. An introductory letter described the purpose of the survey. This was followed by a brief description of the preparation and performance of each commonly used screening test for colorectal neoplasia: faecal occult blood testing (FOBT), fiberoptic sigmoidoscopy (FS), air contrast barium enema (BE) and total colonoscopy (C). The relative accuracy of each exam was not discussed. Six questions followed. The first asked the participant to rank each test in order of overall preference. The second asked the participant to rank each test according to how much that test might cause physical discomfort, the third, inconvenience; the fourth embarrassment, the fifth, the relative danger of the exam. The sixth question asked participants which of the four tests they had previously experienced, along with their gender and age. No further symptom or medical history was obtained and surveys were only numbered consecutively with no personal identifiers. (see appendix for letter and survey)Based upon a related survey concerning subject preference for tests of colonic inflammation , a samplData were analyzed using SPSS 11.0. Analyses focused on comparisons between ranks assigned each test on preference and the other assessed attributes, and included Friedman's test for ranks (to test the hypothesis that ranks differed for different tests) and the Wilcoxon signed-ranks test (to test the hypothesis that pairs of tests were differently ranked.) We also considered whether those rank orders might differ between participants who have and have not received any of these tests, and how gender and age affected preferences.80 of 84 surveys were available for analysis; twenty nine from men and 51 from women. The mean age of the participants was 38.3 years . Eight subjects had previously had a colonoscopy, five a barium enema, seven a sigmoidoscopy and 17 had stool collected for various reasons. Fifty seven subjects had experienced none of the screening tests. The mean rankings for preference among the entire sample are presented in Table Median scores were determined for each domain for both the whole survey group and the naive subgroup. For each domain and in each group the results were the same, with ranks of 4,3,2 &1 for C, BE, FS and FOBT respectively, 1 being most preferred, except for embarrassment in both groups in which C and BE each had a median rank 3.The results hold up for each gender subgroup in all cases except that men didn't consider FOBT significantly less inconvenient than FS. Age was not significantly correlated with ranking of FOBT (that is, the ranking given didn't change with age) by Spearman's rho. Rho values ranged from -0.10 to +0.16, none significant. Splitting the groups into ages 18–39 (n = 39) and 40+ (n = 40), the results are the same for both groups except that for those over 40, preference for FOBT vs. FS and inconvenience of FOBT vs. FS did not reach significance by two-tailed test (p = .079 and p = .057 respectively)A recent review of colorectal cancer screening stated that, "At present there is no preferred CRC screening strategy". This prpatients, were quite different from the group reported herein. Only one of those reports offered all four of the screening modalities that we did in our study [Among previous surveys there are four randomized controlled trials of the use of decision aids that were designed with the intent of altering participation in screening. Three of these presented choices of screening modality or scenario to both intervention and control groups -17. Thesur study , the othur study ,16. Neveur study . In the ur study  and quesAmong some physicians there is a growing popularity for the use of definitive diagnostic testing as a screening tool, that is, colonoscopy . Though But the public has to want to participate in this program and there is little evidence in this current survey and previous studies, especially those done in primary care settings -18, thatThe author(s) declare that they have no competing interests.RLN conceived of the study, designed the questionnaire and supervised its administration.AS organized the domains of preference and performed the statistical analyses.Both authors participated in the writing of the manuscript.The pre-publication history for this paper can be accessed here:


Current journal requirements forcing clinical trials to be registered  are insu


Recently, several members of a vertebrate protein family containing a six trans-membrane (6TM) domain and involved in apoptosis and cancer (e.g. STEAP, STAMP1, TSAP6), have been identified in Golgi and cytoplasmic membranes. The exact function of these proteins remains unknown.We related this 6TM domain to distant protein families using intermediate sequences and methods of iterative profile sequence similarity search.Here we show for the first time that this 6TM domain is homolog to the 6TM heme binding domain of both the NADPH oxidase (Nox) family and the YedZ family of bacterial oxidoreductases.This finding gives novel insights about the existence of a previously undetected electron transfer system involved in apoptosis and cancer, and suggests further steps in the experimental characterization of these evolutionarily related families. A family of vertebrate proteins containing a six transmembrane domain 6TM) has been recently implicated in apoptosis and cancer. Up to date, this family contains four vertebrate members: STEAP, STAMP1/STEAP2, TIARP and TSAP6/pHyde. STEAP  was the first described member of this family and identified as a prostate-specific cell-surface antigen overexpressed in cancer, located at the cell-cell junction of the secretory epithelium of prostate, and found as well in both colon and bladder cancer cell lines ,4. TIARPTM has beEscherichia coli YedZ protein, which binds a single heme and is involved in electron transfer to the molybdopterin cofactor in YedY, its operon neighbour gene [In this work we present evidence of remote homology of this family to other two families: the mainly eukaryotic Nox and the bacterial YedZ Fig. , both inour gene -18. The To do the sequence analysis of the new domain we took advantage of the possibility of connecting distant protein families via intermediate sequences  and methHaemophilus ducreyi) with an E-value of 0.63. This protein belongs to the large YedZ family of bacterial oxidoreductases. The corresponding YedZ global profile detected the STEAP family (most similar member: SpTrembl Q8IUE7, human STAMP1) with an E-value of 0.00087. The global profile of STEAP and YedZ detected the Nox family with an E-value of 0.032 and the corresponding Nox global profile localized the YedZ family with an E-value of 0.007 . These searches detected all of the ACRATA domain-containing families after 15 iterations (using a cut-off of E = 0.005 for the inclusion of retrieved sequences in the profile). None of these profile searches retrieved new unrelated sequences, and reciprocal searches produced convergent results. Therefore, we have concluded that ACRATA is a previously undetected, conserved domain that is commonly found in members of the STEAP, YedZ and Nox protein families. The similarity between these proteins was suspected before [To investigate the consistency of our results we performed iterative database searches using the PSI-BLAST program . We usedd before . HoweverThe complete conservation of two histidines in all ACRATA domain containing proteins Fig.  indicateExperimental evidence show that Nox and YedZ families share heme binding capabilities and also involvement in electron transfer chains -13,16. FE. coli in a number of conditions tested . Very differently, changes in the expression patterns of human genes containing the ACRATA domain could be related with apoptosis or cancer [Although the mechanism of action of the ACRATA domain could be the same in all the proteins containing it, its variable cellular role is made conspicuous by the different effects produced by the modifications in the expression of the corresponding genes. For example, the knock-out of the whole YedYZ operon seems not to affect r cancer -14. The r cancer , or in tr cancer ,12.We have described for the first time a 6TM domain present in three protein families (STEAP, YedZ and Nox): the ACRATA domain. The common functions of the proteins of those families suggest that this domain is involved in electron transfer, mediated by its heme binding capability. We hypothesize that STEAP, STAMP1, TSAP6, and TIARP have this function, and that they form part of electron transfer systems involved in cellular regulation, apoptosis, and cancer. Additional experimental approaches using different members of the ACRATA domain-containing families are required to confirm these hypotheses.STEAP, six-transmembrane epithelial antigen of the prostate;STAMP1, six transmembrane protein of prostate 1;TSAP6, tumor suppressor activated pathway-6;TIARP, Tumor necrosis factor-alpha-induced adipose-related protein;TCTP, Translationally controlled tumor protein;Nox, NADPH oxidase;ROS, reactive oxygen species;TM, Trans-membrane;FAD, flavin adenine dinucleotide;HMM, Hidden Markov Models;The author(s) declare that they have no competing interests.LSP, AMR, and MAA carried out the sequence analysis of the domain.LSP and MAA provided with the initial input of the research.LSP, AMR, AV, CMA, and MAA authored the manuscript.The pre-publication history for this paper can be accessed here:


It is a high priority that health care providers have effective communication skills. It has been well documented that the doctor-patient relationship is central to the delivery of high quality medical care, and it has been shown to affect patient satisfaction, to decrease the use of pain killers, to shorten hospital stays, to improve recovery from surgery and a variety of other biological, psychological and social outcomes. This study sought to quantify the current knowledge of interns in Iran about communication skills.A cross-sectional study using a self-report questionnaire was conducted among interns. Data analysis was based on 223 questionnaires. The internal consistency of the items was 0.8979.Overall, knowledge levels were unsatisfactory. Results indicated that interns had a limited knowledge of communication skills, including identification of communication skills. In addition, there was a significant difference between the mean scores of interns on breaking bad news and sex education. The confidence of males about their communication skills was significantly higher than for females. Analysis of the total scores by age and sex showed that there was a statistically significant main effect for sex and the interaction with age was statistically significant. Free response comments of the interns are also discussed.It is argued that there is a real need for integrating a communication skills course, which is linked to the various different ethnic and religious backgrounds of interns, into Iranian medical curricula. Some recommendations are made and the limitations of the study are discussed. The expectations of the public have been dramatically increased and the majority of them are familiar with their rights in the health care system. As a consequence, it is a high priority that health care providers have effective communication skills. It has been well documented that the doctor-patient relationship is central to the delivery of high quality medical care. It has been shown to affect patient satisfaction, to decrease the use of pain killers, to shorten hospital stays, to improve recovery from surgery and a variety of other biological, psychological and social outcomes -4. Lack However, little is known about the importance of communication skills in the practice and training of doctors in Iran, where the culture differs greatly from that of the West. Sensitivity to religious matters is particularly important in Iranian doctor-patient relationships where Islam is more than a religion; it is a way of life. It controls politics, local laws, behaviour and many other aspects of daily life. It gives guidance in all spheres of human activity from birth to death. Therefore doctors coming into contact with religious patients need to be aware that there are numerous potential barriers to good communication .A major criticism of current medical training in Iran is that communication skills have not been embedded in the curriculum of Iranian medical students, despite the richness and variety of evidence from elsewhere concerning the importance of communication skills. Concerns over poor doctor-patient communication amongst Iranian doctors led to an exploration of the current situation . In thisA quantitative survey was performed at Tehran University of Medical Science (TUMS). A cross-sectional study was conducted using a questionnaire administered to 235 interns. Anonymity was maintained throughout. The subjects received the self-administered questionnaire with a covering letter explaining the project and the subject's rights. 12 subjects did not return the questionnaire and an additional 7 subjects did not give their age and one person did not give his/her sex. Therefore data analysis was based on 223 questionnaires, but covariate-based analysis on fewer. The subjects were asked to complete the questionnaire without referring to source books.The questionnaire consisted of three sections. The first section asked students to give personal details including the demographic items age and gender .The choice of items was based on the communication skills an intern will need. All items were verified and subjected to content validation by three major experts in communication skills. These experts were given copies of the CSKS and the purpose and objectives of the study. They then evaluated the CSKS on an individual basis. Comparisons were made between these evaluations and the authors then made some minor changes within the CSKS. The CSKS had a high internal consistency  is 10 to 100, with 10 indicating low knowledge. Analysis of the total scores produced a mean score of 51.30 [95 per cent confidence interval (CI) 49.05–53.55]. The subjects' performance on the CSKS suggests a knowledge deficit in communication skills. The mean scores for males and females were respectively 53.6 and 48.2 (P = 0.02). The vast majority of interns (78.1%) had not studied a paper on communication skills. When asked whether they had formally attended communication skills courses, 91.4% of interns reported "no". Of the few interns who reported "yes", these interns specified courses such as CPR, injections and semiology . There was a statistically significant main effect for sex [F (1, 212) = 4.90, p = 0.02] and the interaction effect [F (1, 212) = 4.06, p = 0.04) did reach statistical significance. However the effect size was small (eta squared = 0.02). The young male interns were more confident than average, while the young female interns were less confident.Free responses included the following comments:Nobody has trained us about communication skills. Our knowledge in respect of communication skills is very poor. Your items show that we are very far behind other countries. Our universities are not as advanced as other universities'.''I feel we are not familiar with the ABC of communication skills'.'A good guide to communication skills needed'.'I feel communication skills would be an excellent course since it gives us an idea of how we can handle bad news'.'Attending doctors are not totally familiar with the aims and use of communication skills in the clinical setting'.'All our courses only focus on biological issues rather than psychosocial issues'There were a number of limitations to this study.1. The CSKS has not been normed for a population of interns.2. Criterion-related validity of the CSKS was not determined, although content validity was established on the instrument.3. Since it is a self-assessed questionnaire, these may be problems with bias, such as prestige bias.The very high response rate (95%) of this questionnaire may have reflected general interest, or may have resulted from the advantages of self-assessment which itself may improve performance. The results on the CSKS show that basic knowledge of interns in Iran about communication skills is limited. Researchers have reported similar findings in other countries which reveal a deficit in the knowledge of doctors about communication skills . The impThe vast majority of research studies have been conducted on the outcome of communication skills in the practice and training of doctors in western countries. Even here, despite doctors trained in communication skills and the advocacy of the use of a patient-oriented approach, some evidence suggests that there are difficulties in practice ,20. HoweThe results of the study show that there were significant differences between males and females with regard to their reported knowledge of the main communication skills. Women were less confident of their skills. The deficit may partly be an artefact of an inadvertent prestige bias of the male students. The deficit is particularly notable in sex education. There are three possible explanations for this. Firstly, in general, Iranian female interns are very shy to ask patients about sexual issues. Therefore they may feel that sex education skills have no implications for their practice and hence pay less attention to sex education training. Secondly, it may be a systematic error in female respondents, i.e. they may be shy to discuss their knowledge about sex education skills rather than lack knowledge. Thirdly, in the past, sex education was regarded as a taboo in Iran and was not available in schools, especially for girls . This peThe results on the CSKS suggest that there are areas of weakness in the communication skills confidence of interns, particularly in breaking bad news. While it is well recognised that delivering bad news is a difficult task that requires skills and sensitivity , both feThere is a significant difference between the mean score of the interns on breaking bad news. The female interns have reported lower confidence than the male interns. The deficit could be an inadvertent prestige bias of the male students. However, to our knowledge, there is no evidence that underpin such finding. Although Orlander et al's work  demonstrGiven the poor levels of confidence about communication skills, particularly sex education skills, revealed in this study, it is concluded that educational programmes are necessary. In sex education skills training, given the complex interplay of cultural and religious beliefs in Iran, particular attention must be paid to multicultural and religious issues. Therefore, further work is needed on gender education and stereotypes in sex education; learning styles; the 'hidden curriculum'; and how far medical schools make organisational and administrative arrangements on the basis of gender and the implication for female and male interns.The enthusiastic response to the questionnaires may suggest that medicine is accepting the need for developing communication skills within the medical curriculum. Medical education in Iran must respond to this challenge.Finally, our findings may be somewhat limited in generalisability because they are derived from only one medical school in Iran. Self-assessment data may suffer from biases such as prestige bias. Despite these caveats, the authors believe the data to be an accurate reflection of current practice in Iran, based on the Iranian authors training experiences, and consistency with previous accounts.Whilst the approach to this research has been shaped by a government-recognised health need, the authors recognise the need for, and welcome, further examination of these findings from multiple perspectives, especially with regards to ethnicity and social issues. Since not enough attention has been focused on individuals as makers of health as a service rather than customers of health care services, it is strongly recommended, therefore, that medical students be trained in the context of psychosocial issues that may influence health behaviour, as has been indicated by one of the participants. It is particularly important that this type of approach be incorporated into the curricula of medical training. This may assist in transferring from the disease-oriented to the patient-oriented approach and ultimately lead to patients understanding more and taking greater responsibility for their own health.The author(s) declare that they have no competing interests.MT and ST carried out the conception, design, initial analysis and interpretation of the data. MT drafted the paper. ODL was involved in revising the draft critically, revising the statistical analysis and gave final approval of the version to be published. AAZ contributed to the collection of data and the reviewing of the manuscript.The pre-publication history for this paper can be accessed here: