Xenopus laevis.Rho GTPases and their downstream effector proteins regulate a diverse array of cellular processes during embryonic development, including reorganization of cytoskeletal architecture, cell adhesion, and transcription. Changes in the activation state of Rho GTPases are converted into changes in cellular behavior by a diversity of effector proteins, which are activated in response to changes in the GTP binding state of Rho GTPases. In this study we characterize the expression and function of one such effector, XCEP2, that is present during gastrulation stages in Xenopus laevis, a gene encoding a Rho GTPase effector protein (Xenopus Cdc42 effector protein 2, or XCEP2) was isolated, and found to be highly homologous, but not identical, to a Xenopus sequence previously submitted to the Genbank database. These two gene sequences are likely pseudoalleles. XCEP2 mRNA is expressed at constant levels until mid- to late- gastrula stages, and then strongly down-regulated at late gastrula/early neurula stages. Injection of antisense morpholino oligonucleotides directed at one or both pseudoalleles resulted in a significant delay in blastopore closure and interfered with normal embryonic elongation, suggesting a role for XCEP2 in regulating gastrulation movements. The morpholino antisense effect could be rescued by co-injection with a morpholino-insensitive version of the XCEP2 mRNA. Antisense morpholino oligonucleotides were found to have no effect on mesodermal induction, suggesting that the observed effects were due to changes in the behavior of involuting cells, rather than alterations in their identity. XCEP2 antisense morpholino oligonucleotides were also observed to cause complete disaggregation of cells composing animal cap explants, suggesting a specific role of XCEP2 in maintenance or regulation of cell-cell adhesion in early embryos. This loss of cell adhesion could be rescued by co-injection with a morpholino-insensitive version of the XCEP2 mRNA.In a search for genes whose expression is regulated during early stages of embryonic development in Xenopus laevis. XCEP2 is involved in maintenance of cell-cell adhesion, and as such may constitute a regulatory component that could help to balance the need for tissue integrity and plasticity during the dynamic cellular rearrangements of gastrulation.XCEP2 appears to be an essential component in the early developmental program in  Xenopus laevis coordinated changes in cell shape and motility guide the initial formation of the dorsal lip, the initial site of mesodermal involution [Vertebrate gastrulation depends upon exquisite regulation of diverse morphogenetic processes including changes in cell shape, cellular adhesion and migration. For example, in volution ,2. Latervolution . Convergvolution . The cooXenopus laevis and zebrafish, non-canonical Wnt signaling on the dorsal side of developing embryos directly initiates the cellular rearrangements and migration that contribute to convergent extension of involuting mesoderm [Xenopus, resulting in severe gastrulation defects [Drosophila [The molecular basis for initiating and regulating the complex cellular movements of vertebrate gastrulation is only beginning to be discerned. Recent work has shown Wnt proteins as key upstream regulators in gastrulation movements. In mesoderm ,6. Othermesoderm ,8. Inactosophila , vertebrosophila ,11, and osophila ,12-14.Xenopus [Experiments perturbing the activity of Rho GTPases have suggested important regulatory roles for these proteins during gastrulation in Xenopus ,10,12,14Xenopus , cell adXenopus , cell miXenopus , vesiculXenopus  and signXenopus -21. ReguXenopus, when the integrity of the elongating mesodermal sheet must be maintained at the same time that major cellular rearrangements are occuring. Downregulation of C-cadherin-mediated cellular adhesion has been observed to correlate with convergent extension movements of mesodermal cells [Xenopus embryos suggests that analogous upstream regulatory mechanisms may operate during Xenopus gastrulation [Regulation of cell-cell adhesion is thought to be of particular importance during gastrulation movements in al cells . This chal cells . In addial cells -27. Recerulation .Xenopus gene encoding a member of the recently characterized CEP/BORG family of RhoGTPase effector proteins [In this study, the embryonic expression pattern and function of a proteins ,30 is exproteins ,30, and proteins ,32.Xenopus Cdc42 Effector Protein 2, hereafter referred to as XCEP2, is shown here to be developmentally regulated during early embryonic stages, with diffuse expression of mRNA in the animal region that diminishes during late gastrula to neurula stages. Experiments employing morpholino-mediated inhibition of translation suggest that expression of this protein is essential for normal gastrulation movements, and that its activity is required for maintenance of cell-cell adhesion between blastomeres of animal cap explants.Xenopus embryo. The sequence of one of these fragments was found to be highly homologous to the translational start site region of database cDNA sequences encoding Cdc42 effector protein 2. A full length cDNA corresponding to our isolated fragment was amplified using 3' RACE PCR, cloned and sequenced. An alignment of the predicted amino acid sequence for our isolated full length cDNA sequence with human [Xenopus (accession number BC045241) CEP2/BORG1 genes from Genbank revealed significant homology . These differences were found to occur almost exclusively in regions of the protein outside the three conserved domains (CRIB, CI and CII) that define the CEP family of effector proteins. Given the allotetraploid genetic ancestry of Xenopus laevis, and the degree of similarity between the sequences, it is likely that these two sequences constitute pseudoalleles of the same gene. We choose to refer to our isolated pseudoallele as XCEP2A and the database allele as XCEP2B.A differential display approach was employed to reveal genes that are developmentally regulated during the transition from blastula to neurula. This screen allowed for the isolation of numerous cDNA fragments corresponding to differentially regulated mRNAs present in the th human  and XenoIn situ hybridization revealed that XCEP2A mRNA  is distributed uniformly over the animal hemisphere of the embryo at blastula and early gastrula stages , maintenance of this expression level through mid-gastrula stages, with a downregulation of mRNA expression starting at stage 10.5 and becoming more marked by stage 12.5 , MO1 antisense oligonucleotides pseudoalleles elicited significant gastrulation delay as compared to an identical dose of control morpholino oligonucleotide . Given this observation, it is possible that injection of either antisense XCEP2 morpholino significantly reduces the endogenous expression of both pseudoalleles. In addition to the dosage issue in the combined condition (noted above), cross-inhibition of this type could contribute to the observed lack of additive effects in the combined MO1 + MO2 condition.The antisense morpholino oligonucleotides used in these studies were found to elicit specific translational inhibition of XCEP2. Injection of antisense morpholino against our isolated pseudoallele resulted in largescale inhibition of translation of a myc-tagged fusion protein from co-injected mRNA . The absence of effects may not be unexpected, given current models of RhoGTPase effector protein activation. RhoGTPase effector proteins are activated by direct binding to GTP-bound RhoGTPases. As such, the number of activated RhoGTPase molecules may constrain the number of effector proteins bound and activated, regardless of the heightened expression levels of the effector protein.Xenopus embryos suggests that XCEP2 may play a direct role in directing the morphogenetic movements of gastrulation. This interpretation would be consistent with the limited characterization that have thus far been conducted on functional properties of this class of proteins [The observed effects of antisense XCEP2 morpholinos in proteins ,30. HoweIn order to assess the mechanism by which XCEP2 influences gastrulation, the effects of XCEP2 morpholinos on cultured animal cap explants were evaluated. While the initial intent of these experiments was to discern whether XCEP2 morpholinos prevented normal activin-induced convergent extension of animal caps, it soon became clear that morpholino-treated explants had a more fundamental deficiency. Within a 24 hour period, animal cap explants derived from XCEP2 antisense morpholino (MO1) -injected embryos were found to completely lose their integrity . CurXenopus Cdc42 effector protein 2 (XCEP2) in gastrulation movements. XCEP2 is a member of the recently characterized CEP family of Rho GTPase effector proteins [The data presented here indicate a role for the proteins ,30, whicproteins . Effectoproteins ,30. By aproteins ). The exproteins ,30.Consistent with a potential role for this class of proteins in embryonic morphogenesis, we have shown that XCEP2 expression is temporally regulated at gastrulation stages, when major modulations of cellular morphology, cytoskeletal organization, and cellular adhesion are occurring. mRNA for XCEP2 is present prior to mid-blastula transition, persists through mid gastrulation, and is strongly down-regulated by the time the blastopore closes and neurulation begins. This pattern would suggest that XCEP2 protein would be present through the period when active gastrulation movements are occurring. The diffuse spatial pattern of mRNA and, presumably, protein expression of XCEP2 may suggest that XCEP2 functions broadly in cells of the animal and equatorial regions. However, the observed broad spatial distribution of XCEP2 mRNA does not preclude the possibility that the XCEP2 protein may be functionally activated or inactivated at discreet times and locations during early embryonic development.Xenopus gastrulation, delay the closure of the blastopore and inhibit embryonic elongation. The observed rescue with XCEP2 mRNA is strong evidence for the specificity of the antisense morpholino effect. These effects are not due to a loss of mesodermal induction, as brachyury and goosecoid expression do not change in response to antisense XCEP2 morpholinos. This is particularly relevant given recent reports demonstrating a direct link between brachyury expression and control of cellular migration [Furthermore, we show that antisense morpholino oligonucleotides capable of blocking translation of the XCEP2 message interfere with igration .The effects we report require relatively high, although not unprecedented, doses of morpholino antisense oligonucleotide. This dosage requirement may reflect the difficulty inherent in morpholino-mediated translational knockdown of maternally expressed genes. XCEP2 mRNA and (presumably) XCEP2 protein are present prior to midblastula transition. Given this situation, the timing morpholino induced protein downregulation is dependent both upon the effectiveness of translational blockade, and the half life of the protein in the cytoplasm. In this context, it may be essential to impose close to complete translational inhibition in order to reduce protein levels rapidly enough to affect early embryonic events, such as gastrulation.Clearly, specific probes to assess endogenous XCEP2 protein expression will be necessary for fuller characterization of the role of this protein during gastrulation. For this reason, antibodies are currently being raised against the XCEP2 protein. These antibodies will be important in the characterization of the developmental time course of endogenous XCEP2 protein expression, assessment of the subcellular localization of the XCEP2 protein, isolation of potential XCEP2 binding partners, and in assessing and further optimizing the extent of protein down-regulation in morpholino injected embryos.Currently, the specific mechanism by which XCEP2 exerts its role in gastrulation is unknown. However, our preliminary data suggest that XCEP2 may either contribute to a required "ground state" of cellular adhesion or play a role in modulations in the strength of cadherin-mediated cell-cell adhesion that are known to occur during gastrulation ,39,40. MIn embryos Wnt-mediated signals have been shown to activate Cdc42, a process that is required for normal gastrulation movements ,12. In fThe known functional properties of the CEP class of effector proteins, and the characteristics of CRIB domain effector proteins in general, suggest some interesting possibilities relating to the control of cell adhesion during gastrulation. Consistent with the observed functions of the XCEP2 homologs in cultured cells, XCEP2 in embryos may impinge on regulatory circuits downstream of Cdc42 that control actin filament assembly, which in turn may affect diverse cellular processes, including assembly of adherens junctions. Alternatively, XCEP2 may more directly impinge upon cadherin functional activity, perhaps by influencing the association of IQGAP or other molecules with cadherin complexes. In future studies, it will also be important to establish whether embryonic activation of whether there are links between Wnt-mediated activation of Cdc42 and functional activation of XCEP2 and to characterize the mechanism(s) by which XCEP2 contributes to cell adhesion between cells of gastrulating embryos.Xenopus gastrulation. For this reason it will be interesting and important to discern further the role of XCEP2, with regard both to its relationship to intracellular signalling pathways and its effects on cellular behavior during gastrulation.It has become clear in recent years that an integrated network of signals involving Rho GTPase proteins and their effector proteins help to control and regulate the diverse and intricate morphogenetic processes that occur during embryonic development. Less clear are the specific modes of functional interaction between the multiple Rho GTPases and the diversity of potential effector proteins. We have shown that XCEP2 is one component in the complex regulatory puzzle contributing to morphogenetic processes during Adaptor 1-5'-ATGAGTCCTGAC-3' (upper strand)4-CGGTCAGGACTCAT-3'(lower strand).5'-POAdaptor 2 (the 3' phosphate group of the lower strand inhibits DNA polymerase extension)-5'-ACTGGTCTCGTAGACTGCGTACC-3'(upper strand)4-CGGGTACGCAGTC-PO43' (lower strand).5'-PO"Universal" RFDD-PCR primer (complementary to Adaptor 2)-5'-ACTGGTCTCGTAGACTGC-3'"Selection" Primer 4 (complementary to Adaptor 1, with a 3-nucleotide 3' extension)AAG-3' (3-nucleotide extension is underlined)5'-ATGAGTCCTGACCGAXCEP2 cDNA forward-ATGTCCGCCAAG-3'5'-ATTGCAAAGXCEP2* cDNA forward-CGGGATCCTAGATGTCGGCGAAAGCGCCGATATACCTAAAGAG AAG-3'5'-XCEP2 cDNA reverse-5'-AACGTATCCCCTTCCCCA-3'XCEP2A forward (complementary to 5' untranslated region of the cDNA sequence)5'-AACGTATCCCCTTCCCCA-3'XCEP2A reverse (complementary to 5' untranslated region of the cDNA sequence)5'-AAAGAGAAGTAGCCGTAAAGGA-3'XCEP2B forward5'-GCCAAGGCCCCGATATAC-3'XCEP2B reverse5'-CCAATAGCAGGTAGGGAA-3'Brachyury forward5'-GGATCGTTATCACCTCTG-3'Brachyury reverse5'-GTGTAGTCTGTAGCAGCA-3'Goosecoid forward5'-ACAACTGGAAGCACTGGA-3'Goosecoid reverse5'-TCTTATTCCAGAGGAACC-3'ODC forward-5'-GTCAATGATGGAGTGTATG-3'ODC reverse5'-TCCATTCCGCTCTCCTGA-3'Antisense morpholino oligonucleotides, which specifically block translation of targeted mRNAs  were synAntisense XCEP2 MO1 (specific to the XCEP2A, characterized in this study)-CATCTTTGCA-3'5'-GGGCCTTGGCGGAXenopus Genbank sequence accession # BC045241)-Antisense XCEP2 MO2 (specific to XCEP2B, CATCTTTCCA-3'5'-GGGCCTTGGCTGAControl Morpholino oligonucleotide5'-CCTCTTACCTCAGTTACAATTTATA-3'RFDD-PCR primer and adaptor sequences, above) are ligated to restriction digested, double stranded cDNA. Amplification of this cDNA is conducted with the "universal primer", complementary to one of the adaptor sequences, and one of 64 "selection" primers that bind largely to the to the second adaptor sequence but have a 3-nucleotide 3' extension that is complementary only to a subset of cDNA inserts. An extension-inhibiting chemical modification of the lower strand Adaptor 1, consisting of a 3' phosphate group in our modified procedure, and the 3-nucleotide extension of the selection primer largely restricts amplification to cDNA fragments that have different adaptor sequences ligated on opposite ends. Furthermore, any particular selection primer will selectively amplify only a subset of cDNA fragments .Restriction Fragment Differential Display PCR (RFDD-PCR) procedures were patterned closely after the commercially available DisplayProfile kit from QBiogene (Carlsbad, CA), with minor modifications. In this differential display procedure, Adaptors 1 and 2 (see Xenopus laevis embryos was purified using Trizol reagent (Invitrogen, Carlsbad, CA). RNA from Xenopus laevis embryonic stages 7, 8.5, 9.5, 10.5 and 12.5 were prepared. First strand cDNA was synthesized using oligo dT primer and Superscript III reverse transcriptase (Invitrogen), followed by second strand synthesis catalyzed by DNA polymerase I (Roche, Indianapolis, IN). Double stranded cDNA was purified using GeneClean silica resin (Qbiogene, Carlsbad, CA), and then digested with TaqI restriction enzyme (Roche, Indianapolis, IN). An annealed mix of RFDD-PCR of Adaptor 1/Adaptor 2 oligonucleotides was added, and ligated to the TaqI digested cDNA ends using T4 DNA Ligase (Roche). Touchdown PCR using the universal RFDD-PCR primer, 35S-labelled dCTP, and "Selection" Primer 4 was conducted using Accuprime PCR mix (Invitrogen), with the following cycling parameters:Total RNA from staged, duplicate batches of Pre-dwell: 94°C 4 minutes10 cycle touchdown PCR, with a 0.5 temperature decrement each cycle:94°C 30 seconds60°C→ 55°C 30 seconds72°C 1 minute30 cycle standard amplification:94°C 30 seconds55°C 30 seconds72°C 30 secondsPost dwell: 72°C, 5 minutesXenopus homologue of the Cdc42 Effector Protein 2, which we refer to as "XCEP2". The full length cDNA was amplified using gene-specific primers and poly-dT primers using standard 3' RACE procedures [Samples were run on a standard 6% denaturing DNA sequencing gel, and the gel was dried and subjected to autoradiography. Bands showing differential expression were excised from the gel and eluted by heating at 95°C for 15 minutes. Eluted fragments were re-amplified with 30 cycles of standard PCR, using the original primers (above). Direct cycle sequencing of isolated product  revealed that one of the fragments corresponded to the 5' end  of a The RACE amplified full-length fragment isolated from the RFDD-PCR procedure (see above) was cloned into the pCR2.1 vector, using the pCR2.1 TA cloning kit (Invitrogen), and commercially sequenced (MWG Biotech). To construct a vector encoding XCEP2A fused to a C-terminal 6xmyc tag, PCR was conducted using primers directed to the 5' and 3' end of the XCEP2A cDNA sequence (XCEP2A cDNA forward and XCEP2A cDNA reverse) with the cloned, full length XCEP2A as template. The primers used in this PCR mutated the normal stop codon and introduced a BamHI and ClaI site at the 5' and 3' ends of the amplified product, respectively. BamHI/ClaI digested full length cDNA was directionally cloned in the sense orientation into the corresponding into BamHI/ClaI digested pCS2-myc vector, producing the plasmid pCS2-XCEP-myc. In this context, the full length X-CEP2A cDNA encodes a fusion protein with a C-terminal 6x myc tag. A second construct, containing introduced mutations in the XCEP2A antisense morpholino target region, was also derived by PCR. To construct this vector, PCR was conducted using XCEP2* forward and XCEP2 reverse primers with the full-length XCEP2 DNA as template. XCEP2* forward introduced multiple changes in the sequence of the cDNA encoding the translational start site region , producing the plasmid pCS2-XCEP2-anti. In a similar fashion, an RNA expression vector containing the XCEP2 insert in the sense orientation in pCS2+ was also constructed.For transcription of antisense probe for in vitro using the mMessage mMachine kit (SP6) from Ambion (Austin, TX), and were dissolved in distilled water prior to injection.Capped mRNAs were synthesized RT-PCR Primers, above). 25 cycles of PCR were carried out using Accuprime PCR mix (Invitrogen) in 25 ul reactions containing 2 ul of 200 ug/ml reverse transcribed cDNA, 0.3μM of each primer, and a trace of radiolabelled nucleotide to allow for autoradiographic visualization. Parallel reactions containing primers specific to the metabolic gene, ornithine decarboxylase (ODC), were used as a loading control in all semi-quantitative RT-PCR experiments. RT-PCR products were separated electrophoretically on 5% acrylamide gels, which were dried and subsequently subjected to autoradiography. RT-PCR of the XCEP2A allele resulted in stronger amplification than with the XCEP2B primer set. XCEP2B bands required correspondingly longer exposure times (2–4 fold) for detection.Total RNA was isolated from embryos using Trizol (Invitrogen) according to manufacturer's instructions. Reverse transcription was carried out using SuperscriptIII (Invitrogen). Assessment of relative levels of gene expression over developmental time, or under different experimental conditions, was carried out using standard semi-quantitative RT-PCR methods , with thXenopus laevis were purchased from Nasco (Fort Atkinson, WI). Eggs were obtained and fertilized according to standard methods. Staging was determined according to Nieuwkoop and Faber [in vitro transcribed mRNA's were injected into two-cell stage embryos, with injected volumes of approximately 13 nl in doses as described. All injections were carried out using the Nanoject positive displacement microinjector .Adult nd Faber . AntisenMeasurement of blastopore to embryo diameter ratios was accomplished by dividing the measured blastopore diameter  by the embryo diameter. Diameter measurements were obtained using digital images of individual embryos, and the "Ruler" tool of the Adobe Photoshop software.To assess the effects of morpholino antisense oligonucleotides on animal cap explants, morpholino injected and control embryos were allowed to develop until stage 8.5. At stage 8.5 animal caps were dissected, and then cultured in 1xMMR for 24 hours. Non-dissected sibling embryos were allowed to develop until post-neurula stages in order to asses the effectiveness of the morpholino treatment.Proteins from embryos were extracted in 10 volumes of isotonic buffered saline containing 1% NP40. Extracts were cleared by a brief centrifugation, and supernatants were denatured by a 5-minute incubation at 95°C after diluting samples with SDS-PAGE sample buffer. Proteins were separated on 8–16% gradient polyacrylamide gels, and transferred to nitrocellulose. After blocking, blots were probed with monoclonal antibody 9E10 , which rin vitro from linearized pCS2-XCEP2anti plasmid using a digoxygenin nucleotide labeling mix (Roche) and the mMessage mMachine in vitro transcription kit (SP6) from Ambion. In situ hybridization was carried out essentially as described [Digoxygenin-labelled antisense RNA probe was transcribed escribed , using nin situ hybridization methodology in the laboratory, and conducted in situ hybridizations. RWN conceived of the study, designed and conducted molecular biological, embryological and biochemical experiments, and wrote the manuscript. Both authors read and approved of the final manuscript.KKN played a critical role in establishing


Our obstetrics and gynaecology undergraduate teaching module allocates 40–50 final year medical students to eight teaching hospital sites in the West Midlands region. Based on student feedback and concerns relating to the impact of new curriculum changes, we wished to objectively assess whether the educational environment perceived by students varied at different teaching hospital centres, and whether the environment was at an acceptable standard.A Dundee Ready Education Environment (DREEM) Questionnaire, a measure of educational environment, was administered to 206 students immediately following completion of the teaching module.The overall mean DREEM score was 139/200 (70%). There were no differences in the education climate between the teaching centres.Further research on the use of DREEM inventory, with follow up surveys, may be useful for educators to ensure and maintain high quality educational environments despite students being placed at different teaching centres. The undergraduate curriculum at our medical school was redesigned in 1998/99 to bring it in line with recommendations suggested by the General Medical Council (GMC) in Tomorrow's Doctors [Based on previous student feedback reporting differences in educational experiences, together with our concerns relating to the impact of new curriculum changes, we wished to objectively assess whether the educational environment perceived by students varied at different teaching hospital centres, and whether the environment was at an acceptable standard. In particular, was there any potential loss of teaching experience when students were placed away from the principal Teaching Hospital. Thus, the null hypothesis we wished to test was that there was no difference in the learning environment between centres. Several questionnaire-based educational tools are available that set out to 'quantify' the educational environment -4. HowevThe DREEM questionnaire, based on a Likert scale, was administered to the full class of 206 final-year Birmingham University medical students undertaking the exam module in Obstetrics and Gynaecology in 2000. All questionnaires were distributed and returned the same day of the exam, which allowed us to achieve a 100% response rate. Students were told to only comment on their recent 8-weeks experience of Obstetrics and Gynaecology. Statistical analysis was performed using Microsoft Excel and Arcus Quickstat Biomedical Statistical software, and utilised single-sample T test and One-way analysis of variance (ANOVA).The year group comprised 42% male and 58% female. The overall mean DREEM score for the study group was 139/200 (95% CL 136.1 to 141.9), or expressed as percentage of the maximal score, 70% (95% CL 68% to 71%). There was no statistically significant difference between the mean scores for the contributory DREEM domains, which were as follows: perception of learning, 34.52/48 (72%); perception of teaching, 32.05/44 (73%); academic self-perception, 19.46/32 (61%); perception of atmosphere, 34.07/48 (71%), and for social self perceptions, 18.90/28 (68%). The DREEM scores for each hospital, with comparison of all contributory elements of the DREEM inventory, are depicted in Table When converting the raw DREEM score to percentages, two-sided P-value single-sample Student's T test showed no statistically significant difference between hospitals by each DREEM domain, or between each DREEM domain within the same hospital. Greatest variation between hospitals occurred in the Students' Perception of Atmosphere domain, where there were four hospitals beyond the 95% Confidence Limits; this compared to three hospitals beyond 95% Confidence Limits in all other DREEM domains Table s. One-WayWe have used the Dundee Ready Education Environment Measure (DREEM) in 'diagnosing' the educational environment of eight different teaching centres and making comparative analysis between these centres. The overall mean DREEM score was 139/200, or expressed as a percentage, 70% (95% CL 68–71%). The educational learning environment did not vary between centres. The two lowest scoring contributory domains, academic self-perception (61%) and social self-perceptions (68%), were not statistically significantly different from the other three DREEM domains or overall mean DREEM score.This study has benefited by using an established educational measure and obtaining a 100% response rate. No students had been previously taught at the principal teaching hospital as this was solely used for Obstetrics and Gynaecology teaching. However, some of the students (surveyed to be 16/206, 8%) had previously attended the other seven teaching hospital centres due to prior clinical teaching attachments. Thus, previous experiences may have biased the teaching assessment completed by some students. Furthermore, the DREEM questions are of such a nature that it is likely that the environment of the entire curriculum was being assessed. However, by performing the DREEM survey immediately at the end of the obstetrics and gynaecology module, and emphasising reporting only the last eight weeks experience, we believe this maximised the chance that the DREEM measure assessed only the recent hospital teaching site and minimised any recall bias. Other groups  have higThe DREEM domains are unlikely to be independent variables, and may be less of an environment test but more of a measure of the overall motivation and learning attitude of the individual. The Course Valuing Inventory (CVI) score is made up of five domains: worthiness of learning experience, emotional awareness, personal development, cognitive enhancement and task drive. A recent study of first year medical students showed a correlation between higher Course Valuing Inventory (CVI) scores, female gender, stronger self-confidence as a learner, greater motivation to learn and higher DREEM scores .There is no accepted agreement on what is an acceptable DREEM inventory score from published literature. Nevertheless, our DREEM score of 139/200 was higher than other reports. A study of final year medical students in Trinidad reported an overall mean DREEM of 109.9/200 . A largeThe non-significant differences between the DREEM domains and between hospitals were significant findings. This was conveyed to our tutors based at the various teaching centres as a positive and encouraging result. In practical terms, this meant that regardless of hospital capacity or student group size, their education delivery and environment was no different to other centres in the student's curriculum. The DREEM inventory may thus be a useful tool for educators to ensure and maintain high quality educational environments and uniformity in educational delivery despite students being placed at different teaching centres.The author(s) declare they have no competing interests.RV and ET carried out the statisitcal analysis, data interpretation, and drafted the manuscript. JKG conceived and coordinated the study, acquired the results, and made revisions of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


In clinical practice, visual gait observation is often used to determine gait disorders and to evaluate treatment. Several reliability studies on observational gait analysis have been described in the literature and generally showed moderate reliability. However, patients with orthopedic disorders have received little attention. The objective of this study is to determine the reliability levels of visual observation of gait in patients with orthopedic disorders.The gait of thirty patients referred to a physical therapist for gait treatment was videotaped. Ten raters, 4 experienced, 4 inexperienced and 2 experts, individually evaluated these videotaped gait patterns of the patients twice, by using a structured gait analysis form. Reliability levels were established by calculating the Intraclass Correlation Coefficient (ICC), using a two-way random design and based on absolute agreement.The inter-rater reliability among experienced raters  was comparable to that of the inexperienced raters . The expert raters reached a higher inter-rater reliability level . The average intra-rater reliability of the experienced raters was 0.63 (ICCs ranging from 0.57 to 0.70). The inexperienced raters reached an average intra-rater reliability of 0.57 (ICCs ranging from 0.52 to 0.62). The two expert raters attained ICC values of 0.70 and 0.74 respectively.Structured visual gait observation by use of a gait analysis form as described in this study was found to be moderately reliable. Clinical experience appears to increase the reliability of visual gait analysis. Patients exhibiting gait deviations caused by orthopedic impairments are often referred to a physical therapist for treatment. In order to determine treatment goals or to evaluate the effect of a therapeutic intervention, physical therapists visually observe the patient's gait -3. This et al. [et al. [Several reliability studies on observational gait analysis have been described in the literature. These studies included patients with hemiplegia -7, amputet al. , found oIn the Netherlands a gait analysis form has been developed which focuses mainly on orthopedic disorders . Visual The purpose of this present study is to determine the inter- and intra-rater reliability of videotaped observational gait analysis with use of an orthopedic gait analysis form when applied to a cohort of patients suffering from orthopedic impairments. In addition, this study determines how well the raters perform observational gait analysis by comparing their assessments with a criterion, based on the experts' opinion. In order to gain insight into how the results may give guidance to physical therapy treatment, this study also investigates which items on the gait analysis form, that have been considered to be disturbed by visual observation, receive high priority in the physical therapy treatment program according to the physical therapist who performs the visual gait analysis.Thirty videotapes of patients' gait were selected from the archives of the department of Physical Therapy of the University Medical Center Nijmegen, the Netherlands. These videotapes involved patients who had been referred to a physical therapist for gait treatment. It is common practice at this department that prior to gait training therapy the gait of each patient is videotaped according to a standardized protocol.The criteria for inclusion of the videotapes were: (1) the presence of mild to severe gait deviations due to an orthopedic impairment; (2) patient was wearing shorts or underwear to allow for a more accurate observation of the joint movement; (3) ability of a patient to walk 15 meters at least four times, twice in a semi-circle and twice in a straight line on a gymnasium floor; (4) and patient's written informed consent. The first thirty patients who complied with these criteria were included.The group consisted of 15 male and 15 female patients with a mean age of 37.8 years (range: 15 to 62 years). The type of orthopedic impairments varied from status post hip, knee, ankle surgery (n = 8), status post hip or knee prosthesis (n = 6), status post femur, tibia or ankle fracture (n = 3) and traumatic or non-traumatic non-specific hip, knee or ankle pain (n = 13) , anterior and posterior view at each of the three sub-phases of stance and the two sub-phases of swing. All patients were recorded from a lateral view (both sides) while walking 15 meters in a semi-circle (radius approximately 10 m) at a comfortable self-selected walking speed. We used a semi-circle in order to be able to observe the patient's gait in the sagittal plane from one position. The anterior and posterior views were videotaped while the patient walked five meters toward and away from the camera.®). Manufactured videos were reduced into a one-minute film-clip in which the patient's gait could be viewed in the lateral and frontal plane. Subsequently, these videos were converted to analog format again, so that they could be played by a regular video player. Sampling frequency was 24 Hz.The collected videos were edited with use of the computer program adobe premiere 6.0  to the items they scored as disturbed, with respect to a physical therapy treatment program. In other words, which items would receive important attention in the physical therapy intervention if the rater was going to treat this patient for his or her gait disorder.In order to determine the level of performance of observational gait analysis of all experienced and inexperienced raters, we compared their ratings with a criterion. This gives us an indication about how well the raters were capable in performing visual gait analysis. The criterion was attained during a consensus session of the two expert raters: After individually assessing the 30 patients for the second time, the two expert raters jointly observed the videotaped gait of all 30 patients for the third time.Inter- and intra-rater reliability levels were assessed by using Intraclass Correlation Coefficients (ICCs), validated for use with multiple raters and calculated in a two-way random model based on absolute agreement. We used ICCs because it has been shown that with data that are rated as a dichotomy, the ICC is equivalent to measures of nominal agreement, simplifying computation in cases where more than two raters are involved . In addiThe inter-rater reliability among experienced raters was 0.42 (95%CI: 0.38–0.46). This level of reliability is comparable to the inter-rater reliability of in-experienced raters, which reached an ICC value of 0.40 (95%CI: 0.36–0.44). The expert raters reached the highest inter-rater reliability (ICC: 0.54 (95%CI: 0.48–0.60)).There were no differences in inter-rater reliability between the first and second rating session of all three groups separately, based on the overlap of 95% confidence intervals.The average intra-rater reliability of the experienced raters was 0.63 (ranging from 0.57 to 0.70). The inexperienced raters reached an average intra-rater reliability of 0.57(ranging from 0.52 to 0.62). The two expert raters attained ICC values of 0.70 and 0.74 respectively.The agreement between the outcome of the joint assessment of the expert raters (criterion) and those of the individual experienced raters ranged from 0.43 to 0.55 with an average ICC value of 0.48. The inexperienced raterrs attained agreement levels ranging from 0.41 to 0.55, with an average of 0.49. There is no difference in the level of performance of visual gait assessments of experienced or inexperienced raters, when compared to the experts' opinion.The inter-rater reliability per item on the gait analysis form between the two experts is generally moderate to substantial . Only one item, extension movement of the knee during mid stance, had an ICC value for intra-rater reliability of less than 0.6. The experienced raters were able to attain good intra-rater reliability for item 2, posture of the trunk during walking (ICC = 0.81). Three items reached substantial intra-rater reliability (item 4, 5, and 8). Two items of the gait analysis form, pelvis rotation and ankle movement during late stance, were not intra-rater reliable (ICC < 0.40). The inexperienced raters reached the highest intra-rater reliability for the assessment of arm swing during walking (ICC = 0.81). Three items had inadequate intra-rater reliability levels; flexion of the knee in early stance (ICC = 0.36), extension of the knee in mid stance (ICC = 0.22), and ankle movement during the late stance phase (ICC = 0.37).No reliability score was obtained from item 3, which describes a trunk position behind the hips, because this item was observed only once.On average, with respect to all items, in about a quarter of the cases items were judged to be disturbed by the expert and experienced raters . Expert raters showed the least variability between the first and second session; five items showed to have a mean intra-rater reliability level that is considered good (ICC > 0.80).The results of this study suggest that a brief introduction in normal gait kinematics in inexperienced raters gives comparable reliability levels of observational gait analysis in patients with orthopedic impairments compared to experienced physical therapists, who have worked for several years with patients with gait disorders. However, expert raters – those that work significantly more intensive with patients with gait disorders – accomplish higher reliability levels.As mentioned in the methods section, the gait analysis form used in this study is also used in daily practice to guide the treatment of the patient's gait disorder. In the physical therapist's treatment program, some items on the form will obviously receive higher priority than others. The results of this study show that physical therapists mainly focus their intervention on movement disorders of the lower extremity. However, the expert raters also report to give priority to asymmetry of the stance phase and excessive lateral flexion of the trunk during gait. Of the three items in this study that achieved the highest reliability levels, only the movement of the knee received generally a high priority in the treatment program of experienced raters. This implies that experienced raters will mainly focus their treatment on items that have generally a low inter- and intrarater reliability.Structured visual observation of a patient's gait by use of a gait analysis form as described in this study is found to be only moderately reliable, but may be a useful guide to the physical therapist in setting up a gait training or exercise therapy program. Intra-rater levels have shown that visual gait analysis will supply the observer with a fair indication of changes in a person's gait. However, to evaluate the effect of an intervention on a patient's gait we recommend more objective instrumentation which has been proven reliable and valid.The author(s) declare that they have no competing interests.JB carried out the data analysis, participated in the design of the study and drafted the manuscript. CvU participated in the design and coordination of the study, assisted with statistical analysis, and helped to draft the manuscript. SvM participated in the design of the study and supplied the videotapes. JK participated in the design and coordination of the study. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Salmonella enterica serovar Typhimurium aroA strain in two Raf dependent lung tumor mouse models.Serine-threonine kinases of the Raf family (A-Raf, B-Raf, C-Raf) are central players in cellular signal transduction, and thus often causally involved in the development of cancer when mutated or over-expressed. Therefore these proteins are potential targets for immunotherapy and a possible basis for vaccine development against tumors. In this study we analyzed the functionality of a new live C-Raf vaccine based on an attenuated Escherichia coli α-hemolysin and expressed in secreted form by an attenuated aroA Salmonella enterica serovar Typhimurium strain via the α-hemolysin secretion pathway. The effect of the immunization with this recombinant C-Raf strain on wild-type C57BL/6 or lung tumor bearing transgenic BxB mice was analyzed using western blot and FACS analysis as well as specific tumor growth assays.The antigen C-Raf has been fused to the C-terminal secretion signal of Salmonella enterica serovar Typhimurium aroA strain using the E. coli hemolysin secretion system. Immunization of wild-type C57BL/6 or tumor bearing mice provoked specific C-Raf antibody and T-cell responses. Most importantly, the vaccine strain significantly reduced tumor growth in two transgenic mouse models of Raf oncogene-induced lung adenomas.C-Raf antigen was successfully expressed in secreted form by an attenuated Salmonella enterica serovar Typhimurium could form the basis for a new generation of live bacterial vaccines for the treatment of Raf dependent human malignancies.The combination of the C-Raf antigen, hemolysin secretion system and  The Raf proteins (A-Raf, B-Raf, C-Raf) are located upstream of MEK and downstream of Ras and represent an essential part of the mitogenic cascade -3. InterSalmonella enterica serovar Typhimurium aroA strain. Such recombinant live vaccines have been shown to efficiently elicit both, humoral and cellular immune responses against a variety of heterologous antigens [E. coli α-hemolysin (HlyA) secretion system which is fully active in Salmonella [s), which is recognized by the HlyB/HlyD/TolC-translocator, leading to direct secretion of the entire protein into the extracellular medium without the formation of periplasmic intermediates. In addition, fused to the C-terminus of heterologous proteins, the HlyAs leads to efficient secretion of such proteins by the recombinant bacteria. In our case, the whole C-Raf antigen fused to the C-terminal secretion signal of hemolysin was efficiently expressed and secreted by an attenuated Salmonella enterica serovar Typhimurium aroA strain. The effects of this recombinant vaccine were assessed in wild-type C57BL/6 or tumor bearing transgenic BxB mice.Here we describe the development of a C-Raf vaccine on the basis of an attenuated antigens ,8. In orlmonella . This trThe bacterial strains, plasmids, cell lines and mice used in this study are listed in Table Salmonella enterica serovar Typhimurium LB5000  at 2.5 kV, 25 microfarads (μF), and 200 Ohm in a 0.1 cm electroporation cuvette.The plasmids pMOhly1 and pMOhly-Raf were first transformed in competent  E. coli ,11. SubsATGCATCTTGGAAG-3' and antisense primer 3'Raf: 5'-CAACTAGAATGCATGCAGCCTCGGGGA-3' were used to amplify by PCR a 1950 bp DNA fragment representing the entire craf gene from plasmid pUC13-c-raf-1 [NsiI restriction enzyme, the DNA fragment carrying the craf gene was inserted into the single NsiI site of the export vector pMOhly1 [E. coli DH5α (Invitrogen), analyzed and transformed in S. typhimurium SL7207  were centrifuged at 5000 × g at 4°C for 5 min. The supernatant proteins were precipitated with 10% (V/V) trichloroacetic acid (TCA) for 1 h on ice, collected by centrifugation and resuspended in SDS sample buffer. The supernatant proteins were separated on 10% gels by SDS-PAGE [SDS-PAGE . For imm9 bacteria/100 μl phosphate-bufferd saline (PBS) at 5-day intervals. At day 45 after the start of the vaccination, these mice were boosted intravenously (i.v.) with a single-dose of 5 × 105 bacteria/100 μl PBS. The BxB23 mice received a second i.v. boost of 5 × 105 bacteria at day 90 after the start of the vaccination. Induction of Raf-specific immune responses were analyzed at day 50 for C57BL/6 or at day 95 for BxB23 respectively.In order to achieve a broad immune response encompassing both, the mucosal and systemic immunity against C-Raf, we combined oral immunization (p.o.) with an intravenous (i.v.) boost. In these experiments, seven weeks old C57BL/6 or BxB23 mice were immunized, first p.o. three times with 5 × 107bacteria/30 μl phosphate-buffered saline (PBS) at 14-day intervals. The vaccine was applied using a micropipette onto the nares of mice under anesthesia.Seven weeks old BxB23 mice were immunized i.n. four times with 1 × 10Induction of Raf-specific immune responses were analyzed at day 70.8salmonellae in 10 μl PBS at 14-day intervals. The vaccine was applied using a micropipette into the nares of mice without anesthesia.Four months old BXB11 mice were immunized i.n. three times with 1 × 10b haplotype . The EL4 tumor cell line is a murine thymoma cell line of the H-2ype Tab. . 10 μg pAnimals were sacrificed and a single cell suspension of splenocytes was prepared by passage of the spleen through a sieve into RP 10 medium [RPMI medium (Life Technologies) supplemented with glutamine (1%), 50 μM β-mercaptoethanol (ROTH, Wuerzburg), penicillin (10 U/ml, GIBGO), streptomycin (100 U/ml, GIBGO) and 10% fetal calf serum (PAN™, BIOTECH GmbH).4Cl, pH 7.3) for the lysis of erythrocytes. After 2 minutes, 10 ml RP 10 medium was added to stop lysis. After centrifugation, cells were resuspended in 2 ml RP 10 medium and counted.The cell suspension was centrifuged and resuspended in 3 ml lysis buffer (5 mM Tris-HCl, 140 mM NH2, 1 × 106 spleen cells were incubated for 5 hours with 5 × 105 C-Raf overexpressing EL-4 cells (EL-Raf) or 5 × 105 EL-4 cells or with the addition of 10 ng/ml of phorbol myristyl acetate  and 500 ng/ml of ionomycin (Sigma) or with medium alone.In 6 ml FACS tubes and a total volume of 100 μl RP 10 medium in the presence of 30 U/ml recombinant IL-2 and 10 μg/ml Brefeldin A at 37°C, 5% COAfter restimulation, cells were washed with PBS-0.1% bovine serum albumin  and incubated with 1 μl of anti-CD8-CyChrome (Pharmingen Nr. 01082A) in a volume of 100 μl PBS-0.1% BSA for 20 min on ice. Subsequently, cells were washed and fixed for 20 min at room temperature with PBS-2% paraformaldehyde (Sigma). After washing with PBS-0.1% (BSA), cells were permeabilized with PBS-0.1% BSA-0.5% saponin (Sigma, P-B-S) buffer. After incubation for 5 min at room temperature, cells were washed with P-B-S buffer and incubated in a volume of 100 μl at room temperature with polyclonal rat immunoglobulin G (IgG) antibodies (JacksonImmunoResearch) to block nonspecific binding and 1 μl anti-IFN-γ-FITC  for 20 min. After incubation, cells were washed twice with P-B-S buffer and another time with P-B buffer. Cells were resuspended in 400 μl PBS-PFA 0,5% and kept at 4°C until analysis.+ cells.Cells were analyzed by flow cytometry in a FACS Calibur flow cytometer (Becton Dickinson) using CellQuest 3.0 software (Becton Dickinson). Lymphocytes were chosen according to their size and granularity in a forward/side – scatter diagram. Numbers are expressed as percent IFN-γ positive CD8t test. Findings were regarded as significant, if P values were <0.05. Survival curves were compared using a log rank test.The statistical significance of differential findings between experimental groups was determined by Student's Salmonella enterica serovar Typhimurium aroA strain SL7207 secreting the C-Raf antigen was achieved by cloning the human craf cDNA from pUC13-c-raf-1 [craf-hlyAs fused gene and the functional hlyB and hlyD genes required for its secretion  and with Salmonella enterica serovar Typhimurium SL7207 as control in order to test the induction of Raf-specific immune responses. The data showed that 20% of the sera of BxB23 mice immunized with SL7207/pMOhly-Raf contained Raf-specific antibodies .To assess the induction of Raf-specific T-cell responses, mice were sacrificed and Raf specific T-cell responses were assessed using intracellular IFN-γ staining followed by FACS analysis. For this purpose, T-cells were restimulated with C-Raf overexpressing EL-4 cells. Using this technique, we could detect Raf specific CD8Salmonella strains, groups of 6 to 10 heterozygous BxB23 mice at the age of 7 weeks were immunized p.o./i.v. or i.n. with recombinant Salmonella enterica serovar Typhimurium strain (SL7207/pMOhly-Raf) or with SL7207 as control. BxB23 mice normally show an induction of lung adenomas with short latency and at 100% incidence [To test the protective capacity of the immune responses induced by the recombinant ncidence ,16. The craf transgenic strain, which develop lung adenomas after a shorter latency period compared to BxB23 mice [In order to confirm these data we repeated the protection experiments using BxB11, another B23 mice . In thisThese results suggest that the immunization with the vaccine strain SL7207/pMOhly-Raf can achieve a partial protection against tumor growth in the BxB mouse model.The major problems for vaccine development against cancer are the heterogeneity of the tumor cells and the fact that all tumor antigens are self-antigens. Therefore specific T-cells might be anergic or tolerant ,18.However, despite these problems, several cancer vaccines have already reached clinical trials . The sucSalmonella enterica serovar Typhimurium aroA strain as a "live vaccine". In general, recombinant aroA salmonellae are efficient live bacterial vectors that stimulate strong mucosal immunity, humoral and cell-mediated responses with a great potential as live vaccine carriers in both humans and animals [aroA Salmonella vaccines include their safety and easy administration [aroA Salmonella enterica serovar Typhimurium strains were already successfully used as carriers for DNA vaccines against cancer in mice [Here we describe a new strategy for achieving an anti-tumor immune response with a C-Raf vaccine on the basis of an attenuated  animals ,20. Advastration ,22. In a in mice .Salmonella enterica serovar Typhimurium aroA strain using the E. coli hemolysin secretion system. This system allows an efficient antigen secretion and presentation, which are necessary for an optimal immune response against the given heterologous antigen [Salmonella enterica serovar Typhimurium aroA strain SL7207 secreting C-Raf resulted in the induction of a humoral immune response, manifested by the presence of Raf-specific antibodies in some of the vaccinated mice. In addition, C57BL/6 mice immunized with Salmonella enterica serovar Typhimurium SL7207/pMOhly-Raf developed a Raf-specific CD8+ T-cell response. The C-Raf vaccine thus is able to break the peripheral tolerance of the immune system towards C-Raf and to induce a specific immune response. Although the transgenic model is closer to the human setting in comparison to challenge models with tumor cell lines, we can not formally exclude that lung specific expression of the transgene in our BxB model is not sufficient for the induction of peripheral tolerance. Furthermore, side effects due to autoimmunity might occur in humans who have a more generalized expression pattern. However, the lung tissue does not belong to immunoprivileged sites and we have never observed any signs of lung pathology in immunized animals which strongly suggests that the data could, in principle, be translated into the human setting.In this study the C-Raf antigen was delivered in secreted form by attenuated  antigen . In factSalmonella enterica serovar Typhimurium SL7207/pMOhly-Raf. However, we were not able to assess Raf specific CTL responses in these mice due to the high variability and background and therefore we cannot conclude whether protection is really due to cytotoxic T-cells or might be the result of other effects, e.g. an increased intratumoral level of IFN-γ produced by Raf specific CD4+ T-cells. Interestingly, the observed variation occurred only in treated BxB23 mice, not in naïve BxB23 mice. Therefore, the observed effect might be due to a spontaneous induction of an immune response mediated by tumor infiltrating bacteria. It is interesting to note that spontaneous Raf specific immune responses also occur in the human context, which we have recently demonstrated B-Raf V599E specific CTL and B-Raf /B-Raf V599E specific humoral responses in melanoma patients [We demonstrated also a partial tumor protection both in BxB23 and in BxB11 mice after immunization with recombinant patients ,25.+ T-cells are effective in eliminating antigen positive tumor cells. The combined data thus provide proof of concept for the development of Raf-based anti-cancer vaccines.Most importantly, the lack of B-Raf V599E mutations in metastases of melanoma patients with strong B-Raf V599E CD8 response  supportsE. coli hemolysin secretion system is a versatile tool for the delivery of cancer antigens in Salmonella enterica serovar Typhimurium. Moreover, we have recently shown that this secretion system is also fully active in Salmonella enterica serovar Typhi Ty21a, the only Salmonella vaccine strain registered for human use [S. typhi Ty21a could form the basis for a new generation of live bacterial vaccines against cancer.This is the first example demonstrating that the uman use . TherefoSalmonella enterica serovar Typhimurium aroA SL7207 strain via the E. coli hemolysin secretion system. In addition, the immunization of wild-type C57BL/6 or tumor bearing transgenic mice with this C-Raf secreting Salmonella strain provoked specific C-Raf antibody and T cell responses. Most importantly, the vaccine strain induced partial protection against lung cancer in two transgenic mouse models of Raf oncogene-induced lung adenomas.Taken together we have demonstrated that the C-Raf antigen can be successfully expressed and secreted by the attenuated The approach may provide a new strategy for the rational design of cancer therapies.The author(s) declare that they have no competing interests.IG, JF and JT designed the study. IG drafted the manuscript. JF was also involved in writing the report. AS and JF did the FACS analyses. JT constructed the EL4Raf cell line. TP, AS, JF and IG carried out the immunization of mice and the Western blot analyses. WG and URR were involved in providing the conceptual framework for this study. All authors approved the final version of the manuscript.The pre-publication history for this paper can be accessed here:Supplementary figure Click here for file


EMO syndrome, defined as a triad including exophthalmus, pretibial myxedema and osteoarthropathia, is a rare condition in patients suffering from hyperthyreosis.We here describe an interesting case of EMO syndrome associated with unilateral fibromatosis of the hand and an initial stage of generalized myxedema of the skin. To our knowledge a similar case has not yet been described in literature though reports about associated fibromatosis, e.g. located retroperitoneally, already exist. Familiar explanations include its initiation by autoimmune processes or aberrant T-cell cytokine stimulation leading to an overwhelming production of glycosaminoglycans.Interpreting our case in context with previous reports we conclude that associated fibromatosis induced by autoimmune processes may affect a variety of different localizations and therefore requires careful monitoring. A therapeutical attempt by using UVA1 irridation for pretibial myxedema remained without a satisfying regression. EMO syndrome is a rare condition seen in patients suffering from hyperthyreosis. It is defined as a triad of exophthalmus, pretibial myxedema and osteoarthropathia occurring in less than 1% of patients suffering from Graves' disease [In October 2003, a 64-year-old male Caucasian patient was admitted with aggravating pretibial myxedema. Five years ago, he was diagnosed with hyperthyreosis caused by Plummer's disease. Laboratory findings revealed a repressed TSH of 0.01 mU/L (0.4–4.0 mU/L). The patient was treated with radioiodine therapy. Today he suffers from hypothyreosis and a daily substitution of 100 μg L-Thyroxin is performed. Within the months following the radioiodine therapy, an erythema progressing into a manifest pretibial myxedema developed, followed by exophthalmus and fibromatosis of the right hand within the next three years. Surgical orbita decompression due to massive exophthalmus was performed, followed by a subsequent correction of the ocular muscles. Additionally, the fibromatosis of the right hand was excised. Retrospective histological findings revealed a regressive sclerotic fibrosis of firm kollagenous fibers embedded in soft and fat tissue were consistent with a generalized fibrosing process as known in myxedema. Within the last months, decent mucous plaques of the upper limps decreased. The patient now reported progressing congestion of lymph and decreasing flexibility of the lower legs caused by the pretibial myxedema. Clinical examination confirmed massive pretibial myxedema, lymphatic congestion of the lower legs, generalized myxedema accentuating the upper limps, a residual postsurgical nodular tumor of the ulnar aspect of the right hand, exophthalmus, and severe hypertrophic osteopathy of the distal phalanges with clubbed fingers and hippocratic nails . While the congestion of lymph clearly improved, pretibial myxedema remained without any signs of regression.Grave's disease is known to eventually develop after radioiodine therapy  and thisA massive recurrence after excision of thyroid dermopathy, as described in literature, could not be observed in our patient during a follow-up of 2.5 years.Generalized fibrosing processes are thought to be based on an accumulation of glycosaminogylcans. Common explanations comprise its initiation by autoimmune processes, e.g. thyreotropin receptors on fibroblasts, or aberrant T-cell cytokine stimulation, leading to overwhelming glycosaminoglycan production  Fibrosin2 UVA1 (seven treatment sessions). However, we suggest that phototherapy might also be considered as an adjunct therapeutic alternative in persistent EMO syndrome.Therefore, we conclude that concomitant fibromatosis might appear in various localizations requiring elaborated diagnostic procedures and monitoring in all patients affected. We started low-dose UVA1 irradiation of the pretibial myxedema known to be able to degrade pathologic collagenous architecture by inducing dermal matrix-metalloproteinases as well as by decreasing abnormal cytokine liberation following T-cell apoptosis . NeverthThe author(s) declare that they have no competing interests.C.A. attended to the patient, participated in the design of the case report, and drafted the manuscript. A.K. conceived the study. F.B., A.B. and P.A. participated in its design and coordination.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Hypertension is among the most common chronic condition in middle-aged and older adults. Approximately 50 million Americans are currently diagnosed with this condition, and more than $18.7 billion is spent on hypertension management, including $3.8 billion for medications. There are numerous pharmacological agents that can be chosen to treat hypertension by physicians in clinical practices. The purpose of this study was to assess the cost of alternative antihypertensive treatments in older adults with isolated systolic hypertension (ISH).Using the Systolic Hypertension in the Elderly Program (SHEP) and other data, a cost-minimization analysis was performed. The cost was presented as the cost of number-needed-to treat (NNT) of patients for 5 years to prevent one adverse event associated with cardiovascular disease (CVD).It was found that the cost of 5 year NNT to prevent one adverse CVD event ranged widely from $6,843 to $37,408 in older patients with ISH. The incremental cost of the 5 year NNT was lower to treat older patients in the very high CVD risk group relative to patients in the lower CVD risk group, ranging from $456 to $15,511. Compared to the cost of the 5 year NNT of other commonly prescribed antihypertensive drugs, the cost of SHEP-based therapy is the lowest. The incremental costs of the 5 year NNT would be higher if other agents were used, ranging from $6,372 to $38,667 to prevent one CVD event relative to SHEP-based drug therapy.Antihypertensive therapy that is diuretic-based and that includes either low-dose reserpine or atenolol is an effective and relatively inexpensive strategy to prevent cardiovascular events in older adults with isolated systolic hypertension. Use of the diuretic-based therapy is the most cost-effective in patients at high risk for developing cardiovascular disease. Hypertension is among the most common chronic conditions in middle-aged and older adults. Approximately 50 million Americans are currently diagnosed with this condition, and more than $18.7 billion is spent on hypertension management, including $3.8 billion for medications[Treatment of hypertension can significantly decrease the risk of developing CVD ,3. The SCurrently, primary care physicians can choose from numerous pharmacological agents to treat hypertension. The commonly used antihypertensive drug classes include diuretics, beta-blockers, angiotensin-converting enzyme (ACE) inhibitors, alpha-blockers, and calcium channel blockers. Selection of an evidence-based therapy with demonstrated efficacy, safety, and low cost has important economic implications. The purpose of this study was to: 1) assess cost of the SHEP-based antihypertensive treatment to prevent adverse events associated with CVD, including death, stroke, myocardial infarction, and heart failure; and 2) to compare cost of the SHEP-based treatment to the costs of other commonly used antihypertensive agent treatments.The SHEP trial is a randomized, double-blind, placebo-controlled clinical trial sponsored by the National Heart, Lung, and Blood Institute and the National Institute on Aging that tested the efficacy of diuretic-based stepped-care antihypertensive drug treatment of isolated systolic hypertension (ISH) to prevent strokes .The study subjects consisted of community-dwelling men and women 60 years and older who had isolated systolic hypertension, defined as an average systolic blood pressure (SBP) ≥ 160 mm Hg and an average diastolic blood pressure (DBP) < 90 mm Hg over 2 baseline visits. The primary endpoint of the trial was combined nonfatal and fatal stroke over a 5-year period. Secondary endpoints included nonfatal myocardial infarction (MI) plus fatal coronary heart disease (CHD) and major cardiovascular disease (CVD) morbidity and mortality. A total of 2,365 and 2,371 persons were randomized into the treatment and placebo group of the study respectively.4. Fasting blood samples were analyzed at a central laboratory, including serum glucose, lipid levels, creatinine, uric acid, sodium, and potassium.Subjects who met the preliminary blood pressure (BP) eligibility criteria at the initial contact visit were referred to SHEP clinics for the baseline visits. At the baseline visits, subject's demographics, medical conditions, health behaviors, and cardiovascular risk factors were obtained. Methods of these measurements have been reportedOf the 4,736 SHEP participants, 4,189 were included in this analysis. The 547 participants were excluded either because of missing data concerning CVD risk factors (n = 283) or with previous CHD or stroke (n = 264). These 547 excluded subjects had similar age, sex, race, and other characteristics as those who were included in this analysis.<160 mm Hg and for those with SBP between 160 and 179 mm Hg to have a reduction of at least 20 mm Hg. All participants were given chlorthalidone, 12.5 mg/d, or matching placebo (step 1 and dose 1 medication). Drug dosage (step 1 and dose 2 medication) was doubled, 25 mg/d, for participants failing to achieve the SBP goal at the follow-up visits. If the SBP goal was not reached at the maximal dose of step 1 medication, atenolol, 25 mg/d, or matching placebo was added (step 2 and dose 1 medication). When atenolol was contraindicated, reserpine, 0.05 mg/d, or matching placebo could be substituted. When required to reach the blood pressure goal, the dosage of the step 2 drug could be doubled (atenolol 50 mg/d or reserpine 0.10 mg/d, step 2 and dose 2 medication). Potassium supplements were given to all participants who had serum concentration below 3.5 mm0l/L at two consecutive visits. The SHEP participants were followed up monthly until SBP reached the goal or until the maximum level of stepped-care treatment was reached [A stepped-care treatment approach was used, with the goal for individuals with SBP >180 mm Hg to reduce to  reached ,7The present analysis focused on five types of events: 1) death; 2) first-occurring major cardiovascular event, including stroke, MI, or heart failure; 3) first-occurring stroke; 4) first-occurring MI; and 5) first clinical diagnosis of congestive heart failure (CHF). The adjudication and clarification of the events was done by a panel of three physicians blinded to treatment assignment and blood pressure status. Members of the panel reviewed the documentation of new cardiovascular events over the study period and adjudicated outcome events according to predetermined criteria. [a priori global score for the risk of developing future cardiovascular events, according to the Multiple Risk Factor Assessment Equation jointly proposed by the American Heart Association and the American College of Cardiology.[Information at the baseline on age, sex, total cholesterol, high density lipid (HDL) cholesterol, systolic blood pressure, diabetes (diabetic vs. non-diabetic), and smoking (current vs. never or past smoking) were used to calculate an rdiology. The equaThe methods of economic evaluation include cost-effectiveness analysis, cost-utility analysis, and cost-benefit analysis, which can be used to assess the trade-off between costs and benefits in choices of antihypertensive treatment regimens. The primary aim of this analysis was to examine cost of the diuretic-based antihypertensive drug intervention in the SHEP trial. A cost-minimization analysis is a special type of cost-effectiveness analysis. It can be used to compare cost difference among competing alternatives of antihypertensive drug treatments when these treatments are medically equivalent. In this study, we used cost-minimization analyses to compare costs and incremental costs of NNT for 5 years to prevent one adverse event related to CVD among antihypertensive treatment regimens. The perspective of this economic evaluation was that of a national health insurance system.We used the number-needed-to-treat as an unit of common outcome measure in the analysis. The number-needed-to-treat to prevent one adverse outcome has become a widely used measure of treatment benefits derived from the results of clinical trials. The NNT is the reciprocal of the absolute risk reduction (ARR) which is the difference between the proportions with the adverse event in the treatment and placebo groups. The 95% confidence interval of NNT was calculated based on the regression-based method described by Laupacis et al. The cost specified in the analysis includes the drug acquisition cost of SHEP treatment from the perspective of a national health insurance system. According to the SHEP treatment protocol, the stepped-care was classified into four types of drug treatments: 1) the Step 1 and Dose 1: chlorthalidone 12.5 mg/d; 2) the Step and Dose 2: chlorthalidone 25 mg/d; 3) the Step 2 and Dose 1: chlorthalidone 25 mg/d plus atenolol 25 mg/d or reserpine 0.05 mg/d; and 4) the Step 2 and Dose 2: chlorthalidone 25 mg/d plus atenolol 50 mg/d or reserpine 0.1 mg/d. Direct drug acquisition costs were calculated based on the minimum average wholesale prices (AWP) within drug manufacturers in the year 2000. All drug1 × C1 + W2 × C2 + W3 × C3 + W4 × C4EC = WThe W1, W2, W3, and W4 represent proportions of the participants using the Step 1 and Dose 1, the Step 2 and Dose 2, the Step 2 and Dose 1, and the Step 2 and Dose 2 medication, respectively. C1, C2, C3, and C4 represent the drug acquisition cost of the Step 1 and Dose 1, the Step 2 and Dose 2, the Step 2 and Dose 1, and the Step 2 and Dose 2 medication, respectively. A Monte Carlo method was performed to estimate the average cost and its standard deviation.To compare the cost of the SHEP-based therapy to other antihypertensive drugs, it was assumed that all antihypertensive drugs in the comparisons have equal efficacy in terms of the NNT for 5 years to prevent one CVD related event. The NNT was calculated based on the method. All drug costs were expressed as dose-specific cost per patient in 1-year and/or 5-year. Using the approach, costs were calculated for each representative drug based on equipotent doses in terms of blood pressure reduction.  The non-In this analysis, we focused on the drug acquisition cost for antihypertensive management. Therefore, the monitoring cost for antihypertensive treatment was not included. Total treatment cost includes antihypertensive drug cost and monitoring cost. The monitoring of treatment in ambulatory care settings including physician visits and laboratory tests have an estimated cost of $284 per patient per year.  Total coTable Results of the 5 year NNT to prevent one adverse event and its associated cost by event type are shown in Table Table In Table The result of an economic evaluation essentially shows the cost per benefit gained from adapting a specific treatment. The effective and efficient use of resources has been increasingly emphasized from society, health plans, and health care providers. This cost-minimization analysis incorporating outcome data from the SHEP trial presents information treatment cost for older patients with ISH. We found that a long-term, low-dose and diuretic-based antihypertensive therapy is relatively inexpensive and effectively prevents adverse events associated with cardiovascular diseases, especially in older patients who had a high CVD risk profile.Our findings indicate that the total and incremental treatment costs of antihypertensive drugs in ambulatory care settings range widely among drug classes as well as within drug classes. This analysis suggests that diuretic-based antihypertensive treatments are the least expensive, whereas atenolol (beta-blocker) is less costly than enalapril (ACE inhibitor) and nifedipine , and terazosin  is the most expensive drugs in terms of the 5 year NNT to prevent one CVD event. It appears that use of the SHEP-based drug therapy offers greater economic benefits for controlling isolated systolic hypertension in the elderly than other antihypertensive drug treatments. Using a decision analysis model that simulated clinical decisions and outcomes that would occur when primary care physicians follow the JNC IV hypertension management guidelines, it was found that a newer class of calcium channel blockers can slightly increase the proportion of patients who achieve and maintain hypertension control, but at a substantially higher cost than with a generic diuretic drug. For our analyses, we presumed that all drugs offer equivalent therapeutic benefits. This assumption may have introduced a conservative bias into our primary findings. In fact, randomized controlled trials directly comparing active treatments for hypertension reported that calcium antagonists and doxazosin were inferior to low-dose diuretics or other agents in preventing cardiovascular events, suggesting that the cost-effectiveness of diuretic-based treatments may be even more favorable than estimated in the present study. -17 FurthWith regard to costs projected in our study, it is noteworthy to consider that compared to the SHEP treatments, costs of treatments based on more recently developed antihypertensive agents (than reported here) are likely to be even higher than estimated in the present analyses.The results of this study are limited to men and women 60 years and older who have isolated systolic hypertension and no presumed contraindication to any one class of antihypertensive medications. One limitation to our study relates to the fact that comparisons were based on costs of monotherapies, while combination therapies are frequently needed to control blood pressure.The number-needed-to-treat to prevent one adverse outcome has become a widely used measure of treatment benefits in medical community, which is easy for physicians to understand. The shortcomings of NNT are that the outcome measure of an effect is with one dimension- survival probability and that it measures the specified outcome at a single point in time. Therefore, a measure of NNT can not capture an outcome in effectiveness of the intervention with two dimensions: time and survival probability. These limitations may not allow us to take time and discounting on cost and effect into account in this study.Based on our findings, antihypertensive therapy that is diuretic-based and that includes either low-dose reserpine or atenolol represents a cost-effective regimen in preventing or delaying cardiovascular events in older adults. Use of the diuretic-based therapy is the most cost-effective in patients at high risk for developing cardiovascular disease. These results suggest that clinicians should consider using diuretics plus low-dose reserpine or atenolol as first-line therapy in patients with isolated systolic hypertension who are greater than 60 years old when there are no contraindications among these patients.ACE: angiotensin-converting enzymeALLHAT: Antihypertensive and Lipid Lowering treatment to prevent Heart Attack TrialARR: absolute risk reductionAWP: average wholesale priceBP: blood pressureCHD: coronary heart diseaseCHF: congestive heart failureCVD: cardiovascular diseaseDBP: diastolic blood pressureHDL: high density lipidISH: isolated systolic hypertensionJNC IV The Sixth Report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure.NNT: number-needed to treatSBP: systolic blood pressureSHEP: Systolic Hypertension in the Elderly ProgramThe author(s) declare that they have no competing interests.GC, LF, WM and MP participated the development of the analytic framework. GC performed all data analyses. GC, LF, WM and MP drafted and revised the manuscript. All authors approved the final manuscript.


Helicobacter pylori diagnosis and susceptibility profile directs the applicability of recommended treatment regimens in our setting. To our knowledge, there is no published data on the culture and local susceptibility pattern of Helicobacter pylori in the Philippines.52 dyspeptic adult patients undergoing endoscopy from the Outpatient Gastroenterology clinic of the University of the Philippines-Philippine General Hospital underwent multiple gastric biopsy and specimens were submitted for gram stain, culture, antimicrobial sensitivity testing, rapid urease test and histology. Antimicrobial susceptibility testing was done by Epsilometer testing (Etest) method against metronidazole, clarithromycin, amoxicillin, and tetracycline.H. pylori infection (mean age of 44 years ± 13), 70% were males. H. pylori culture showed a sensitivity of 45% (95% CI [29.5–62.1]), specificity of 98% (95%CI [81.5–100%]), positive likelihood ratio of 19.93 (95% CI [1.254–317.04]) and a negative likelihood ratio of 0.56 (95% CI [0.406–0.772]). All H. pylori strains isolated were sensitive to metronidazole, clarithromycin, amoxicillin and tetracycline.Sixty percent (60%) of the study population was positive for Knowledge of the antibiotic susceptibility patterns in our setting allows us to be more cautious in the choice of first-line agents. Information on antibiotic susceptibility profile plays an important role in empiric antibiotic treatment and management of refractive cases. Helicobacter pylori is a gram-negative bacterium that colonizes the gastric mucosa of more than half of the world's population [H. pylori infection and the subsequent development of chronic active gastritis, peptic ulcer disease, gastric cell carcinoma and B cell MALT lymphoma has been well established[® . A lower prevalence rate of 42% was reported by Daez et. al. in 2002 (unpublished data) among 375 patients undergoing endoscopy at the Philippine General Hospital utilizing the rapid urease test and histopathology.pulation ,2 Since tablished. The pritablished. The pretablished,6 In theH. pylori infections. However, isolation of the organism by culture has been highly variable. Success rates depend on the technical expertise of the microbiology laboratory, ranging from 30% to 73%[Microbiological isolation of the organism is the theoretical gold standard for the detection of 0% to 73%. FailureH. pylori such as urease broth tests, urea breath tests, serologic methods and stool antigen detection. However, the increasing prevalence of resistant strains makes culture and antibiotic sensitivity testing valuable to determine alternative treatment regimens after failure of initial eradication regimen.In clinical practice, gastric biopsy with culture is not routinely performed due to the availability of more rapid diagnostic tests in the detection of H. pylori infections in the empiric setting.In the Philippines, due to methodological difficulties in isolating the organism, detection of the organism by culture methods has not been popular. Realizing the increasing prevalence of antimicrobial resistance in other countries and its potential negative impact on the efficacy of many treatment eradication regimens, it is important in clinical practice to determine the prevailing local antibiotic susceptibility patterns when choosing appropriate eradication regimens for H. pylori infection among patients with dyspepsia, to determine the sensitivity and specificity of culture technique in the detection of H. pylori infection, and to determine the antibiotic susceptibility patterns of H. pylori organisms isolated by culture among Filipino patients.This study aims to evaluate the use of culture in the diagnosis of This is a prospective, cross-sectional study involving adult patients with dyspepsia, who had independently been determined to have clinical indications for an endoscopy at the out-patient gastrointestinal clinic of the Philippine General Hospital, a tertiary training university hospital in Manila. Eligible patients were enrolled in the study after informed consent to undergo the required diagnostic testing of endoscopy samples.Patients were excluded if they were less than 18 years old, had a history of proton pump inhibitors (PPI) intake within 2 weeks, H2 antagonists within 1 week and antibiotic intake within 1 month prior to inclusion in the study.The nature and purpose of the study were discussed with the patient until fully understood. All patients with dyspepsia undergoing endoscopy who fulfilled the inclusion criteria had a complete history and physical examination. Data were obtained using a data collection form. Participants underwent upper gut endoscopy as clinically indicated. Pre-procedure preparations for Esophagogastroduodenoscopy were performed according to standard methods. Biopsy of gastric tissue were collected from the antrum and body of the stomach and specimens were sent for (1) histopathologic study, (2) gram staining, (3) culture and sensitivity and (4) rapid urease broth test. Those interpreting results of the above diagnostic tests were blinded.Helicobacter pylori infection was defined as those patients independently assessed by their attending physician based on clinical symptoms and a positive test for any of the two diagnostic tests (histology and rapid urease test). In the evaluation of the diagnostic performance of H. pylori culture, the above clinico-laboratory case definition were used as the comparator reference standard.A patient with H. pylori colonies are typically small, flat and translucent to grey. On days 4 to 5, all plates with no characteristic colonies, were subcultured to a fresh HBAP to promote growth of slow or fastidious strains and incubated for an additional 3 to 5 days. Plates were examined until the tenth day before reporting a negative growth. Suspected H. pylori colonies were tested for urease, oxidase and catalase production. A modified gram stain was performed on a methanol-fixed smear using crystal violet for 1 minute followed by a water wash and then a safranin counter stain for 30 minutes prior to a final washing with tap water The smear was air-dried and examined under oil immersion.Two pieces of gastric tissue were obtained and placed in 0.2 mL sterile saline and transported to the microbiology laboratory for processing. The biopsy specimen was placed in a sterile petri dish and minced with 2 sterile scalpel blades. Specimens were inoculated in both 7% Horse Blood + Brain Heart Infusion Agar (HBAP) and Brucella Blood Agar with Skirrow's supplement (5% defibrinated Sheep's Blood with Trimethoprim (5 mg/L), Vancomycin 10 mg/L and Polymixin B (2500 units/L)) Plates wH. pylori is confirmed by the presence of a gram negative curved bacilli and a positive test for urease, oxidase and catalase production. Five H. pylori isolates that survived the shipping and handling were verified at University of Virginia Center for Studies of Diseases Due to H. pylori.The presence of H. pylori (NCTC # 12822) and the University of Virginia Center for Studies of Diseases Due to H. pylori metronidazole resistant culture strain #Cp 2124 and clarithromycin resistant strain #Cp 5535. In the absence of a resistant control for amoxicillin and tetracycline, susceptibility breakpoints set by NCCLS and large clinical trials were used[Epsilometer test (Etest, AB Biodisk, USA) was used to determine the minimal inhibitory concentrations (MIC). MIC values were read as the intercept of the elliptical zone of inhibition with the graded strip for the Etest. Strains were considered resistant when the MIC was >8 g/ml for metronidazole, >1 g/ml for clarithromycin and >0.5 g/ml for amoxicillin. These breakpoints were used based on the recommendations from the National Committee for Clinical Laboratory Standards (NCCLS) and a large clinical trial ,11. For were used,11,13.The Rapid Urease Test (RUT) was performed by placing 0.5 ml of 8% (weight/vol) unbuffered urea in distilled water (pH 6.8) in a clear 0.7 ml Eppendorf tube, to which one drop of 1% phenol red (free acid) suspension was added. The urea solutions was stored at 4°C and prepared on the day of use to ensure color stability. Two gastric biopsy specimens from the antrum and body were placed in the tube. A positive test was indicated by a rapid color change of the media surrounding the biopsy from yellow to magenta followed by a rapid generalized color change throughout the media. A negative result was indicated when there was no change in color appreciated after 2 hours of observation.Specimens were sent to the pathology lab and gastric tissues were fixed and stained with Giemsa and Hematoxylin-Eosin dye. A specimen was read as positive if curve bacilli organisms were seen on microscopy. Pathologists were blinded to the results of the other diagnostic tests.H. pylori culture were expressed as sensitivity and specificity rates, positive and negative predictive values and likelihood ratios with a 95% confidence interval.Demographic data was described using rates and percentages for categorical variables. For continuous variables, means and standard deviations were used. Measures of accuracy for H. pylori infection based on the pre-defined case definition. The mean age for H. pylori infected individuals was 44 years ± 13. Seventy percent were males with a male:female ratio of 2:1. The majority of infected patient were married (80%), and had reached only up to the secondary level of education (70%). Fifty-five percent were unemployed. Seventy-five percent of infected patients had access to piped water. No significant differences among the demographic characteristics of H. pylori positive and negative cases were observed.Among 52 patients with dyspepsia, 31 (60%) were positive for H. pylori infection (histopathology, rapid urease test (RUT) and culture) , specificity of 98% (95%CI [81.5–99.8%]), positive likelihood ratio of 19.93 (95% CI [1.254–317.04]) and a negative likelihood ratio of 0.56 (95% CI [0.406–0.772]). The positive predictive value was 97% (95% CI [74.7–99.7%])) and the negative predictive value was 55% (95% CI [39.8–69.7%]).All included patients underwent the 3 diagnostic tests for the diagnosis of ) figure . FourteeH. pylori organisms were isolated from 52 clinical specimens. The mean number of incubation time was 3.8 days ± 1 day. All isolates grew on primary plates. All isolates were highly sensitive to amoxicillin (mean MIC of 0.016 ug/ml by Etest)), tetracycline (mean MIC of 0.164 ug/ml SD ± 0.16 SD by Etest), metronidazole (mean MIC of 0.061 ug/ml SD ± 0.04 by Etest) and clarithromycin (mean MIC of 0.016 SD ± 0 by Etest) . A study of the World Health Organization's Programme for appropriate Health Care Technology (ATH) has shown a correlation between the occurrence of multi-resistant bacteria and antibiotic consumption patterns. The Philippines has the highest percentage in 1983 of antibiotic utilization among countries (including USA, Japan, United Kingdom) surveyed (>25%). However, majority of the people whether rich or poor allot minimum expenses for medical care at 2.7% and 1.2%, respectively[H. pylori has been well documented in other studies, the absence of primary resistance in our results may also be an underestimate of the true prevalence of H. pylori resistance because of the smaller sample size compared with published literature[H. pylori in the Philippines.Potential reasons for the absence of resistant pectively. Such paurveyed >%. Howeveiterature. Only wiH. pylori infection approximates that in published literature abroad. In the absence of standard disk diffusion zone sizes for regimens used in H. pylori eradication regimen except for metronidazole, further establishment of the susceptibility pattern of locally occurring isolates by comparing zone size breakpoints with Etest, agar dilution method and as well as molecular genotyping of resistant strains will be the future direction of this pilot study.Knowledge of the antibiotic susceptibility patterns in our setting allows us to be more cautious in the choice of first-line agents. The use of culture technique in the diagnosis of H. pylori infection, information on antibiotic susceptibility profile plays an important role in empiric antibiotic treatment and management of refractive casesWhile the use of culture is not an ideal test for the rapid diagnosis of H. pylori, manuscript writing and editingRVD, LJB, CSA contributed in the microbiologic isolation of EDL, MOD, VD contributed in the specimen processing of biopsy samples, manuscript writing and editingRLG contributed in the research design planning, manuscript content and final editing