The aim of this study was to evaluate whether the HER2 expression in breast cancer is retained in metastases. The HER2 expression in primary tumours and the corresponding lymph node metastases were evaluated in parallel samples from 47 patients. The HercepTest was used for immunohistochemical analyses of HER2 overexpression in all cases. CISH/FISH was used for analysis of gene amplification in some cases. HER2 overexpression (HER2-scores 2+ or 3+) was found in 55% of both the primary tumours and of the lymph node metastases. There were only small changes in the HER2-scores; six from 1+ to 0 and one from 3+ to 2+ when the metastases were compared to the corresponding primary tumours. However, there were no cases with drastic changes in HER2 expression between the primary tumours and the corresponding lymph node metastases. The literature was reviewed for similar investigations, and it is concluded that breast cancer lymph node metastases generally overexpress HER2 to the same extent as the corresponding primary tumours. This also seems to be the case when distant metastases are considered. It has been noted that not all patients with HER2 overexpression respond to HER2-targeted Trastuzumab treatment. The stability in HER2 expression is encouraging for efforts to develop complementary forms of therapy, for example, therapy with radionuclide-labelled Trastuzumab. There is a need for new therapy modalities to improve the survival time for patients with disseminated breast cancer. One approach is to target the antibody Trastuzumab (Herceptin™) to the HER2 receptor when it is overexpressed . HER2 a. HER2 isBinding of Trastuzumab to HER2 blocks growth-stimulating intracellular signalling, decreases the cellular repair capacity after chemo- and radiotherapy, and possibly also improves the apoptosis capacity . Thus, tHowever, it has been shown that less than half of the patients with a high expression of HER2 respond to Trastuzumab treatment whether the antibody is given alone or in combination with chemotherapy . The reaTissue sections were obtained from the Scandinavian Breast Cancer Group study, SBG 9404 . Blocking serum was applied for 15 min and followed by incubation with rabbit anti-human c-erbB-2 oncoprotein  diluted 1 : 500. Sections were then incubated with the biotinylated secondary antibody and were visualised using the peroxidase substrate 3-amino-9-ethyl-carbazole (AEC) (Sigma A-5754) as chromogen. Finally, the sections were counterstained with Mayer's haematoxylin and mounted with Aquamount .The histological and immunohistochemical staining was carried out as previously described  gene amplification were carried out to validate the immunohistochemistry. This verified that the primary tumours with high HercepTest scores (2+ or 3+) were also gene amplified. For a few cases, methodological comparison was also made between FISH and CISH, and an exact match was found. Samples that showed erbB2 gene amplification with FISH also showed amplification with CISH while samples, which did not show amplification with FISH, did not show amplification with CISH.For the FISH assay, we applied the PathVysion HER-2 DNA Probe Kit . Briefly, formalin-fixed paraffin-embedded tissue specimens were placed on slides. The DNA was denaturated and allowed to hybridise with two fluorescent probes. The chromosome 17-marker probe gave green fluorescence and the erbB2 marker probe gave intense orange fluorescence. The cell nuclei were stained with DAPI giving blue fluorescence. The three fluorescence signals were observed sequentially in the same field of view using a microscope, Leica DMLB, equipped with appropriate filters. The scoring conditions followed the recommendations given by Vysis Inc. Gene amplification was at hand when the number of orange spots per cell was significantly higher than the number of green dots per cell.The CISH analysis was carried out according to the description by n=47), the quality of both the primary tumour and the metastases samples was high.In six patients, the primary tumours and the lymph node metastases deviated severely with respect to their HercepTest score. In all six cases, the samples with lowered HercepTest scores (five metastases and one primary tumour) also had a distorted histological and morphological appearance. In addition, FISH analyses revealed that the samples with lowered HercepTest scores were of such poor quality that the chromosome 17 marker could not be identified. Further, the DAPI stainings were blurry and did not stain the nuclei as expected. We were unable to obtain more material from these patients and they were therefore excluded from the analysis. In the remaining analysed cases  or there was no staining at all (n=13). There were not so many cases with intermediate staining intensity, faint staining (n=8) and moderate staining (n=3). This pattern was similar in the lymph node metastases, which had strong staining in 22 cases, moderate staining in four cases, faint staining in two cases and no staining in 19 cases.The intensity in staining in the primary tumours was, in most cases, either strong (The major results from the HER2-score analyses are summarised in The literature was reviewed for recently published studies on HER2 expression in primary breast tumours and corresponding metastases (see The studies referred to in It is noted that the percentage HER2 positive primary tumours and metastases were higher in the present study than in the previously published studies . This isThe results in the present study and in seven recently published similar studies clearly showed that breast cancer metastases generally overexpress HER2 in a manner similar to the corresponding primary tumours. This stability in HER2 expression seems surprising in the light of the genomic instability that characterises most malignant tumours, including breast cancers. Tumours are formed via multistep carcinogenesis  involvinThe arguments given above about the lack of influence of genomic instability on HER2 expression are also of interest when HER2 targeted therapy is considered. It could be expected that an efficient therapy based on targeting of HER2 would tend to induce a selection for cells with low or no HER2 expression. However, as discussed above, such cells might have a decreased growth potential and, during therapy, even be triggered to apoptosis. Thus, it is possible that HER2 is a suitable target also if treatment induced selection is considered.The observed stability in HER2 expression is also supported by older studies on breast cancer (e.g. As mentioned in the Introduction, the treatment of disseminated breast cancer needs improvement and it is necessary to consider new therapy methods. Less than half of the patients with high HER2 expression respond to Trastuzumab treatment . The reaTargeting against HER2 not only with Trastuzumab but possibly also with radiolabelled Trastuzumab could be an improvement. Radionuclide therapy is a promising modality for treatment of tumours of haematopoietic origin, while the success for treatment of solid tumours has so far been limited (New knowledge is continuously emerging with respect to receptor targeting since pharmacokinetics and cellular processing of different types of targeting agent are increasing and the research dealing with molecular design of targeting agents is rapidly expanding. The development of peptides and small proteins with specificity against tumour cells is one strategy . Liposom


HLA-DRB1 gene in the class II region. The possibility that other genes in the MHC independently influence susceptibility to multiple sclerosis has been suggested but remains unconfirmed.Variation in the major histocompatibility complex (MHC) on chromosome 6p21 is known to influence susceptibility to multiple sclerosis with the strongest effect originating from the DRB1*1501 risk haplotype. We then refined this analysis by extending the genotyping of classical HLA loci into independent cases and control subjects.Using a combination of microsatellite, single nucleotide polymorphism, and human leukocyte antigen (HLA) typing, we screened the MHC in trio families looking for evidence of residual association above and beyond that attributable to the established HLA-C gene. Extending analysis of the classical loci confirmed that this residual association is partly due to allelic heterogeneity at the HLA-DRB1 locus, but also reflects an independent effect from the HLA-C gene. Specifically, the HLA-C*05 allele, or a variant in tight linkage disequilibrium with it, appears to exert a protective effect (p = 3.3 × 10−5).Screening confirmed the presence of residual association and suggested that this was maximal in the region of the HLA-C gene influences susceptibility to multiple sclerosis independently of any effect attributable to the nearby HLA-DRB1 gene. Ann Neurol 2007Variation in the  It is well established that the major histocompatibility complex (MHC) on chromosome 6p21 contains at least one gene that influences susceptibility to multiple sclerosis.DRB1*1501 allele.DRB1*1501 is generally overrepresented in cases.DRB1*1501 allele is carried on a particularly extensive haplotype,DR15 haplotype found in white Europeans. As a result, many variants from flanking genes, even some located quite a distance from DRB1, have sufficient LD with DRB1*1501 that they invariably also show evidence for association with the disease in any population where association with DRB1*1501 can be demonstrated.DRB1*1501 and DQB1*0602 has been particularly taxing because the LD between these closely mapped genes is especially tight in those populations where the disease is frequent, that is, white Europeans and their migrant descendants. However, recent studies in the admixed African American population indicate the supremacy of the DRB1*1501 allele.18In virtually every population studied, multiple sclerosis is found to be associated with the DR15 haplotypes are less common, and by using large cohorts, it has been possible to demonstrate that the DRB1*0301 allele also confers susceptibility to multiple sclerosis, thereby confirming allelic heterogeneity at the DRB1 locus.DRB1*1501 allele may be modulated by other DRB1 alleles.20In the presence of one susceptibility allele it is difficult to identify effects attributable to a second allele,DRB1 also influence susceptibility to the disease.DRB1 supports the existence of secondary loci,DRB1. Confirmation of linkage in this region has been established only in more recent studies involving many hundreds of families.DRB1 does not exclude the presence of secondary loci. Several authors have attempted to identify secondary loci using more powerful association-based methods. Two groups have typed dense microsatellite maps of the region and both found evidence for a secondary locus maximal in a region close to HLA-A: one group identifying the marker D6S1683 just telomeric of HLA-A,HLA-A extending from MOGCA to D6S265 marker.HLA-C (marker C1_3_2) also showed evidence for an independent effect.DRB1*1501, although this study was limited by a high genotyping failure rate (40%) and, more importantly, a distribution of markers leaving regions close to the classical loci essentially unexplored.DRB1 and the large correction required for multiple testing. Unfortunately, none of the published studies has used sufficient samples to compensate for these statistical penalties, and thus none is able to provide unequivocal evidence supporting any particular secondary locus.The relation between the MHC and multiple sclerosis is further complicated by the accumulating evidence suggesting that MHC loci mapping outside The 480 trio families  and 721 sporadic cases participating in our study were recruited from across the United Kingdom. All subjects involved in this study gave written informed consent and provided a venous blood sample from which DNA was extracted and normalized. Comparing data from the five classical loci in the trio family index cases (n = 480) with those from the sporadic cases (n = 721) showed that there was no statistically significant difference between these two cohorts. The clinical details for each set of cases are summarized in Table 31HLA-A, -B, -C, -DRB1, and -DQB1) were available for the donor and UKT cohorts, whereas these were available for only three loci in the 1958 birth cohort . Only white individuals from these cohorts with complete data were included: 408 for the donor cohort, 2,201 for the UKT cohort, and 1,051 for the 1958 birth cohort . There was no evidence for any statistically significant difference among these three cohorts in a pairwise comparison of the classical loci. We also compared each of the three control cohorts with the nontransmitted alleles from the 480 trio families. Again, there was no evidence for any statistically significant difference.The fully anonymous control data used in the extension analysis were derived from three sources: local organ donors, national organ donor records held by UK Transplant (UKT), and the 1958 birth cohort. Ethical permission for using these data was obtained from the appropriate respective research ethics committees. Data from all five classical loci , GDB Human Genome Database (http://www.gdb.org), and Ensembl . From Ensembl BLAST (http://www.ensembl.org/Multi/blastview) and electronic polymerase chain reaction (ePCR) analysishttp://www.ncbi.nlm.nih.gov/sutils/e-pcr), with emphasis on markers previously suggested to be of relevance in multiple sclerosis,To generate a screening set of microsatellites, we first identified an exhaustive list of such markers (n = 248) lying within the extended MHC using publishedHLA-DRB1 and HLA-DQB1 was performed in all the UK trios and 60% of the US trios.HLA-DRB1*15 alleles were subtyped into HLA-DRB1*1501, 1502, or 1503. The primer sequences and combinations for each PCR reaction are given in Supplementary In the screening of trio families, four-digit (medium-resolution) typing of DRB1 and the other classical loci was confirmed in both the trio family and extension data sets; estimates for Global D' and Cramer's V  are summarized in Table DRB1. It should be noted that although the extent of LD between class I loci and DRB1 is modest, this level of LD is sufficient to produce an association signal at class I loci secondary to DRB1 effects. Indeed, this is precisely how the association between the MHC and multiple sclerosis was first recognized. Allele counts and frequencies for the five classic loci in the screening and extension data sets are provided in Supplementary LD between Transmission disequilibrium testing (TDT) of data from trio families was performed using the TDTPHASE program, part of the UNPHASED suite.HLA-DRB1 and HLA-DQB1) in a cohort of 930 trio families : 480 from the United Kingdom and 450 from the United States. In addition, we also typed 50 microsatellite markers and 3 class I HLA loci  in the same 480 UK trios. As expected, multiple markers showed highly significant evidence for association . The results generated in this subgroup analysis are also listed in Supplementary HLA-C (pcorrected = 0.04) and rs3132552 (pcorrected = 0.006), a synonymous coding polymorphism in the corneodesmosin gene. Because rs3132552 lies just 151kb telomeric of HLA-C, it was not surprising to find substantial LD between these loci in the UK trios . Testing rs3132552 in the DRB1*1501-negative trios from the United States and United Kingdom independently demonstrated that the marker shows association in both populations . These data demonstrate that there is significant residual association within the MHC region above and beyond that attributable to the well-established association with DRB1*1501. However, the power available in this modest DRB1*1501-negative subgroup, together with the extensive LD between the various MHC loci, makes it impossible to establish with any confidence which locus is primarily responsible for the observed effect.To refine the relation between the MHC and multiple sclerosis, we first screened the region by genotyping 110 SNPs and two class II HLA loci  in an additional 721 sporadic UK multiple sclerosis patients and established UK control data from a cohort of 3,660 individuals (see Subjects and Methods). Given that the US trio families provided independent evidence implicating the HLA-C region, we analyzed these new data from additional UK samples together with those from the 480 UK index cases used in the first screening experiment in the form of an extension analysis as opposed to a replication study.DRB1 is overwhelmingly the most significant, with the majority of this effect attributable to the DRB1*1501 allele (puncorrected = 4.5 × 10−88). After excluding all individuals carrying DRB1*1501, analysis of the remaining data continues to show highly significant evidence for association with the most significant effect still appearing to come from the DRB1 locus . Therefore, we next excluded all individuals carrying DRB1*03 alleles. Analysis of the remaining data shows that significant evidence for association is still evident but only at HLA-C and DRB1 . Even though this allele is relatively uncommon, we also excluded all individuals carrying the DRB1*0103 allele. In this final stratified analysis, statistically significant association is only apparent at the HLA-C locus  and one HLA-C allele (*05) exert independent effects on susceptibility to multiple sclerosis. To determine the risk associated with each DRB1 susceptibility allele in haplotypes with and without HLA-C*05, we reanalyzed the full data set three times, first excluding all individuals carrying DRB1*03 or *0103, next excluding all individuals carrying DRB1*1501 or *0103, and then finally after excluding all individuals carrying DRB1*1501 or *03. Table DRB1 and HLA-C alleles, as compared with haplotypes carrying no associated allele at either locus. The relative risk associated with haplotypes carrying HLA-C*05 without DRB1 risk alleles is consistent across the three analyses and is significantly less than 1 in each case, confirming the protective nature of this allele. The data suggest that the risk associated with the DRB1*1501 allele is reduced but not abolished by the presence of an HLA-C*05 allele on the same haplotype. Unfortunately, the frequency of the other combined haplotypes (DRB1*03 with HLA-C*05 and DRB1*0103 with HLA-C*05) was too low to provide sufficient statistical power to make a judgment about whether the HLA-C*05 allele alters the risk associated with the secondary DRB1 risk alleles.Consequently, to further refine the nature of the secondary MHC association identified in our screening experiment, we typed the five classical HLA loci  were to be applied.Adequate correction for multiple testing is particularly important in studies that involve systematic screening and stratification because the number of tests performed is generally large.Association testing can be confounded by population stratification and other phenomena that lead to inadequate matching of cases and control subjects. Our use of trio families and transmission disequilibrium testing protects against these confounders in the screening phase. However, the extension phase experiments used case–control analysis; therefore, results emerging from these efforts could have been confounded by such errors. The classical loci are highly polymorphic and provide a powerful means to test for stratification. The absence of any difference between the three UK control cohorts or the two sets of UK cases is therefore extremely reassuring in this respect. Perhaps even more reassuring is the absence of any difference between the unrelated UK control cohorts and nontransmitted alleles from the UK trio families, suggesting that our cases are drawn from the same genetic background as the control subjects and making it unlikely that hidden population stratification accounts for our observations. However, replication of the findings in independent cohorts will be necessary to fully exclude this possibility.DRB1 susceptibility alleles means that only 47% of control subjects and 22% of cases will ultimately be informative for the study of HLA-C. Large initial cohorts would need to be selected to provide realistic power. The absence of any statistically significant difference in an underpowered study should not be misinterpreted as evidence against this effect. Just as it proved difficult to establish that DRB1*1501 is responsible for the primary effect in this region, it may prove even more difficult to refine this secondary effect in detail. It remains possible that the observed association is secondary to LD with a nearby but as yet untyped variant. The absence of any residual main effects at DQB1, HLA-B, and HLA-A make it unlikely that the observed association results from these loci despite their strong LD with HLA-C.Efforts to further replicate our findings will require considerable resources because exclusion of individuals carrying HLA-C is, in fact, the locus primarily responsible for our observations, this would implicate novel pathways in disease pathogenesis, in particular, the innate immune system. HLA-C molecules, loaded with nonamer peptides, act as ligands for the killer cell immunoglobulin-like receptors (KIRs). KIR receptors contain two or three immunoglobulin-like domains and a short  or long  cytoplasmic tail, corresponding to an activating or inhibitory action on the natural killer cells and T-cell subsets on which they are expressed. Whereas the ligands for activating KIRs remain elusive, and may have relatively weak binding affinities, those for the inhibitory KIRs are well defined. The subset of HLA-C alleles with Ser at position 77 and Asn at position 80 (C1 group) bind to the KIR2DL2 and KIR2DL3 receptors, whereas those with Asn at position 77 and Lys at position 80 (C2 group) bind to the KIR2DL1 receptor. HLA-C peptide binding specificity may further influence interaction with KIRs.HLA-C alleles according to this functional categorization, suggesting that the protective effect seen for HLA-C*05 is specific to this allele, and not a consequence of its group function. Alternately, as the effect appears to be allele rather than functional group specific, it may indicate that susceptibility to multiple sclerosis is conferred by an aspect of HLA-C function that is independent of its interaction with KIR.If HLA-C*06 with susceptibility to psoriasis provides a clear precedent for the involvement of HLA-C in complex inflammatory disease. In a Sardinian study of psoriasis, HLA-C*05 was found to be significantly underrepresented (protective) in patients, although it was not established whether this effect is independent of or secondary to the overrepresentation of HLA-C*06 and/or haplotypes containing risk alleles at the nearby corneodesmosin (CDSN) gene,KIR2DS1 and KIR2DS2 genes was reported to be a novel risk factor for psoriasis and an interaction between HLA-C and KIR observed with the overall combination of activating and inhibitory genotypes influencing susceptibility.KIR genes has also been reported for a range of other infectious and autoimmune diseases.KIR gene cluster as a candidate susceptibility locus in multiple sclerosis subsequent to our finding of association with HLA-C. The fact that the KIR gene cluster lies on chromosome 19 where modest evidence of linkage was observed in the recent high-density screen for linkage in multiple sclerosisDRB1*1501 have been excluded.24The established association of HLA-C, or a locus in tight LD with it, confers additional effects on susceptibility to multiple sclerosis, substantially adding to our understanding of the MHC region in this disease and offering a clear roadmap to further experiments that will refine these observations in larger data sets.In conclusion, we show that the class I gene


Background and purpose Tantalum rod implantation has recently been proposed for treatment of early stages of femoral head osteonecrosis. The purpose of our study was to report the early results of its use in pre- and post-collapse stages of the disease.Methods We studied prospectively 27 patients who underwent tantalum rod implantation for treatment of nontraumatic femoral head osteonecrosis between December 2000 and September 2005. Patients were evaluated radiologically and clinically using the Steinberg classification and the Harris hip score (HHS). Disease stage varied between stages II and IV. Mean follow-up time was 38 (15–71) months.Results 1 patient (1 hip) died 15 months after surgery for reasons unrelated to it. 13 of 26 hips remained at the same radiographic stage, and 13 deteriorated. Mean HHS improved from 49 to 85. 6 patients required conversion to total hip arthroplasty. When the procedure was used for stages III and IV, both radiological outcome and revision rates were worse than for the stage II hips. There was, however, no difference in postoperative HHS between patients at pre- and post-collapse stages at the time of initial evaluation. Survivorship, with revision to THA as the endpoint, was 70% at 6 years.Interpretation The disease process does not appear to be interrupted, but there was a significant improvement in hip function initially in most hips. Tantalum rod implantation is a safe “buy-time” technique, especially when other joint salvage procedures are not an option. Appropriate patient selection and careful rod insertion are needed for favorable results. The femoral head is the most vulnerable site for development of osteonecrosis. The incidence is increasing: every year 10,000–20,000 new cases are diagnosed in the USA and 5–12% of total hip arthroplasties performed each year are due to osteonecrosis . AlthougThe joint-preserving procedures include core decompression with or without bone grafting , rotatioWe studied prospectively 27 patients (32 hips) who underwent tantalum rod implantation for treatment of nontraumatic femoral head osteonecrosis between December 2000 and September 2005. Although the preferred joint-preserving procedure at our institution is vascularized fibular graft, we proposed tantalum rod to patients who were medically unfit or unwilling to undergo this longer procedure with long rehabilitation, or to young patients with post-collapse disease who were reluctant to have a total hip replacement. The study was approved by the ethics committee of our institution  and all patients gave their informed consent.The study included 23 male and 4 female patients with a median age of 36 (15–55) years. 1 patient (1 hip) died 15 months postoperatively for reasons unrelated to surgery (sickle cell crisis), and was excluded from the study. Osteonecrosis was idiopathic in 8 patients, secondary to cortisone intake in 15, due to alcohol intake in 2, and secondary to sickle cell anemia in 1 patient. None of the patients had any prior treatment for the osteonecrosis. The preoperative stage was stage II in 9 patients, stage III in 7, and stage IV in 10 patients. Mean follow-up was 38 (15–71) months. No patients were lost to follow-up.5 patients had bilateral tantalum rod implantation. For these 5 patients, 1 of the hips was selected using a computer-generated random sequence in order to fulfill the requirement of data independence for statistical analysis purposes.Patients were evaluated preoperatively and at the end of the follow-up period, both clinically and radiologically using the Harris hip score (HHS) and Steinberg classification . The meaTwo independent observers other than the surgeons did the radiographic evaluation. In cases of discordance, a unanimous final judgment was made. We made no attempt to examine the intraobserver or interobserver variability. Throughout the study, clinical evaluation was done by a single observer (not a surgeon).Finally, an MRI-based volumetric method was used to quantify the size of the lesion on the preoperative MRI scans. We used a semi-automated technique in which the area with abnormal signal was outlined for each MRI slice using the tracing tool of the ANALYZE image analysis software, version 8.1 . We have reported details of the technique elsewhere .The implant used was a porous tantalum rod (10 mm in diameter) . It was inserted through a small lateral incision under biplanar fluoroscopic guidance, and it was aimed at the osteonecrotic lesion to support the subchondral bone. The tip of the rod was advanced not more than 5 mm from the articular surface. We did not place any bone graft in the femoral head. Although according to the technique originally described the lateral part of the rod should abut the lateral femoral cortex, we used shorter rods (either 70 or 75 mm) so that the rod ended distally at the cancellous bone of the trochanteric area. With this method, if a later arthroplasty is required, the initial cortical defect will have been filled with bone. The distal threads and the high frictional stability of the implant prevent backing out.There were no perioperative complications. Postoperative rehabilitation included partial weight bearing for 6 weeks.We used the Wilcoxon signed ranks test to compare the differences in pre- and postoperative Steinberg classification and HHS. The Wilcoxon-Mann-Whitney test was used to compare postoperative Steinberg and HHS scores between primary and secondary etiology groups. 95% bootstrap confidence intervals (CIs) for differences in median values follow p-values were calculated using the Wilcoxon-Mann-Whitney test. Spearman’s rho (ρ) was calculated for the assessment of the correlation of age to postoperative Steinberg and HHS scores and to assess the correlation between lesion size and outcome. The endpoint for the survival analysis was hip arthroplasty and the results were presented with a Kaplan-Meier survivorship curve. The standard error of the cumulative survival was calculated using Greenwood’s formula. All reported p-values are two-tailed with p < 0.05 being considered significant. Analyses were conducted using SPSS version 15.0  and S-plus version 8.0 .13 of the 26 hips did not have any radiographic progression of the disease at the end of the follow-up. The preoperative Steinberg staging of these 13 hips was: 5 stage II , 2 stageThere was better radiological outcome for patients at precollapse stage at the time of operation (4 of 9 stage II hips deteriorated) when compared to those at post-collapse stages (9 of 17 stage III and IV hips deteriorated) (p = 0.02). Interestingly, there was no statistically significant difference in postoperative HHS between these two groups of patients ). Moreover, HHS at last follow-up was not statistically significantly greater  in patients with unchanged Steinberg stage, when compared to those with radiographic progression  ) .There were no significant differences in the radiological outcome ) and the clinical outcome ) between the subgroup of patients with a serious underlying disease  and those without. Furthermore, we could not find any significant difference in the postoperative Steinberg stage ) and HHS ) when we compared the primary and the secondary etiology groups of patients.There was no significant correlation between age and postoperative Steinberg  or HHS score (r = –0.15).It is notable that when all hips were grouped together, despite radiological deterioration over time ) there was a clinical improvement compared to the preoperative status of the patients ). The mean HHS increased from 49 (SD 19) preoperatively to 85 (SD 15) postoperatively. The mean estimated survival time of the implant was 59 months . There were no cases of tantalum rod breakage, loosening, or subsidence.We found a statistically significant positive correlation between the preoperative volume of the osteonecrotic lesion and the postoperative radiographic stage of the disease (ρ = 0.73). This correlation was noted not only in hips that had been treated prior to femoral head collapse (ρ = 0.89) but also in the post-collapse group of hips (ρ = 0.66). There was no correlation between the preoperative volume of necrotic femoral head and postoperative HHS (ρ = –0.41).During the study period, 6 of the 26 hips underwent revision to a total hip arthroplasty at an average of 29 months . Only 1 of the 6 hips was stage II at the time of the index operation ; the othPreservation of an osteonecrotic femoral head depends on prevention of collapse of the structurally compromised necrotic bone. Non-surgical management with partial weight bearing can only be selected for early stages and very small lesions. Even in such cases, it has been proven to be ineffective in 80–90% of patients . Other nA meta-analysis of studies with core decompression or nonoperative treatment  demonstrRadiographic progression after core decompression has been well documented in the literature. In a 2–6-year follow-up study, Cortical bone grafting of the femoral head may offer structural reinforcement, particularly if the graft penetrates deeply into the superior-central or lateral aspect of the lesion . SupportEven with this demanding technique, outcomes seem to be compromised with time, leading to a hip arthroplasty in many patients .Recently, trabecular metal rods implanted in the femoral head with or without the addition of autologous growth factors have been advocated as an alternative to prevent collapse in stages I–III. Because of the surface characteristics of these metal implants (pore dimensions and 3D structure similar to the cellular structure of bone), high-volume bone ingrowth is encouraged and collapse is prevented. The porous tantalum is a highly biocompatible material with a porosity of 75% and pore size of 430 µm. Pores are fully interconnected and the microtexture of the material (dodecahedron) permits rapid bony ingrowth. Elastic modulus falls between that of cortical and subchondral bone and compressive strength is between that of cortical and cancellus bone. Initial stability at implantation is high due to its high coefficient of friction.2 for the porous tantalum, whereas the fibular graft had a flexural stiffness that ranged from 11 to 17 Nm2. In another laboratory study, The use of tantalum rods for the treatment of femoral head osteonecrosis was first proposed in 1997 . The invSince then, very few clinical studies have been published. Despite the radiographic deterioration that was seen in 13 of 26 hips, HHS improved. We found no significant difference in postoperative HHS between patients at pre- and post-collapse stages at the time of the initial evaluation. Moreover, postoperative HHS was similar in the group of patients with radiographically stable lesions over the course of the study and those with radiographic deterioration. There was a correlation between the volume of the lesion and the postoperative Steinberg stage, but not between volume and the final clinical score.The frequency of radiologic progression observed in our study was less than that reported for core decompression alone. This indicates that the tantalum rod implant provides a meaningful structural support for the subchondral plate in addition to the core decompression achieved by the porous nature of the material.We did not find any correlation between disease etiology and postoperative clinical and radiological scores . This contrasts with a recent study  in whichIn summary, we found that use of the porous tantalum rod is a relatively easy, minimally invasive, and reproducible procedure that can provide, at least in the short term, satisfactory functional improvement at both pre- and post-collapse stages of hip osteonecrosis. To a lesser extent, it can preserve the structural integrity of the femoral head. This is best achieved in hips at the pre-collapse stage.It would be interesting for future studies to do randomized comparisons between the outcomes of tantalum rod implantation and core decompression alone, or with implantation of fibular strut allograft.


Surrogate markers of protective immunity to malaria in humans are needed to rationalize malaria vaccine discovery and development. In an effort to identify such markers, and thereby provide a clue to the complex equation malaria vaccine development is facing, we investigated the relationship between protection acquired through exposure in the field with naturally occurring immune responses  to molecules that are considered as valuable vaccine candidates.-up study of this village. Of the 30 immune responses measured, only one, antibodies of the IgG3 isotype directed to merozoite surface protein 3 (MSP3), was strongly associated with clinical protection against malaria in all age groups, i.e., independently of age. This immunological parameter had a higher statistical significance than the sickle cell trait, the strongest factor of protection known against Plasmodium falciparum. A single determination of antibody was significantly associated with the clinical outcome over six consecutive years in children submitted to massive natural parasite challenges by mosquitoes (over three parasite inoculations per week). Finally, the target epitopes of these antibodies were found to be fully conserved.We analyzed, under comparative conditions, the antibody responses of each of six isotypes to five leading malaria vaccine candidates in relation to protection acquired by exposure to natural challenges in 217 of the 247 inhabitants of the African village of Dielmo, Senegal (96 children and 121 older adolescents and adults). The status of susceptibility or resistance to malaria was determined by active case detection performed daily by medical doctors over 6 y from a unique followP. falciparum infection in young children, our results provide the encouraging indication that these antibodies should be possible to elicit by vaccination early in life. Since these antibodies have been found to achieve parasite killing under in vitro and in vivo conditions, and since they can be readily elicited by immunisation in naïve volunteers, our immunoepidemiological findings support the further development of MSP3-based vaccine formulations.Since anti-MSP3 IgG3 antibodies can naturally develop along with protection against  Using data from malaria cases in Senegal, Pierre Druilhe and colleagues found that antibodies of the IgG3 isotype directed to merozoite surface protein 3 (MSP3) were strongly predictive of clinical outcome. Plasmodium falciparum, a parasite transmitted to people through the bites of infected mosquitoes. In the human body, the parasites replicate in liver cells before changing into so-called “merozoites.” These infect red blood cells, where they replicate rapidly before bursting out and infecting more red blood cells. This massive increase in the number of parasites in the body causes a fever and can also damage vital organs. Although individuals can protect themselves against being bitten by mosquitoes, a vaccine is urgently needed to reduce the global burden of malaria. Vaccines help the immune system fight infectious diseases. When a disease-causing organism (pathogen) enters the human body, the immune system produces antibodies. These are proteins that recognize molecules (antigens) on the pathogen and that enlist other parts of the immune system to kill the invader. This process is often slow the first time around, so people can be ill for a while. However, the immune system “remembers” the experience and responds much quicker to subsequent attacks by the same pathogen. Vaccines, which contain antigens from pathogens, prepare the immune system so that it responds quickly and effectively to a pathogen's first attack.Malaria kills about one million people—mainly children—every year. Most of these deaths are caused by An effective vaccine against merozoites would limit the severity of malaria and prevent many deaths, but scientists do not know which of the antigens on merozoites stimulate a protective immune response. Each candidate therefore has to be tested in long, expensive field trials. Young children who live where malaria is endemic  have frequent attacks of malaria but gradually develop natural immunity to the disease so that after the age 10 y, even though there are always parasites in their blood, they rarely become ill. If researchers knew which malaria antigens and which parts of the immune response are involved in natural immunity, they could use this information in vaccine design. In this study, the researchers have investigated which type of antibody  and which of five potential malaria antigens is associated with protection against clinical malaria.In 1990, the researchers started a unique study in Dielmo, Senegal, West Africa, a small village where malaria is transmission is very high all year round. For 7 y  medical staff visited every villager daily to look for clinical signs of malaria. Diagnoses of malaria were checked by looking for parasites in blood samples. One year into the study, the researchers tested blood from each participant for antibodies that recognized the candidate malaria antigens. When they analyzed their data, they found that only one immune response—the production of antibodies of the IgG3 isotype directed against an antigen called merozoite surface protein 3 (MSP3)—was strongly associated with clinical protection against malaria in all age groups. The researchers also found that some of the children developed protection against malaria when they were very young. Furthermore, in children, production of anti-MSP3 IgG3 in 1991 was associated with protection against malaria for the next 6 y.These findings show that the production of IgG3 antibodies that recognize MSP3 is strongly associated with the development of long-lasting natural protection against malaria. They also show that both protection and these antibodies can develop in very young children. Malaria transmission is unusually high in Dielmo, so these findings may not necessarily be relevant to regions where transmission is lower. In addition, although other research has shown that antibodies of this type can kill parasites in test tubes and in animals, it is possible that these antibodies are markers of, rather than causes of, protective immunity against malaria. Nevertheless, these findings support the continued development of MSP3-based vaccines, particularly since they suggest that early vaccination has the potential to protect infants against life-threatening attacks of malaria.http://dx.doi.org/10.1371/journal.pmed.0040320.Please access these Web sites via the online version of this summary at malaria and on vaccination (in English and Spanish)The MedlinePlus encyclopedia contains pages on malaria Information is available from the World Health Organization on malaria and on immune responses to malaria (in English and Spanish)The US Centers for Disease Control and Prevention provide information on malaria and malaria vaccine developmentInformation is available from the Malaria Vaccine Initiative on  A malaria vaccine is urgently needed ,2; howevP. falciparum , p = 0.183; and for anti-MSP2 (3D7), 0.71 [0.05–9.14], p = 0.785).In contrast, in the children group, a significant association of IgG3 anti-MSP-3 antibodies with clinical protection was observed in the following 12, 24, and 36 mo of follow-up  and remained marked by year 4 of the study . The differential number of attacks was still significant by year 6 of the follow-up  when the model was compared with and without the antibody response. Hence, the two methods of analysis provided convergent conclusions indicative of the long-term clinical significance of a single IgG3 anti-MSP3b determination, in terms of the resistance of children to malaria attacks.The above results were confirmed using a nonlinear model fitted to the variables demonstrated to be significant in the previous analysis, i.e., age, haemoglobin AS phenotype, and anti-MSP-3 IgG3. The maximum difference between the mean number of malaria attacks identified in children with anti-MSP-3 IgG3, and children without such a response (4.3-fold), was detected 3 y after sampling , which revealed 16 nucleotide substitutions in the same 45 strains (ten within the Th2R and six within the Th3R regions), all resulting in amino acid substitutions, in agreement with previous reports [Since MSP3 appeared at this point to be a strong surrogate marker of clinical protection, we deemed it important to investigate whether this antigen exhibits the polymorphisms reported for many malarial vaccine candidates –50. The  reports .Anopheles mosquitoes [A detailed analysis of the malarial clinical status, based on daily medical records actively collected from each inhabitant of Dielmo, Senegal, demonstrated a highly significant relationship between clinical protection acquired through natural exposure and the production of cytophilic anti-MSP-3 antibodies, notably IgG3. The stronger relationship of this marker with protection than that conferred by sickle cell trait  indicates that its contribution to the control of parasites is highly significant and therefore demonstrates its promise as a vaccine candidate. Above all, the clinical value of this surrogate marker, i.e., its association with protection, persisted out to six years in individuals who are exposed to approximately 260 inoculations per year of parasites by squitoes .Except for one study in which two antigens were investigated in parallel , most imIn such epidemiological investigations, age is a confounding factor, since protection builds up over time with exposure, and hence with age ,54. ThisFollowing the report that the isotype distribution of antimalarial antibodies was a critical parameter of malaria immunity , variousThe long-term clinical effect of a single anti-MSP3 antibody determination over a number of years in participants that are receiving malaria inoculations by mosquitoes every two days is impressive, particularly as it remained significant over six consecutive years. This finding has not been reported for any other antigen, to our knowledge. The conceptual importance and implications for vaccine development merit further investigation in other settings. The longer-term association in children than in adults is likely related to two factors: (i) children can be divided into two groups, a small subset acquiring protection early in life who remain protected in the ensuing years (this is associated with our surrogate marker) and the majority who do not, and (ii) most adults are continuously protected against malaria, but some may briefly lose this protection and reacquire it after a short, self-resolving attack. A long-term effect cannot be expected in these occasional situations of brief loss of existing protection.http://www.who.int/vaccine_research/documents/en/malaria_table.pdf) and a concomitant increase in development costs. The second reason is that the elucidation of the malaria genome, although welcome, nonetheless generates knowledge about ∼5,300 proteins—500-fold more molecules than was previously known. These potential antigens must now be assessed for their suitability for inclusion in a malaria vaccine, a task that is not achievable through individual vaccine efficacy trials with each one.Malaria vaccine discovery, which has relied for several decades on preclinical in vitro and in vivo animal models, is now increasingly reliant on clinical trials in humans. For two major reasons this is driving vaccine development towards an ever more unmanageable situation. One reason is that since none of the preclinical models is recognised as being of undisputable predictive value, each candidate must presently be taken into lengthy, time-consuming and costly clinical trials. From an initial number of 12 vaccine candidates, this translates, with various antigen presentation systems, into 58 planned clinical trials  to various pre-erythrocytic antigens among 217 inhabitants from Dielmo  was measured by ELISA against LSA1-, LSA3-, and SALSA-derived peptides, two recombinant proteins (R32 LR and R32Tet), and a peptide (NANP4) derived from the CS protein.(6.9 MB EPS)Click here for additional data file.Figure S2For each antigen, the median value of antibody responses was determined. Individuals with antibody responses above the median (>median) were considered high responders (closed circles). Individuals with antibody responses below the median (<median) were considered low responders (open circles). Following a single determination of antibodies to each antigen in blood samples, the mean number of malaria attacks recorded in Dielmo over 6 y was calculated for each year of the study and for each group of children (either high or low responders to each antigen). The two subgroups had the same mean age, so that there was no age imbalance between children with and without malaria attacks. The mean cumulative numbers of malaria attacks are illustrated for anti-MSP3b (A), anti-MSP1 (B), anti-AMA1 (C) and anti-MSP2-3D7 (D) IgG3 responses. Error bars indicate SD.(1.1 MB EPS)Click here for additional data file.Table S1ORs and 95% CIs were calculated to evaluate the relationship between two binary variables . The results are given as an indication of effect size with ORs greater than 1 indicating that the no malaria attack condition was more likely to happen in the group with antibody responses specific for the antigen tested. C:NC indicates the cytophilic to noncytophilic ratios ; NA (not available) indicates situations where ORs could not be calculated.(1.2 MB EPS)Click here for additional data file.Table S2A subgroup of Dielmo inhabitants present during 6 y of survey after blood sampling was identified. Presence or absence of anti-MSP3 IgG3 responses was tested with regard to the cumulative number of malaria attacks identified each year. The indications in favour of a potential association between anti-MSP3 IgG3 responses and resistance to malaria attacks recorded during 6 mo to 6 y following blood sampling are given as age-adjusted ORs and 95% CIs determined for children and adults separately.(482 KB EPS)Click here for additional data file.http://www.ncbi.nlm.nih.gov/) accession number AF024624.The primer positions in the MSP3seqence are based on GenBank (


Using 3D generic models to statistically analyze trends in biological structure changes is an important tool in morphometrics research. Therefore, 3D generic models built for a range of populations are in high demand. However, due to the complexity of biological structures and the limited views of them that medical images can offer, it is still an exceptionally difficult task to quickly and accurately create 3D generic models  based on medical image stacks (a stack is an ordered collection of 2D images). We show that the creation of a generic model that captures spatial information exploitable in statistical analyses is facilitated by coupling our generalized segmentation method to existing automatic image registration algorithms.The method of creating generic 3D models consists of the following processing steps: (i) scanning subjects to obtain image stacks; (ii) creating individual 3D models from the stacks; (iii) interactively extracting sub-volume by cutting each model to generate the sub-model of interest; (iv) creating image stacks that contain only the information pertaining to the sub-models; (v) iteratively registering the corresponding new 2D image stacks; (vi) averaging the newly created sub-models based on intensity to produce the generic model from all the individual sub-models.After several registration procedures are applied to the image stacks, we can create averaged image stacks with sharp boundaries. The averaged 3D model created from those image stacks is very close to the average representation of the population. The image registration time varies depending on the image size and the desired accuracy of the registration. Both volumetric data and surface model for the generic 3D model are created at the final step.Our method is very flexible and easy to use such that anyone can use image stacks to create models and retrieve a sub-region from it at their ease. Java-based implementation allows our method to be used on various visualization systems including personal computers, workstations, computers equipped with stereo displays, and even virtual reality rooms such as the CAVE Automated Virtual Environment. The technique allows biologists to build generic 3D models of their interest quickly and accurately. Spatial information of biological structures has been used to analyze their functions and to relate their shape changes to various genetic parameters -4. In paIn order to create valid 3D generic models from 2D image stacks, more attention should be paid to two essential steps - image segmentation and image registration. Image registration is the process to find a 3D transformation that can map the same anatomical region from one subject into another one. This process is essential in clinical and research applications because researchers often need to compare the same anatomical region scanned using different modalities or at different time points . Image sWith the tremendous advancements in medical imaging technologies such as CT, PET, MRI, and fMRI, we are now able to capture images of biological structures and their functions more clearly than ever before. Additionally, advanced technologies from other fields such as computer vision, computer graphics, image processing and artificial intelligence have been used to analyze 2D medical images of various modalities . HoweverDue to the difficulties with automating the segmentation task, enhanced manual segmentation software is still widely used. Various image processing algorithms have been produced to minimize user interactions and increase segmentation accuracy . However2D image stacks of mice whole-body micro-computed tomography (μ-CT) scans were provided by the Morphometrics Laboratory at the University of Calgary. Eight male and eight female laboratory mice from the same strain (AWS) were scanned. The female mice were 54 to 61 days old and weighed 16 to 21 grams; the male mice were 61 days old and weighed 20 to 25 grams. All individuals were scanned at a resolution of 35 μm. Each slice of the volumetric dataset is 1024 × 1024 pixels and the intensity of each pixel ranges from 0 to 255  scanning subjects to obtain image stacks; (ii) creating individual 3D models from the stacks; (iii) cutting each model to generate a sub-model of the user's interest; (iv) making image stacks that contain only the information pertaining to the sub-models; (v) iteratively registering the corresponding new 2D image stacks from the previous step; (vi) averaging the newly created sub-models based on intensity to produce the generic model from all the individual sub-models. All the algorithms are implemented using Java and C++ based on functionalities from open source toolkits VTK , ITK  image registration. The global affine transformation from the previous step might create some remaining local shape variations. Therefore, in order to sharpen the blurry average images, a non-rigid image registration can also be used after step 2. An intensity-based deformable 3D registration algorithm which uses a mean square metric, a linear interpolator, a B-spline based transform and a LBFGS (limited memory Broyden-Fletcher-Goldfarb-Shanno update) optimizer inside ITK is applied for further deformable image registration.3. We use a similar iterative image registration protocol to the one mentioned in   are registered. At this point, we can use intensity based image averaging technique as described in .The global median of the averaged image intensities is used to apply the marching cube algorithm to the averaged image stacks  to extraWe have developed a generalized virtual dissection-based method for the creation of generic models from 2D image stacks of a group of individuals. To illustrate our novel generic models creation technique, whole body scans of eight female mice and eight male mice are used to create averaged 3D models of the left mandible. For each subject, the left mandible 3D model is created using our cutting tools and the corresponding 2D image stack that contains only information of the left mandible is also generated.Once we have 16 left mandible models, we register the image stacks for both male and female mice. Corresponding pixels in the images of the female/male group are averaged to create an averaged image stack. Within the averaged image stack, the blurry image areas result from the misaligned sections. Therefore, the sharper the averaged images are, the better the registration process is. We use the ratio of the number of pixels with intensity 255 to the number of pixels with non-zero intensity to measure the performance of the registration process  is different from the regular vector product. However, in ITK, we can convert the versor product into the Euclidean matrix format. The 3D rotation matrix and the translation vector can be calculated from the versor product and can be saved for further analysis.In the above equation, 3D affine transformation can be represented as:X is a vector and represents a point in the 3D space, A is a 3 × 3 matrix and represents the affine transformation matrix, C is a vector and represents the transformation center, and T is a vector and represents the 3D translation. X' is the new position for X after the affine transformation. The affine registration from ITK that we utilized consists of rotation, scaling, shearing and translation in the 3D dimension. There are (3+1) × 3 parameters in this transformation. The first 3 × 3 parameters define A, the last 3 parameters define the translations for each dimension. The center of the transformation is automatically calculated from the programs and is also available.where B-Spline based non-rigid transformation ,6,9,13 wUsing our cutting tools to build models from 2D image stacks allows beginners in medical fields to learn anatomy intuitively and enjoy the process of separating the biological structures from the virtual body model before dealing with real subjects. Quickly and accurately creating various 3D averaged models can satisfy the requirements for a large number of models in virtual crash testing, therapy planning, and customizing replacement body parts. Large scale morphological studies that require quantification of anatomical features can be really tedious and might be very detailed and only focused on a few important measurements. Our method facilitates morphological studies by allowing anatomical structures to be measured and compared rapidly and in more detail. These tools help put morphological analysis at a similar level to other studies such as genetic and molecular studies where a large amount of data and measurements can be dealt with relatively quickly.The issue of homology, which refers to biological structures that have the same function, is also addressed through our method. If we measure an average and do quantitative comparisons, we would want to compare the same anatomical region. This requires the two models being compared to be first registered correctly with each other, such that if one area of interest is picked in one model, it refers to the same region in the other model. The iterative registration employed in our approach can, to a large extent, reduce the misalignments. The method we developed optimizes the functionalities and technologies of existing toolkits and the resulting software package allows biologists to build their generic models more quickly and accurately.As our virtual dissection tools are implemented in Java, they can run on both regular display systems and on the state-of-the-art CAVE Automated Virtual Environment , which iWe have developed a new technique that uses virtual model cutting and iterative image registration to create generic models from 2D image stacks of a group of individuals. Our system allows biologists to build generic 3D models quickly and accurately. However, particularly complicated morphological structures, such as highly branched and convoluted designs that typify vascular or nervous networks, still pose a challenge to our generalized and enhanced method toward generic model creation. It is difficult to use the current manual virtual dissection tools to remove such sub-models from initial, unprocessed scans. More convenient and intuitive manual virtual dissection methods will be developed in our future research. Producing deformable models based on the current tools will also be an area of further development. Those deformable averaged models can then be used for automatically segmenting the anatomical structures. More advanced automated segmentation algorithms that utilize generic models will be studied to enable higher throughput analyses of anatomical structures in both medical and more general biological contexts. Quantification of 3D shape variations will also be studied based on our generic model building technique.http://www.visualgenomics.ca/~mxiao/research.html. The current version of the software has been tested on Unix Solaris 10 and Windows XP with .NET Framework 3.5. In order to run the program from our jar files, at least Java 1.6 need to be installed. ImageJ as well as shared  libraries of VTK and ITK should also be installed.The implementation of our method is available for free downloading at Detailed installation and the user's guide are also available on the project website. VTK, ITK and ImageJ are all open source and freely available software toolkits.The authors declare that they have no competing interests.MX, JS, OEMP, EJS, BH and CWS participated in writing the manuscript and designing the technique. MX developed the computing framework. BH and CWS directed the research. All authors read and approved the final version of the manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2342/10/5/prepub


To assess the feasibility and uptake of a diabetes screening programme; to examine the effects of invitation to diabetes screening on anxiety, self-rated health and illness perceptions.Randomised controlled trial in two general practices in Cambridgeshire. Individuals aged 40–69 without known diabetes were identified as being at high risk of having undiagnosed type 2 diabetes using patient records and a validated risk score . 355 individuals were randomised in a 2 to 1 ratio into non-invited (n = 238) and invited (n = 116) groups. A stepwise screening programme confirmed the presence or absence of diabetes. Six weeks after the last contact (either test or invitation), a questionnaire was sent to all participants, including non-attenders and those who were not originally invited. Outcome measures included attendance, anxiety (short-form Spielberger State Anxiety Inventory-STAI), self-rated health and diabetes illness perceptions.95 people (82% of those invited) attended for the initial capillary blood test. Six individuals were diagnosed with diabetes. Invited participants were more anxious than those not invited , and those diagnosed with diabetes were considerably more anxious than those classified free of diabetes . Non-attenders had a higher mean treatment control sub-scale  and a lower mean emotional representation sub-scale  than attenders. No differences in the other five illness perception sub-scales or self-rated health were found.Screening for type 2 diabetes in primary care is feasible but may be associated with higher levels of short-term anxiety among invited compared with non-invited participants.ISRCTN99175498 Type 2 diabetes meets many of the criteria for suitability for screening. It is increasingly common and creates a substantial burden of suffering and health service use . . Participants with a random capillary glucose result ≥ 5.5 mmol/l were classified as screening-test-positive and were asked to re-attend for a fasting capillary blood glucose test at a later date. Individuals whose random capillary glucose result was < 5.5 mmol/l were classified as not having type 2 diabetes and were informed of their result. In the second stage, participants whose fasting capillary glucose result was ≥ 12 mmol/l were informed that they had type 2 diabetes. Participants with a fasting result ≤ 5.5 mmol/l were informed that they did not have diabetes. Those participants with a fasting glucose of 5.5 – 12 mmol/l were classified as requiring a further confirmatory 75 g oral glucose tolerance test, which was undertaken at the same visit. Those whose 2-hour capillary glucose was ≥ 11.1 mmol/l were informed that they had type 2 diabetes. Those who tested positive were informed of the consequences of the diagnosis of diabetes in a subsequent consultation with their general practitioner. The thresholds for fasting and 2-hour glucose in this study were those recommended by the World Health Organisation in 1999 for diagnosis using capillary glucose testing . A flow Six weeks after the last contact (either test or invitation), a questionnaire assessing anxiety, self-rated health and illness perceptions was sent to all participants, including non-attenders and those who were not originally invited. A freepost return envelope was included, and repeat duplicate questionnaires were sent to non-responders at 4 and 8 weeks following the initial mailing.Anxiety was measured using the short form of the Spielberger State Anxiety Inventory (STAI). This consists of six, 4-point Likert scales, and has been shown to correlate well (r > 0.9) with the results of the longer questionnaire from which it was derived , which i1. Identity 2. Timeline acute/chronic (e.g. a statement that agrees that diabetes is chronic would be: "diabetes lasts a long time")3. Timeline cyclical (e.g. "the symptoms of diabetes come and go in cycles")4. Consequences of diabetes 5. Personal control of diabetes 6. Treatment control 7. Emotional representation 8. Causes of diabetes The primary outcome, STAI, was scaled to have the same range (20 to 80) as the original longer form. STAI and IPQ scores were assessed for internal consistency using Cronbach's alpha. Unpaired t-tests were used to compare mean scores between groups for state anxiety, illness representations and self-reported health. Attendance rates were summarised with exact 95% confidence intervals. Pearson's chi-squared test was used to compare response rates between groups. Linear regression was used to assess whether STAI was associated with baseline characteristics or other psychological measures. Analyses were completed using SPSS (version 12.0) and StatXact (version 4.0). All tests were two-sided and assessed at the 5% level of statistical significance. 140 non-invited and 70 invited participants responding to the questionnaire (60% response rate), would have provided 80% power to detect a five-point difference in mean STAI (SD = 12) between non-invited and invited arms using a two-sided unpaired t-test at the 5% level of significance (nQuery version 4.0 software).This trial was approved by Cambridge LREC (00/071). Participants provided written consent.Baseline characteristics for invited and non-invited participants are shown in Table 95 people attended the initial screening test appointment  out of the 116 patients invited. Those who attended did not significantly differ from non-attenders in terms of age, sex, and BMI. However, attenders were more likely to have been prescribed antihypertensive or steroid medication (data not shown). The rate of attendance through the complete step-wise screening process was 77% (95% CI: 68% to 84%).Of the 95 people who attended for the random capillary glucose test, 43 (45%) had a glucose ≥ 5.5 mmol/l and were asked to re-attend for a fasting appointment. 37 (86%) of these people returned, of which 23 (62%) were declared free of diabetes, 13 (35%) required a 2-hour oral glucose tolerance test, and one person had a fasting glucose > 12 mmol/l and was labelled as having type 2 diabetes. 5 (39%) of those undergoing a 2h-OGTT test had a 2 h glucose of ≥ 11.1 mmol/l and were labelled as having diabetes. In total, 6 individuals (5% (95%CI: 2% to 11%) were given the diagnosis of diabetes as a result of the screening programme.A randomised evaluation of loss and gain frames in an invitation to screening for type 2 diabetes – effects on attendance, anxiety and self-rated health. In submission, Eval Health Prof, 2008), so they were treated as a single group.The overall response rate to the questionnaire was 68%. There was no difference in baseline characteristics between responders and non-responders, and no difference in the response rate between those invited and not invited to screening. The STAI six-item anxiety scale showed a Cronbach's alpha of 0.84, while the IPQ sub-scales showed a range of internal consistency (0.38 to 0.90). The alpha values were lowest for the timeline cyclical and treatment control sub-scales, with alphas of 0.40 and 0.38 respectively. There was no significant difference in attendance or anxiety between those invited with loss and gain frame invitations  differed significantly from those who did attend in two sub-scales of their illness perceptions of diabetes Table . Non-attNone of the baseline characteristics  were associated with anxiety (data not shown). Of the psychological outcomes, consequences, emotional representations and illness coherence sub-scales of the IPQ were correlated with raised anxiety. Overall, mean anxiety level was higher among patients in Practice A than Practice B, but this was largely due to the fact that all those labelled with type 2 diabetes were from Practice A .This study has shown that it is feasible and acceptable to screen for type 2 diabetes in two general practices in Cambridgeshire using a risk score to select a high-risk population and capillary glucose measurement to test for diabetes. This finding was replicated in the ADDITION (Cambridge) controlled trial . There wAttenders did not significantly differ from non-attenders, except that they were more likely to have been prescribed either antihypertensive or steroid medication. It is possible that attenders were more likely to have already been labelled with a chronic disease (such as hypertension) and had become used to returning regularly to the practice for monitoring, testing and treatment, and this in turn made them more motivated or less anxious about attending for screening for diabetes. Our results for age and sex contrast with findings from other screening studies where those who attend for health screening are more likely to be female  and oldeBeing invited to screening was associated with raised anxiety Evidence of causality is strengthened by the apparent dose-response relationship as participants who progressed further through the screening process exhibited higher levels of anxiety compared to those who did not progress so far or were not invited. There was also a significant difference in anxiety between test-positives and those who tested negative at the first stage of screening, suggesting an adverse effect of the label of diabetes. These results were gathered by questionnaire at least 6 weeks after the last appointment, implying that these are medium-term changes in anxiety rather than an immediate short-term effect of the invitation or test.A systematic review of the psychological impact of being presented with risk information  found thSince the results in this study would seem to indicate that being invited for screening is associated with anxiety, it was important to examine which variables were associated with greater anxiety. Only the consequences, emotional representations and illness coherence sub-scales were positively correlated with raised anxiety, which implies that those with raised anxiety had more negative feelings about diabetes and its consequences. The provision of information at the invitation and screening stages about the potential for controlling diabetes and its complications may therefore be useful in reducing anxiety.Non-attenders had significantly higher treatment control and lower negative emotional perceptions than attenders. This suggests that non-attenders are not sufficiently concerned about the significance of diabetes risk to attend, for example, they believe that diabetes can be controlled effectively by treatment and/or do not associate diabetes with negative emotions. There were no differences in the results of the IPQ sub-scales between those who were invited and those not invited to screening, implying that invitation to screening and its sequelae did not alter the participants' illness perceptions of diabetes. It is therefore likely that the observed differences between attenders and non-attenders were present at baseline and were not caused by screening, that is to say, the differences in the IPQ sub-scales were predictors rather than consequences of attendance. These findings have implications for the design of future diabetes prevention and invitation materials concerning screening for type 2 diabetes, as previously discussed.This was a fairly small trial with a limited set of measures yet had the advantage of being population-based and unlike all previous studies addressing this topic, included individual randomisation to screening or control group. Although we included only two GP practices, the study population was similar to the general population of England and Wales for age, sex and BMI as assessed by the Health Survey for England in 1994 , though We used capillary glucose measurements for diabetes screening and diagnosis which are approved by the WHO , but useThe questionnaire in this study was administered at only one time point and the study design relied on randomisation into invited and non-invited groups to quantify the effects of invitation to screening on the variables measured. This approach has the advantage of avoiding the possible accommodation effect of repeat administration of questionnaires, an issue that may have limited interpretation of an earlier diabetes screening study . Single This paper describes the first individually-randomised controlled trial to assess the psychological impact of screening for diabetes at six weeks. Evidence from this study suggests that one-off screening in general practice is feasible and acceptable. However, in contrast to previous studies, our data imply that being invited to screening may be associated with increased anxiety six weeks later, particularly among those given a new diagnosis. While increased anxiety may not be an entirely negative consequence, it merits further consideration in order to optimise the balance of benefits and harms of any future screening programme.Anglo-Danish-Dutch study of Intensive Treatment in People with Screen Detected Diabetes in Primary Care)STAI: (Short-form Spielberger State Anxiety Inventory); IPQ: (Illness Perception Questionnaire); ADDITION: (The authors declare that they have no competing interests.ADDITION-Cambridge study group. PP performed the data collection and statistical analysis and drafted the manuscript. RKS contributed to the writing of the manuscript. ATP contributed to the analysis and helped to draft the manuscript. SJG conceived the study and supervised the design, coordination and data analysis and helped to draft the manuscript. All authors read and approved the final manuscriptThis paper was written on behalf of the The pre-publication history for this paper can be accessed here:


Physical activity is associated with reduced risks of many chronic diseases. Data collected on physical activity in large epidemiological studies is often based on paper questionnaires. The validity of these questionnaires is debated, and more effective methods are needed.ref). This study evaluates repeated measures of physical activity level (PAL) and the feasibility of using a Java-based questionnaire downloaded onto cell phones for collection of such data. The data obtained were compared with reference estimates based on the doubly labeled water method and indirect calorimetry (PALcell). Results were compared with reference data obtained from the doubly labeled water method and indirect calorimetry (PALref). Results were also compared against physical activity levels assessed by two regular paper questionnaires completed by women at the end of the 14-day period (PALquest1 and PALquest2). PALcell, PALquest1, and PALquest2 were compared with PALref using the Bland and Altman procedure. Using a Java-based cell phone application, 22 women reported their physical activity based on two short questions answered daily over a 14-day period  with narrow limits of agreement (2SD = 0.30). Compared with PALref, the mean difference was also small for PALquest1 and PALquest2 ; however, the limits of agreement were wider . The test for trend was statistically significant for PALquest1  as well as for PALquest2  when compared with PALref. The mean difference between PAL A Java-based physical activity questionnaire administered daily using cell phones produced PAL estimates that agreed well with PAL reference values. Furthermore, the limits of agreement between PAL obtained using cell phones, and reference values were narrower than for corresponding estimates obtained using paper questionnaires. Java-based questionnaires downloaded onto cell phones may be a feasible and cost-effective method of data collection for large-scale prospective studies of physical activity. Physical activity is associated with reduced risks of several chronic diseases such as cardiovascular diseases, diabetes, and certain cancers ,2. To fu2H and 18O) in the body water pool from which energy expenditure is calculated[In free-living subjects the best method of assessing PAL and AEE is to combine measurements of total energy expenditure assessed using the doubly labeled water method with measurements of basal metabolic rate assessed using indirect calorimetry -5. In thalculated. The metalculated and is ualculated.Other available methods of assessing energy expenditure due to physical activity are activity records, heart rate monitors, and accelerometers ,5. The aThe primary aim of this study was to validate PAL values aggregated over 14 days against reference estimates based on the doubly labeled water method and indirect calorimetry. The aim was also to evaluate the feasibility of collecting repeated measures of PAL through a Java-based questionnaire downloaded onto cell phones. Two traditional paper questionnaires were also used to compare the validity of the PAL estimates based on questionnaires administered daily over cell phones with the validity of the PAL estimates based on paper questionnaires administered retrospectively.ref ) covering this 14-day period was calculated as total energy expenditure divided by basal metabolic rate.Twenty-two healthy women were recruited to a study on energy metabolism and physical activity by means of advertisements in the local press between September 2007 and February 2008. The women were employed in different areas of work, such as office work, nursing, and childcare. On the morning of the first day, basal metabolic rate was measured. Each woman was then given a dose of stable isotopes and asked to collect urine samples during the subsequent 14-day period to measure total energy expenditure.  A reference PAL  How physically active have you been during work/daytime today? and (2) How physically active have you been during leisure time/evening today? . AnswersThe download of the Java-based cell phone application was initialized through a WAP-push message. The Web client is built on Ajax in the Wicket application. The Web and application server was developed on Spring to build and run Java application and Hibernate to perform object relation mapping and query to the database. The database for collected information was MySQL, and Linux was used as the operating system. All data traffic was through https (hypertext transfer protocol secure). The procedure is described in Basal metabolic rate was measured in the following manner. On the morning of the first day of the study, each woman’s carbon dioxide production and oxygen consumption were measured after an overnight fast and 45 minutes of rest by indirect calorimetry during a 20-minute period using a ventilated hood system . The women came to the hospital by car to keep physical activity to a minimum. Carbon dioxide production and oxygen consumption were converted to basal metabolic rate through the equation by Weir .2H20 per kg body weight and 0.23 g H2                    180 per kg body weight) after having collected two to three background urine samples. Women collected another six urine samples on days 1, 4, 6, 8, 11, and 15 after the day of dosing. Isotopic enrichments of dose and urine samples were analyzed as described by Lof et al [2H and 0.03 for18O. Carbon dioxide production was calculated as described by Coward [To obtain measures of total energy expenditure, each woman was given an accurately weighed dose of the stable isotopes deuterium and oxygen-18 , that is AEEcell, AEEquest1, and AEEquest2, and were calculated as  minus basal metabolic rate. Reference estimates of AEE (AEEref) were calculated as total energy expenditure minus basal metabolic rate.For each woman, PALcell, PALquest1 , and PALquest2 versus PALref was evaluated using the Bland and Altman procedure [Values are given as mean ± SD. Significant differences between mean values were identified by repeated measures analysis of variance (ANOVA) with subsequent post hoc analyses using Tukey’s multiple comparison tests after having ascertained that the values were normally distributed. The agreement between PALrocedure . This pr2, and 2 had a BMI greater than 30 kg/m2. None of the women were smokers, and all but 4 had a university or college degree. At baseline, 5 women (23%) reported that they never exercised, 5 women (23%) reported that they exercised 1 to 2 times per week, while 12 women (54%) reported that they exercised 3 times a week or more.Characteristics of women who participated in the study are shown in cellwas based on 13 and 12 days of reporting, respectively. None of the women asked to terminate the reporting before the end of the 14-day period, and at the end of the study, all women expressed little or no burden of completing the reporting during the 14-day period.Of the 22 women, 14 (64%) had a cell phone that supported the Java-based application, and 8 (36%) borrowed a cell phone from the study center. During the 14-day period, all women but 2 answered all questions. Of the 2 women who did not answer all questions, one failed to report once, and the other, twice. For these women, PALcell was 1.82. This value was not statistically significantly different from PALref , which was 1.83. The Bland and Altman plot for PALcell in comparison with PALref is shown in cell and PALref was small (0.014), and the limits of agreement were 2SD = 0.29. The test for trend was not statistically significant. The regression equation was y = -0.58 x + 1.05;r = -0.38;P = .08.                    quest1was 1.84 while the average PALquest2was 1.90 (ref. The Bland and Altman plots for PALquest1and PALquest2in comparison with PALrefare shown in quest1and PALquest2compared with PALref(0.004 and 0.07 respectively). However, the limits of agreement were wider than for PALcell . The test for trend was statistically significant for PALquest1 as well as for PALquest2. Thus, both questionnaires (in particular questionnaire 2) overestimated higher PAL values while they underestimated lower PAL values.The average PALwas 1.90 . None ofcell, AEEquest1, and AEEquest2versus AEEref are shown in AEEPAL obtained using cell phones varied considerably from day to day during the 14-day study period . The intThis study describes a novel approach to collecting data on physical activity using a Java-based physical activity questionnaire administered repeatedly through cell phones. The results indicate that measuring physical activity through cell phones is a promising method of assessing PAL that could be used in large-scale epidemiological studies.The method generated high compliance and high acceptance among the participants. On average, PAL obtained using cell phones agreed well with reference estimates of PAL obtained using the doubly labeled water method and indirect calorimetry. Also the PAL values assessed by means of the two paper questionnaires were in good agreement with reference estimates. However, the limits of agreement for the difference between PAL obtained by cell phone and reference PAL were narrow (2SD = 0.29), while the corresponding limits for the two paper questionnaires were much wider (2SD = 0.51 for questionnaire 1 and 2SD = 0.90 for questionnaire 2). Furthermore, both questionnaires produced biased results , overestimating PAL of physically active women while underestimating PAL of less active women.Only two previous studies have compared PAL estimates obtained from paper questionnaires to reference estimates for healthy adults based on the doubly labeled water method and indirect calorimetry. In these studies, paper questionnaires underestimated PAL by 0.12 units (6%)  or overecell compared with AEEref. These limits of agreement are narrower than for most paper questionnaires that have been evaluated previously using reference estimates [When expressing the results as AEE, the cell phone questions overestimated reference estimates of AEE by only 2% on average. For comparison, in the recent review by Neilson et al , only eistimates .Our results showed a minor tendency  for PAL to be underestimated for physically active women using the cell phone questionnaire . This maMany paper questionnaires are designed to assess AEE. When comparing AEE between individuals, it should be expressed in kilojoules per kg body weight since smaller individuals have smaller AEE. This is a concern in epidemiological studies where body weight is often self-reported, creating a risk of bias. PAL is independent of body weight. In this study, PAL was chosen since our overall goal was to develop a procedure for use in epidemiological settings. Reference estimates of PAL were assessed as total energy expenditure divided by basal metabolic rate. Total energy expenditure consists of basal metabolic rate (50-70%), energy expenditure due to physical activity (20-40%), and diet-induced thermogenesis (5-10%). Thus, in the calculations of PAL, diet-induced thermogenesis was included. However, the cell phone and questionnaire estimates of PAL were calculated using published values for PAL  and MET                 Only 2 of the 22 women missed a reporting occasion , indicating a high compliance for the study method. The high compliance may be due to the SMS sent as a reminder. The technique also provides a possibility for the study center to monitor whether the participant answers the cell phone questions during the study period. These features are an advantage compared with ordinary paper-based physical activity records. None of the 22 women expressed any concerns or that having to report daily had interfered with daily life.The two questions were downloaded onto cell phones using a Java-based application; thus the participants had to have access to a cell phone with Java support. Out of the 22 women, 14 (64%) were able to download the Java-based questionnaire onto their own cell phone while 8 (36%) borrowed a cell phone from the study center. The same distribution of access to cell phones supporting a Java-based questionnaire was seen in another unpublished study. The advantage with Java in Web-based applications is that more advanced applications can be used, and the cost of data transfer is lower than the cost of sending an SMS. Furthermore, the Java program can also store data if the online connection is poor and then send the data once the signal is stronger. Once the program had been downloaded on the participant’s cell phones, the time and effort from the study center was minimal as all communications were automatically managed by the application server. As the cell phone questionnaire included only two questions, the questions could be asked through SMS or interactive voice response (IVR) in order to increase the accessibility, though these techniques allow less advanced interface and are more dependent on connection to the cell network. However, both these techniques are accessible to all cell phones and have been used to communicate with patients in intervention and therapy studies ,21,43.The strengths of this study are that this is the first study to have developed and evaluated a cell phone based method of assessing PAL, and that gold standard methods were used as reference methods. One possible limitation of the PAL cell phone estimates is that they were based on self-report. Still, the mean difference ± 2SD between PAL obtained using cell phones and reference estimates was smaller than earlier studies that reported evaluations of PAL or total energy expenditure that had been assessed using objective methods like accelerometry ,44. The In this study high within-subject variation in relation to between-subject variation was noted for the cell phone estimates. The high within-subject variability is a natural consequence  of subjects having different levels of physical activity from day to day, but it may also indicate that the cell phone questionnaire may not have captured all of the information about subjects’ physical activity. We aimed to develop a simple procedure to classify physical activity levels of subjects suitable for epidemiological studies that could predict health outcomes many years later. For this purpose, short-term (day-to-day or hour-to-hour) variation is likely not a problem. However, our procedure may need modification to be useful for intervention studies since such studies may require more detailed information, such as the type and duration of physical activities performed. We cannot exclude the possibility that the low between-subject variation was to some extent due to the fact that our study participants were a relatively small group of women. However, this study is a first step toward developing and evaluating a unique cell phone strategy to assess PAL. These first results in a group of healthy Swedish women are promising, and the next phase should include evaluations in other populations including men and different age groups.Using the procedure described by Bland and Altman, the cell phone estimates produced narrow limits of agreement when compared with reference estimates. We can only speculate why, but it may be due to the fact that the cell phone questions were answered every evening, reducing the reporting error due to loss of memory. Another reason may be that the procedure takes advantage of a technique (cell phones) that is associated with “instant answers” which also may reduce reporting error. Paper questionnaires may provide subjects with more time to reflect on their answers and also to consider their total activity level, which may make subjects more prone to adjust their answers in order not to be “too inactive,” for example.Noteworthy is that the more detailed paper questionnaire used in this study was not superior to the use of only one rating question in assessing total physical activity. This finding supports the result from a recent study in rheumatoid arthritis patients, where good agreement was found for PAL values obtained through two questions on physical activity compared with reference estimates . Future In this study, large day-to-day variations in PAL were shown, indicating the need for repeated measurements within subjects to decrease this variation. When repeating the questions, it was possible to obtain average PAL estimates typical for individuals. Furthermore, this study shows that cell phones are useful tools for repeated collection of PAL values. This procedure for gathering information about physical activity has a great potential for large-scale prospective epidemiological studies. The cell phone-based procedure could also be used for data collection of other variables in epidemiological settings where variation could be decreased by repeated measurement, such as assessment of energy intake or other health-related variables.In conclusion, a Java-based physical activity questionnaire administered using cell phones produced average PAL estimates that agreed well with PAL reference values. Furthermore, the limits of agreement between PAL obtained using cell phones and reference values were narrower than for the corresponding estimates obtained using paper questionnaires. Java-based questionnaires downloaded onto cell phones may be a feasible and cost-effective method of data collection for large-scale prospective studies of physical activity.