Osteoarthritis (OA) is a debilitating disease with poorly defined aetiology. Multiple signals are involved in directing the formation of cartilage during development and the vitamin A derivatives, the retinoids, figure prominently in embryonic cartilage formation. In the present study, we examined the expression of a retinoid-regulated gene in murine models of OA.Col1a1, Col10a1, Sox9 and Crabp2) in individual joints was determined using TaqMan quantitative PCR on RNA from microdissected articular knee cartilage.Mild and moderate forms of an OA-like degenerative disease were created in the mouse stifle joint by meniscotibial transection (MTX) and partial meniscectomy (PMX), respectively. Joint histopathology was scored using an Osteoarthritis Research Society International (OARSI) system and gene expression  in the MTX animals and about five-fold  in the PMX animals in comparison to the contralateral control joint.For MTX, there was a significant increase in the joint score at 10 weeks  in comparison to sham surgeries. PMX surgery was slightly more severe and produced significant changes in joint score at six , eight  and 10  weeks. The expression of Together, these findings suggest that the retinoid signalling pathway is activated early in the osteoarthritic process and is sustained during the course of the disease. FRZB or GDF5, have been confirmed to have causal roles [Osteoarthritis (OA) is a degenerative joint disease (DJD) that impacts multiple joint tissues , but is typically associated with a deterioration of articular cartilage. Although numerous factors have been suggested to be important contributors to the development and progression of this disease, very few, with the possible exception of al roles . OA is cIn the past few years, genetic links to OA have been established, and the first mutations in the collagen type II gene involved in the disease described ,3. More in vitro) has been shown to decrease the number and size of cartilage nodules formed. More interestingly, treatment of mature chondrocytes with retinoic acid causes them to assume an immature phenotype [Col2a1 expression [In vivo, intra-articular injection of retinoic acid leads to chondrocyte dedifferentiation and DJD [Mmp13 [One class of molecules that is important in development and homeostasis are the metabolites of vitamin A, the retinoids ,9. In thhenotype -17. Thispression  and an ipression  that leapression ,20. In v and DJD . More reD [Mmp13 .in vivo [in vitro and in vivo, where retinoic acid was observed to promote premature closure of the growth plate [The changes in aggrecan metabolism seen in OA are similar to those produced by treatment of cartilage with retinoic acid. Bovine cartilage explant cultures treated with retinoic acid exhibit increased degradation of proteoglycans . In rat in vivo ,26. A swth plate ,28.Crabp2 in the articular cartilage. In a recent study, Crabp2 was found to be elevated in DJD in a rat model of OA [Crapb2 to be significantly increased in early OA, indicating that the retinoic acid pathway may play a role in OA pathophysiology.To examine the status of the retinoic acid signalling pathway in OA, we have used two murine DJD models and quantified the expression of a retinoic acid-regulated gene, including el of OA ,30. We fSurgeries were performed on 10-week-old male C57BL/6NCr1 mice . After a one-week period of acclimation after arrival, mice were sorted into random groups using a lottery system. For each experimental time point a minimum of five mice were evaluated. Mice were induced with 4% isoflurane and 1 L/minute oxygen. Once mice were anaesthetised they were transferred to a mask and maintained on 2% isoflurane and 0.8 L/minute oxygen. The surgical area was shaved and a three-part preparation was applied containing hibitane soap , isopropyl alcohol and betadine solution .During surgery, the body temperature of the animals was maintained by placing them on a warm circulating water pad. Standard sterile techniques were used throughout the surgery. A small sterile drape was fitted over each mouse to expose only the area of interest, the medial side of the left knee. The surgery began with a small skin incision starting from the distal femur and extending to the proximal tibia on the medial side of the knee. The subcutaneous tissue was dissected throughout the length of the skin incision. The deep fascia that connects the parapatellar fascia to the biceps femoris was separated exposing the medial side of the joint including the medial collateral ligament (MCL) and the joint capsule. In the partial meniscectomy (PMX) surgical paradigm both the MCL and the joint capsule were incised to reveal the medial meniscus. The medial meniscus was gently held and retracted in a way that allowed for identification and transection of the meniscotibial ligament Figure . The medIn the meniscotibial transection (MTX) surgical paradigm there was no medial dissection of the joint capsule or transection of the MCL. In this regard, the MTX model is similar to the destabilisation of the medial meniscus (DMM) model recently described by Glasson and colleagues . Once thAt the time of processing, mice were euthanased by carbon dioxide inhalation and both stifle joints were harvested and fixed in 4% paraformaldehyde. After a minimum of 48 hours fixation the joints were decalcified for 96 hours in 26% formic acid . Once decalcified the knees were paraffin embedded and serially sectioned in a sagital plane starting on the medial edge of the joint. Slides were stained with safranin-O and the medial side of tibial plateaus were subsequently scored according to Pritzker and colleagues from a minimum of three slides . The secCol1a1, Sox9) or off the shelf TaqMan Gene Expression Assays . Relative expression was determined using the relative quantitation method with a standard curve and gene expression was normalised to 18S abundance.At predetermined endpoints the mice were euthanased and the knees were carefully dissected to expose the cartilage surface of both tibial plateau and the femoral chondyles. Under an operating microscope, using a pair of micro-rongeurs, the articular cartilage was gently scraped away from the underlying subchondral bone and transferred into Qiazol . The cartilage was subsequently homogenised in a microcentrifuge tube using a disposable plastic pestle and stored at -80°C. RNA was isolated from the samples according to the manufacturer's guidelines and for real-time quantitative PCR (RT-qPCR) the RNA was reverse-transcribed using the High Capacity cDNA Reverse Transcription Kit . Gene expression was quantitated using qPCR on an ABI 7500 Fast system with either custom TaqMan MGB probe or primer sets  with Bonferroni's or Tukey's post-hoc tests as indicated. With the exception of the analysis of Crabp2 expression as determined from microarray analysis was elevated in the OA joint four weeks post-surgery [Crabp2 is regulated by retinoic acid and is often induced upon activation of this pathway [Crabp2 induction during OA. Further, we desired to develop and validate murine cartilage-sparing models of OA to facilitate the use of genetically modified lines and enable molecular analysis of gene expression in the articular cartilage. For these purposes, surgeries were tailored to produce less joint instability with the intent of producing a more slowly progressing disease than the standard anterior cruciate ligament (ACL) models as has been recently described for the DMM and other models [In an earlier study of a rat surgically induced model of DJD, Appleton and colleagues reported that -surgery . Crabp2  pathway . We werer models ,34.MTX and PMX surgeries were performed and joint histology was scored at various times up to 10 weeks post surgery. In the MTX group there was a significant increase in the joint score by 10 weeks , although increases at two and six weeks were not significant Figure . In the Col1a1 and Col10a1 are both elevated in OA [Sox9, a transcription factor important in chondrocyte differentiation and matrix production [Col1a1 was found to be elevated more than three-fold at all times in the MTX/PMX joint in comparison to the sham control . Previous studies have shown that ed in OA -38. We aoduction . With thl Figure . In conty Figure , with a l Figure . InteresCrabp2 expression is regulated by retinoic acid and its increase in expression is consistent with activation of this pathway. CRABP2 has been shown to function both to suppress retinoic acid receptor activity by sequestering ligands and also as a vehicle to deliver ligands to the retinoic acid receptors, thereby enhancing ligand-mediated retinoic acid receptor transcriptional activity [Crabp2 enhances retinoic acid receptor transcriptional activity [Crabp2 null animals present with minor limb defects including an extra post-axial digit that (based on more recent reports) would suggest decreased retinoid signalling in the absence of CRABP2 [Crabp2 is generally linked to activation of the retinoid pathway either directly, because expression of Crabp2 is regulated by retinoic acid, or indirectly, because increased CRABP2 would increase retinoic acid receptor transcriptional activity. Inappropriate activation of the retinoic acid signalling pathway is expected to enhance cartilage degradation and/or chondrocyte dedifferentiation, both of which are observed in the osteoarthritic process.activity -45. Moreactivity ,42,44. Ff CRABP2 ,46,47. IAdamts5 and another major aggrecanase Adamts4, addition of retinoic acid was still found to promote release of aggrecan through cleavage in the CS-2 domain [Adamts5 and Mmp13 expression [As mentioned above, retinoic acid has been commonly used to promote degeneration in cartilage explants, and this has been associated with increased activity of various aggrecanases. Knockout animals of the gene encoding the aggrecanase ADAMTS5 are protected to a great extent from OA in joint instability models, indicating that this enzyme may play a major role in cartilage catabolism, at least in mouse models of OA -50. Inte2 domain . Retinoipression ,22, and pression  suggest Crabp2, other components of the retinoid signalling pathway were found to be significantly elevated in the joints of the aforementioned rat OA joint instability model, including genes encoding proteins involved in retinoic acid synthesis (Aldh1a3) and retinol transport and delivery (Lrat and retinol dehydrogenase) and a putative retinoic acid target gene (Stra3) [Crabp2 expression in mouse models of DJD suggest that retinoic acid may play a fundamental and perhaps unappreciated role in the osteoarthritic process.In addition to  (Stra3) . TogetheCrabp2 is robustly expressed in early OA, it may represent a marker for detection of early OA. Further, polymorphisms in FRZB and GDF5 have been linked to OA, and similar to the retinoid signalling pathway, they all play a role in endochondral ossification and modulation of their activity may impact maintenance of the articular chondrocyte [As ndrocyte . In thisndrocyte .The joint instability models presented herein in contrast to more aggressive models involving for instance ACL transection, present with OA-like pathology but still appreciable articular cartilage 10 weeks after surgery, thereby enabling the use of molecular approaches to quantify gene expression changes in early OA .Crabp2 is significantly elevated in early DJD, and may be a useful marker to follow early changes in cartilage in response to joint instability or in OA. Manipulation of the retinoic acid signalling pathway may prove useful in modifying the clinical course of OA.The expression of the retinoic acid-regulated gene ACAN: aggrecan; ACL: anterior cruciate ligament; ANOVA: analysis of variance; DMM: destabilisation of the medial meniscus; DJD: degenerative joint disease; ECM: extracellular matrix; MCL: medial collateral ligament; MTX: meniscotibial transection; OA: osteoarthritis; PMX: partial meniscoectomy; RT-qPCR: reverse transcription quantitative polymerase chain reaction.The authors declare that they have no competing interests.IW performed experiments, contributed to experimental design, writing of the manuscript and data interpretation. MC contributed to experimental design and carried out the experiments. FM and TMU were involved in experimental design, data interpretation and writing of the manuscript.


Organisms that are sensitive to nitrofurantoin express a nitroreductase. Since bacterial resistance to this compound results primarily from mutations in the gene encoding nitroreductase, the resulting loss of function of nitroreductase results in a selectable phenotype; resistance to nitrofurantoin. We exploited this direct selection for mutation to study the frequency at which spontaneous mutations arise .N. gonorrhoeae FA1090 genome by using a bioinformatic search with the deduced amino acid sequence derived from the Escherichia coli nitroreductase gene, nfsB. Cell extracts from N. gonorrhoeae were shown to possess nitroreductase activity, and activity was shown to be the result of NfsB. Spontaneous nitrofurantoin-resistant mutants arose at a frequency of ~3 × 10-6 - 8 × 10-8 among the various strains tested. The nfsB sequence was amplified from various nitrofurantoin-resistant mutants, and the nature of the mutations determined. Transition, transversion, insertion and deletion mutations were all readily detectable with this reporter gene.A nitroreductase- encoding gene was identified in the nfsB is a useful reporter gene for measuring spontaneous mutation frequencies. Furthermore, we found that mutations were more likely to arise in homopolymeric runs rather than as base substitutions.We found that  Neisseria gonorrhoeae (GC) is an obligate human pathogen. In order to manifest the diversity of diseases that it is able to cause, GC must produce a variety of cell surface antigens such that the appropriate antigen(s) is (are) expressed in the appropriate environment at the appropriate time. Since each of the anatomical sites that GC can infect has unique physiological properties, its success in establishing itself in a new niche requires that it rapidly adapt to its new environment. To do this, it has evolved a variety of genetic mechanisms that result in high frequency antigenic variation of its surface components. These include: intramolecular recombination for pili antigenic variation  . N. gonorrhoeae strains used in this study are described in Table N. gonorrhoeae were grown on GCK agar  [2PO4 (4 g), K2HPO4 (1 g), NaCl (5 g)/L of ultra-pure water (pH 7.5). LB agar and broth were prepared from powder obtained from US Biologicals. Plasmids used in this study are described in Table lements) . GCP broChromosomal DNA for PCR reactions was prepared from bacterial cultures by resuspending a small amount of cells in 5:l 1 M NaOH. The solution was neutralized by adding 5:l of 0.5 M Tris-HCl (pH 7.5). The suspension was further diluted in 90:l of purified water, and 1:l of this solution was used as a template for PCR. Plasmid DNA isolations were carried out according to the alkaline lysis procedure .Polymerase chain reactions (PCR) were performed using various enzyme systems, based either on Taq or Pfu polymerases using chromosomal or plasmid DNA as a template. The primers used for various PCR reactions are described in Table nfsB sequences were obtained using Primers S1 and S2.DNA sequencing was performed by Macrogen, Inc.  or the DNA sequencing facility at the Center for Biosystems research at the University of Maryland. All All procedures were performed using methods described in Sambrook et al. . When biE. coli cells (strain DH5α-mcr) were prepared using the procedure of Inuoe [Transformation-competent of Inuoe , and stoN. gonorrhoeae, piliated bacteria were resuspended to light turbidity in 1 ml GCK+ 10 mM MgCl2 + Kellogg's supplement + 0.42% NaHCO3. DNA was added to the culture, and the bacteria were incubated at 37°C with shaking for 2-6 hours. Bacteria were plated onto GCK agar plates containing the appropriate antibiotic, and the plates incubated for 36-48 hrs. When transformations were performed under nonselective conditions, a spot transformation procedure was used [2 + 0.42% NaHCO3 + Kellogg's supplement. The cell suspension was diluted 1:10, and additional two-fold serial dilutions were then carried out 9 times. An aliquot (5:l) of each suspension was spotted onto a GCK agar plate. To each spot, 5:l of DNA were added. After incubation overnight at 37°C with 4% CO2, individual colonies were isolated and streaked for isolation on GCK agar plates. The next day, individual colonies were inoculated onto GCK and spectinomycin-containing GCK agar plates. This procedure was repeated until spectinomycin-sensitive colonies were obtained. The correct replacement of the desired DNA fragment by the transformation process was verified by PCR amplification of the desired region, and restriction digestion analysis of the PCR amplicon, or direct DNA sequencing of the PCR amplicon.For transformation of was used . For tranfsB gene from strain FA1090 was amplified by PCR using primers NP1 and NP2. The amplicon was purified, digested with BamHI and cloned into the BamHI site in pK18, resulting in plasmid pNFSB. To alter the poly adenine sequence at the 5' end of the gene from AAAAA to AAGAA, PCR primers NfsB-BsmI-F and NfsB-BsmI-R were designed. The resulting amplicon was digested with BsmI, ligated, and introduced into E. coli by transformation, giving pEC1. Plasmid pEC1 was amplified via PCR using the primers dwnstrm-F and dwnstrm-R, allowing for the insertion of a BsrGI site. A spectinomycin resistance cassette was amplified from pMP45Σ using primer Omega-ABC, and ligated into the BsrG1 site, resulting in pEC3. This plasmid was used to transform strain FA1090 to spectinomycin resistance, resulting in strain NfsB-BsmI-Ω. The spectinomycin resistance cassette was removed using the spot transformation procedure [nfsB gene(FA1090-NfsB(mod)). The correct construction was verified by DNA sequence analysis of a PCR amplicon. The DNA sequences for nfsB from the various strains have been submitted to GenBank with the following accession numbers: F62, GU112780; MS11, GU112781; FA19, GU112782; and PID2, GU112783. Point mutations in nfsB that resulted in nitrofurantoin resistance are identified in GenBank as accession numbers: GU112770; GU112771; GU112772; GU112773; GU112774; GU112775; GU112776; GU112777; GU112778; and GU112779.The rocedure  with pEC8 gonococci were spotted onto media containing various amounts of nitrofurantoin . The inoculated plates were incubated overnight, and the MIC was defined as the amount of nitrofurantoin needed in the plate to completely inhibit the growth of the organisms in 24 hours.The minimum inhibitory concentration (MIC) of nitrofurantoin for several gonococcal strains was determined by a plate dilution method. Approximately 103 and Kellogg's supplement to exponential growth phase, and aliquots (~1 × 108 cfu) plated onto GCK plates containing 3:g/ml nitrofurantoin. Viable counts were determined by plating cells onto GCK agar plates. Mutation frequencies were defined as the number of colonies obtained on nitrofurantoin-containing media divided by the number of colonies obtained on GCK media.Cultures of GC were grown in GCP broth + 0.42% NaHCO3 and Kellogg's supplement at 37°C with shaking to a turbidity of 100 klett units. Cells were collected by centrifugation , washed with PBS, and resuspended in 5 ml 100 mM Tris-HCl, pH 7.5. Cells were lysed by sonication using a Branson sonicator with the microprobe, set on full power, using 5 10 sec pulses (Suspensions were incubated on ice for 1 min between pulses). The sonicates were clarified by centrifugation  and the supernatants collected. Protein concentrations of each sample were measured with the BioRad protein assay  using BSA as a standard, and samples were normalized to the same protein concentration in 100 mM Tris-HCl, pH 7.5. Samples containing 800:l lysate and 0.1 mM nitrofurazone were placed in a quartz cuvette, and the reaction initiated by adding 100:l NADPH (2 mM stock). A control reaction was performed using water instead of nitrofurazone. Reactions were incubated at room temperature and absorbance was measured every 30 sec at 400 nm.Nitroreductase activity was measured by a modification of the method of Whiteway et al. . CultureE. coli nfsB in the gonococcus was identified by submitting the entire E. coli nfsB protein sequence to http://blast.ncbi.nlm.nih.gov/Blast.cgi using the tblastn program. The database option was set to "nucleotide collection," and limited to Neisseria gonorrhoeae. The database option was set to "bacteria," and the number of best-scoring sequences to show was set to 250. The top scoring hits from unique genera were aligned using ClustalW http://www.ebi.ac.uk/Tools/clustalw/.A homolog of N. gonorrhoeae possesses a nitroreductase, it should be sensitive to antimicrobial agents that are activated by nitroreductases and it should be possible to isolate mutants that become resistant to these activated antimicrobials due to the organism's loss of nitroreductase activity. We determined the MIC for nitrofurantoin, an antimicrobial agent that is activated by nitroreductases, for several different gonococcal strains. Plates were analyzed after 24 hrs incubation, and the concentration of nitrofurantoin that completely inhibited the ability of GC to form visible colonies was determined. The data indicated that the MIC for nitrofurantoin was approximately 3:g/ml for all strains tested (data not shown). When plates were incubated for an additional 24 hrs, a small number of colonies arose, and these were presumptive nitrofurantoin-resistant mutants. By comparing the number of colonies found on an agar plate after 48 hours incubation in the presence of nitrofurantoin to the number of colonies obtained when a similar aliquot was inoculated onto media lacking the antimicrobial agent, we were able to calculate the spontaneous mutation frequency to resistance to this agent. The data  that would result in an amino acid substitution in NfsB at residue 192 . Since these proteins were essentially identical, it suggests that the variability in spontaneous mutation frequencies observed in these strains could reflect different DNA repair capacities for the various strains.The A spectrophotometric assay was performed to measure nitroreductase activity in GC. Lysates from wild type and nitrofurantoin resistant mutants were assayed for nitroreductase activity using a spectrophotometric assay that detects the loss of the substrate, nitrofurazone, using a method adapted from Whiteway et al. . The datnfsB, the DNA sequence of this region was determined from spontaneous nitrofurantoin resistant mutants by amplifying the nfsB region from this strain. The data indicate that FA1090(M1) possessed a small insertion of 7 nucleotides about midway through the coding sequence, producing a frame shift mutation in nfsB. This genetic data supported the hypothesis that the nitrofurantoin resistant phenotype is due to the loss of nitroreductase activity. Conclusive evidence that this gene was responsible for nitrofurantoin resistance was obtained by deleting the coding sequence for this gene from FA1090 and then demonstrating that FA1090NfsB-BsmIS lacked nitroreductase activity (data not shown).In order to verify that the nitrofurantoin resistance observed in the mutant was due to alterations in nfsB gene in these strains. Most of these mutants (90%) possessed the insertion of an adenine in a run of 5 adenines near the beginning of the gene, suggesting a bias for base insertion during DNA replication at this position. This gene contains three other polynucleotide runs of five nucleotides distal to the start codon; 2 poly adenines and one polythymine. Interestingly, even though we were able to isolate base insertions at each of these three clusters, none of the clusters showed the elevated propensity for generating spontaneous mutations.We isolated numerous independent spontaneous nitrofurantoin resistant mutants and determined the DNA sequence of the E. coli and DNA used to transform N. gonorrhoeae. Spectinomycin resistant transformants were identified, and DNA sequence analysis of a PCR amplicon derived from the constructed strain indicated that the derivative of FA1090, FA1090-NfsB(mod) contained the desired sequence modification. Nitroreductase assays of this strain indicated that it possessed wild-type NfsB activity (data not shown).To eliminate the bias introduced by the 5 bp polyadenine run at the 5' end of the gene, this DNA sequence was altered to remove the poly-A tract, while preserving the corresponding amino acid sequence. The plasmid, pEC3 was constructed as described in figure nfsB using Primers S1 and S2. The experimental design employed should allow for the identification of six different types of mutations, four that would be manifested within the coding sequence of nfsB  and mutations outside of the coding sequence, presumably mutations that effected nitrofurantoin uptake, or in the regulation of nfsB expression. The data presented in Table We isolated spontaneous nitrofurantoin resistant mutants of strain FA1090-NfsB(Mod), by plating this strain on GCK agar containing 3:g/ml nitrofurantoin. We determined the genetic basis of 107 individual independently isolated mutants that arose from this plating by amplifying the desired region using Primers NP1 and NP2, and determining the DNA sequence of nfsB identified 23 possible bases where a single base change would result in the production of a stop codon. We identified 33 mutations that resulted from this type of base change. The distribution of the mutations obtained suggested that no hot spot for mutation existed in any of these sequences . The inability to isolate second-step nitrofurantoin resistant mutants suggested that the gonococcus only contained a single nitroreductase. We obtained biochemical data to support this conclusion, where mutants that were resistant to nitrofurantoin lost the ability to reduce nitrofurantoin. Since cell lysates that did not contain the co-factor NADPH had no nitroreductase activity, it indicated an absolute requirement for this co-factor. However, we did not determine if other molecules such as NADH could substitute for the NADPH requirement, or if the organism might have other nitroreductases that utilized a cofactor other than NADPH.We determined that nfsB. While we were able to identify mutations that would result in amino acid substitutions in the region involved in FMN binding [We determined the nature of spontaneous mutation by analyzing where mutations occurred in  binding , the majThe results of the spontaneous mutation frequency plating experiments and the subsequent genetic analysis showed that nitrofurantoin resistance is a potential target for analyzing mutation in the gonococcus. The fact that almost all mutations originally examined resulted in an extension of a polyadenine run of 5 adenines was surprising, as it is thought that this sequence is too short to participate in strand slippage. Furthermore, the absence of slippage at two other polyadenine runs of 5 in other locations indicates that sequence context is important in strand slippage.nfsB as a reporter system allowed us to assess the nature of spontaneous mutation in an unbiased fashion. If one removes the high frequency of errors that occurred in the polynucleotide run of adenines, the propensity of errors directed towards transitions and transversions occurred at a similar frequency to insertion or deletion mutations. However, the high rate of insertions and deletions is in contrast to what was observed by Schaaper and Dunn [lacI gene of Escherichia coli saw that single base insertions and deletions only made up 4.2% of their observed mutations. While we observed that single base insertions and deletions accounted for ~40% of our observed mutations in a background where a run of five adenines was removed, if the bias observed at this sequence was included, insertions would have made up about 75% of all observed mutations. The implication of this finding would suggest that homopolymeric runs should have a tendency to increase, and that they should dominate the types of mutations seen in the gonococcus. This is precisely what is observed. The mechanism by which gonococcal DNA polymerase allows this to occur, and the inability of the gonococcus to efficiently correct insertions indicates that gonococcal DNA repair is somewhat different from that seen in E. coli.The use of and Dunn , who in rpoB in the gonococcus or meningococcus. These studies have analyzed numerous strains for the rate of spontaneous resistance to rifampicin, and find that in general, this rate is between ~1 × 10-8 - 1 × 10-9 [-6 - 8 × 10-8). We believe that the higher mutation frequencies that we observed relate to the nature of the selection procedure employed. Mutation screens designed to detect rpoB mutants are constrained in that they must result in the production of a functional protein. Our screening procedure allowed us to detect any mutation that results in the loss of function of the target, and hence is able to identify insertions and deletions, as well as point mutations. We believe that the elevated mutation frequency that we observed for nfsB, relative to that observed by others for rpoB was due to the presence of the polyadenine sequence in nfsB and our ability to detect frame shift mutations.Most of our understanding of DNA repair in the Neisseria has come from studies focused on understanding the contribution of various DNA repair proteins in preventing mutations in 1 × 10-9 -36. Our E. coli. Interestingly, none of the mutations that we identified were contained in any of the key residues that they demonstrated to be interacting with nitrofurantoin. However, a significant number of the amino acid substitutions that we identified would be expected to have dramatic structural implications.Race and coworkers  have solnfsB is a useful reporter for measuring spontaneous mutation frequencies. Its ability to detect elevated mutation frequencies in very short polynucleotide runs indicates that any gene that contains a short polynucleotide run has the potential to phase vary.In summary, we found that SD and DS conceived of the scientific concept that formed the basis of this manuscript. EC performed the experiments and participated in the data analysis. DS wrote the manuscript.


Sfpi1 gene consists mostly of C:G to T:A transitions at a CpG site and is likely to be of spontaneous origin. To work out a mechanism underlying the association between radiation exposure and the AML induction, we have hypothesised that replicative stress after irradiation accelerates the ageing of haematopoietic stem cells (HSCs), and the ageing-related decline in DNA repair could affect the spontaneous mutation rates.The essential aetiology of radiation-induced acute myeloid leukaemia (AML) in mice is the downregulation of the transcription factor PU.1. The causative mutation of the PU.1-endocing Mathematical model analysis was conducted to examine whether and to what extent the cell kinetics of HSCs can be modified after irradiation. The haematopoietic differentiation process is expressed as a mathematical model and the cell-kinetics parameters were estimated by fitting the simulation result to the assay data.The analysis revealed that HSCs cycle vigourously for more than a few months after irradiation. The estimated number of cell divisions per surviving HSC in 3 Gy-exposed mice reached as high as ten times that of the unexposed.The mitotic load after 3 Gy irradiation seems to be heavy enough to accelerate the ageing of HSCs and the hypothesis reasonably explains the leukaemogenic process. There is substantial evidence that ionising radiation can cause various types of malignancies. Among them, leukaemia is noteworthy because of the high relative risk and the short latency . The finSfpi1 gene on the retained homologue  in some strains of mice serves as a well-established animal model to explore this issue. The incidence increases with doses up to 3 Gy and the symptoms of the affected mice are similar to those found in human AML  because they accumulate DNA damage after repeated rounds of replications and those mutations are associated with stem cell ageing . With evIn this study, we have used a mathematical model analysis to examine whether and to what extent radiation cause replicative stress in HSCs. The differentiation process of haematopoietic cells was expressed in a mathematical model. The cell-kinetics parameters were estimated by fitting the simulation result to the experimental data of CAFC (cobblestone area forming cell) and CFU-G/M (colony forming unit-granulocyte/macrophage) assays. The analysis showed the upregulated cell kinetics for HSCs, which seemed to be sufficient to accelerate ageing of them. Possible leukaemogenic processes have been discussed based on the results, and we have found the most plausible explanation is that the replicative stress-related stem cell ageing plays a role elevating spontaneous mutation rates of HSCs and their progeny.0 phase, which can be quantified by a repopulation assay such as day-28 CAFC. Multipotent progenitor (MPP) in in vivo multilineage potential and are thought to share similar cell kinetics. Day-14 CAFC can be an indicator of these cell types as well as cycling HSCs . Temporal variations of CMPs and GMPs can be described in the same manner.Multipotent progenitor is a progeny of HSC and produces two types of progenitors, common lymphoid progenitor and CMP. The rate of change of MPP frequency is then written as r were estimated from literature data of BrdU incorporation in vivo . The irradiated mice as well as age-matched controls were killed at 30, 90, 150, and 300 days post irradiation and bone marrow cells were collected from the femora. Animal experiments were performed with the approval of the Research Ethics Committee of Oita University of Nursing and Health Sciences in conformity with applicable laws and regulations.Male C3H/HeN mice were purchased from Japan Clea and maintained under conventional conditions. At 8–10 weeks of age, they were exposed to 3 Gy of 200 kV X-rays filtered with 0.5 mm Cu and 0.5 mm Al  containing 12.5% foetal calf serum , 12.5% horse serum (ThermoTrace), 10−5 M hydrocortisone (Sigma-Aldrich), 100 U ml−1 penicillin, and 100 μg ml−1 streptomycin (Sigma-Aldrich). The cells were overlaid on MC3T3-G2/PA6 stromal cells (provided by the RIKEN CELL BANK) grown in a collagen-coated 96-well flat-bottom microplate . Six dilution steps of the overlaid cells were made with 3-fold intervals from 1000 to 243 000 cells in 150 μl culture volume, and 16 wells were used for each dilution. Cultures were maintained at 33°C, 5% CO2, and fed weekly by a half medium change. On days 14 and 28 of culture, the wells were inspected for cobblestone areas with an inverted microscope TMF . A well was scored as positive if it contained more than one cobblestone area that comprised at least five phase-dark cells. The limdil function of the statmod package for R was used to find the maximum likelihood solutions of the frequencies of the CAFCs.For the CAFC assay, bone marrow cells of individual mice were suspended in 4 bone marrow cells in 1.1 ml were prepared for each mouse and were incubated at 37°C, 5% CO2. On day 10 of incubation, the number of colonies containing more than 50 cells was counted on an inverted microscope.Part of the bone marrow cells from each mouse was used for the CFU-G/M assay. The cells were diluted with IMDM (Sigma-Aldrich) containing 2% foetal calf serum and were mixed with methylcellulose medium MethoCult GF M3534 . Two dishes of the semi-solid culture containing 1.5 × 10In the simulation of the recovery process, we first tested for the priority to generate downstream cells rather than self-renewal of HSCs, that is, increasing the branching probabilities for HSCs differentiating into MPPs. This was not effective even in the short term, however, and just exhausted HSCs. Upregulating the transfer/mitotic rates of HSCs and MPPs resulted in quick recovery of CFU-G/M, but the frequency of HSCs was suppressed to a low level at all times if the self-renewal probability of HSC was unchanged. These results show that it is necessary to enhance the self-renewal of HSCs before the committed progenitors are restored. It is also noteworthy that the frequencies of committed cells can vary drastically in response to a subtle change in HSC kinetics, indicating that continuous, fine adjustments are required for the parameters.Phase I:HSCs and MPPs respond to haematopoietic demands until CFUs-G/M are restored. The probability of HSC self-renewal is increased. Transfer/mitotic rates of HSC and MPP are high.Phase II:Cell kinetics is downregulated to prevent the overgrowth of CFUs-G/M. The probability of the HSC self-renewal is suppressed. Transfer/mitotic rates are decreased, but still higher than the normal state.Phase III:HSCs increase gradually whereas the CFU-G/M frequency remains constant. The probability of the HSC self-renewal is moderately increased, but CMP self-renewal is suppressed. Mitotic rates of HSCs and MPPs are gradually increased with time.The number of cell divisions in each cell compartment was calculated in the course of the simulated recovery process. We used CAFC assay to get a measure of multipotent haematopoietic cells. The CAFC assay is validated in many applications (e.g., In the experiment, day-14 CAFCs and day-28 CAFCs revealed long-term suppression after irradiation, although CFUs-G/M did not show noticeable changes. This is because committed cells are preferentially restored to minimise their influence on circulating blood cells, which are essential for life. The mathematical simulation has showed a quick recovery of CFUs-G/M by vigorously cycling HSCs and MPPs just after irradiation, but the emergent situation settled down as the frequencies of committed cells reached a normal level. Once CFUs-G/M were restored, their frequency remained almost constant and the CAFCs started recovering slowly. It is a contradictory situation where HSCs and MPPs proliferate without affecting the frequencies of the progeny cells. The simulation has found that this can only be achieved by upregulating the kinetics of HSCs/MPPs gradually while suppressing the self-renewal of committed progenitors. The frequency of MPP is suboptimal in that condition even if the cells cycle more rapidly than in the normal state. This indication is consistent with the reported late effects of x-irradiation on CFU-S . On the −10 mutations per base pair replicated per generation (Sfpi1 mutations of AMLs are confined to specific positions of exon 5 (Sfpi1 mutations occur at the rate of up to 10−9 per cell division in normal cells.Given the number of cell divisions, it is possible to estimate the spontaneous mutation frequency. Although spontaneous mutations arise from various pathways during normal metabolism, they are fixed through the DNA replication process. If one assumes a constant mutation rate per cell division, mutational events in a cell population follow poisson statistics. Mutation rates in eukaryotic cells are generally ⩽10neration . As the Sfpi1 (Sfpi1 has to coincide with the deletion of chromosome 2 to form the malignant cell.Sfpi1 is a consequence of radiation exposure. Mutation frequency of a single gene after 3 Gy exposure in vivo is estimated to be 10−5–10−4 based on an extrapolation from high-dose data in lacZ transgenic mice (Sfpi1 mutations required for the AML development are just an inactivation of the gene. Evidence of a total gene deletion or removal of exon 5 developing into the leukaemia (There are some possible explanations that reconcile this inconsistency. One is to reconsider the fundamental hypothesis, presuming the mutation of nic mice . In eithSfpi1 can bring phenotype alteration enhancing neutrophil progenitor development, but the gene dosage effect is masked in vivo by changes in cytokine expression (Another possibility is that cells with hemizygous deletion of chromosome 2 acquire a growth advantage and just one or two spontaneous mutagenic events are enough with a large population base of those cells. Haploinsufficiency for The most plausible explanation will be to allow for the increase in the mutation rate associated with the ageing of stem cells. Our simulation predicts that most of the surviving HSCs are recruited into the cell cycle after 3 Gy exposure, and that the percentage of cycling HSCs remains at ∼60%. This contrasts with the cell kinetics of HSCs in the steady state, in which two-thirds of the population are quiescent. Owing to the increased cycling fraction, together with the enhanced mitotic rate, the surviving HSCs in the exposed mouse undergo the extensive rounds of replication. This will make the cells senescent and the mutation accumulation would be accelerated.Atr gene has showed that even homeostatic proliferation can promote stem-cell dysfunction and ageing (et al has found that the gene expression profile changes as HSCs age, and among the downregulated genes are those involved in the preservation of genomic integrity, such as chromatin remodelling and DNA repair (There is considerable evidence that continuous replicative stress accelerates the ageing of stem cells, and that aged stem cells are characterised by compromised genome maintenance. Serial transplantation assays in mice have shown that forced stem cell expansion results in deterioration of HSCs (Sfpi1 gene. When a substantial fraction of haematopoietic cells are killed by radiation, surviving HSCs vigourously cycle to reconstitute haematopoiesis. Enhanced replicative stress promotes their ageing and mutation rates gradually increase. Random mutations thus accumulate in HSCs during repeated replications unless the mutations bring on a loss of stem cell function. If a leukaemia-specific mutation occurs in an Sfpi1-hemizygous cell in due course, the cell becomes a leukaemic stem cell. As the leukaemic stem cells can arise not only from HSCs, but also from CMPs and GMPs (We therefore propose a model for murine myeloid leukaemogenesis as shown in and GMPs , those oAccording to this model, the induction pattern of AML could be modified by changing the cell kinetics of haematopoietic cells. For example, the latency of AML development will be longer and the lifetime incidence will decrease if cycling rates of HSCs are slowed down after irradiation. This has actually been showed by dietary restriction in mice. Under caloric restriction, the cycling fraction of haematopoietic stem/progenitor cells is low (Finally, we should note the limitations of our mathematical analysis. We consulted the literature for realistic parameter values, but available data were limited and arbitrary values had to be assigned to some parameters. In addition, there are factors not taken into account in the analysis; that is spontaneous cell death/inactivation, cell kinetics change because of natural ageing, strain difference in cell kinetics, and radiation effects on stromal cells. The accumulation of empirical data will enable a more realistic simulation integrating those factors. However, despite these limitations, it is certain that surviving HSCs have to cycle at enhanced rates to recover and maintain haematopoiesis after irradiation. We therefore conclude that the acceleration of HSC ageing because of continuous replicative stress is a major effect of irradiation in murine leukaemogenesis.


Background and purpose Revision surgery for periprosthetic femoral fractures around an unstable cemented femoral stem traditionally requires removal of existing cement. We propose a new technique whereby a well-fixed cement mantle can be retained in cases with simple fractures that can be reduced anatomically when a cemented revision is planned. This technique is well established in femoral stem revision, but not in association with a fracture.Patients and methods We treated 23 Vancouver type B periprosthetic femoral fractures by reducing the fracture and cementing a revision stem into the pre-existing cement mantle, with or without supplementary fixation.Results 3 patients died in the first 6 months for reasons unrelated to surgery. In addition, 1 was too frail to attend follow-up and was therefore excluded from the study, and 1 patient underwent revision surgery for a nonunion. The remaining 18 cases all healed with radiographic union after an average time of 4.4 (2–11) months. There was no sign of loosening or subsidence of the revision stems within the old cement mantle in any of these cases at the most recent follow-up after an average of 3 (0.3–9) years.Interpretation Our results support the use of the cement-in-cement revision in anatomically reducible periprosthetic fractures with a well-preserved pre-existing cement mantle. This technique is particularly useful for the elderly patient and for those who are not fit for prolonged surgical procedures. The most commonly occurring fractures around a femoral stem are of Vancouver type B . TreatmeThere are many advocates for the use of uncemented long stems for revision . PotentiRegardless of the choice of replacement stem, revision surgery for periprosthetic fractures around a cemented prosthesis normally necessitates the laborious task of removing the existing cement mantle. An alternative is to retain cement that is well fixed at the cement-bone interface and to use the “cement-in-cement” technique for femoral component revision. To reduce the intraoperative time and complications—particularly in high risk, more elderly patients—we performed cement-in-cement stem revisions, with or without internal fixation, on a series of patients with Vancouver type B periprosthetic fractures. The objectives of this study were to assess the validity of this technique and to evaluate patient outcomes.A review of the hip surgery registry at our unit identified 23 patients (mean age 79 (54–92) years, 15 men) who underwent cement-in-cement revisions for Vancouver type B periprosthetic femoral fractures between September 1995 and March 2005.1 patient had an American Society of Anaesthesiologists (ASA) score of 1, 13 were ASA 2, 7 were ASA 3, and 2 were ASA 4. There were 3 Vancouver type B1, 17 type B2, and 3 type B3 fractures. 14 fractures involved primary hip replacements and 9 followed previous revision surgery to the femoral stem . 3 patients underwent simultaneous acetabular revisions: 1 for a mal-aligned cup and 2 for aseptic loosening. The mean time of fracture from original implantation was 6 (0.4–23) years. 22 fractures involved Exeter stems and 1 involved a Charnley stem. The original cement type was known in 11 cases, all being Simplex. The remaining 12 prostheses were not implanted in our unit and the type of cement was not known.Patients were selected for cement-in-cement revision by the treating surgeon on the grounds of their age, comorbidities, fracture configuration, and the radiographic and intraoperative appearance of the bone-cement interface. Most patients were elderly or had multiple comorbidities, requiring a short surgical procedure. The 3 B1 fractures were of transverse or short oblique patterns. These were considered to be unstable fractures that would benefit from the added stability of long stem revision, as it has been shown that there is a high failure rate associated with fixation alone of B1 fractures . The B2 Following an extended posterior approach to the hip and stem removal, it was common to find some loose, comminuted fragments of cement proximally. These were removed and well-fixed cement was left attached to the fracture fragments but debulked as necessary with a high-speed burr or power reamers to allow the subsequent insertion of a larger, longer stem. This was used to bypass the fracture site in 20 of the 23 cases. In the remaining 3 cases, a standard-length stem was used and a lateral plate and/or cortical strut allograft was used to bridge the fracture site . In caseA cement restrictor was inserted distally, with a temporary transfemoral wire placed distal to this to limit migration of the plug during cement pressurization. The fracture was then reduced and stabilized with circlage wires or cables. Additional plates or strut graft were used in 11 of the 23 cases to help stabilize the femur. Autograft was applied to the fracture site in 7 cases to promote fracture union. The revision stem was then cemented into the remaining existing cement mantle using third-generation techniques with Simplex cement. Stem insertion was earlier (within the first 2–3 min) than in routine primary surgery.It was common for small amounts of cement to escape from gaps in the fracture site due to the high pressures generated during stem insertion. Rather than attempting to occlude such escape, this was allowed so as to limit the escape to this area alone; attempts to seal such vents risk causing the cement to be forced out of the fracture site at other additional areas, which might impair fracture union. Such escaped cement was then removed before wound closure.Patients were reviewed clinically and radiographically at 6 weeks, 3 months, 6 months, and then annually. Outcomes were assessed in terms of complications, time to union, and function. Union was determined radiographically by the presence of callus bridging the fracture on 2 radiographic views .On average, patients were discharged home or to rehabilitation units from hospital 11 (5–28) days postoperatively. The mean time to partial weight bearing in these patients was 3.6 (1–13) days. The mean time to full weight bearing was 31 (3–120) days.3 patients died during the first 6 postoperative months, before union could be recorded adequately, and they were thus excluded from the analysis. 1 patient, a 90-year-old man with multiple comorbidities, was seen at 6 weeks but was not subsequently reviewed as he was too infirm to travel from his nursing home. He was reported to have had no problems with his revision until his death 2.5 years later, but as he was not assessed radiographically he was also excluded from our analysis. The remaining 19 fractures in 19 patients were followed up for a mean period of 3 (0.3–9.3) years.1 nonunion occurred, leading to a plate fracturing at the unhealed fracture site 7 months after revision. This patient had extruded cement removed from the fracture site at the time of revision. In addition, the revision stem was not bypassing the most distal fracture line, leading to mechanical failure of the construct. This stem was successfully revised again using the cement-in-cement technique, to a longer Exeter prosthesis. The fracture united and has been problem-free for 3 more years.Of the 18 fractures followed up to successful union, the mean time to radiological union was 4.4 (2–11) months .At the latest radiographic follow-up, there was no evidence of prosthetic loosening or lucency at the new cement-cement interface. The revision polished tapered stems were found to have subsided by an average of 1.0 (0-3.2) mm at a mean follow-up time of 3 years, which is consistent with this design of stem . There wThere has been ongoing debate over the use of the cement-in-cement technique for over 30 years, based on laboratory  and clinThe largest series of periprosthetic fractures to date is the review by We found 1 nonunion in 19 patients with a mean follow-up of 3 years, and no cases of loosening. These results, while from a small sample size, are favorable compared to the previous reports.Concern has been expressed that cement extrusion may inhibit fracture healing  and in cAlthough it is difficult to make comparisons between the outcomes in our small cohort of 23 patients with the outcomes in more extensive studies , one of It is worth noting that all except 1 of the stems revised were Exeter polished taper stems, and all were revised to Exeter stems (long or standard size). The procedure in which the original stem was a Charnley was not complicated by revising to a different stem, as the original mantle is generally reamed up to accommodate a larger revision stem. This particular case showed union at 6 months. It cannot be ascertained from this study whether using alternative revision stems would achieve satisfactory results, although other stem designs have been used with success in published series of cement-in-cement revision hip replacement .Although the study was limited by sample size, the results suggest that there is a valid role for the use of the cement-in-cement revision technique for periprosthetic fractures. This method is particularly recommendable for patients who are not suited to lengthy procedures, with simple, reducible fractures associated with a well-fixed cement mantle; it has significant advantages in terms of simplification of operative technique and enhancement of speed of recovery.


Oxidative stress is associated with increased cardiovascular morbidity and mortality particularly in patients with end stage kidney disease. Although observational data from the general population has shown dietary antioxidant intake is associated with reduced cardiovascular morbidity and mortality, most clinical intervention trials have failed to support this relationship. This may be a consequence of not using an effective antioxidant dose and/or not investigating patients with elevated oxidative stress. The SPACE study, conducted in haemodialysis patients, reported that 800 IU/day of alpha tocopherol significantly reduced cardiovascular disease endpoints. A recent time course and dose response study conducted in hypercholesterolaemic patients that found 1600 IU/day of alpha tocopherol was an optimal dose. There is no such dose response data available for haemodialysis patients. Therefore the aim of this study is to investigate the effect of different doses of oral alpha tocopherol on oxidative stress in haemodialysis patients with elevated oxidative stress and the time taken to achieve this effect.2-isoprostanes) and alpha tocopherol. The optimum time period to significantly decrease plasma F2-isoprostanes will be determined from this study. In the dose response study 60 patients will be randomised to receive either placebo, 100, 200, 400, 800 or 1600 IU/day of natural (RRR) alpha tocopherol for a time period determined from the time course study. Blood will be collected at baseline and every two weeks and analysed for plasma F2-isoprostanes and alpha tocopherol. It is hypothesised that doses ≥ 800 IU of vitamin E will be required to significantly decrease plasma F2-isoprostanes.The study will consist of a time-course followed by a dose response study. In the time course study 20 haemodialysis patients with elevated oxidative stress will take either 1600 IU/day natural (RRR) alpha tocopherol for 20 weeks or placebo. Blood will be collected every two weeks and analysed for a marker of oxidative stress (plasma FThis study will determine the time and dose required for alpha tocopherol to significantly decrease oxidative stress in haemodialysis patients. Data will be used to plan a large randomised controlled trial to assess the effects of alpha tocopherol on cardiovascular outcomes in haemodialysis patients.ACTRN12609000608268 Oxidative stress is an important contributor to the development of atherosclerosis and hence cardiovascular morbidity and mortality . ObservaChronic kidney disease is associated with high levels of oxidative stress, and treatment with haemodialysis can further increase oxidative stress levels, which may contribute to high level of cardiovascular disease in these patients ,12. ThesAlpha tocopherol is the main form of vitamin E and this has been the most commonly studied dietary antioxidant supplement in clinical trials. In one of the few trials to demonstrate a beneficial cardiovascular affect, the Secondary Prevention with Antioxidants of Cardiovascular disease in End stage renal disease (SPACE) study assessed the effects of 800 IU of alpha tocopherol on cardiovascular outcomes in 196 haemodialysis patients . In thisA recognised weakness of many of the large vitamin E cardiovascular trials is a lack of oxidative stress data assessing whether the supplementation was pharmacologically effective. In addition there has been evidence from meta-analyses indicating potential safety concerns with higher doses of alpha tocopherol .2-isoprostanes, a biomarker of oxidative stress [Roberts et al (2007) conducted a time course and dose determining study in hypercholesterolemic patients. They reported that 1600 IU of alpha tocopherol was required over 16 weeks to significantly reduce plasma Fe stress . No suchThis investigation will involve two sequential components, a time course, followed by a dose response study. The studies will be conducted at the Renal Medicine Department of the Royal Brisbane and Women's Hospital, Brisbane, Australia, which services a population of approximately 1.2 million people.The Royal Brisbane and Women's Hospital Research and Ethics Committee approved the studies. The Ethics Committee will promptly receive all adverse event reports along with a final report.2-isoprostane levels > 500 pg/mL. This is based on a study that compared plasma F2-isoprostane levels in chronic haemodialysis patients (1662 ± 289 pg/mL) with matched healthy controls (396 ± 18 pg/mL) [For both studies, inclusion criteria are; age > 18 and < 85 years, incident and prevalent dialysis patients, who are undergoing regular three times per week, hospital based haemodialysis and who have elevated oxidative stress. Elevated oxidative stress will be defined as having total plasma F8 pg/mL) ,25 and t8 pg/mL) .Patients will be excluded if they are currently taking alpha tocopherol or have been taking it in the previous three months for at least one week, and if they are unable, or unwilling to give informed consent. In addition, patients who are participating in another investigational study, or are within 30 days of completing an investigational study, or patients who are taking warfarin, desimpramine, chlorpromazine and chloroquine will also be ineligible.Eligible patients will be screened from the haemodialysis unit and will receive a copy of the patient information and consent form. An investigator will then explain the study during a clinical consultation. The patient will then be asked to take the information and consent form away with them and make a decision before their next haemodialysis visit. If they agree to participate, they will sign the consent form with an independent person signing as a witness.An individual not associated with the trial will perform the randomization for both studies using a computer generated random number system.Twenty participants will be recruited for the time-course study with ten randomly allocated to a treatment group and ten to a placebo group. Both groups will be required to take two capsules per day (one in the morning and one at night) each containing either a placebo or 800 IU of natural (RRR) alpha tocopherol  for 20 weeks.Following the time course study, sixty participants will be recruited for the dose-response study with placebo, 100 IU/day, 200 IU/day, 400 IU/day, 800 IU/day and 1600 IU/day natural (RRR) alpha tocopherol groups each consisting of ten people. If patients from the time course study agree to participate in the dose-response study they will undergo a 12-week period (wash out) where they will not be consuming alpha tocopherol. Dose-response study participants will be required to take two capsules per day (one in the morning and one at night) containing either a placebo or alpha tocopherol, to provide the respective IU/day doses.The placebo will contain the emulsifying agent (wheatgerm oil) used in the alpha tocopherol supplement. Cognis Nutrition & Health Pty Ltd, Sydney, Australia, will supply all capsules.All vitamin E and placebo capsules will be identical. The allocation will be provided coded to the person who dispenses the medication thus ensuring the dispenser and patient are blinded to the allocation.A medical co-investigator will assess whether reported adverse events are related to the study medication and will also assess all signs, symptoms and complaints. All adverse events, regardless of relationship to the study, will be recorded and will include; the specific condition or event and direction of change, whether the condition is pre-existing, the dates and times of the occurrence, the severity and significance to study, the action taken and the outcome.Serious adverse events recorded will include death, in-patient hospitalisation, persistent or significant disability or incapacity, cancer, or be life threatening. Additionally, important medical events that may not result in the above stated outcomes but may require medical or surgical intervention will also be reported.The first primary objective is to determine the time point when oxidative stress is significantly reduced in haemodialysis patients taking 1600 IU/day of alpha tocopherol. Based on the Roberts et al. (2007) study in hypercholesterolemic patients, it is hypothesised that this will occur at 16-weeks .Using this information, the second primary objective is to determine the optimal dose of alpha tocopherol required to reduce oxidative stress in patients undergoing haemodialysis. Based on the SPACE trial, it is hypothesised that ≥ 800 IU of alpha tocopherol will be required to significantly reduce oxidative stress .For both studies, secondary objectives will be to determine the effects of alpha tocopherol on additional measures of oxidative stress and antioxidant status. The additional measures will be plasma protein carbonyls, malondialdehyde and antioxidant enzyme activities of superoxide dismutase, glutathione peroxidase and catalase.2-isoprostanes to assess their eligibility (> 500 pg/ml) to progress to the next phase of the study.After obtaining written, signed and witnessed informed consent patients will have blood taken from the dialysis line, at the start of dialysis, to measure total plasma F2-isoprostanes and alpha tocopherol.Once randomized, patients will be booked into a clinic appointment for baseline trial measures. This will occur on a mid-week dialysis day. At the first trial visit, additional data will be obtained from the medical records  and measures of height, weight and blood pressure will be recorded. Additional blood will be collected pre-dialysis for routine blood chemistry and for measures of plasma F2-isoprostanes and alpha tocopherol.Participants recruited into the time-course study will be required to consume 1600 IU of alpha tocopherol orally each day for 20 weeks or an identical placebo. Blood samples of 8 mL will be taken once every fortnight from the dialysis line, at the start of dialysis, during a normal mid-week dialysis visit and analysed for plasma F2-isoprostanes and alpha tocopherolParticipants recruited for the dose-response study will be allocated after randomisation to consume either a placebo, 100 IU, 200 IU 400 IU, 800 IU or 1600 IU of alpha tocopherol orally each day for the duration determined by the time-course study. Blood samples of 8 mL will be taken at the completion of the time period from the dialysis line, at the start of dialysis, during their normal mid-week dialysis visit and analysed for plasma FPatients will be required to return the container each month with unused capsules inside. Adherence to therapy will be assessed by duplicate table counts of the remaining capsules conducted independently by two study investigators.Patients will be withdrawn from the study at their request, without prejudice, as documented and explained at the time of consenting. Patients who withdraw will be invited to consent to follow-up testing for the remainder of the trial to enable an intention to treat data analysis.EDTA blood (approx. 8 mL) will be collected from the sample line shortly after the patient has commenced dialysis. If there is considerable time (> 10 mins) between blood collection and separation then the blood sample will be placed on ice. Centrifugation will be at 600 × g for 10 mins with plasma aliquots then stored at -80°C.2α) will be extracted from plasma according to a modified method of Taylor and colleagues [2α-d4  before methanolic NaOH solution is added. The solution will be vortexed, caped and placed in a water bath at 37°C for 60 min. This is followed by further dilution with distilled water and then acidification to pH 3 with hydrochloric acid before being extracted with hexane. Following gentle rotation and centrifugation at 3000 × g, hexane extracts will be removed and discarded. The aqueous layer will then be extracted twice with ethyl acetate, with extracts pooled and dried under nitrogen. Once dry, the samples will be reconstituted with acetonitrile, vortexed and transferred into 300 μL glass inserts and dried ready for derivatisation. The derivatization method will be based on that used by Mori and colleagues [2-isoprostane pentafluorobenzyl ester. Samples will then be treated with pyridine, bis(trimethylsilyl)trifluoroacetamide and trimethylchlorosilane and incubated at 45°C for 20 min to yield the trimethylsilylethers. After incubation, hexane will be added to each sample, mixed and placed in the auto sampler. Samples will then be analyzed using a Varian 320 MS/MS with a Varian 450 gas chromatograph using Varian MS Workstation - system control software version 6.9.1 . Using a 10 μL Hamilton syringe with 1.2 μL of sample injected for each analysis. The injector will operate at 250°C with a Varian FactorFour Capillary Column - VF - 5 ms 30 m × 0.25 mm ID DF = 0.25 using helium as the carrier gas at a flow rate of 1.0 ml·min-1 with pressure maintained at 13.9 psi. The column oven will start at 160°C held for one minute then increase to 300°C over 7 min then holding for 10 min for an overall run time of 18 min with the peaks eluting around 10.3 min. Running in negative chemical ionization mode the reagent gas will be methane with an ion source pressure of 7.00 Torr and argon as the collision gas at a pressure of 2.00 mTorr. The mass size of our compounds is expected to be 569 and 573 for isoprostanes and the internal standard, respectively.Total isoprostanes  of the initial values will be regarded as significant. To our knowledge, there is little data to support this assumption as no studies have assessed the relationships between plasma F2 isoprostanes and long-term health outcomes. Data from the Roberts et al. (2007) study found an approximately 50% reduction in plasma F2-isoprostane levels after 16 weeks vitamin E treatment in hypercholesterolemic patients [The primary outcome measure for both studies will be the total plasma F89 pg/mL ,25. A depatients . With th2-isoprostanes and alpha tocopherol, which occur during the intervention. The time-course study will use general linear modelling to analyse the levels of plasma F2-isoprostanes at bi-weekly intervals and levels of alpha tocopherol will be compared at baseline and week 20. In the dose-ranging study we will use general linear modelling to compare the changes in plasma F2-isoprostane levels between the intervention and placebo group at baseline and at the completion of the study. All statistical analyses will be performed using STATA statistical software .The analysis of both the time-course and dose-ranging study will focus on analysing the changes in the levels of plasma FDespite observational studies suggesting alpha tocopherol may protect from cardiovascular disease, most interventional studies have failed to support this contention. There may be many reasons for this discrepancy including the fact many patients treated in such studies may not have had elevated levels of oxidative stress. In addition, the administered alpha tocopherol may not have lowered oxidative stress. This time course and dose response study of alpha tocopherol in haemodialysis patients will provide data to support further large randomised controlled trials in this area.The authors declare that they have no competing interests.AR, YJC, RGF and JSC are responsible for the design of this trial, and the construction of the protocol. All authors contributed to, and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2369/10/32/prepub


Few data are available to guide programmatic solutions to the overlapping problems of undernutrition and HIV infection. We evaluated the impact of food assistance on patient outcomes in a comprehensive HIV program in central Haiti in a prospective observational cohort study.2, CD4 cell count <350/mm3 (in the prior 3 months) or severe socio-economic conditions. A total of 600 individuals (300 eligible and 300 ineligible for food assistance) were interviewed before rations were distributed, at 6 months and at 12 months. Data collected included demographics, BMI and food insecurity score (range 0 - 20).Adults with HIV infection were eligible for monthly food rations if they had any one of: tuberculosis, body mass index (BMI) <18.5kg/mAt 6- and 12-month time-points, 488 and 340 subjects were eligible for analysis. Multivariable analysis demonstrated that at 6 months, food security significantly improved in those who received food assistance versus who did not ; BMI decreased significantly less in the food assistance group than in the non-food group . At 12 months, food assistance was associated with improved food security  and BMI . Food assistance was associated with improved adherence to monthly clinic visits at both 6 (P < 0.001) and 12 months (P = 0.033).Food assistance was associated with improved food security, increased BMI, and improved adherence to clinic visits at 6 and 12 months among people living with HIV in Haiti and should be part of routine care where HIV and food insecurity overlap. Food insecurity and undernutrition are increasingly recognized as factors that are important in the health and livelihoods of individuals living with HIV infection in poor settings ,2. HIV iAttention to adequate nutrition during HIV care has the potential to contribute to improved clinical HIV-related outcomes, improved nutritional outcomes for the individual, as well as improved coping strategies and ability of individuals to contribute to livelihoods at the household level. Although the qualitative effect of food on relieving hunger is not in doubt, the quantitative benefits of food assistance on individuals or on families has rarely been studied in the context of HIV . As a re3 in the prior three months, BMI less than 18.5 or severe socioeconomic circumstances . A standard pre-determined WFP family ration was provided by prescription monthly. The ration contained 50 gm of cereal, 50 gm of dried legumes, 25 gm of vegetable oil, 100 gm of corn-soya blend and 5 gm of iodized salt for each of 3 family members  per person per day.The study was a prospective observational cohort study of 600 people living with HIV enrolled in HIV care in Partners In Health (PIH) programs in rural Haiti. PIH is a non-profit organization working in conjunction with the Ministry of Health of Haiti to provide comprehensive primary healthcare services, including HIV care, in two departments in rural Haiti. In May 2006, PIH entered into collaboration with the World Food Programme (WFP) to provide food rations for beneficiaries living with HIV. Because available rations were limited, beneficiaries of the program were determined by a set of criteria agreed upon in advance by WFP and PIH program staff, including clinicians, social workers, and ethicists. Adults received twelve months of food assistance if they had HIV and any one of: co-infection with active TB, CD4 count less than 350 cells/mmThree PIH clinic sites were included in the study . At each site the first 100 individuals eligible for food assistance and first 100 ineligible for food assistance by the criteria defined above were invited to participate in the study. Individuals were eligible for interview if they were living with HIV, were being assessed by the clinical team for eligibility for the food program, were over the age of 18 years and were not pregnant at the time of interview.3 or with World Health Organization clinical criteria to begin treatment. Pregnant women are offered ART for their own health when CD4 count is less than 350 cells/mm3 or at 28 weeks of gestation for prevention of mother-to-child transmission. Weight is measured routinely during patient monthly visits to clinic. Height was measured for adults at the beginning of the WFP collaboration using a clinic-installed stadiometer to allow calculation of BMI by clinic staff. In addition to medical care, attention is paid to the socioeconomic causes and contributors to disease and ill-health, and social assistance programs make small grants for commerce or housing repair available. All care is provided free of charge to patients [Combination ART is offered to those with HIV infection and CD4 counts less than 350 cells/mmpatients .Individuals were interviewed before rations were distributed and at 6 months and 12 months after food assistance began. Data collected in surveys included demographics, education level, BMI, food insecurity score and quality of life. Additional information was abstracted from the respondent's electronic medical record including CD4 count, timely attendance at prescribed monthly clinic visits, weight, BMI and pick up of prescribed food rations. Food insecurity score was measured using the Household Food Insecurity Access Scale (HFIAS) . In thisDuration on ART was determined for each respondent at the time of entry into the study and the respondent analyzed in this category throughout the study: 'never on ART', 'on ART < 12 months, 'on ART ≥12 months'. Active TB infection was an absolute indication for receiving food assistance in the PIH program. Since no active TB patient would be found in the 'non-intervention' group, and because TB contributes to weight loss independently of HIV infection, subjects that had active TB during the period of the study were excluded from the final analysis. Subjects that were either enrolled in or discontinued from food rations by the clinical team during the period of the study were also excluded from the final analysis . Individuals were also excluded from analysis if they became pregnant during the study. BMI analysis was limited to those with weight available (N = 4). We also performed a sensitivity analysis using an 'intention to treat' approach, including all subjects based on their enrollment status at the time of baseline evaluation. For those with missing food security items, the response was replaced by the median value from all respondents in the same phase of the study . We also performed a sensitivity analysis using the E-M algorithm to impute missing food security items at 6 and 12 months.Baseline data were summarized using mean/standard deviation (SD) or percentage and compared between the 'no food assistance' group and the 'food assistance' group using two-sample t-tests or Chi-square tests. Continuous outcomes of change from baseline were summarized using mean/standard error (SE). In the univariate analysis, Wilcoxon rank sum tests were used to compare continuous outcomes while repeated measures logistic regression with Generalized Estimating Equations (GEEs) were used to compare dichotomized outcomes. In the multivariable analysis, linear regression and repeated measures logistic regression analysis were used to compare the change from baseline between the two groups controlling for other factors. All analyses were performed using SAS version 9.2 .Between May and July 2006, 600 adults were enrolled across the three clinical sites. At 6- and 12-month follow ups, 488 and 340 individuals were eligible for the analysis  age of 36 years 10); 60% were female  was 5.49 vs. 2.82 (P < 0.0001) for the food assistance vs. the non-food group, and at 12 months (out of 12 visits) was 9.73 vs. 8.34 (P = 0.007).There was no statistical difference in role-functioning quality of life (QOL) between the groups at 6 months. At 12 months, mean role-functioning QOL score increased in the food assistance group (3.72) and decreased in the non-food group (-3.80), however the difference did not reach significance level (P = 0.13). Performance-functioning QOL had a slightly greater increase at 6 months in the food assistance group compared to the non-food group . There was no difference at 12 months between the two groups .Among those on ART, at 6 months, those receiving food assistance reported fewer difficulties taking their medications compared to those who did not receive food . At 12 months, although a difference remained between the groups (11.0% vs. 18.8%), it did not reach statistical significance level (P = 0.068). There were no significant differences between groups at either 6 or 12 months in terms of ability to save money in case of disaster, spending on agriculture or education, or spending on livestock. There was also no statistical difference in outcomes of died or abandoned care at 6 or 12 months.3 at the 6-month evaluation; one had TB and 12 were pregnant at the 12-month survey. When compared to the 125 individuals that did not change status from 'no food' to 'food assistance', they had slightly less improvement in food security (-0.10 vs. -0.20) and slightly worse BMI (-0.72 vs. -0.61) at 6 months, but neither was significant . These individuals were excluded from the final analysis  of these changes occurred during the second half of the study in the 6-to-12-month period that coincided with the 'lean season' in Haiti. It is possible that individual's socioeconomic status worsened during this time, prompting the team to enroll individuals in food assistance based on these criteria. In addition, as the program timeline progressed, rations that had not yet been assigned within the total number of program rations available may have prompted an informal relaxation of the socioeconomic criteria for eligibility. Of the 129 individuals that switched status from 'no food assistance' to 'food assistance', seven had BMI < 18.5 and eight had CD4 count < 350 cells/mmIn establishing the multiple regression analysis model we used existing literature, including the conceptual framework of Egge et al ,2,8,21 tThis study finds that providing food assistance to individuals with HIV and food insecurity in central Haiti improves BMI, food security and adherence to clinic visits. We also observed a significant improvement in ability to take ART at 6 months and a trend for improvement in this variable at 12 months. Although many studies have evaluated the impact of micronutrient supplementation on HIV disease progression, to our knowledge, this is the first study demonstrating a quantitative clinical benefit of macronutrient supplementation on HIV clinical outcomes.Low BMI may result from chronic inadequate food intake, wasting as a result of HIV or other infections, or in some individuals may be a normal anthropometric variation. Low BMI has previously been independently associated with poor outcomes among individuals with HIV infection even while on ART -24. In tOf note, the pre-determined WFP ration distributed to support people living with HIV in Haiti provides approximately 45% of the caloric requirement of a family of three, although the median number of people eating at the households in this study was six. Furthermore, the ration is not targeted specifically to individuals with HIV infection, but is rather a family support. Despite these issues, that by virtue of quantity and quality might be expected to attenuate the effect of food support on the individuals we studied, food rations were protective against weight loss in the short-term and associated with weight gain in the long-term for the individuals with HIV.Seventy-two percent of the participants in this study spent most or all of their income on food and mean baseline food insecurity was very high . This is consistent with national statistics for the region . In our This current study also found that individuals receiving food assistance were significantly more likely to attend scheduled clinic visits than those not receiving food assistance. Loss to follow up of individuals not yet on ART, and those on ART, is a critical challenge to HIV care in resource-poor countries. Studies have shown early loss to follow up of as many as 21% of individuals newly started on ART at 6 months ,36. In aLack of sufficient food in the diet has further negative effects on HIV care by impacting ability to take antiretroviral medications in a number of ways, including causing symptoms of nausea while taking medications on an empty stomach, increasing drug toxicity, and/or forcing competing choices between expenditures on food and transportation or other health-related needs. Cantrell and colleagues demonstrated that food supplementation was associated with improved adherence to ART in an HIV clinic in Zambia , but thaHIV program managers had flexibility to enroll individuals in food assistance throughout the period of this study because the study was observational. This happened in particular between months 6 and 12 of the study as WFP made an increased number of rations available and evaluation of socioeconomic status became more inclusive. In order to examine the food rations effect in an "uncontaminated" fashion, we focused our analysis on comparing the group of subjects that were eligible for and remained in the food assistance program from baseline to the group of subjects that were never eligible for food assistance. Subjects not receiving rations at baseline but who were enrolled for rations during the study (and vice versa) were excluded from the analysis . We also performed a sensitivity analysis that was 'intention to treat', including subjects based on their enrollment status at the time of baseline evaluation. This analysis demonstrated that at 6 and 12 months food assistance was associated with improvements in food security (P < 0.001 and P = 0.03) and better adherence to clinic visits (P < 0.0001 and P < 0.0001) compared to no food assistance. There were trends towards difference in BMI similar to those found in the as-treated analysis, but these did not have statistical significance (P = 0.07 and P = 0.16). A further sensitivity analysis was performed using an alternate method for imputing missing data. Using the E-M algorithm at 6 and 12 months , food asThe study has several limitations. It was an observational study and subjects were not randomly selected to receive the intervention. We attempted to control for measured differences between the groups using multivariable analysis however other factors not controlled in the analysis may have influenced the associations among food security, food assistance and other outcomes. With regards to unmeasured bias, subjects in the intervention group were 'worse-off' or 'more vulnerable' than the subjects in the control group by virtue of the design of the food assistance program, which aims to help those that are considered the most vulnerable. The food assistance group had lower weight, higher food insecurity and was more likely to be on ART than the no food assistance group before the study began and may have had other unmeasured differences that could have systematically influenced the outcome. Since those that are 'more vulnerable' may be expected to do worse over the course of 12 months than those in the 'less vulnerable' control group, we believe that the differences at baseline would have biased the study result to the null. If the effect we had observed was simply a phenomenon of "regression toward the mean," we would have expected the food insecurity score to be quite similar between the two groups at follow-ups. However, the food insecurity score was lower in the food group than the non-food group at both follow-up times (8.94 vs. 9.65 at 6 months and 8.95 vs. 9.16 at 12 months); therefore, the effect cannot be simply explained by regression to the mean. The fact that our study found significant differences in outcomes, despite non-randomization of the intervention, suggests that the effect of the intervention is real.This study demonstrates that food assistance is associated with improvements in clinical outcomes among people with HIV infection and food insecurity in central Haiti. Food assistance as part of comprehensive healthcare is associated with significant improvements in BMI, food security, and adherence to clinic visits at 6 and 12 months among people living with HIV. Food assistance should be standard of care in regions where HIV and food insecurity overlap.ART: antiretroviral therapy; BMI: body mass index; HFIAS: Household Food Insecurity Access Scale; HIV: human immunodeficiency virus; PIH: Partners In Health; QOL: quality of life; TB: tuberculosis; SD: standard deviation; WFP: World Food ProgrammeThe authors declare that they have no competing interests.LCI, KAF and JGJ contributed to the design of the study, analysis of the data and to writing the manuscript. YC contributed to analysis of data and writing of the manuscript. All authors read and approved the final manuscript.


Previously we have conducted a randomised controlled trial (RCT) to evaluate the effect of a brief cognitive behavioural program with a vocational approach aiming to return disability pensioners with back pain to work, as compared to no intervention. One year after the intervention, 10 participants (22%) who received the program and 5 (11%) in the control group reported to have entered a return to work process. The aims of this study were to evaluate long-term effects of the intervention, and compare this effect to 2 reference populations not participating in the original trial.Three groups of disability pensioners were investigated: 1) Disability pensioners having back pain (n = 89) previously participating in the RCT , 2) 342 disability pensioners having back pain, but refusing to participate in the study and 3) 449 disability pensioners having other musculoskeletal disorders than back pain. Primary outcome was return to work, defined as a reduction in payment of disability pension.Only 2 of 89 (2.3%) participants from the RCT had reduced disability pension at 3-years follow-up, both from the control group. None of the participants that had been in a process of returning to work after 1 year had actually gained employment at 3-years follow-up. In the 2 groups not participating in the previous RCT, only 4 (1.2%) and 8 (1.6%) had returned to work after 3 years respectively.The number of pensioners who returned to work was negligible in all groups regardless of having participated in a cognitive behavioural intervention or not. The large number of individuals leaving work prematurely due to ill health is alarming. In Norway, the number of disability pensioners increased by 26% from 1996 to 2003 [The time from leaving work and until disability pension (DP) is granted will in Norway take at least 3 years . HoweverDP is a permanent allowance in Norway. However, the pensioners are allowed to earn a basic income of about 8100 EUR per year without cuts in pension . If thisWe have recently conducted a randomized controlled trial (RCT) to evaluate the effect of a brief cognitive behavioural program with a vocational approach aiming to motivate individuals receiving DP due to back pain to re-enter work . The stuThe 87 participants in our previous RCT was a highly selected sample (only 20% of the invited population). We were concerned that our sample was more or less motivated to return to work compared to the population they were recruited from, and had the opportunity to investigate whether our sample differed from this population regarding a return to work. As there is a substantial overlap between LBP diagnoses and other diagnoses related to musculoskeletal pain ,16 we alThe primary aim of the present study was to investigate whether disability pensioners reporting to have entered a return to work process at 1-year follow-up  actuallyThree groups of disability pensioners were investigated. 1) Disability pensioners having back pain (n = 89) previously participating in the RCT  or to a control group (n = 44)) , 2) 342 Inclusion criteria were age under 55 years, receivers of DP for at least 1 year due to back pain or another musculoskeletal disorder. Register data including age, gender, duration of DP and current DP status 1 and 3 years after the intervention was available.The eligible population of disability pensioners was recruited from the National Insurance Administration (NIA). Recruitment and follow-up of the study groups are shown in Figure The study was approved by the Norwegian Ethics Committee for Medical Research, Health Region West, and performed according to the Helsinki Declaration.The main outcome was return to work defined as reduced payment of DP measured by register data from the NIA. Reductions in DP payment will in Norway only take place when the pensioner increases his income.SPSS version 15.0 was used for the statistical analysis. Descriptive statistics were used to describe socio-demographic and insurance data  of the study groups. Analysis of variance and Tukey HSD Post Hoc Test was used to examine differences in socio-demographic data between the respective groups. Differences in payment of DP were counted and compared manually due to the low numbers.Only 14 out of 899 disability pensioners (1.6%) were registered by reductions in payment of DP at 3-years follow-up.At 3-year follow-up none of the participants in the intervention group had returned to work. Hence, the 15 participants reporting to have entered a return to work process after 1 year had not succeeded in getting employed after 3 years. Two participants in the control group had a reduction in DP at 3-years follow-up. One had 50% reduction, and the other 20% reduction (Table Four 1.2%) of the non-participants with back pain were registered with only minor reductions in payment of DP after 3 years. In the musculoskeletal group, only 1 pensioner (0.2%) had fully returned to work (100% reduction in DP) and 8 pensioners (1.6%) had less than 50% reduction in payment of DP at 3-years follow-up (Table .2% of thThe RCT participants (n = 89) did not differ from the non-participants (n = 342) or the musculoskeletal group (n = 449) regarding age  = 1.2, p = 0.3) (Table This study confirms that it is a great challenge to re-enter work after becoming a disability pensioner. Of 899 disability pensioners with musculoskeletal disorders, only 1 single person had returned to full time work, while 14 had small reductions in payment of DP at 3-year follow-up. This result also included a group of disability pensioners who had participated in a previous cognitive behavioural program with a vocational approach. Despite a positive trend regarding progress towards work after 1 year, none of these individuals actually returned to work within the next 2 years. The poor results are in line with other reports on re-employment efforts among disability pensioners .Despite no effect in the main outcome, the initial conclusion after 1-year follow-up suggested a modest success as several participants reported to have entered into a work-related process. We expected that a proportion of the participants who had been in such a process would actually have returned to work at the 3-years follow-up. This assumption is supported by findings in other studies showing a moderate effect of brief interventions in terms of returning sick listed persons with back pain to work ,19. HoweWork-related interventions for this chronic group should maybe have been more extensive and individually adjusted than the brief intervention we offered. A strategy based on a closer collaboration with possible work places, and a more comprehensive program in general, including more extensive support from all parts involved in the process, might have increased the success rate. Another important factor for success in this field is the individual's positive beliefs and expectancy regarding recovery and employment -22. PrevPensioners being in a work related process might have experienced only a small, if any, increase in income, and this fact has been described as de-motivating . A more Failure of lasting results might also be due to the insurance policy. An initial attempt to try out for work will often be made with economic support from the social insurance offices. Transformation to a permanent job paid for by an employer has been shown to be difficult, as employers prefer to hire employees without a history of sick leave and disability if given the option.We found no difference in long time re-employment between different diagnostic groups or between intervention and control groups. Neither age, gender, time upon DP, nor being submitted to intervention or not, seemed to influence the negative result. We found a difference between the groups regarding duration of DP varying from 9.8 to 11 years, but this small difference has probably no practical impact. We regard the long time span of disability in itself as the main obstacle to a successful return to work, not the diagnosis or vocational interventions provided. It is tempting to speculate whether the pensioners concluded that they were comfortable with their life situation as it was. Most have many health complaints  and may We used reduction in payment of DP as an indicator of having returned to work. If disability pensioners exceed the limit of income he or she is allowed to earn, the pension will be reduced correspondingly. In Norway will be no other reason for the pension to be reduced or removed. Thus, we could assume that a reduction in DP payment meant that the pensioner earned more than the allowed amount. A possible limitation in our study was that we did not have access to more detailed information about the pensioners. We do not know whether the 3 study groups were comparable regarding factors like expectancy, level of education and previous occupation. Also, considering the small number of participants who had entered a return to work process after 1 year, we find that a qualitative study outlining the pensioners' experiences of the unsuccessful return to work process could have yielded additional important information to the outcome.The results of our study showed that return to work after 3 years in a chronic group of disability pensioners was negligible, whether they had received a brief intervention or not. The modest, but promising trend at 1-year follow-up did not yield a lasting effect. These disappointing findings add to the impression that returning long-term disability pensioners to work is a very challenging task. Prevention or more comprehensive interventions at much earlier stages in the disability process seem necessary to prevent long-term DP in those who have a potential for a return to work.The authors declare that they have no competing interests.LHM contributed to conception and design of the study, data interpretation, and drafted the manuscript. LIS and JSS conceived of the study, participated in its design, and revised various versions of the manuscript. HRE conceived of the study and made substantial contribution to the conception and design of the study, the interpretation of data, and revised various versions of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here: