Placental malaria (PM) is an important cause of maternal and foetal mortality in tropical areas, and severe sequelae and mortality are related to inflammation in the placenta. Diagnosis is difficult because PM is often asymptomatic, peripheral blood smear examination detects parasitemia as few as half of PM cases, and no peripheral markers have been validated for placental inflammation.In a cohort of Tanzanian parturients, PM was determined by placental blood smears and placental inflammation was assessed by histology and TNF mRNA levels. Maternal peripheral blood levels of several immune mediators previously implicated in PM pathogenesis, as well as ferritin and leptin were measured. The relationship between the levels of these soluble factors to PM and placental inflammation was examined.Peripheral levels of TNF, TNF-RI, TNF-RII, IL-1, IL-10, and ferritin were elevated during PM, whereas levels of IFN-γ, IL-4, IL-5 and IL-6 were unchanged and levels of leptin were decreased. In receiver operating characteristic curve analysis, IL-10 had the greatest area under the curve, and would provide a sensitivity of 60% with a false positive rate of 10%. At a cut off level of 15 pg/mL, IL-10 would detect PM with a sensitivity of 79.5% and a specificity of 84.3%. IL-10 levels correlated with placental inflammatory cells and placental TNF mRNA levels in first time mothers.These data suggest that IL-10 may have utility as a biomarker for inflammatory PM in research studies, but that additional biomarkers may be required to improve clinical diagnosis and management of malaria during pregnancy. Plasmodium falciparum is a major cause of mortality for mothers and their offspring, and is most frequent and severe during first pregnancies [Placental malaria (PM) due to gnancies . PM is cgnancies . In histgnancies . Chronicgnancies . In areagnancies ,5.P. falciparum DNA in peripheral blood is frequently positive when peripheral blood smear is negative. However, PCR can detect dead parasites, free parasite DNA, or DNA in phagocytic cells, and PCR-detection is not associated with pregnancy outcomes [Antenatal diagnosis of PM by Giemsa-stained blood smears fails to identify a substantial proportion of PM cases , possibloutcomes . Antigenoutcomes . A recenoutcomes , suggestPlacental samples, peripheral blood and clinical information were provided by Tanzanian women aged 18 to 45 years delivering at the Muheza Designated District Hospital, Muheza, Tanga region, in an area of intense malaria transmission. These women were participating in a birth cohort study known locally as the Mother-Offspring Malaria Studies (MOMS) Project. Women signed an informed consent form before joining the study, and women with known HIV or HIV-related sequelae in their offspring were excluded. Routine microbiological testing for other infectious diseases was not performed at the study site. Clinical information was collected by project nurses and assistant medical officers on standardized forms. Study procedures involving human subjects were approved by the International Clinical Studies Review Committee of the Division of Microbiology and Infectious Diseases at the US National Institutes of Health, and ethical clearance was obtained from the Institutional Review Boards of Seattle Biomedical Research Institute and the National Institute for Medical Research in Tanzania.Peripheral blood was collected in citrate phosphate dextrose around the time of delivery, and plasma was separated and frozen at -80°C. The placenta was collected at delivery, and a full thickness biopsy from the middle third of the placental disc was taken. Tissue was fresh frozen in liquid nitrogen and stored at -80°C. Placental blood samples were obtained by manual compression of the placental tissue in a grinder. Placental parasitaemia was defined as the identification of any parasites in a placental blood slide by microscopy. Thick and thin smears were prepared; thin smears were fixed with methanol. Blood slides were stained for 10 minutes in 10% Giemsa, washed in tap water, air-dried, then examined using light microscopy at 1000 × magnification. Ten thousand red cells were examined in the thin smear before concluding that a placental blood slide was negative.®, BioRad, Irvine, CA) and custom-made assay kits as previously described [Plasma levels of cytokines, cytokine receptors, ferritin and leptin were analyzed using a multiplexed, bead-based platform  for none or very few inflammatory cells present, (+) for inflammatory cells present. Histological analysis was performed by a single observer (A.M.).(control CT-gene CT). The oligonucleotide primers used for PCR reactions included: TNF Forward CACGCTCTTCTGCCTGCT; TNF-α Reverse CAGCTTGAGGGTTTGCTACA; KRT7 forward: GGCTGAGATCGACAACATCA; KRT7 reverse: CTTGGCACGAGCATCCTT.Quantitative PCR was performed as described elsewhere . BrieflyStudent's t-test was used for the analysis of maternal age and birth weight within primigravid (first pregnancy) and multigravid (second and later pregnancy) groups. Mann-Whitney test was used to examine cytokine levels. Linear regression coefficients were calculated using simple regression analysis. Receiver operating characteristic (ROC) curve and area under the curve (AUC) analyses were performed with IL-10 and other soluble factors levels as continuous variables using JROCFIT and JLABROC4 algorithms that are available online at the URL . SensitiPeripheral plasma samples used for these studies were provided by 660 women delivering singleton live-born babies in Muheza, Tanzania. Clinical data are shown in Table Comparison of concentrations of cytokines and other soluble factors in maternal peripheral blood stratified for PM and parity is shown in Table The soluble factors that were significantly elevated in peripheral blood during PM were analyzed by ROC curve analysis to determine their utility as biomarkers to detect PM Table . IL-10 hThe ability of IL-10 elevations above various threshold values to discriminate infected from uninfected women was examined Table . An IL-1Peripheral blood smear analysis has low sensitivity to detect PM. PCR based and antigen capture tests for the diagnosis of PM have increased sensitivity but cannot detect inflammation, which is related to poor pregnancy outcomes. This study suggests that peripheral IL-10 levels may be a useful tool to identify women with inflammatory PM and therefore those likely to have poor pregnancy outcomes. Using a cut-off level of 15 pg/mL, IL-10 levels would detect PM with a sensitivity of 79.5% and specificity of 84.3%. IL-10 may have utility in longitudinal studies, examining the burden of malaria over gestation, when the placenta is not available for microscopic analysis. Future studies should measure IL-10 levels throughout gestation to assess relationships to antenatal parasitemia and to pregnancy outcomes.Toxoplasma gondii [Trypanosoma cruzi [Leishmania major [Schistosoma mansoni schistosomulas [IL-10 is a key cytokine both in protection and immunopathology during malaria. High levels of IL-10 observed during malarial episodes may be beneficial by reducing the inflammatory response, but may be detrimental by decreasing antiparasitic cellular immune responses. IL-10 is an anti-inflammatory cytokine that acts in part by blocking monocyte/macrophage production of inflammatory cytokines such as IL-6, TNF, and IL-l . Animal a gondii , Trypanoma cruzi  and Leisia major  and the osomulas . These eosomulas .P. falciparum are also cleared by phagocytosis and killed by oxidative products of nitric oxide released by macrophages [The blood stages of rophages . IL-10 hrophages ,20 and prophages . Both inrophages ,23 whilerophages .PM results from the accumulation of parasites that bind to CSA in the intervillous spaces of the placenta ,25. In rThe present data indicate that peripheral ferritin levels are also elevated during PM. Ferritin is a positive acute phase protein and is known to increase during infection and injury. In non-pregnant individuals, ferritin levels increase during both asymptomatic and symptomatic malaria, and the highest levels have been recorded in individuals with severe disease . Serum fIn summary, these data suggest that the peripheral IL-10 level may be useful as a biomarker of inflammation due to PM. Future studies should measure antenatal levels of IL-10, and assess its relationship to parasitemia and pregnancy outcomes, and its utility for monitoring interventional trials. The sensitivity and specificity of peripheral IL-10 levels at delivery suggest that they may not be sufficient to be used clinically as diagnostic tools. Additional biomarkers of PM, placental inflammation and PM-related poor outcomes are needed to improve the clinical management of this major public health problem.TKM, MF, and PED designed and managed the MOMS Project. ERK and JDK performed the multiplex cytokine assay. AM performed PCR and histology studies. AM analyzed the data and wrote the manuscript with assistance from other authors.The author(s) declare that they have no competing interests.


Selective serotonin reuptake inhibitors (SSRI) decrease platelet-function, which suggests that SSRI use may increase the risk of post-surgical bleeding. Few studies have investigated this potential association.We conducted a population-based study of the risk of re-operation due to post-surgical bleeding within two weeks of primary surgery among Danish women with primary breast cancer. Patients were categorised according to their use of SSRI: never users, current users , and former users . We calculated the risk of re-operation due to post-surgical bleeding within 14 days of initial surgery, and the relative risk (RR) of re-operation comparing SSRI users with never users of SSRI adjusting for potential confounders.389 of 14,464 women (2.7%) were re-operated. 1592 (11%) had a history of SSRI use. Risk of re-operation was 2.6% among never users, 7.0% among current SSRI users, and 2.7% among former users. Current users thus had an increased risk of re-operation due to post-operative bleeding  = 1.4, 3.9) compared with never users. There was no increased risk of re-operation associated with former use of SSRI .Current use of SSRI is associated with an increased risk of re-operation due to bleeding after surgery for breast cancer. Selective Serotonin Reuptake Inhibitors (SSRI) decrease platelet serotonin storage and platelet-function in humans ,2 and arThe prevalence of patients using SSRI is increasing. According to The Danish Medicines Agencys' data, usage has increased from 38.3 Defined Daily Doses/1000 inhabitants in 2004 to 50.3 in 2008 [We therefore conducted a population-based study with prospectively collected administrative data to examine the association between use of SSRI and re-operation due to post-surgical bleeding in a large cohort of Danish women undergoing breast cancer surgery.We conducted this population-based cohort study among residents of Northern Denmark, which has a total population of 1.7 million inhabitants. The Danish National Health Service provides tax-supported healthcare to all residents of the country and refunds part of patient expenditures on a wide range of prescribed drugs, including SSRI. A unique civil personal registration (CPR) number has been assigned to all Danish citizens since 1968 by the Danish Civil Registration System. This number encodes gender and date of birth [All hospitalizations are registered to individual patients in the National Registry of Patients, which has covered all Danish hospitals since 1977. Data from the National Registry of Patients on the inhabitants of Northern Denmark have been merged into a research database at Aarhus University ,11. ThisInformation on re-operation due to post-surgical bleeding within 14 days of primary breast cancer-directed surgery was retrieved from the National Registry of Patients in accordance with the Danish Classification of Surgical Procedures and Therapy . All breast cancer patients were treated with either mastectomy (code KHAC) or breast-conserving surgery (code KHAB).All pharmacies in Northern Denmark use computerised accounting systems connected with the National Health Service to record a customers' CPR number, type and quantity of medication dispensed , and prescription data according to the drug's Anatomical Therapeutic Classification (ATC) . Data on prescriptions for refundable drugs are transferred electronically to the National Health Services and to the research database at Aarhus University, with complete coverage in the region since 1998. In Denmark, anti-depressants are available only by prescription. We identified prescriptions for the following SSRI anti-depressants: Fluoxetine N06AB03, citalopram N06AB04, paroxetine N06AB05, sertraline N06AB06, fluvoxamine N06AB08, and escitalopram N06AB10. Patients were categorised according to their use of SSRI: never users, current users , and former users .To account for factors that may be associated with SSRI anti-depressant prescription and post-surgical bleeding, we obtained data on age at breast cancer surgery and on preoperative use of platelet inhibitors, vitamin K antagonists, oral anti-coagulants, non-steroidal anti-inflammatory drugs (NSAID)  non-SSRFollow-up began at the date of primary breast cancer surgery and continued until fourteen days after the operation or 1 April 2007. We analysed the data first by tabulating contingency tables for the main study variables, from which we calculated the risk of re-operation due to post-surgical bleeding according to use of SSRI. We computed the crude risk difference and risk ratio with their 95% confidence intervals associating SSRI prescription with post-operative bleeding. We then stratified the contingency tables according to each of the possible confounding variables to examine the strength of confounding. We also fitted multiple logistic regression models to the data to compute the odds ratio and associated 95% confidence intervals that controlled for confounding by identified confounders. Given that re-operation for post-surgical bleeding was rare in all combinations of the independent variables, these adjusted odds ratios provide an estimate of the adjusted risk ratios (aRR). In the final model we included variables that changed the exposure odds ratio by 10 percent . All datStudies based on registry data do not require formal ethical approval under Danish law. However, handling of data was approved by the Danish Data-protection Agency.Characteristics of the cohort of 14,464 breast cancer patients according to SSRI prescription are presented in Table Risk of re-operation due to post-surgical bleeding after breast cancer surgery is presented in Table The elevated risk of re-operation among current users of SSRI remained after adjustment for age  when compared with never users. Similarly, the near-null association between former use of SSRIs and re-operation due to post-surgical bleeding remained after adjustment for age . Further adjustment for other medications or the selected potential confounding comorbid diseases did not substantially affect the risk ratio estimates of association.We hypothesized that current use of SSRI would increase the risk of re-operation after breast cancer surgery due to postoperative bleeding, and the results of this investigation were consistent with that hypothesis. While current users of SSRI had almost tripled the risk of re-operation due to postoperative bleeding, the absolute change in risk was only from 2.7% to 7.0%.The present study is population-based with complete prescription and follow-up data. Recall-bias was eliminated by use of prescription records generated before the initial breast cancer surgery date and all data were obtained prospectively from population-based registries that have a completeness approaching 100% . This inOur comparison of post-surgical bleeding risk in SSRI users versus never SSRI users may raise a concern about non-comparability of the baseline risk of post-surgical bleeding related to indications for SSRI use. For example, patients with a history of depression, which is the primary indication for SSRI use, may tend to have a less healthy lifestyle than patients without a history of depression with respect to tobacco use, diet, alcohol use, and exercise ,19. DeprA limitation of the current study is that it includes no information on SSRI prescription compliance. For example, women defined as current users may not have taken the medication up to the day of surgery. Our information on SSRI use relied on registration of dispensed prescriptions and the Danish health care system covers only a proportion of the costs of prescribed medicine. Dispensed prescriptions for which patients must pay part of the cost are likely to be actually used. Furthermore, women on anti-depressant medication with the additional distress of a newly diagnosed breast cancer would be unlikely to stop their anti-depressant medication. Earlier studies on orthopaedic surgery and coronary artery bypass surgery ,6, have The overall risk of re-operation due to postoperative bleeding in breast cancer patients observed in our study was of the same magnitude as that observed in a small single centre cohort (0.8-4.1%) , which aThe clinical relevance of SSRI in relation to bleeding has been discussed for almost two decades ,2,23. BrOur results show overall low risk of re-operation due to post-surgical bleeding among breast cancer patients regardless of whether they are current, former, or never users of SSRI medications. The consequence of stopping SSRI medication used to treat depression in women undergoing treatment for breast cancer may be overwhelming, and may even delay any subsequent cancer-directed treatment. Thus, the potential consequences of stopping the treatment -- taken together with the low overall risk -- suggests that physicians and patients should discuss the risks and benefits of discontinuing treatment with SSRI before breast cancer surgery.Use of SSRI is associated with an increased risk of re-operation due to bleeding after surgery for breast cancer, from about 3% to about 7%. However, the magnitude of the risk difference does not merit pausing antidepressive medicine in psychologically vulnerable patients without a complete discussion of the risks and benefits.(SSRI): Selective serotonin reuptake inhibitors; (CPR): Civil Personal Registration; (ICD): International Statistical Classification of Diseases and Related Health Problems; (NSAIDs): non-steroidal anti-inflammatory drugs; (TCAs): tri-cyclic anti-depressants; (TeCA): tetracyclical anti-depressants; (ATC): Anatomical Therapeutic Chemical.The authors declare that they have no competing interests.RG put forward the hypothesis; made substantial contributions to conception and design and interpretation of the data; and drafted the paper: abstract, introduction, and discussion. DCF was involved in study conception and design; performed the statistical analysis, data interpretation and conclusions; drafted the methods and results section; reviewed and edited the paper. HHH & LP provided the datasets and statistical analysis and support. TLL was involved in revising the manuscript critically for important intellectual content and consistency regarding analysis, interpretation and conclusions. HTS was involved in study conception, design and data collection and revised the manuscript critically for important intellectual content and consistency regarding analysis, interpretation and conclusions. NK supported the development of the hypothesis, helped to draft the paper and was involved in the clinical interpretation of the results and revision of the manuscript for important intellectual content.All authors read and approved the final manuscript.ATC codes for potential confounding drugs:Platelet inhibitors: , dipyridamol B01AC07; clopidogrel B01AC04);Oral anti-coagulants: ;NSAIDs: ;Anti-depressants: Tri-cyclic anti-depressants (TCAs): Desipramine N06AA01; imipramin N06AA02; imipramine oxide N06AA03; clomipramin N06AA04; opipramol N06AA05; trimipramine N06AA06; lofepramine N06AA07; amitriptylin N06AA09; nortriptylin N06AA10; protriptyline N06AA11; doxepin N06AA12; dosulepin N06AA16; amoxapine N06AA17; Tetracyclical anti-depressants (TeCA): Maprotilin N06AA21, Mianserin N06AX03; Other anti-depressants: Duloxetin N06AX21, venlafaxin N06AX16, Mirtazapin N06AX11, Reboxetin N06AX18, Isocarboxazid N06AF01, Moclobemid N06AG02.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2482/10/3/prepub


Daphnia  provides a model experimental system for the study of the developmental mechanisms and evolutionary processes associated with predator-induced polyphenisms. In D. pulex, juveniles form neckteeth in response to predatory kairomones released by Chaoborus larvae .Numerous cases of predator-induced polyphenisms, in which alternate phenotypes are produced in response to extrinsic stimuli, have been reported in aquatic taxa to date. The genus D. pulex can generally be divided into the embryonic and postembryonic developmental periods. We therefore examined which of the genes in the embryonic and first-instar juvenile stages exhibit different expression levels in the presence or absence of predator kairomones. Employing a candidate gene approach and identifying differentially-expressed genes revealed that the morphogenetic factors, Hox3, extradenticle and escargot, were up-regulated by kairomones in the postembryonic stage and may potentially be responsible for defense morph formation. In addition, the juvenile hormone pathway genes, JHAMT and Met, and the insulin signaling pathway genes, InR and IRS-1, were up-regulated in the first-instar stage. It is well known that these hormonal pathways are involved in physiological regulation following morphogenesis in many insect species. During the embryonic stage when morphotypes were determined, one of the novel genes identified by differential display was up-regulated, suggesting that this gene may be related to morphotype determination. Biological functions of the up-regulated genes are discussed in the context of defense morph formation.Previous studies suggest that the timing of the sensitivity to kairomones in It is suggested that, following the reception of kairomone signals, the identified genes are involved in a series of defensive phenotypic alterations and the production of a defensive phenotype. Daphnia, commonly called the waterflea, is considered to be a model case for elucidating the ecological and developmental underpinnings of this process . The. The6].  insects ,28-30. BD. pulex defense morph formation, it is thought that morphogenetic factors such as those that have been identified in many arthropods are expressed downstream of physiological regulation , morphogenetic genes , endocrine genes  and neuronal genes [tyramine beta-monooxygenase (TBM) and dopamine beta-monooxygenase (DBM)]. BLASTX searches http://blast.ncbi.nlm.nih.gov/Blast.cgi confirmed that the predicted D. pulex sequences are homologues of the candidate genes  genes were tested as reference genes for real-time qPCR. Since GAPDH was considered to be the most stable gene and its expression levels were closest to those of the candidate genes (data not shown), GAPDH was used as the reference gene. This result was consistent with a previous report of expression stability in D. magna following exposure to the drug, ibuprofen [The relative expression levels of the candidate genes were quantified using real-time quantitative RT-PCR to examine whether these genes were differentially expressed after exposure to the predator kairomone in the embryonic stage (stage 4) and the postembryonic instar (first-instar). 18S ribosomal RNA, buprofen .abd-A and FOXO) were not amplified by PCR, probably because the designed primer sites were inappropriate. Since a number of traits in first-instar juveniles change in response to defense morph formation , the candidate genes were thought to be up-regulated in the preceding developmental stage (i.e. embryonic stage 4), although the expected results were not obtained. It is therefore suggested that the up-regulated genes are involved in neckteeth development, which is most pronounced at the second- and third-instar [Daphnia genome database can be used for the analysis of the molecular mechanisms responsible for the defense morph formation.Real-time qPCR revealed that 21 of the 31 candidate genes were up-regulated in the presence of kairomones in first-instar juveniles. Conversely, none of the candidate genes were up-regulated by more than 1.5-fold at the embryonic stage 4  and the two gene candidates thought to be associated with JHAMT (Met) and InR (IRS-1), we reperformed the real-time qPCR using biological replicates to analyze the detailed expression profiles of kairomone-responsive genes whose functions were suspected of being involved in defense morph formation , we excluded TBM from further analyses.To further clarify differences in the expression of the six candidate genes showing the most marked up-regulation , has lost the function of specifying segmental identity during embryogenesis [Hox3, expression in D. pulex has been reported in the nuchal area (where the neckteeth subsequently form) and in the mandibular mesoderm during the early- and mid-embryonic stages, respectively [Daphnia neckteeth cannot be considered to be homologous to appendages, it is possible that the molecular mechanisms for appendage development are co-opted for neckteeth development.Compared to conditions without kairomones, the expression levels of s Figure . Hox3 isogenesis ,45-47. Aectively , suggestExd and esg respectively encode a homeobox transcription factor and a zinc finger transcription factor, and both are known to determine the proximal segmental identity of appendages (coxa and trochanter) in Drosophila melanogaster [dac, a known selector gene for the femur and tibia in D. melanogaster, and Dll, which defines tarsus and pretarsus, did not produce as conspicuous a response as exd and esg in first-instar juveniles  . Indeed, further analyses of this hypothesis will provide us with insights, not only into defense morph formation in Daphnia, but also into the evolution of appendage morphology in arthropods.nogaster . Furtherppendage , was alspendages . It has pendages . This isDD1, DD2 and DD3) showed marked up-regulation in response to kairomone exposure . For the single fragment for which the functional sequence could not be predicted by the gene predictor, the full sequence was determined by rapid amplification of cDNA ends (RACE)-PCR. Of these 22 sequences, we performed real-time quantitative RT-PCR to confirm their responsiveness as described for the candidate genes above. As a result, three genes  domain. DOMON domains are ubiquitous among plants and animals, and exist in a variety of proteins, including dopamine beta-monooxygenase, in which this domain was originally found [D. pulex, DD1 is thus considered to be a novel gene containing a DOMON domain. In addition to the aforementioned dopamine beta-monooxygenase (DBM), representative proteins containing a DOMON domain also include tyramine beta-monooxygenase (TBM), which is involved in the biosynthesis of biogenic amines [DD1 were completely different to those of DBM and TBM (data not shown), and the expression profiles of DD1 also did not correspond to those of DBM and TBM  tail, which is not typically present in bacterial transcripts and suggests that the identification of DD2 was not the result of contamination. Interestingly, in addition to kairomone responsiveness, the expression levels of DD2 were more than 100-fold higher in the first-instar juveniles than in the embryos   was exposed to nerve growth factor (NGF) [GTD-P is involved in the cellular proliferation observed during defense morph formation in D. pulex.P) Table . Althougor (NGF) . As it wD. pulex embryos and first-instar juveniles. Most of the differences in gene expression induced by the kairomone exposure were observed in postembryonic juveniles, while a single novel gene, DD1, was up-regulated in the embryonic stage. Taken together, a putative physiological and developmental cascade for the defense morph formation consisting of the following steps is suggested: 1) Kairomone reception by embryos, 2) Physiological changes through endocrine mechanisms including JH and insulin pathways, 3) Morphogenesis triggered by pattern formation genes , Tsukuba, Japan. The NIES clone, originally from Lake Kasumigaura in Japan, was reared in the laboratory at 20°C in aged tap water and fed unicellular green algae  over generations. Using an established rearing method [Daphnia clone were maintained in 1 L beakers in a temperature- and photocycle-controlled incubator .The g method , populatChaoborus flavicans larvae were collected from a pond at NIES and maintained in dechlorinated tap-water at a density of 10-15 larvae/L for more than 7 days in a temperature- and photocycle-controlled incubator on a diet of D. pulex.Fourth-instar Chaoborus larvae, the water was filtered using a Whatman GF/C filter  to remove any daphnid juveniles and particles larger than approximately 1 μm, before being stored in plastic bottles at -20°C. For the experiments, the frozen water was thawed in an incubator (23°C) and used as a rearing medium for D. pulex (Chaoborus-conditioned medium). Dechlorinated tap-water, in which Chaoborus had never been reared, was filtered using a Whatman GF/C filter for use as control medium. The experimental and control media were changed every day.After incubation with D. pulex were severally used to detect genes specifically expressed in response to the exposure to predator kairomones, with particular emphasis on the expression of those genes that manifested in defense morph formation. We focused on these developmental stages, because, after exposure to kairomones during the embryonic stages of development, fate determination into the defense morph should occur during embryonic stage 4 and morphogenesis of the crest and neckteeth should occur in the first instar . To obtain total RNA from first-instar juveniles, egg-bearing females were reared in the kairomone and control media . After one-week of rearing, newly-born juveniles were harvested and continuously incubated in the kairomone media. From the next day onward, only first-instar juveniles were collected everyday. Harvested individuals were then frozen in liquid nitrogen and preserved at -80°C. Total RNA was extracted from approximately 2,000 daphnia for each treatment (kairomone and control) using an SV Total RNA Isolation System . To ensure reproducibility, we conducted an additional series of induction experiments and repeated the RNA extraction procedure for quantitative RT-PCR. In addition, to control for any possible artifacts due to freezing of the kairomone medium or any elution from the plastic bottles used for sample storage, we also tested individuals that had been exposed to control medium that had been frozen and melted in the plastic bottles.To obtain total RNA from stage 4 embryos, very early stage embryos (stage 1-2) were removed from the maternal brood chambers and treated with either the kairomone or control media. After incubation at 20°C for 24-48 h, stage 4 embryos were collected and total RNA was extracted from about 100 individuals for each treatment (kairomone or control), using RNAqueousDaphnia defense morph formation  were obtained using euGene's Arthropod genomes http://insects.eugenes.org/arthropods/, and aligned by the CLUSTALW program from GenomeNet http://align.genome.jp/. Amino acid alignments were made principally with the flour beetle (Tribolium castaneum), the pea aphid (Acyrthosiphon pisum), several species of Drosophila, and the tick (Ixodes scapularis). Based on the aligned sequences, conserved regions were identified and used to perform TBLASTN searches against the wFleaBase http://wfleabase.org/ to identify D. pulex homologues. Subsequently, the predicted coding sequences generated by gene prediction software on wFleaBase  were used as the sequences for the candidate genes . The resultant cDNA samples were amplified by PCR in reaction mixtures (20 μl) containing 20 combinations of arbitrary 12-mers with a HindIII site , the anchor primer (20 μM), and AmpliTaq Gold polymerase . The PCR conditions were as follows: one cycle at 94°C for 5 min, followed by 40 cycles at 94°C for 30 sec, 40°C for 2 min and 72°C for 30 sec, and a final extension at 72°C for 5 min. The PCR products were separated on a non-denaturing 12% polyacrylamide gel and visualized by ethidium bromide staining to identify differential bands between the two experimental conditions. To ensure that the results were reproducible, duplicate PCRs and electrophoresis runs were performed.Differential display (DD) was employed to identify genes that were involved in defense morph formation, but which were not detected using the candidate gene approach. The DD assays were performed according to a previously-described method with slight modifications ,55,56, uhttp://www.ebi.ac.uk/Tools/InterProScan/.Differential cDNA bands were excised from the gels and re-amplified by PCR. Subsequently, the cDNA fragments were cloned into pGEM-T vector (Promega) according to the manufacturer's instructions before being sequenced with Big Dye terminator kit on a Model 3100 DNA sequencer (Applied Biosystems). To identify homologous sequences and to estimate gene function, similarity searches were performed with obtained sequences using the wFleaBase, NCBI BLAST database and the European Bioinformatics Institute (EBI) InterPro database DD2 fragment, rapid amplification of cDNA ends (RACE)-PCR was performed using synthesized RACE primers (5'-CCGTTACTCTTTAGGAGGAGACCGCCCC-3' for the 5'-RACE and 5'-TAGGATAGGTGGGAGGCTTTGAAGCGGG-3' for the 3'-RACE) and the SMART RACE cDNA Amplification Kit  to obtain the full-length sequence of DD2. The amplified cDNA fragments were cloned and sequenced as described above, and the full-length sequence thus obtained was subjected to an NCBI BLAST search.Since the homologous gene was not found using the gene predictor for the obtained Among the identified genes obtained by the candidate gene approach and by the differential display, the relative expression levels associated with the two experimental conditions (kairomone and control) were quantified and compared using real-time quantitative RT-PCR. Total RNAs of stage 4 embryos and first-instar juveniles that had been exposed to the kairomone and control media were extracted and reverse-transcribed using the conditions described above, except that random hexamer primers were used instead of the oligo-dT primer. Relative quantification of cDNAs was performed using a SYBR Green I chemistry system and ABI Prism 7500 sequence detection system (Applied Biosystems). As endogenous controls of constitutive expression, GAPDH  (Accession No. FJ668125), actin (Accession no. AJ245732) and/or 18S ribosomal RNA (Accession no. AF014011) genes were used. Primers for both target and control genes were designed using Primer Express software .Click here for fileTable S3: Protein ID and primers for real-time qPCR.Click here for file


Osteosarcoma is the most common type of solid bone cancer and the second leading cause of cancer-related death in pediatric patients. Many patients are not cured by the current osteosarcoma therapy consisting of combination chemotherapy along with surgery and thus new treatments are urgently needed. In the last decade, cancer stem cells have been identified in many tumors such as leukemia, brain, breast, head and neck, colon, skin, pancreatic, and prostate cancers and these cells are proposed to play major roles in drug resistance, tumor recurrence, and metastasis. Recent studies have shown evidence that osteosarcoma also possesses cancer stem cells. This review summarizes the current knowledge about the osteosarcoma cancer stem cell including the methods used for its isolation, its properties, and its potential as a new target for osteosarcoma treatment. Osteosarcoma is the most common type of solid bone cancer, mainly arising in children and young adults. About 6 in every million children and 2 in every million adults will develop osteosarcoma . OsteosaOsteosarcoma is believed to arise from mesenchymal stem cells (MSCs) or osteoprogenitor cells due to a disruption in the osteoblast differentiation pathway ,6. GenetSince the 1970s, combination chemotherapy along with limb-sparing surgery has been the main treatment for osteosarcoma. The most commonly used chemotherapeutic regimen includes pre- and post-operative cisplatin and doxorubicin with or without high-dose methotrexate . Many paThe cancer stem cell hypothesis proposes that within a heterogeneous tumor there is a small subpopulation of cells called "cancer stem cells (CSCs)" that are responsible for forming the bulk of the tumor -16. They+CD38-) to normal hematopoietic stem cells [+CD38- leukemic stem cell population but not the CD34+CD38+ or CD34- population was able to form AML in SCID mice.The first evidence of the existence of CSCs came from studies of hematological malignancies. In 1994, Lapidot and colleagues showed evidence that only a small percentage of acute myeloid leukemia (AML) cells were capable of initiating leukemia in mice . They foem cells ,18. InteFollowing the success in hematological malignancies, FACS and magnetic-activated cell sorting (MACS) for stem cell surface markers including CD34, CD138, CD20, CD90, CD133, and CD44 have now been widely employed to identify CSCs in a number of cancers . HowUsing the above mentioned assays, the presence of CSCs has now been identified not just in hematological malignancies but also in a number of solid tumors including breast, brain, skin, lung, colon, pancreatic, liver, head and neck, and prostate cancers . Overall+ glioma stem cells are less sensitive to radiation and undergo less radiation-induced apoptosis than CD133- glioma cells both in vitro and in vivo. In fact, radiation enriches the percentage of CD133+ glioma stem cells relative to other tumor cells [+ glioblastoma stem cells are more resistant to the chemotherapeutic agents temozolomide, carboplatin, paclitaxel and etoposide compared to CD133- cells [+/CD24-/low breast CSC population [Similar to stem cells, evidence suggests that CSCs are resistant to cancer therapies including radiation and chemotherapy. For example, CD133or cells . CD133+ 3- cells . Neurobl3- cells ,30. Pairpulation . CSC chapulation . Therefopulation ,19.Although CSCs may be resistant to chemotherapy, evidence from studies of leukemia has shown that it is possible to find drugs that specifically inhibit the growth of CSCs. For example, the anthracycline idarubicin in combination with the proteasome inhibitor MG-132 induced apoptosis of AML stem cells in vitro and in vivo with no effect on normal hematopoietic stem cell viability .+ AML patients 60 years and older who are not eligible for other cytotoxic therapies [+nestin+ brain cancer stem cells in mice [Several methods have been proposed to target the CSC . One metherapies . Another in mice . Inducti in mice . HoweverSince the proposal of the CSC hypothesis, many studies have been performed to identify the osteosarcoma CSC. Currently, there are three methods that have now been employed to enrich for osteosarcoma CSCs including: (1) the sphere culture assay (or sarcosphere assay), (2) cell sorting for CD133, high ALDH activity, SP cells, or CD117 in combination with Stro-1, and (3) identification of cells that express the embryonic stem cell gene Oct4. This review will summarize each of these methods below.Gibbs et al. (2005) were the first to show that osteosarcomas possess cells with CSC characteristics . When gr+ population (3-5%) in the human osteosarcoma cell lines MG-63, Saos-2, and U20S with stem cell characteristics [- cells, these cells had an increased percentage of cells in G2/M phase, were Ki67-positive and had increased in vitro growth, indicating that they are more proliferative. CD133+ cells, but not CD133- cells, were capable of forming spheres in culture and had an increased ability to form colonies in a soft agar assay. Cells obtained from spheres formed by CD133+ cells were capable of forming new spheres containing both CD133- and CD133+ cells, indicating that CD133+ cells can differentiate into CD133- cells. Spheres initially formed from CD133+ cells and passaged 4 to 6 times showed increased expression of Oct4 and CD133. In addition to expressing CD133, the human osteosarcoma cell lines Saos-2, OSA-1, OSA-2, and OSA-3 also express nestin, a marker for neural stem cells and brain CSCs, suggesting that nestin and CD133 might be used as co-markers for identifying osteosarcoma CSCs [CD133 (prominin-1) is a pentaspan membrane glycoprotein used initially as a marker for neuroepithelial stem cells and has been subsequently used as a marker for many CSCs including brain and colon CSCs -45. Receoma CSCs .+ Saos-2 cells do possess a small side population (0.97%) [SP cells are capable of effluxing the DNA-binding dye Hoechst 33342 using ATP-binding cassette (ABC) transporters. This ability to efflux Hoechst dye was first identified as a characteristic of normal haematopoietic stem cells ,48 but h (0.97%) . These rALDHs are a group of cytosolic enzymes that oxidize intracellular aldehydes into carboxylic acids . High AL+Stro-1+ (DP) cells. DP K7M2 and 318-1 mouse osteosarcoma cells were more resistant to the chemotherapeutic doxorubicin than CD117-Stro-1- (DN) and parental cells. Both mouse and human DP osteosarcoma cells had increased expression of ABCG2 and CXCR4 compared to DN cells. DP mouse 318-1, K7M2, and P932 and human KHOS, BCOS, and MNNG/HOS osteosarcoma cells had increased tumorigenicity when subcutaneously injected into nude mice compared to DN cells derived from the same cell line. 318-1 DP cells produced tumors not just with DP cells but also DN and single positive, suggesting that 318-1 DP cells not only self-renew but also can differentiate and reform all of the cells within the tumor. When 318-1 DP cells were injected into the femoral bone marrow cavity of NOD/SCID mice, they had increased primary tumor take and metastasis to the lung. These lung metastases had more cells positive for the markers CD117, Stro-1, ABCG2, and CXCR4 than the primary bone tumor [CD117(c-kit) is the receptor for stem cell factor and a known proto-oncoprotein. It is also one of the markers used to isolate CSCs from ovarian cancer ,65. Strone tumor , suggest+ cells from xenograft tumors also expressed the MSC markers CD105 and ICAM-1. Moreover, GFP-enriched cells were more than 100 fold more tumorigenic than GFP-depleted cells, capable of forming subcutaneous tumors with less than 300 cells in NOD/SCID mice and metastasizing to lung. These cells could also differentiate and form Oct4/GFP- cells.Oct4 is a central determinant of embryonic stem (ES) cell identity and one of four transcriptional factors which, when introduced together, were sufficient to reprogram differentiated fibroblasts to confer pluripotency indistinguishable from ES cells . Based oOverall, the methods mentioned above show evidence that a subpopulation of osteosarcoma cells do exist with cancer stem cell characteristics. One interesting common feature of the CSCs derived from the different isolation methods is that they all have increased expression of genes required for ES cell maintenance (Oct4 and Nanog) ,45,50,63CSCs have been proposed to arise either from the transformation of normal stem cells to cancerous stem cells or from the dedifferentiation and transformation of progenitor or terminally-differentiated cells to tumor cells with stem cell-like characteristics . OsteosaTaking into account the CSC hypothesis, we propose that MSCs might be the cells of origin for osteosarcoma CSCs. Therefore, further understanding of the MSC may aid in the understanding of the osteosarcoma CSC. Currently the markers for isolating MSCs are controversial and not as defined as the hematopoietic stem cell (reviewed in ). One ofNormal stem cells are found within niches (microenvironments) that support the stem cell. Stem cells and niche cells interact with each other via adhesion molecules and molecular signals that are important for maintenance of stem cell self-renewal, differentiation, and quiescence . For exa+CD133+ brain CSCs were found next to capillaries in brain tumors and adhere to endothelial cells [+ human medulloblastoma cells with endothelial cells into mice increased tumor formation [Like normal stem cells, CSCs also require a microenvironmental niche to maintain stemness. CSCs may form their own niche or take over normal stem cell niches ,76,77. Tal cells . Co-injeormation . If CSCsormation ,37. OS99ormation . TherefoThe osteosarcoma CSC niche has not been defined. However, if osteosarcoma CSCs arise from MSCs, it is feasible that they may reside within the proposed MSC niche, a perivascular niche (reviewed in ). The loSince the local environment affects the behavior of CSCs, studying osteosarcoma CSCs in the context of its local environment, the bone, may be important for determining how to target osteosarcoma CSCs for treatment. The bone is a unique environment with properties that could alter the behavior of a CSC. For example, the bone is a hypoxic environment . Activat+Stro-1+ mouse osteosarcoma cells compared to CD117-Stro-1- cells in the femur of NOD/SCID mice. However, no one has studied human osteosarcoma CSCs in an orthotopic model. This is most likely because there are currently very few reports of untransformed human osteosarcoma cell lines that are commercially available and able to grow within this model [The orthotopic osteosarcoma model is produced by injecting osteosarcoma cells into the long bones of immuno-compromised mice. Despite the importance of the local environment in CSC behavior, to date, only one group has published results looking at the growth of potential osteosarcoma CSCs in an orthotopic model . Adhikaris model . FurtherThere is compelling evidence that osteosarcoma tumors possess cancer stem cells. This will have a great impact on the design and evaluation of novel treatments for osteosarcoma. The current treatment, chemotherapy together with surgical removal, can only cure around 70% of osteosarcoma patients because of chemoresistance ,12. OsteIn order to develop CSC-targeted therapy, it is important to be able to specifically isolate the CSCs. Although the methods utilized to detect the osteosarcoma CSC show populations with enriched stem cell-like characteristics, no specific markers for the osteosarcoma CSC have been established. One immediate question is: What are the correct markers to isolate the osteosarcoma CSC? Further understanding of the MSC, a putative cell-of-origin for the osteosarcoma CSC, could aid in successful specific isolation of the osteosarcoma CSC.Once we specifically isolate the osteosarcoma CSC, another question is: How can these cells be targeted and killed? One way to detect therapeutic targets in CSCs is to determine how these cells differ genetically from other non-CSCs using microarray analyses. One recent study found that MG-63 spheres have increased expression of the DNA repair enzyme genes MLH1 and MSH2 compared to adherent cells and increased resistance to the common osteosarcoma therapeutics cisplatin and doxorubicin . TreatmeAlthough the CSC may be a great new target for cancer therapy, one major problem with the CSC as a therapeutic target is that it has many similar properties to normal stem cells. This leads to the third question: How do osteosarcoma CSCs differ from normal stem cells? It will be important to monitor the effect of proposed CSC therapeutics on normal stem cells to ensure a limited amount of non-specific toxicity. Further understanding of the osteosarcoma CSC will aid in determining how to target it. Microarray analyses can determine genes that are upregulated in the osteosarcoma CSC compared to non-CSCs but not in the normal stem cell population. High-throughput screening could identify drugs that CSCs are sensitive to, while leaving the normal stem cells unharmed. Ultimately, the development of new therapies targeting the osteosarcoma CSC requires the monitoring of any effect on normal stem cells as a potential side-effect.The authors declare that they have no competing interests.VS and LQ both reviewed the literature and decided upon the content of this review. VS wrote the first draft and both VS and LQ edited the manuscript. All authors read and approved the final manuscript.


Anti-TNF therapies represent a breakthrough in the treatment of severe psoriatic arthritis. However, little is known about long-term drug persistence with these treatments in patients with psoriatic arthritis in routine clinical practice. The aim of this study was to assess persistence with first-course and second-course treatment with anti-TNF agents in a prospective cohort of psoriatic arthritis patients and to identify factors associated with and reasons for drug discontinuation.A total of 566 patients with psoriatic arthritis were registered with the British Society for Rheumatology Biologics Register . Treating physicians completed 6-monthly follow-up questionnaires detailing changes to anti-TNF therapies. Persistence with treatment was examined using Kaplan–Meier survival analysis. Reasons for withdrawal were classified as due to inefficacy, adverse events or other reasons. Univariate and multivariate Cox proportional hazard models were developed to examine potential predictors of withdrawals due to inefficacy or adverse events, using a range of demographic, baseline disease-specific and therapeutic variables.At baseline, the mean (standard deviation) age of patients was 45.7 (11.1) years, 53% were female and the mean disease duration was 12.4 (8.7) years. Persistence data were available for a mean (standard deviation) follow-up of 2.3 (0.9) person-years. In total, 422 patients had completed at least 12 months of follow-up, 75.5% of whom remained on their first anti-TNF drug while 9.5% discontinued due to inefficacy, 10.0% due to adverse events and 5.0% due to other reasons. During the period of follow-up, 178 patients received a second anti-TNF therapy. The survivor function on second anti-TNF for switchers was 74% at 12 months.Psoriatic arthritis patients show high persistence rates with both initial and second anti-TNF therapies. The development of anti-TNF therapies has dramatically improved the management of a range of autoimmune diseases, including psoriatic arthritis (PsA). TNF acts in the early stages of the inflammatory process, during which it can stimulate T-cell activation and induce the expression of IL-2, IFNγ receptors, proinflammatory cytokines (such as IL-1 and IL-12) and proinflammatory chemokines (such as IL-8) [The currently available anti-TNF agents  have been studied in a number of randomised controlled trials that assessed their efficacy and safety in PsA -7. A recThere are only limited data available examining long-term persistence with these therapies in patients with PsA who are managed in routine clinical practice . The preSubjects included in the study were selected from the British Society for Rheumatology Biologics Register (BSRBR) . The BSRThe British Society for Rheumatology guidelines for PsA published in February 2005 recommend that anti-TNF drugs should be reserved for patients with active PsA (defined as ≥ 3 tender joints and ≥ 3 swollen joints) despite adequate therapeutic trials of at least two standard disease modifying anti-rheumatic drugs individually or in combination . During At the time of initiation of the anti-TNF therapy, the rheumatologist or rheumatology nurse specialist completed a consultant baseline questionnaire that collected data on the patient's age, gender, diagnosis and disease duration, and information about current disease activity, including swollen and tender joint counts (based on the 28-joint count), the erythrocyte sedimentation rate and/or the C-reactive protein level. The 28-joint Disease Activity Score (DAS-28) was then calculated . DetailsEach patient completed a patient baseline questionnaire that included details about their current work status, ethnicity, smoking history, the Health Assessment Questionnaire (HAQ) adapted for British use , and theThe rheumatology consultants/nurse specialists were sent a 6-monthly postal follow-up questionnaire for 3 years and then annual follow-ups thereafter. This consultant follow-up questionnaire recorded details of all anti-TNF therapies received, including start and stop dates and reasons for discontinuation. The reasons for stopping were documented by the clinician and were classified into lack of efficacy, adverse events (AEs), or other reasons. In addition, data were recorded for calculation of the DAS-28. Details of all AEs resulting in drug discontinuation were classified using the MedDRA system organ classification .Analysis was limited to those patients for whom at least one consultant follow-up questionnaire had been returned, as data on drug persistence were otherwise not available. Persistence with anti-TNF therapies was examined for both the first and second treatment courses using Kaplan–Meier survival analysis. For the purpose of this study, drug persistence was defined as 'the length of time from initiation to discontinuation of therapy' . PatientUnivariate and multivariate Cox proportional hazard models were used to identify factors associated with drug discontinuation of the first course of anti-TNF therapy. Separate models were developed for overall discontinuation, for discontinuation due to inefficacy and for discontinuation due to adverse events. The following covariates were examined in the models: baseline demographic variables (age (years), therapy start year, gender, current smoking status (yes/no), whether the patient had additional baseline co-morbidities (yes/no)), baseline disease-specific variables  >20 mg/l and/or erythrocyte sedimentation rate >28 mm/hour – 28-tender joint count and 28-swollen joint count, HAQ, disease duration (years)) and therapeutic variables ). In the multivariate analyses, we used the composite DAS-28 score as a potential predictor rather than its individual components. The results are presented as hazard ratios (HRs) with corresponding 95% confidence intervals (CIs). All calculations were undertaken using STATA version 9.0 .The study was approved by the North West NHS Multicentre Research Ethics Committee, and all subjects gave their written consent for participation.A total of 596 biologically naive PsA patients were registered with the BSRBR. Persistence data were available for 566 patients with PsA (95%). Of these, 316 (55.8%) patients commenced treatment with etanercept, 162 (28.6%) patients started with infliximab and 88 (15.6%) patients started with adalimumab.The baseline characteristics of the PsA patient cohort and the three anti-TNF groups are presented in Table At baseline, 37.0%, 30.7% and 32.3% of patients had no co-morbidity, one co-morbidity or more than one co-morbidity, respectively. The most frequent co-morbid condition was hypertension, which was present in 29.2% of the patients. Baseline co-morbidities were classified by body systems into cardiovascular (33.9%), pulmonary (13.9%), endocrine (6.7%), gastrointestinal (13.6%) and central nervous system disorders (21.6%). There was no significant difference between the three agents in the presence of baseline co-morbidities.The mean (standard deviation) follow-up for all patients was 2.3 (0.9) person-years. The mean length of follow-up for patients receiving infliximab was 2.5 person-years, for etanercept was 2.1 person-years and for adalimumab was 1.6 person-years – reflecting the time that these drugs became readily available on the market. Kaplan–Meier survival analyses showing rates of discontinuation for inefficacy or AEs are presented in Table Details of AEs leading to withdrawal of treatment for the three anti-TNF agents are presented in Table Table For discontinuation due to inefficacy, the univariate analyses suggested that patients who smoked , who had higher baseline HAQ scores  or who were receiving infliximab  were more likely to discontinue treatment due to inefficacy. In the multivariate analysis in which we simultaneously controlled for all of the potential explanatory variables, only treatment with infliximab rather than etanercept was found to be significantly associated with drug discontinuation for inefficacy , although there was also a nonstatistically significant trend towards better survival in patients receiving concomitant disease-modifying anti-rheumatic drug therapy and in those with higher baseline disease activity.For drug discontinuation due to AEs, the presence of baseline co-morbidities  and the use of infliximab rather than etanercept  were associated with significantly higher drug discontinuation rates. There were no significant associations between any of the other potential predictors.A total of 178 patients received a second course of therapy with an alternative anti-TNF drug. Not surprisingly, the mean (standard deviation) length of observation was shorter for the second course of anti-TNF therapy (1.3 (0.8) person-years) compared with that of the first course (2.3 (0.9) person-years). Drug discontinuation rates in those patients who switched treatment to a second anti-TNF therapy are presented in Table The present observational study, reflecting clinical practice in the UK, has shown that persistence with anti-TNF agents in PsA is good, with an estimated 1-year drug survival of 82%. Over a mean follow-up period of 2.3 person-years, 30.8% of patients discontinued their first anti-TNF treatment, mainly because of lack of efficacy (12.4%) or AEs (11.4%). The survivor function on second anti-TNF for switchers was 74% at 12 months.Previous studies examining persistence with biological therapies in patients with PsA have only reported on persistence with the first anti-TNF treatment. An earlier study from the Spanish BIOBADASER register found that 88% of patients with PsA (n = 570) continued with their first anti-TNF drug for 12 months, compared with 83% of RA patients  . More reTo our knowledge, three other studies have formally explored possible predictors of drug discontinuation in patients with PsA treated with anti-TNF therapies. Kristensen and colleagues suggested that concomitant use of methotrexate and high C-reactive protein levels were associated with treatment continuation in a study of 261 PsA patients using anti-TNF drugs . Gomez-RData from the BSRBR reflect routine clinical practice in one of the largest prospective cohorts of patients with PsA. The register is therefore well suited for an investigation of drug persistence and switching between anti-TNF therapies. There are potential limitations to the present study, however, which should be considered when interpreting the findings. First, treatment decisions were not randomised but were left to the discretion of the treating physician, based on clinical opinion as to whether a patient's treatment had failed and the initial anti-TNF therapy should be discontinued. Second, as the BSRBR was originally developed as a RA register, there are certain aspects of the patient's PsA that were not captured – such as the pattern of joint involvement  and the Psoriasis Area and Severity Index scores, which may also have influenced the physician's decision on choice of anti-TNF agent as well as judgements about lack of efficacy.A full set of domains to assess improvements in PsA patients would ideally include outcomes that measure skin involvement, dactylitis, enthesitis, spine involvement and radiological outcomes. For feasibility reasons, however, the short form of the Disease Activity Score the DAS-28) was used, which has been shown to be discriminant between anti-TNF drugs and methotrexate in PsA patients . Physica was usedThe present observational study found that, for patients starting anti-TNF therapy between 2002 and 2006, there was better persistence with etanercept when compared with infliximab, which is in accordance with data published earlier from the Swedish  and the Firstly, infliximab was the first anti-TNF drug to be marketed and, during the early years of this study, more patients were receiving infliximab as their first anti-TNF therapy, despite the drug not being licensed for use in PsA. These early patients were likely to be those with the most severe disease, and as a consequence they may have had a different response to those recruited later; the potential risk of channelling bias can therefore not be discounted . AdjustiFurthermore, and perhaps most importantly, only 22% of patients treated with infliximab were receiving the licensed dose for PsA (5 mg/kg), with the remaining 78% receiving the dose of 3 mg/kg recommended for use in RA. This is probably a result of the drug not receiving its license for use in PsA until 2004 and national guidelines not being issued until 2005, and thus most physicians may have applied the same dosing regimen used for RA to these patients. In keeping with this, 73% of patients with PsA included in the present study received infliximab before the date the drug was licensed for this specific indication. All of these reasons may explain the observed differences in drug survival, and therefore no definite conclusions about differential drug efficacy should be made from the present analysis.The present study has provided important insights into persistence rates with anti-TNF therapies in a large unselected population of patients with PsA. We have demonstrated that persistence in patients with PsA is at least as good as in those with RA, and that the main predictor of stopping for AEs is the presence of baseline co-morbidities. Our results have also shown that the survivor function in those patients receiving a second anti-TNF therapy was 74% at 12 months. Further studies are needed to explore the clinical effectiveness and cost-effectiveness of sequential use of different anti-TNF therapies in patients with PsA.AE: adverse event; BSRBR: British Society for Rheumatology Biologics Register; CI: confidence interval; DAS-28: 28-joint Disease Activity Score; HAQ: Health Assessment Questionnaire; HR: hazard ratio; IFN: interferon; IL: interleukin; PsA: psoriatic arthritis; RA: rheumatoid arthritis; TNF: tumour necrosis factor.The British Society for Rheumatology commissioned the Biologics Register (BSRBR) as a UK-wide national project to investigate the safety of biologic agents in routine medical practice. DPMS and KLH are principal investigators on the BSRBR. The British Society for Rheumatology receives restricted income from UK pharmaceutical companies, presently Abbott Laboratories, Amgen, Schering Plough and Wyeth Pharmaceuticals. This income finances a wholly separate contract between the British Society for Rheumatology and the University of Manchester, who provide and run the BSRBR data collection, management and analysis services. The principal investigators and their team have full academic freedom and are able to work independently of pharmaceutical industry influence. All decisions concerning analyses, interpretation and publication are made autonomously of any industrial contribution. Members of the Manchester team, British Society for Rheumatology trustees, committee members and staff complete an annual declaration in relation to conflicts of interest.All authors were involved in protocol development, data analysis, interpretation of the findings and preparation of the final manuscript. AAS also carried out data extraction and prepared the first draft of the manuscript. DPMS and KLH are the principal investigators on the BSRBR, and KDW is the study coordinator.


As important regulators of developmental and adult processes in metazoans, Fibroblast Growth Factor (FGF) proteins are potent signaling molecules whose activities must be tightly regulated. FGFs are known to play diverse roles in many processes, including mesoderm induction, branching morphogenesis, organ formation, wound healing and malignant transformation; yet much more remains to be learned about the mechanisms of regulation used to control FGF activity.Drosophila proteins, Thisbe (Ths) and Pyramus (Pyr), which share homology with the FGF8 subfamily of ligands in vertebrates. Ths and Pyr proteins are secreted from Drosophila Schneider cells (S2) as smaller N-terminal fragments presumably as a result of intracellular proteolytic cleavage. Cleaved forms of Ths and Pyr can be detected in embryonic extracts as well. The FGF-domain is contained within the secreted ligand portion, and this domain alone is capable of functioning in the embryo when ectopically expressed. Through targeted ectopic expression experiments in which we assay the ability of full-length, truncated, and chimeric proteins to support cell differentiation, we find evidence that (1) the C-terminal domain of Pyr is retained inside the cell and does not seem to be required for receptor activation and (2) the C-terminal domain of Ths is secreted and, while also not required for receptor activation, this domain does plays a role in limiting the activity of Ths when present.In this work, we conducted an analysis of the functional domains of two We propose that differential protein processing may account for the previously observed inequalities in signaling capabilities between Ths and Pyr. While the regulatory mechanisms are likely complex, studies such as ours conducted in a tractable model system may be able to provide insights into how ligand processing regulates growth factor activity. Drosophila FGF ligands are all predicted to be much larger proteins with molecular masses of approximately 80 kD  for 12mins on an orbital shaker on high. The Heptane/Formaldehyde was removed and replaced with MeOH. The embryos were rinsed in MeOH 4 times and stored at -20°C for short term or -80°C for long term. Embryos were blocked in 1x western blocking reagent (Roche) for 30mins RT, and primary antibody incubations were performed overnight. Primary antibodies used were: anti-Even skipped rabbit  and anti-βgal rabbit . Secondary antibody was applied for 1-2 hrs. at RT: anti-rabbit 1:200 .35 Methionine, to assay the unmodified, full-length size of the proteins. Transcend™ Non-radioactive Translation Detection System  containing biotinylated lysine in the Transcend tRNA was used in conjunction with the TnT Coupled Reticulocyte kit to transcribe and translate pUASt-HA-Pyr(1-348) and pUASt-HA-Pyr(1-466), which were subsequently run on 10% SDS-PAGE and detected with anti-HA by Western Blot.TnT T7/T3 Coupled Reticulocyte Lysate System  was used with pBS-Pyr and pBS-Ths, incorporating SST and AS conceived of the study and designed the experiments, which were performed by ST. ST and AS wrote the manuscript. All authors read and approved the final manuscript.


Background: To evaluate the clinical and economic impact of a                    specialty care management program among patients with multiple sclerosis.Methods: This retrospective cohort analysis included patients aged                    ≥18 years with ≥2 claims of multiple                    sclerosis diagnosis and ≥1 multiple sclerosis medications from 1                    January 2004 to 30 April 2008. The outcome metrics included medication adherence                    and persistence, multiple sclerosis-related hospitalization, and multiple                    sclerosis-related cost. Multivariate analyses were performed to adjust for                    demographics and clinical characteristics.Results: Among the 3993 patients identified, 78.3%                    participated in the program and 21.7% did not. Over                    12 months, medication adherence and persistence improved among                    participants but deteriorated among non-participants . Multiple                    sclerosis-related hospitalization decreased from 9.6% to                    7.1% for participants, whereas it increased from 10.1%                    to 12.0% for the non-participant group                    (p < 0.001). Multiple                    sclerosis-related medical spending (non-pharmacy) decreased among participants,                    but it increased among non-participants . Total multiple                    sclerosis-related cost for both groups increased over time                    .Conclusions: This program was associated with improved medication                    adherence and persistence, reduced multiple sclerosis-related hospitalization,                    and decreased multiple sclerosis-related medical costs. Unfortunately, the cost                    savings in the medical component did not offset the increased pharmacy                    expenditures during the 12-month follow-up period. The cause is                relatively unknown, the pathophysiological mechanisms are diverse, and the disease                course is highly variable.5                In addition to the disease uncertainty and complexity, patients with MS experience a                wide range of symptoms including fatigue, cognitive dysfunction, bowel and bladder                dysfunction, weakness, spasticity, sexual dysfunction, and visual                        problems.5Multiple sclerosis (MS) is a chronic and progressive inflammatory disease of the                central nervous system that affects approximately 211,000 to 400,000 people in the                United States of America.7                Although the treatment efficacy has been established, adherence to pharmacotherapy                remains one of challenges because of difficult side-effects. To meet this need and                improve the management of MS, a specialty care management program has been                introduced in one of the largest commercially insured populations in the USA since                July of 2005. The intervention includes mailing medication and disease-specific                patient education materials directly to the patients. Nurses make assessment calls                at the beginning and follow-up assessments at months 3, 6, and 12, and every                12 months thereafter. They serve as a liaison to the pharmacy, a source                of medical information, and a cheerleader to encourage adherence despite frequent                difficult medication side-effects. The program also includes refill reminder calls.                These calls enforce the importance of medication compliance, inquire about                patient’s status and develop a relationship with the patient.Despite the availability of disease-modifying therapy (DMT) since 1993, the                management of MS remains challenging. With no cure available, treatment and disease                management focus on slowing progression and preventing relapse as well as                controlling symptoms.12 However, relatively little published evidence about MS care                management programs is available. Some earlier studies have shown the feasibility of                programs for patient education, exercise, specialist nurse, depression disease                management, and energy conservation among people with MS.17 Most of these studies were                symptom specific and few were conducted on a wide scale. There is a need to                understand the impact of this type of program and facilitate informed                decision-making for review and implementation. This study aims to address this                existing gap. The primary study hypothesis is that the specialty care management                program is associated with medication adherence and persistence, risk of MS-related                hospitalization, and MS-related cost of care. To our knowledge, this is the first                study to evaluate the effectiveness of a specialty care management program on these                clinical and economic outcomes among patients with MS.Many studies have reported the potential benefits of care management programs in                other therapeutic areas such as diabetes, congestive heart failure, coronary artery                disease, and asthma.SM), which includes 13                    geographically dispersed US commercial health plans, providing coverage for                    approximately 24.2 million members. The administrative data set consists of                    integrated medical claims, pharmacy claims, and eligibility files. The study                    database was developed in compliance with Health Insurance Portability and                    Accountability Act of 1996 regulations (HIPAA).This is a retrospective cohort study using administrative claims data from the                    HealthCore Integrated Research Database  code for MS (ICD-9-CM:                    340.xx) and at least one medical or prescription claim for MS medication between                    1 January 2004 and 30 April 2008. MS medications of interest included interferon                    beta-1a (both intramuscular (IM) and subcutaneous (SC)), interferon beta-1b,                    glatiramer acetate, mitoxantrone, and natalizumab. For patients enrolled in the                    specialty care management program (participant group), the index date was                    defined as the start date of program participation. As other patients                    (non-participant group) did not have a program participation start date, an                    index date was randomly assigned through Monte Carlo simulation with regard to                    the participant patients on treatment history (length of time from the first                    observed MS treatment to program participation) to maximize the comparability of                    treatment history between participant and non-participant groups for subsequent                    evaluation. Non-participants included those who elected to obtain their                    medications from other sources, those who resided in geographical regions where                    the care management was not implemented at the time of this study, and/or those                    who participated in other care management programs which cannot be identified in                    the database. All patients were required to have                    ≥12 months of continuous health plan enrollment prior to                    and after index date.The study population included those members aged 18 or above with at least two                    medical claims with an 19 Medication persistence                    referred to the duration of time from initiation to discontinuation of                            therapy,19 where discontinuation was defined as failing to obtain any MS                    medication within 60 days after the depletion of the previous days’                    supply. MS-related hospitalization was measured through identification of                    inpatient hospitalizations from medical claims in which there were any claims                    containing an ICD-9-CM code for MS. Total MS-related cost of care reflected the                    total allowable amount reimbursed by the health plans, which consisted of both                    medical and pharmacy components. The medical component included cost incurred in                    inpatient, emergency room, and outpatient settings associated with an ICD-9-CM                    code for MS. The pharmacy component referred to the cost of MS medications                    filled or administered .Three outcomes were assessed: medication adherence and persistence, MS-related                    hospitalization, and total MS-related cost of care during the                    12 months post-index period. Medication adherence was measured using                    medication possession ratio (MPR), defined as the ratio of the total days supply                    of medication dispensed during the period to the total number of days over the                    post-index 12-month period.All outcome measures were compared between the participant and non-participant                    groups. Statistical differences between groups were assessed using Wilcoxon                    rank-sum test for continuous variables and Pearson Chi-square test for                    categorical variables. An a priori two-tailed level of significance  was set at the 0.05 level for all analyses.Multivariate analyses were performed to evaluate the association between the                    intervention of the specialty care management program and each outcome while                    controlling for potential confounders. Generalized linear models were                    constructed using various response probability distributions and link functions,                    depending on the distribution of the outcomes and goodness of fit of the models.                    For instance, gamma distribution and log link function were used for total cost                    of care, while binomial distribution and logit link function were used for                    hospitalization. Covariates were chosen a priori for all models based on                    clinical relevance and baseline differences, which included age, gender,                    geographical region, plan type ,                    preferred provider organization (PPO)), Deyo–Charlson co-morbidity                    index, co-morbid conditions , the first MS medication on or after index date , length of treatment history (number of days                    between the first observed MS medication and index date), and baseline health                    care utilization .n = 3125) participated in                the specialty care management program and 21.7%                (n = 868) did not. Overall, the                mean age was 46.3 ± 9.6 years and                approximately three-quarters of the cohort (75.2%) were female.                Interferon beta-1a IM was the most common MS medication used (37.3%),                followed by glatiramer acetate (31.4%), interferon beta-1a SC                (16.6%), interferon beta-1b (13.5%), natalizumab                (0.8%), and mitoxantrone (0.5%). The participants and                non-participants were comparable in terms of age, Deyo–Charlson                co-morbidity index, and baseline co-morbid conditions (p < 0.001). A total of 3993 patients with MS were identified. Of them, 78.3%                    .                In multivariable regression analysis controlling for pre-index characteristics                (including pre-index MPR), the participant group on average had 0.18                : 0.16–0.19) higher MPR than                the non-participant group. Similarly, the participants were more persistent on MS                therapy (time from initiation to discontinuation of therapy) than the                non-participants . When pre-index                characteristics were controlled for in multivariate analysis, the participants on                average had a mean medication persistence of 50.6 (95% CI:                43.1–58.2) days longer than the non-participants. During the 12-month period after the index date, medication adherence was                significantly better in the participant group than the non-participant group . Multivariable                logistic regression analysis also showed the consistent trend that the participants                were significantly less likely to have a MS-related hospitalization during the                12-month period after index date . During the 12-month period prior to the index date, participant and non-participant                groups had comparable MS-related hospitalization rates . Summing up the                MS-related medical and pharmacy costs, the participant group had a larger increase                in MS-related total cost of care from pre- to post-period than the non-participant                group                 decreased by US$264 among participants, while it increased                US$1536 among non-participants                                while the adherence deteriorated among non-participants. This observation was                further substantiated with similar findings in persistence on medication. Given                equivalent persistence at the baseline, the participants’ persistence on                MS medication improved during the 12-month period after index date, whereas the                non-participants’ persistence worsened in the same period (                +29.4 days vs −9.2 days). Adherence has been crucial for                obtaining the beneficial effects of treatment, as is true for other therapeutic areas.p = 0.64).                This reduction in MS-related hospitalizations represents a successful improvement in                clinical benefits of the program, as these hospitalizations serve as an indicator of                severe MS exacerbation and acute care service. The reduced risk also coincided with                improved adherence and persistence among participants in the same period, which                suggested that the improved clinical outcomes might be a result of better control in                disease progression.During the 12-month period after enrollment, the MS-related hospitalization rate for                the participant group dropped from 9.6% to 7.1%, whereas it                increased from 10.1% to 12.0% in the non-participant group.                This considerable reduction in hospitalization risk among participants was                encouraging, especially given that both groups had comparable MS-related                hospitalization rates during the baseline period  to 84.0% (participants) of the MS-related total cost                of care, which was in line with other studies showing that pharmacy costs accounted                for the majority of the MS-related total cost.10,23–25 One could speculate that the improved                medication adherence and persistence, reduced risk of MS-hospitalization, and                decreased MS-related medical spending observed in this study might have affected                patients’ quality of life, productivity, and disease progress. With                decreasing medical care utilization indicated by both reducing MS-related                hospitalizations and medical spending, patients’ mobility level and                functional status may have been better controlled and disease progress may have                slowed down. As a result, we hypothesize that the long-term health of these MS                patients would be more stable than if they had not participated in the program. The                reduction of medical utilization also leads us to believe that the MS patients would                have fewer MS-related absences from work and family-related responsibilities.                However, further research will be needed to substantiate these hypotheses.This study did not evaluate patient-reported outcomes. Thus, the effect of the                program on other outcomes such as functional status and absenteeism was unclear.                Other studies had shown the potential impact of care management program on quality                of life, lost work/school days, and patient satisfaction in other therapeutic                    areas.To validate the robustness of the study findings, we performed several sensitivity                analyses with different methods. These methods included a) using all-cause                hospitalization instead of MS-related hospitalization; b) using all-cause total cost                instead of MS-related total cost; c) excluding patients over 65 years                old; and d) excluding patients on mitoxantrone and natalizumab. All results of these                sensitivity analyses were consistent with the primary results and did not alter the                overall conclusions of this study.The study findings are subject to several limitations. First, randomization of                    intervention (care management) was not performed due to the nature of                    observational study. Although the comparability between participants and                    non-participants in unobserved characteristics , disability                    level) cannot be assumed, the fact that over time change comparison still                    reflected the potential impact of the program increases the chance that the                    current results are robust. Second, misclassification or measurement error could                    occur in administrative claims, but it is unlikely that such error would be                    systematically different across cohorts. Third, the disenrollment information                    from the program was not available. The participant group might have included                    those who only partially participated or dropped out sometime after enrollment                    in the program, and hence the program effect observed in this study could be                    underestimated. Fourth, it could be argued that the results might not be                    generalizable to non-participants due to difference in unmeasured                    characteristics such as self-motivation and health-consciousness. The                    participants may already have been more motivated so that the management program                    was an add-on. Nonetheless, this does not refute the observation that the                    program affects participants as an aggregate, who accounted for                    78.3% in this commercially insured population.To our knowledge, this study was the first to evaluate the impact of a MS specialty                care management program on clinical and economic outcomes. Our findings suggested                that this program was associated with improved medication adherence and persistence,                reduced risk of MS-related hospitalization, and decreased MS-related medical costs                (excluding pharmacy) over time. This represented an important metric of program                success. It was also associated with higher MS-related total cost of care, which was                primarily driven by high pharmacy costs. The cost saving in medical utilization,                unfortunately, did not offset the increased expenditure in pharmacy during the                12-month period after the program intervention.