FN1) is a glycoprotein involved in cellular adhesion and migration processes. The aim of this study was to elucidate the role of FN1 in development of renal cell cancer (RCC) and to determine a prognostic relevance for optimal clinical management.Fibronectin 1  as biological control and the ΔΔCt method for calculation of relative quantities.212 renal tissue samples  were collected from patients undergoing surgery for renal tumors and subjected to total RNA extraction. Detection of FN1 mRNA expression was found to be increased approximately seven fold comparing RCC and corresponding kidney control tissues . Furthermore, tissue specific mean FN1 expression was increased approx. 11 fold in clear cell compared to papillary RCC . Patients with advanced disease had higher FN1 expression when compared to organ-confined disease . Applying subgroup analysis we found a significantly higher FN1 mRNA expression between organ-confined and advanced disease in the papillary and not in the clear cell RCC group . There was an increased expression in RCC compared to oncocytoma .Mean tissue specific FN1 mRNA expression is higher in RCC compared to normal renal tissue. FN1 mRNA expression might serve as a marker for RCC aggressiveness, indicating early systemic progression particularly for patients with papillary RCC.To our knowledge, this is the first study to show that  Renal cell carcinoma (RCC) is a common urologic tumor and accounts for about 3% of all human malignancies . The annFibronectin (FN) seems to play an important role in both inhibition and promotion of cellular attachment by interacting with different receptors.An essential step in local disease progression and in the formation of metastases is the invasion of tumour cells into the extracellular matrix. Cell adhesion molecules and extra-cellular matrix proteins support either an increase or a decrease in the ability of tumours cells to adhere to surrounding tissue. Among the extracellular matrix proteins identified, FN is a glycoprotein that is involved in cellular adhesion and migration processes including embryogenesis, wound healing, blood coagulation, host defense, and metastasis. The molecule is widely distributed in healthy membrane, in the lamina propria, in vessel structures, nerves and smooth-muscle cell layers [FN is not clearly known [FN in different malignancies [FN protein was found [FN is still unclear [l layers . Howeverly known ,5. Few sgnancies . For exaas found . Elevate unclear -11.FN1 in the development of RCC using mRNA expression analyses. 212 renal tissue samples from patients undergoing surgery for renal tumors were analysed using quantitative real time PCR. FN1 mRNA expression was significantly increased in RCC compared to corresponding normal renal tissue. Furthermore, our data suggest FN1 as marker for progressive disease especially in papillary RCC.The aim of this study is to elucidate a possible role of Tumors and corresponding tumour free tissue of 126 patients subjected to kidney surgery between 2001 and 2005 collected from the Eberhard Karls University Tuebingen were included in the present investigation. All tumors were freshly obtained from the urological department's operation room. The ethical committee of the institution approved the study. Two pathologists evaluated all specimens with respect to tumor stage, grade and histological subtypes. Tumor stages were assessed according to the UICC 2002 issue of the TNM system . HistoloRenal proximal tubular epithelial cells (RPTEC) were obtained from Lonza  and cultured according to the manufactures recommendations.FN1 (Assay ID: Hs00365058_m1), GUSB (Hs00939627_m1), RPL13A (Hs03043885_g1) and HPRT1 (Hs99999909_m1). The human GUSB, RPL13A and HPRT1 transcripts served as endogenous controls; cDNA derived from RPTEC primary cell transcripts served as biological control. Additional no-template, no reverse transcription and blank controls were included in each run.Total RNA was isolated from 20 cyro sections (each 20 μm) using TriReagent (Ambion) according to the manufacturers' instructions. Furthermore, of each tissue sample two sections were stained with hematoxylin-eosin and evaluated by a pathologist (J.U.B). Total RNA was reverse-transcribed into single-strand complementary DNA (cDNA) using the High Capacity cDNA Reverse Transcription Kit . Quantitative real time PCR analyses were performed in duplicate with an ABI 7900 Fast Sequence Detection System using TaqMan gene expression assays and universal PCR master mix according to the manufacturer's specifications (Applied Biosystems). The TaqMan assays were Data were evaluated using the SDS 2.3 Manager, dataAssist V1.0 software and the ΔΔCt method ,16. The FN1 in RCC, 109 tumour RNA samples and corresponding histopathological normal renal tissue of 86 of these patients were analysed using quantitative real time PCR. Furthermore, 17 tissue samples of oncocytomas were examined. Expression of FN1 was observed in all analysed samples. Mean tissue specific FN1 mRNA expression was found to be increased approximately seven fold comparing RCC and corresponding kidney control tissues . There was no significant difference in FN1 expression between normal renal tissue and oncocytoma. However, there was an increased expression in RCC compared to oncocytoma  and advanced disease (pT≥3 and/or N+/M+). Both, organ-confined and advanced disease showed significantly higher levels of FN1 mRNA expression compared to normal renal tissues  and advanced disease (pT≥3 and/or N+/M+) showed significant higher levels of FN1 mRNA expression compared to normal renal tissue. Moreover, patients with advanced disease had higher FN1 expression when compared to organ-confined disease. This supports the hypothesis that FN contributes to renal carcinogenesis and/or RCC progression.In the present study, a significantly increased expression of FN as a candidate marker for aggressive RCC. The first studies focusing on different RCC cell lines showed altered FN secretion and the possibility to influence mobility and invasiveness of these malignant cells [There have been previous functional studies suggesting nt cells -26.FN1 mRNA expression in clear cell RCC compared to papillary RCC. Our results generally suggest that an increased FN1 expression is correlated to a more progressive disease in RCC. Moreover, higher FN1 expression in clear cell RCC as compared to papillary RCC support the common belief that papillary tumors are less aggressive [FN1 mRNA expression between organ-confined and advanced disease in papillary RCC but not for clear cell RCC. Papillary RCC can be sub classified into types 1 and 2 as well as into less and more aggressive tumors. FN1 might be used to differentiate between papillary RCC with less or more aggressive potential.A further interesting finding in our study was a significant increase in gressive . On the gressive . This stFN1 mRNA expression is significantly higher in RCC compared to normal renal tissue and oncocytoma. FN1 mRNA expression might serve as a marker for RCC aggressiveness, indicating early systemic progression particularly for patients with papillary RCC. Therefore, our data on FN1 encourages further investigations to enlighten the future role of FN1 in RCC.To our knowledge, this is the first study to show that The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/10/503/prepub


Systemic and local inflammation plays a prominent role in the pathogenesis of atherosclerotic coronary artery disease, but the relationship of whole blood gene expression changes with coronary disease remains unclear. We have investigated whether gene expression patterns in peripheral blood correlate with the severity of coronary disease and whether these patterns correlate with the extent of atherosclerosis in the vascular wall.Patients were selected according to their coronary artery disease index (CADi), a validated angiographical measure of the extent of coronary atherosclerosis that correlates with outcome. RNA was extracted from blood of 120 patients with at least a stenosis greater than 50% (CADi≥23) and from 121 controls without evidence of coronary stenosis (CADi = 0).P<0.003). Prominent differential expression was observed especially in genes involved in cell growth, apoptosis and inflammation. Using these 160 genes, a partial least squares multivariate regression model resulted in a highly predictive model . The expression pattern of these 160 genes in aortic tissue also predicted the severity of atherosclerosis in human aortas, showing that peripheral blood gene expression associated with coronary atherosclerosis mirrors gene expression changes in atherosclerotic arteries.160 individual genes were found to correlate with CADi (rho>0.2, In conclusion, the simultaneous expression pattern of 160 genes in whole blood correlates with the severity of coronary artery disease and mirrors expression changes in the atherosclerotic vascular wall. Coronary artery disease, a multifactorial chronic disease, is the leading cause of death in Western countries. Despite considerable advances in the prevention and treatment of coronary artery disease and its complications, morbidity and mortality remains high. In half of patients with coronary artery disease, the first manifestation is death Systemic and local inflammation has been shown to play a prominent pathologic role in atherosclerotic coronary artery disease Microarray analysis of peripheral blood cells is a practical approach to study gene expression changes that may reflect not only genetic predisposition but also presence and activity of disease, environmental modifier effects, and treatment responses Patients and control subjects were recruited from individuals that had undergone catheterization in the Duke University Hospital Cardiac Catheterization Laboratory and participated in a proteomics study to discover candidate proteins that are differentially displayed in populations with and those without angiographic coronary artery disease matched men (n = 106), who were matched for age and ethnic group, 2) unmatched men (n = 82), who did not fulfill the matching criteria and 3) unmatched women (n = 53). The severity of coronary artery disease was scored using the Duke Coronary Artery Disease Index (CAD-Index) Patient selection, design and results from the main proteomics study have been reported previously Inclusion criteria for the coronary artery disease patient population (cases) were: age between 40 and 65 and coronary artery stenosis of >50% in at least one major coronary artery. Inclusion criteria for the control population (controls) were: age between 40 and 65 for matched men cohort only, no angiographically detectable coronary artery stenosis on cardiac catheterization within the last two years, normal left ventricular ejection fraction and normal regional wall motion. Exclusion criteria for controls were typical signs of angina, or any history or evidence of myocardial ischemia on stress testing, myocardial infarction or unstable angina, any history of peripheral arterial or cerebrovascular disease, or significant vascular stenosis on noninvasive imaging or angiography. Exclusion criteria also included myocardial infarction within one month (for cases), diabetes, uncontrolled hypertension (systolic blood pressure >180 mmHg or diastolic blood pressure >100 mmHg) or with end-organ damage, renal insufficiency (creatinine >2.0 mg/dL or BUN>40 mg/dL), active malignancy, significant valvular heart disease, NYHA Class III or IV heart failure, cigarette smoking >2 packs per day, total cholesterol >300 mg/dL or triglyceride >400 mg/dL, anemia , and hypotension (systolic blood pressure <90 mmHg and diastolic blood pressure <50 mmHg).The blood samples (2.5 mL) were collected in PAXgene™ Blood RNA tubes and total RNA was isolated using the standardized RNA Kit  RNA was then quantified by absorbance at A260 nm and the purity was estimated by the ratio A260 nm/A280 nm. RNA integrity was confirmed by non-denaturing agarose gel electrophoresis. RNA was stored at −80°C until further analysis. The quality of 19 RNA samples was insufficient for microarray analysis due to degradation. The genomic studies were conducted in the Novartis Genomics Factory, Basel, Switzerland.http:/www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE12288). As quality control, RT-PCR was performed on 8 selected genes in 2×20 subjects from the ‘matched men’ cohort.Genome-wide transcript profiling was assessed using human HGU133A oligonucleotide expression probe arrays , comprising 22,483 probe sets. The experiments were done according to the recommendations of the manufacturer To test whether the expression pattern in peripheral whole blood is representative for atherosclerosis in general, we have examined the capability of expression of genes derived from the peripheral blood cell study to predict the severity of atherosclerosis in human aortas. Gene expression data was generated using RNA extracted from a unique collection of freshly harvested human aortas with varying degrees of atherosclerosis (n = 67 donors). Donor identification, RNA extraction and micro-array methods (Affymetrix U95Av2) as well as gene expression signatures that differentiate between atherosclerotic disease states in human aortas have been reported previously Spearman rank correlation between CAD-index and gene expression was calculated (Partek Genomics Suite Version 6.3). An absolute correlation coefficient (rho) >0.2 was considered clinically relevant, corresponding to a p-value of 0.003 (n = 222). Among the 22,483 probe sets of the Affymetrix HGU133A chip, about 60 probe sets can be expected to have an absolute rho>0.2 by chance . Student's t test, parametric correlation and rank correlation according to Spearman were performed with the statistical software package S-Plus Version 6.Projections to Latent Structures (PLS) analysis including Orthogonal Signal Correction (OSC) (SIMCA-P Version 10.0) was used to identify gene sets that discriminate between increasing CAD-indices or the three classes  of atherosclerosis in the aorta samples. To reduce gene selection bias, models were subsequently repeatedly built based on data from two cohorts to predict CAD in the third cohort. In addition, extensive cross-validation by leave-one-out technique and validation by response permutation was applied to 7 groups of approximately 32 subjects to reduce bias in creating a predictive gene set.Demographic data, medical history and medication of the study population are summarized in Clinical laboratory parameters were available for all subjects . Hematoc2 = 0.228).Traditional risk factors, including body weight, smoking, and systolic and diastolic blood pressure did not correlate significantly with the extent of coronary disease in a rank correlation analysis. Total cholesterol (but not LDL-cholesterol) level was found to be inversely related with the CAD-index , which may in part reflect the higher use of statins and better blood lipid control in cases. In addition, other parameters were found to positively  or negatively  correlate with CAD-index (rho>0.2). Of note, important clinical markers such as LDL-C (rho = 0.02), CRP (rho = −0.12) and homocysteine (rho = 0.02) exhibited a poor correlation with CAD-index, which could result from treatment of affected individuals. In a multivariate correlation analysis, the combination of risk factors and biochemical markers only poorly predicted the extent of coronary artery disease ; RNA from the remaining 19 subjects did not pass quality control due to degradation.P<0.003). All probesets correlating with CAD-Index are listed in In a univariate analysis, 160 genes were found to correlate with CAD-Index with an absolute rank correlation coefficient (rho) >0.2  and CAD-index as fixed factors or random effects, respectively) . However2 = 0.764 (p<0.001)  . Predict2cum of 0.776, indicating an excellent predictive ability.In order to test for robustness of the model, the PLS analysis was performed separately for each of the three cohorts, with the model repeatedly constructed using two cohorts (training sample) and tested in the third cohort (test sample). While the controls remain quite stable in the range of -2 standard deviations, the t1-scores of the cases were located mainly in the +2 standard deviation range and increase with increasing CAD-Index . This reFTL, FKBP8, TUBA3, PNPLA2, UBXD1, MARCH2, ITPK1, PINK1, in order of contribution; listed in bold in 2 = 0.752). Adding traditional risk factors and biochemical markers do not significantly improve this model (r2 = 0.782).A Variable Importance in the Projection (VIP) of each gene for the separate PLS analyses of the three cohorts compared to the PLS analysis including all subjects was calculated. The VIP of the first 24 genes shows only little variation between the three cohorts suggesting a rather high stability of the prediction model . A set oSince the genes whose expression contributes to prediction of CAD were studied within circulating leukocytes, we sought to define whether they actually reflect a molecular process that is ongoing within atherosclerotic arteries or not. Furthermore, as a test of reproducibility of the contribution of these 160 genes to predicting atherosclerotic disorders, we have investigated whether the in situ expression pattern of our 160 genes derived from peripheral blood could also adequately predict the severity of aorta atherosclerotic lesions. To achieve this goal, we have used gene expression data extracted from a large set of human aortas obtained from heart donors (n = 67), an independent human model of atherosclerosis. Excluding genes that are not present on the microarray used in the aorta expression study, the expression pattern of the remaining genes accurately separated the aorta samples according to the severity of atherosclerosis . These rIn this large-scale expression analysis of peripheral whole blood cells, we have found 160 genes whose expression correlates with the severity of angiographically documented coronary artery atherosclerosis. Taking into account that the CAD-Index is a semi-quantitative estimate of the extent of coronary atherosclerotic disease, which implies variation across subjects even with the same degree of disease, the prediction based on expression pattern of these genes is robust. Our findings are also robust as assessed by internal validation and consistency across three distinct subgroups. Importantly, the in situ expression pattern of the 160 genes derived from the peripheral blood analysis was also predictive of the severity of atherosclerosis in human aorta tissue. This provides validation of the association of this set of genes with atherosclerosis and support for the concept that peripheral blood gene expression reflects pathophysiology in the vascular wall. Taken together, the molecular signature in peripheral blood for varying degrees of coronary artery disease is remarkably consistent with that seen in the atherosclerotic arterial wall, providing valuable new information of the pathways and their genes that are involved in the atherosclerotic process.Peripheral blood is easily accessible and routinely used for diagnostic laboratory analysis and thus is a good resource for additional tests that might define extent of coronary artery disease. Several inflammatory markers, including high sensitivity C-reactive protein (CRP) are associated with cardiovascular risk, independently from traditional risk factors MAN2ARXRALGALS9PSG3CEPBAARGHAP4MADH5AIF1ELAVL2STXBP2KCNMB1PDE4DEPHB2GGA3PLAURNPR3TNFRSF5 (CD40) KCNMB1, NEDD4L, ADD1 and NPR3) have been implied in genetic susceptibility for hypertension PDE4D) ADD1) FTL) in atherosclerosis FTL gene expression in peripheral cells of CAD patients might in at least in part reflect a general pro-inflammatory state that leads to degenerative changes Most of the differentially expressed genes in the present study are involved in bone marrow cell differentiation, cell growth or growth arrest, apoptosis, cell adhesion and matrix modulation, and inflammatory and immune response, processes known to modulate atherosclerosis. Since blood samples were taken in stable patients, our finding that circulating blood cells differentially express many pro-inflammatory genes supports the paradigm that inflammation is an important process in patients with coronary artery disease. Expression patterns of the same genes were found to correlate with the extent of atherosclerosis in human aortas as well, indicating that gene expression patterns in peripheral blood cells associated with coronary artery disease to some extent mirror gene expression changes in the atherosclerotic vessel wall. Indeed, many of the genes shared by our predictive models modulate monocyte or macrophage function, including We intentionally did not separate peripheral blood cells or leukocyte subtypes. There is currently little pathophysiological evidence that the study of leukocyte subgroups would add to our predictive model and the isolation process could, in itself, affect the gene expression pattern. Using whole blood cells not only allows aggregate RNA expression analysis per patient without the need to pool rare subtypes, but is also more practical from a clinical perspective. Leukocyte levels in all groups were very similar, although it cannot be excluded that the percentage of specific subtypes differ between groups, and hence that different numbers of subtypes are responsible for the observed effect. Peripheral whole blood might also include differential expression signatures from reticulocytes, platelets or rare hematopoietic progenitors.In a recent paper, Wingrove et al reported 526 differentially expressed genes (>1.3-fold expression) from a genome-wide microarray analysis of peripheral blood mononuclear cells of 27 cases with angiographically documented CAD and 14 controls Of the 160 genes we found to be correlated with the extent of CAD, only 19 were significantly differentially expressed between all cases and controls, while gene expression was significantly different for 90 genes when comparing 20 patients with the least predicted CAD-index to 20 patients with the highest predicted CAD-index. Most of our cases only have mild to moderate disease, with only a minority having extensive disease. Thus, in part as a result of our proteomics-driven patient selection, there is likely to be a very gradual transition from controls to cases, with the distrubution of cases being skewed towards the lower end of CAD-index. We therefore assumed that the difference between controls and cases was not likely to be very large, hence our preference for a correlation-based analysis. Furthermore, since the average age of the controls was 52 years, it is highly likely that some degree of coronary atherosclerosis is present in these subjects. Interestingly, patients with normal angiograms but with microvascular dysfunction may also demonstrate peripheral monocyte activation, although not to the extent seen in patients with angiographically documented coronary artery disease In conclusion, the combined predictive value of differentially expressed genes in peripheral blood correlates with the extent of coronary atherosclerosis. Importantly, the expression pattern of the same genes is also correlated with the extent of disease in atherosclerotic aortas. While these findings need prospective validation in further populations, our findings also suggest that gene expression profiles might represent a novel and promising non-invasive test to assess the presence and extent of coronary artery disease. Although the extent of angiographic disease is a strong predictor of clinical outcome, further studies in larger and unselected populations will also be needed to examine the potential role of gene expression patterns in predicting outcome and to address potential confounding factors.


Schistosoma japonicum infection and iron homeostasis. We aimed to determine if host iron status has an effect on schistosome maturation or egg production, and to investigate the response of iron regulatory genes to chronic schistosomiasis infection. Wild-type C57BL/6 and Transferrin Receptor 2 null mice were infected with S. japonicum, and sacrificed at the onset of chronic disease. Transferrin Receptor 2 null mice are a model of type 3 hereditary hemochromatosis and develop significant iron overload providing increased iron stores at the onset of infection. The infectivity of schistosomes and egg production was assessed along with the subsequent development of granulomas and fibrosis. The response of the iron regulatory gene Hepcidin to infection and the changes in iron status were assessed by real-time PCR and Western blotting. Our results show that Hepcidin levels responded to the changing iron status of the animals, but were not significantly influenced by the inflammatory response. We also show that with increased iron availability at the time of infection there was greater development of fibrosis around granulomas. In conclusion, our studies indicate that chronic inflammation may not be the primary cause of the anemia seen in schistosomiasis, and suggest that increased availability of iron, such as through iron supplementation, may actually lead to increased disease severity.Schistosomiasis is a significant parasitic infection creating disease burden throughout many of the world's developing nations. Iron deficiency anemia is also a significant health burden resulting from both nutritional deficit as well as parasitic infection in these countries. In this study we investigated the relationships between the disease outcomes of  Schistosomiasis is a significant parasitic infection creating considerable disease burden in many tropical and developing nations. Currently, some 74 countries are endemic for schistosomiasis with an estimated 600 million people residing within regions in which the parasite is prevalent Schistosoma mansoni, S. japonicum and S. haematobium, which have different geographic distributions throughout Africa, South America and Asia. Despite differences in disease arising from the site of patent infection per se, but rather from the intense granulomatous response to antigens secreted by the larvae within the schistosome eggs that have become lodged within host tissue Schistosomiasis in humans is caused primarily by 3 species, S. japonicum from the Philippines, for which several studies have demonstrated an inverse relationship between infection intensity and hemoglobin Several recent cross-sectional multi-variant population cohort studies have confirmed the association of anemia and the disease state of schistosomiasis. These studies have shown that when confounding factors such as nutritional deficiencies, co-infection of other parasites, age, sex, and weight are accounted for, schistosomiasis infection correlates inversely with hemoglobin levels, and can be significantly linked to the associated anemia AI has become the most commonly theorized cause of the anemia following a number of studies that have shown that increased levels of pro-inflammatory cytokines, in particular IL-6, are produced during chronic schistosomiasis Hepcidin 1 (Hamp1) and Hepcidin 2 (Hamp2). While the individual role of hepcidin 2 has not yet been confirmed, hepcidin 1 has been shown to function as the primary iron regulatory molecule. Since anemia in schistosomiasis is attributed primarily to AI, and a key mediator of AI is hepcidin The central regulator of iron homeostasis is hepcidin, a 25 amino acid peptide expressed and secreted by hepatocytes. Hepcidin is up-regulated in mammals by iron overload and is suppressed by iron deficiency HAMP) or upstream regulators of hepcidin such as hemojuvelin (HJV), HFE and transferrin receptor 2 (TFR2) lead to low levels of circulating hepcidin in relation to iron stores and consequent iron loading Transferrin Receptor 2 null mice (−/−Tfr2), a model of type 3 hereditary hemochromatosis and shown that they have dysregulation of hepcidin resulting in iron overload Tfr2 dysfunction on iron homeostasis demonstrate increased iron absorption within the gut along with increased expression of iron transport genes Hereditary hemochromatosis is a genetically heterogeneous group of iron overload disorders caused by mutations in molecules involved in the regulation of iron homeostasis. Mutations in the gene encoding hepcidin . We next assessed the level of transferrin receptor 1 (Tfr1) protein in livers. Tfr1 is responsible for the uptake of transferrin-bound iron into hepatocytes and its expression level is inversely regulated by the cellular iron content. Tfr1 protein was significantly lower in uninfected −/−Tfr2 control mice  higher serum transferrin saturation (TS), as representative of circulating iron, than wild-type animals with 96% and 48% saturation respectively (S. japonicum infected mice TS was significantly (P<0.05) lower in both −/−Tfr2 and wild-type animals when compared to their respective uninfected controls groups, a reduction of 76% and 14% respectively. Together these results show that infection significantly reduces the iron status of both strains of mice. The iron over-loaded status of the −/−Tfr2 mice, as shown by the Perls' quantification, Tfr1 protein levels and TS provided the background to compare the effects of iron availability on schistosome maturation, egg production, and liver pathology.Uninfected ectively . In S. j−/−Tfr2 mice than in wild-type mice, there was no statistically significant difference in the number of mature females  and Orosomucoid ) are acute phase response genes expressed by hepatocytes and are representative of hepatic inflammation −/−Tfr2 infected mice, but not in uninfected control mice  displayed a downward trend in infected mice of both strains, while Hepcidin 2 (Hamp2) expression was significantly reduced in both strains with infection . Decreased hepcidin expression results in increased iron absorption, the opposite of what is required for inflammation to result in AI In this study we show that schistosomiasis infection resulted in a substantial reduction in iron indices, with infected wild-type and S. mansoni infection Friis and co-workers investigated the use of macro-nutrient supplements in treatment of Kenyan school children suffering chronic et al suggested that AI is likely to be a significant contributor to the anemia seen in schistosomiasis following their findings that anemia was commonly present in the absence of iron deficiency (ID) et al, subjects with anemia who were classified as iron replete based on their SF may have actually been ID.Within the schistosomiasis literature the presence of anemia is often attributed to AI because of the failure of occult blood loss to account for the anemia In another cohort study, Cherian and co-workers investigated the relationship of hepcidin and iron status in refugee children −/−Tfr2 mice have a greater amount of bioavailable iron for invading schistosomes than wild-type mice at the time of infection. While we did not measure iron absorption and thus iron availability directly during infection in our study, previous studies on Tfr2 dysfunction in mice have demonstrated increased iron absorption through the gut along with increased expression of iron transport genes Hepcidin expression concurrent with S.japonicum infection. It is therefore likely that elevated dietary iron absorption occurs concurrently with infection, providing ongoing increased iron availability within the −/−Tfr2 host animals. Despite the high requirements of invading schistosomules for host iron, and an expectation that the greater availability of iron in −/−Tfr2 mice might lead to higher burdens in those mice, there were no differences in adult worm numbers between wild-type and −/−Tfr2 mice. The progression toward iron depletion in schistosomiasis may be initiated with the onset of egg-laying, which occurs approximately 4 weeks post infection for S. japonicum, resulting in transfer of iron from the host into the eggs. If on-going iron availability is a rate-limiting factor in the progression toward some aspect of chronic disease, then the level of a host's pre-infection iron stores may contribute to the rate of onset of severe symptoms, i.e. the larger the available iron pool, the faster the onset of chronic symptoms. In the same manner, iron supplementation to resolve the anemia associated with schistosomiasis may actually increase the development of long term pathology by providing on-going increased iron availability.−/−Tfr2 mice with high iron indices develop a significantly increased fibrotic response surrounding the granulomas when compared to infected wild-type mice. Activated hepatic stellate cells (HSC) and alternatively activated macrophages are responsible for the deposition of extracellular matrix resulting in hepatic fibrosis surrounding the granulomas et al have also shown a direct correlation between the hepatic iron concentration (HIC) and the activation of HSCs Ironically the anemia associated with schistosomiasis may actually provide a degree of protection for hosts against the more severe long term pathological consequences of chronic infection. The development of fibrosis surrounding granuloma formation can lead to significant health consequences resulting in morbidity and mortality. We showed that infected Denic and Agarwal propose that iron deficiency provides an evolutionarily-selected survival advantage Hepcidin responds to the decreased iron status. Furthermore, we have shown than increased iron available from pre-infection stores and possible ongoing iron absorption leads to increased fibrotic deposition within the liver. Together these results suggest that increasing the iron status of infected subjects may actually result in an increased disease burden. In light of this the utilization of dietary iron supplementation to resolve anemia associated with chronic schistosomiasis may result in more severe long term disease outcomes.The disease outcomes associated with schistosomiasis have a significant impact throughout many of the world's developing nations. The anemia associated with schistosomiasis is often labeled as anemia of chronic inflammation: however our findings suggest that this is likely not to be the case. We show that the iron regulatory gene −/−Tfr2 mice on the C57BL/6 background were bred in-house and have been described previously All mice  were maintained on standard laboratory chow . Wild-type C57BL/6 mice were purchased from the Animal Resource Centre, Perth, Australia. Oncomelania hupensis hupensis snails, infected with Chinese (Anhui) strain of S. japonicum, were imported from the Institute of Parasitic Diseases, Shanghai, China. Mice were infected percutaneously with 30 cercariae shed from snails, and adult worms were perfused 6 weeks after challenge as described previously Liver tissue was fixed in 4% formalin for 24 hours prior to embedding in paraffin wax and sectioning. Fibrosis was detected in sections by staining for collagen with Sirius red which was performed by the histology department at QIMR. Iron was detected in sections using the Perls' Prussian Blue staining method. Briefly, liver sections were deparaffinized and rehydrated. Slides were incubated in Perls' Staining Solution , washed, counterstained with Nuclearfast Red, dehydrated, cleared in xylene and mounted using Depex mounting medium. Transferrin saturation (TS) was measured using an iron and iron binding capacity kit . Sections were scanned using the Aperio ScanScope XT  with doubler inserted for x40 scanning. Sirius Red and Perls' Prussian Blue stains were quantified within the sections using the ImageScope Version 10.0.36.1805 software and the ColorDeconvolution_v9 algorithm calibrated to the sections.β-actin, Hamp1, Hamp2, Tfr1, Tfr2Orm , Saa . All targets were normalized to the respective β-actin levels by subtracting the threshold cycle (CT) of β-Actin from the CT of the target (ΔCT). Results were then transformed to log base 2 to represent fold change and normalized to 1 for comparison.Total RNA from mouse liver was isolated using Trizol  and treated with DNase to remove any genomic DNA. One µg was reverse transcribed into cDNA with Superscript III . Real-time reaction mixes contained cDNA transcribed from 15 ng RNA, 200 nM each primer and LightCycler 480 SYBR Green Mix . Reactions were performed on the LightCycler 480  using the following conditions: 2 minutes denature at 95°C followed by 45 cycles of 95°C for 15 seconds, 55°C for 15 seconds and 72°C degrees for 15 seconds. The fluorescence due to SYBR green binding to double stranded DNA was measured during the extension steps. Melt curve analysis was routinely performed to monitor primer dimer levels by raising the temperature from 50°C to 99°C at 1 degree/minute whilst monitoring fluorescence continuously. All experiments were performed in duplicate, and a single batch of cDNA was used for Tissues were homogenized in phosphatase inhibitor lysis buffer  containing 10 µg/mL DNase. Twenty five micrograms of total liver homogenates were electrophoresed on a 10% SDS-PAGE and then transferred onto Hybond-C+ membrane. Blots were blocked in 10% skim milk powder, 0.5% Tween 20 in PBS (blocking buffer) overnight at 4°C. One µg/mL each of anti-TfR1 (Invitrogen) or anti-actin (Sigma) antibody, diluted in blocking buffer, was applied to the blot for 1 hour at room temperature. The blots were washed extensively with 0.1% Tween 20 in PBS and then incubated with anti-rabbit or anti-mouse horse-radish peroxidase for 1 hour at room temperature. Blots were washed extensively and Immobilon Western chemiluminescent HRP substrate (Millipore WSBLKS0500) was applied for 5 minutes to the blot and then exposed to film.


While the addition of cetuximab to radiotherapy improves clinical outcomes in locoregionally advanced head and neck squamous cell cancers, there are a small number of reports of severe radiation dermatitis occurring with this therapeutic combination. We present the case of a 69 year old male who developed severe radiation dermatitis following treatment with cetuximab and radiotherapy for a locoregionally advanced head and neck squamous cell cancer. The addition of cetuximab to radiotherapy improves local control and overall survival in locoregionally advanced head and neck squamous cell cancer. However, evidence is emerging that severe radiation dermatitis can occur in patients receiving cetuximab. We present the case of a 69 year old male who developed severe radiation dermatitis following treatment with cetuximab and radiotherapy for a locoregionally advanced head and neck squamous cell cancer.2 weekly during radiation, with a single dose of 400 mg/m2 the week prior to radiation).A 69-year-old Caucasian male developed a severe rash two weeks after starting treatment with concurrent cetuximab and radiotherapy for a loco-regionally advanced right buccal head and neck squamous cell cancer (HNSCC). Two months earlier, magnetic resonance imaging (MRI) identified a 3.8 × 1.4 × 3.6 cm right buccal mass with a 1.7 cm left sided midcervical lymph node. There was no other disease on fluorodeoxyglucose positron emission tomography (FDG-PET). His disease was considered unresectable and cisplatin was contra-indicated because of sensori-neural industrial hearing impairment. He commenced radiotherapy (66 Grays in 33 fractions) and cetuximab . The rash was treated with twice daily dressings and silver sulphadiazine cream and he was discharged home after three weeks. FDG-PET performed three months later showed complete metabolic remission.Cetuximab is a chimeric monoclonal antibody against epidermal growth factor receptor (EGFR), a receptor tyrosine kinase that is up-regulated in more than 90% of HNSCCs. In a piInterestingly, there was no significant increase in grade 3 radiation dermatitis for the cetuximab group (23% versus 18%) in this study. However, multiple reports have subsequently emerged of severe radiation dermatitis complicating treatment with concurrent cetuximab and radiotherapy for HNSCCs . RadiatiIt should also be noted that most patients receiving radiotherapy alone for HNSCCs experience some degree of radiation dermatitis. NonetheThe case presented here, in addition to others in the recent literature, highlights the risk of severe radiation dermatitis in patients with HNSCCs treated with cetuximab and radiotherapy. Clinicians must be aware of this risk, and implement appropriate strategies for prevention and treatment of radiation dermatitis in these patients.Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The author declares that they have no competing interests.AA was solely responsible for writing and editing this manuscript


MSMB promoter has been linked to an increased risk of developing prostate cancer. The promoter location of the risk allele, and its ability to reduce promoter activity, suggested that the rs10993994 risk allele could result in lowered MSMB in benign tissue leading to increased prostate cancer risk.Microseminoprotein-beta (MSMB) regulates apoptosis and using genome-wide association studies the rs10993994 single nucleotide polymorphism in the MSMB expression in benign and malignant prostate tissue was examined using immunohistochemistry and compared with the rs10993994 genotype. Urinary MSMB concentrations were determined by ELISA and correlated with urinary PSA, the presence or absence of cancer, rs10993994 genotype and age of onset. MSMB levels in prostate tissue and urine were greatly reduced with tumourigenesis. Urinary MSMB was better than urinary PSA at differentiating men with prostate cancer at all Gleason grades. The high risk allele was associated with heterogeneity of MSMB staining and loss of MSMB in both tissue and urine in benign prostate.These data show that some high risk alleles discovered using genome-wide association studies produce phenotypic effects with potential clinical utility. We provide the first link between a low penetrance polymorphism for prostate cancer and a potential test in human tissue and bodily fluids. There is potential to develop tissue and urinary MSMB for a biomarker of prostate cancer risk, diagnosis and disease monitoring. The risk allele is common, with a frequency of ∼30–40% in Europeans and 70–80% in men of African ancestry, and confers an increased risk of prostate cancer of 1.3 . This association is observed in both Europeans and African-Americans and appears to be independent of age of diagnosis and tumour grade MSMB proximal promoter region reduced promoter activity is suggested to occur via altered transcription factor binding sites such as CREB MSMB mRNA in cell lines was reduced in cells homozygous for the risk allele, which suggests a hypothesis whereby reduced MSMB expression in individuals with the rs10993994 risk allele could lead to reduced regulation of cell growth and an increased risk of tumourigenesis Two genome wide association studies (GWAS) have identified a tag small nucleotide polymorphism (SNP), rs10993994, on 10q that is strongly associated with an increased risk of developing prostate cancer Several groups have studied MSMB expression in prostate cancer and all have found higher levels of MSMB in benign and normal tissue or serum when compared with material from tumours Prostate cancer is the most common solid male cancer in the UK. At present the best diagnostic tool is serum PSA while urinary PCA3 mRNA is also used as a useful diagnostic test particularly for men with raised PSA and an initial negative biopsy We have used immunohistochemical studies of MSMB in benign prostate tissue to demonstrate a relationship between MSMB protein expression and the rs10993994 genotype. We have developed a urinary ELISA assay to examine the association between urinary PSA, MSMB levels and MSMB genotype to determine if this has potential for clinically use as a screening or diagnostic tool.Full ethical approval was obtained for all human sample collections from either the West Midlands Multi-Centre Research Ethics Committee or the Trent Multi-Centre Research Ethics Committee. All samples were obtained with written consent and analysed anonymously.BRCA1/2 germline mutations at The Institute of Cancer Research (05/MRE07/25) BRCA1 and BRCA2. Median age, PSA and Gleason scores of the cohorts used are given in Tissue, blood and urine samples from patients with diagnosed prostate cancer were obtained from Addenbrooke's Hospital, Cambridge, UK  and The Institute of Cancer Research . Normal/benign patients were recruited as part of the IMPACT study, a study of prostate cancer risk in men with Not all patients had good quality tissue for IHC, DNA for genotyping, serum PSA measurements and urine available. To overcome this, patients were split into three cohorts to answer specific questions. 168 patients with good quality tissue were used for the IHC study depicted in Only 145 patients had DNA which could be used for genotyping as shown in BRCA1/2 germline mutations (n = 215). All urine samples were obtained prior to any digital rectal exam, aliquoted and frozen immediately at −80°C. Where available age, PSA and Gleason score at diagnosis was determined for each patient.For the MSMB ELISA shown in Genotyping was completed as previously described All immunohistochemistry (IHC) was performed using a Bondmax automated stainer and anti-MSMB antibody (Abcam) (1∶400) on tissue from series 1 and 2. For the multi-normal/tumour array a 48 core TMA from Stretton Scientific was used. Nuclei were counterstained with hematoxylin and slides imaged using an Aperio scanning system. Pathology was confirmed by a specialist uro-pathologist (AW) prior to the scoring of any immunohistochemical staining. Scoring was performed by two observers, one of whom was a uro-pathologist (AW), and a consensus obtained. Sequential hematoxylin and eosin stained sections were used to confirm pathology. Staining was graded as; ‘none’ - no staining, ‘weak’ - not all epithelial cells stained and pale staining, ‘moderate’ – most or all epithelial cells stained moderately, ‘strong’ - all epithelial cells stained strongly . Where sSerum and urinary PSA  & creatinine assays were performed by the NIHR Cambridge Biomedical Research Centre Core Biochemistry Assay Laboratory, Addenbrookes Hospital using the ProStratus Free/Total PSA DELFIA kit  and Dimension RXL analyser . For the MSMB ELISA all procedures were performed with shaking at room temperature. Wells were washed between each step three times with 0.05% PBS-Tween and a BioTek Elx50 platewasher. Ninety-six well plates (Fisher Scientific) were coated overnight at 4°C in mouse anti-PSP94 antibody  diluted in PBS. Wells were blocked with 1% BSA (Sigma) for 1 hour before 50 µl of sample or standard was added to each well in duplicate and incubated for a further 2 hours. Serial dilutions (25−0.78 µg/ul) of recombinant PSP94  were used as a control on each plate. Goat anti-MSMB (R&D systems 0.2 µg/ml) was added to each well for 1 hour followed by a1 hour incubation with donkey anti-goat-HRP . To visualize 100 µl TMB (Sigma) was added to each well for 15 mins. The reaction was stopped by acidifying with 50 µl HCl and absorbance measured at 450 nm using a Lucy II spectrophotometer.The dynamic range of the ELISA was determined to be 0.01–100 µg/ml although the non-linear range was much greater. Any plate exhibiting >20% variability in the standard curve was deemed to have failed and repeated. Intra- and inter-assay variability was 8.8% and 22% respectively. Effects on protein stability were shown to be <20% after testing by freeze thawing 3 times before assaying or incubating pre-diluted rPSP94 -3 for 4 hours at room temperature prior to assaying. Recovery from different fluids was determined to be <20% by diluting rPRDX-3 in either PBS, 0.1% bovine serum albumin (Sigma) or 10% goat serum (Dako Cytomation). MSMB concentration was determined by interpolation of values from non-linear regression of the sigmoidal standard curve. The concentration of all urine samples was normalised using creatinine measured using a creatinine assay kit (R&D Systems) and the slope of standard curve determined by known standards (20–0.3125 mg/dL) and linear regression. Only samples which could be interpolated from the standard curves without extrapolation were included in the analysis.All statistics including linear regression and interpolation for the ELISA assay were performed using GraphPad Prism 5. To compare sensitivity and specificity of measurements in the tumour cohort compared to the benign cohort receiver-operator curves (ROC) were performed. A 1-way ANOVA test with a Kruskal-Wallis correction was used to determine if tissue or urinary MSMB levels were significantly different between the benign and tumour cohorts when the tumour cohort was stratified according to Gleason grade or genotype. A Mann Whitney two-tailed student t-test was used for all pairwise comparisons. For all experiments a p-value of 0.05 was considered significant.Other than in benign prostate no normal or tumourigenic tissues demonstrated any MSMB staining suggesting that at the protein level in tissue MSMB is highly prostate specific . In partConsistent with previous reports benign prostate glands showed significantly higher levels of staining compared with tumourigenic regions in our cohort , staining being weakest in men homozygous for the high risk allele (TT) and strongest in CC homozygotes . There wWe determined urinary levels of MSMB in 215 normal/benign men with no history of prostate disease and 89 men with prostate cancer . ConsistThis is the first study to describe the relationship between the risk allele of the tag SNP rs10993994 and MSMB levels in benign and tumour prostate tissue and to correlate the protein level with genotype and aggressiveness of prostate cancer. The almost complete loss of MSMB in PIN  suggestsMSMB promoter region to an increased risk of developing prostate cancer There is strong evidence linking the rs10993994 SNP in the Several groups have examined MSMB levels in serum demonstrating prognostic value after radical prostatectomy BRCA1 and BRCA2 mutation  In total 168 tumour patients were studied but not every patient had tissue, DNA and urine available. From the cohort 168 had tissue which included benign which was used in (0.73 MB EPS)Click here for additional data file.Figure S2MSMB is prostate specific in normal and tumour tissues. A multi-normal tissue TMA was stained for MSMB using immunohostochemistry and a Bondmax autostainer. The array contained multiple cores from the following tissues; skin, breast, thyroid gland, oesophagus, kidney, bladder, lung, colon, liver, ovary, uterus and stomach. Representative images from testes (seminoma tumour), breast and prostate are shown. MSMB staining is brown, nuclei are blue.(7.19 MB EPS)Click here for additional data file.Figure S3The correlation between genotype and age/PSA at diagnosis and immunohistochemical staining and age/PSA at diagnosis. n =  the number of individuals examined in each group. p-values were calculated using a 1-way ANOVA with a Kruskal-Wallis correction.(1.21 MB EPS)Click here for additional data file.Figure S4(A) Urinary MSMB levels were compared by ELISA and a 2-tailed t-test. (B) Urinary MSMB values were analysed according to the age of the individual (years) at the time of sample collection. (C) PSA measurements for the benign cohort stratified according to the rs10993994 genotype. (D) Urinary MSMB does not alter with BRCA mutations. Benign men in Cohort 3 were genotyped for mutations in BRCA 1 and BRCA 2 which are known to confer risk of developing prostate cancer. BRCA genotype was compared with urinary MSMB. Control - no mutation, BRCA 1 - mutation in BRCA 1, BRCA 2 - mutation in BRCA 2. For all graphs n =  number of samples analysed and p-values were calculated using a 1-way ANOVA with a Kruskal-Wallis correction.(2.03 MB EPS)Click here for additional data file.


Pharmacological and genetic studies have suggested that the metabotropic glutamate receptor 5 (mGluR5) is critically involved in mediating the reinforcing effects of drugs of abuse, but not food. The purpose of this study was to use mGluR5 knockout (KO), heterozygous (Het), and wildtype (WT) mice to determine if mGluR5 modulates operant sensation seeking (OSS), an operant task that uses varied sensory stimuli as a reinforcer. We found that mGluR5 KO mice had significantly reduced OSS responding relative to WT mice, while Het mice displayed a paradoxical increase in OSS responding. Neither KO nor Het mice exhibited altered operant responding for food as a reinforcer. Further, we assessed mGluR5 KO, Het and WT mice across a battery of cocaine locomotor, place preference and anxiety related tests. Although KO mice showed expected differences in some locomotor and anxiety measures, Het mice either exhibited no phenotype or an intermediate one. In total, these data demonstrate a key role for mGluR5 in OSS, indicating an important role for this receptor in reinforcement-based behavior. Metabotropic glutamate receptor 5 (mGluR5) is a receptor that is thought to regulate anxiety and drug reward, though the mechanisms remain uncertain We have recently described a method of assessing non-drug reinforcement by “self-administration” of varied sensory stimuli, a method we termed “operant sensation seeking” (OSS) Analysis of FR-1 operant responding for OSS revealed a significant main effect of mGluR5 genotype on active lever responding for OSS  = 9.5, p<0.01), but not inactive lever responding  = 3.1, n.s.; To test another measure of reward-related behavior, we performed place conditioning with cocaine. First, a group of mice were conditioned using 5 mg/kg cocaine . We founmGluR5 deletion has been reported to increase locomotor activity in a novel environment and strongly attenuate the locomotor activating effects of cocaine Following NOF, a subset of mice were further habituated to the open field, then given an injection of cocaine  and the light-dark exploration test. Considering the previously established anxiolytic phenotype of mGluR5 null mutant mice, EPM and light-dark exploration testing were conducted under low lighting conditions to enhance our ability to detect a potential anxiogenic phenotype in the heterozygous mice . We founWe have previously demonstrated that C57Bl/6J mice self-administer varied visual and auditory cues in operant sessions without any prior training, a phenomenon we term “operant sensation seeking” (OSS) A previous report found that mGluR5 KO mice failed to self-administer cocaine, although Het mice were not investigated A recent paper identified a role for the mGluR5 receptor in Pavlovian to instrumental transfer; that is, when mGluR5 was blocked during Pavlovian conditioning training, the cue later failed to serve as a reinforcer in an operant test of conditioned reinforcement It is important to consider potential differences in locomotor activity that could influence phenotypes such as OSS. To determine if altered mGluR5 expression levels also affected locomotor activity relevant to OSS, we tested locomotor activity in a novel environment and after habituation. Following habituation, we also tested the locomotor sensitivity to cocaine. mGluR5 deletion has been reported to increase novelty-induced locomotor activity mGluR5 has been strongly implicated in mediating anxiety behavior in rodent models A modified Irwin screen indicated that in most regards, there were no differences between mGluR5 genotypes. Among these tests was the reaching reflex, which assesses a mouse's ability to reach toward an edge using a visual cue. Over 90% of all mice were able to reach prior to vibrissae contact, indicating that there are no gross alterations in vision in any of the genotypes. A notable finding in the Irwin screen was the decreased body weight observed in mGluR5 knockout mice. This difference has been reported previously and was attributed to alterations in appetite induced by mGluR5 deletion In conclusion, we found that OSS was highly susceptible to changes in mGluR5, as both the Hets and KOs had altered responding. We also found that different traits associated with the mGluR5 receptor are differentially vulnerable to variance in the grm5 gene. Unlike OSS, haploinsufficiency of the grm5 gene did not significantly affect anxiety-like behavior. Similarly, we found that conditioned place preference for cocaine was similar across genotypes for 5 mg/kg, although there was a trend for Het and KO to have greater preference for 1 mg/kg. Considering that variation in the human mGluR5 gene (GRM5) has been linked to alcohol dependence Protocols were performed in compliance with the Guide for the Care and Use of Laboratory Animals  and were approved by the Institutional Animal Care and Use Committee, Vanderbilt University .grm5) null mutant, heterozygous, and wildtype littermate mice ad libitum unless noted. Mice were handled for at least three days prior to the beginning of experiments. All procedures were approved by the Animal Care and Use Committee at Vanderbilt University.mGluR5  housed individually within sound-attenuating chambers (MED Associates ENV-515). Each chamber of the apparatus was identical with the exception of the floor material (bar floor vs. wire mesh floor). These floor materials have previously been described as imparting different preferences in mice 31 male mice were tested for open field locomotor activity in a novel environment as previously described 34 male mice were tested on the elevated plus maze followed by the light-dark exploration test, then subjected to a modified Irwin screen. The maze consisted of two white closed arms protected by black acrylic walls 15 cm tall, bisected by two white open arms with raised edges and was lit by incandescent lighting (9–11 lux per arm). Sessions were recorded by a ceiling-mounted video camera connected to a computer using video acquisition and analysis software . Mice were allowed one hour to acclimate after being transported to the experimental room. Experimental sessions were five minutes in duration, and the apparatus was cleaned with 30% ethanol between each trial. Data were represented as % open arm time divided by total arm time . Locomotor activity was represented as distance traveled throughout the entire maze.48 hours following elevated plus maze testing, mice were tested in the light-dark exploration test. Eight light-dark apparatuses consisted of inserts  placed into the open field chambers described in Experiment 3. The lighting was ∼30 lux in the bright chamber and ∼2 lux in the dark chamber. After transport to the experimental room, mice were given one hour to acclimate before testing. Each test was 5 minutes and began by placing the mouse into the light chamber facing away from the entrance to the dark chamber. Latency to enter the dark side, time spent in each chamber, and the number of transitions was measured.At least one week following the light-dark exploration test, mice were screened for physiological and reflexive measures in a modified Irwin battery post hoc testing comparing each genotype to wildtype.Genotype differences were compared by between-subjects ANOVA followed by Dunnett's multiple comparisons tests when significant overall ANOVA effects were found. In the instance that multiple sessions were also performed on each subject, two-way repeated measures ANOVA was performed with session as the repeated factor. In the case of heterogeneous variance, Games-Howell multiple comparisons were employed. For the modified Irwin screen, measurements that were not done on a three-point scale were compared by one-way ANOVA (genotype) followed by Dunnett's Supporting Information S1post hoc tests comparing Het and KO to WT. N = 3-7, **p<0.01.Methodology for and Measurement of D1 and mGluR5. Levels of D1 (A) and mGluR5 (B) receptor protein in nucleus accumbens of naïve mGluR5 WT, Het, and KO mice. Data analyzed by 1 way ANOVA followed by Dunnet's (PDF)Click here for additional data file.


Nerve growth factor β (NGFB) is involved in cell proliferation and survival, and it is a mediator of the immune response. ProNGF, the precursor protein of NGFB, has been shown to induce cell death via interaction with the p75 neurotrophin receptor. In addition, this neurotrophin is differentially expressed in males and females. Hence NGFB is a good candidate to influence the course of multiple sclerosis (MS), much like in the murine model of experimental autoimmune encephalomyelitis (EAE).NGFB gene in up to 1120 unrelated MS patients and 869 controls. Expression analyses were performed for selected MS patients in order to elucidate the possible functional relevance of the SNPs.Ten single nucleotide polymorphisms (SNPs) were genotyped in the NGFB mRNA seems to be expressed in sex- and disease progression-related manner in peripheral blood mononuclear cells.Significant association of NGFB variations with MS is evident for two SNPs. NGFB variation and expression levels appear as modulating factors in the development of MS. HLA-DRB1 region. Recent studies point to complex allelic interactions of the HLA-DRB1 locus[e.g. ciliary neurotrophic factors.Multiple sclerosis (MS) was shown to depend on genetic components in various twin-, family- and association based studies. This coRB1 locus underscoNGFB) gene known to be involved in many cell regulatory pathways including cell survival and proliferation as well as in immune regulatory processes[NGFB is a prominent candidate gene to influence MS development.Here, we focussed on the nerve growth factor beta , 622 of which showed relapsing remitting (rr), 252 secondary progressive (sp) and 249 primary progressive (pp) course according to the Poser criteria as well as 869 healthy blood donors  resided in the Rhein-Ruhr area (Germany) as detailed in previous studies[® assays . Power analyses for this study were performed using GPower software assuming small effect size of 0.20 of the variation, α = 0.05 and degree of freedom (DF) = 2 (data not shown)[Genotyping was performed via polymerase chain reaction (PCR) with subsequent restriction fragment length polymorphism (RFLP) analyses or via TaqManot shown).® SYBR® Green one-step RT-PCR (Qiagen). The quantification of NGFB RNA was analysed using the ΔΔCt method. Oligonucleotides used were F-GCT TTC TAT CCT GGC CAC A, R-CAG GGA CAT TGC TCT CTG AG for the analysed NGFB as well as F-AGG TCG GAG TCA ACG GAT TTG, R-AAG CAG CCC TGG TGA CCA G for GAPDH as reference system. Statistical analyses were performed using t-tests .Fresh peripheral blood mononuclear cells (PBMCs) were obtained from MS patients in stable phases of the disease. 66% of patients received immunomodulatory treatment . RNA of 23 male patients (of which eight suffered from rr (three Ø treatment), eleven from sp (three Ø treatment) and four from pp MS (one Ø treatment)) as well as ten females (of which five showed rr (two Ø treatment) and five sp (two Ø treatment) MS course) was isolated using the RNeasy kit (Qiagen). Expression analyses were performed using QuantiTectNGFB gene with subsequent expression analysis in 23 male and ten female MS patients. A map of the genotyped SNPs in the NGFB gene is depicted in figure NGFB gene, since this exon encodes the precursor protein proNGF exclusively. rs6330 showed significant association in allele ) and genotype frequencies (p = 0.0126) when comparing healthy controls and MS patients. Interestingly, the strongest association was observed mainly for male rr MS patients for allele ) and genotype frequencies (p = 0.0230) when stratifying the cohorts by sex and disease progression, although the C allele was in general increased in all MS sub-cohorts , this allele could represent a risk factor due to its possible role in inducing increased cell death via enhanced interaction with the p75 neurotrophin receptor. Interestingly, male MS patients seem to have a higher risk than female MS patients  to its DNA motif[unc = 0.01 and a significant effect in the C dominant model for the genotype distribution punc = 0.0075  MS cohorts in order to provide convincing statistical power. Under the assumption of enhanced NGFB expression due to the higher affinity of the VDR binding in case of the C allele, we propose a protective effect of the C allele due to higher NGFB levels and its cell proliferating and survival mediating properties, thus preventing chronic disease progression. Although rs11102930 is reported to alter the binding affinity of the VDR, the expression regulatory influence has still to be shown via in vitro reporter assays. Our analyses of the rs11102930 genotype on the expression profile revealed no correlation of the genotype patterns for SNP rs11102930 and NGFB levels. Yet, male rr MS patients have a 2.5 fold elevated expression in comparison to male sp + pp MS patients as shown in figure rs11102930 is located in the promoter region of the DNA motif. VDR hasDNA motif and it aDNA motif. For ourOur results indicate a disease modulating role of NGFB for MS progression as shown in the EAE model. The preThe complex LD patterns for the 52.32 kilobase pair long sequence (see HapMap data) and the haplotype analysis for the 10 tested SNPs make predictions of putative interactions of rs6330 with rs11102930 largely impossible  for the 10 tested  Provided are the data for the genotyping assays of the 10 tested SNPs in the NGFB gene as well as the corresponding statistical analysis including the Haplotype block representation.Click here for file


Intravaginal practices are commonly used by women to manage their vaginal health and sexual life. These practices could, however, affect intravaginal mucosal integrity. The objectives of this study were to examine evidence for associations between: intravaginal practices and acquisition of HIV infection; intravaginal practices and vaginal infections; and vaginal infections and HIV acquisition.st January 2008. Relevant articles were selected and data extracted in duplicate. Results were examined visually in forest plots and combined using random effects meta-analysis where appropriate. Of 2120 unique references we included 22 publications from 15 different studies in sub-Saharan Africa and the USA. Seven publications from five studies examined a range of intravaginal practices and HIV infection. No specific vaginal practices showed a protective effect against HIV or vaginal infections. Insertion of products for sex was associated with HIV in unadjusted analyses; only one study gave an adjusted estimate, which showed no association . HIV incidence was higher in women reporting intravaginal cleansing but confidence intervals were wide and heterogeneity high . HIV incidence was higher in women with bacterial vaginosis  and Trichomonas vaginalis .We conducted a systematic review of prospective longitudinal studies, searching 15 electronic databases of journals and abstracts from two international conferences to 31A pathway linking intravaginal cleaning practices with vaginal infections that increase susceptibility to HIV infection is plausible but conclusive evidence is lacking. Intravaginal practices do not appear to protect women from vaginal infections or HIV and some might be harmful. Trichomonas vaginalis, which can increase the risk of HIV acquisition Intravaginal practices comprise a variety of behaviors that women use to positively manage their health and sexual life Biological and epidemiological data have been integrated to hypothesize a pathway linking intravaginal practices with acquisition of HIV infection, which might be mediated by changes in vaginal flora or vaginal infections that disrupt mucosal integrity  [1], , [8]Of the 22 included publications , seven iTypes of intravaginal practices were described in 14 publications from eight studies . The mos2 92.0%). Only two studies provided adjusted effect estimates 2 76.6%) and adjusted analyses including only two studies  2 0%) Five studies reported on associations between any kind of intravaginal practice and incident HIV-1 infection in analyses that included 4,169 women with more than 253 HIV infections (number not reported in one study) in seven publications  [7], [142 0%) 2 49.2%) Associations between intravaginal practices and incident bacterial vaginosis or disturbance of intravaginal flora were reported in six studies involving 6,503 women in analyses 2 81%) and adjusted analyses .There was strong and consistent evidence of associations between bacterial vaginosis and HIV and between trichomoniasis and HIV in both unadjusted and adjusted analyses . EvidencThis systematic review and meta-analysis of 22 publications from 15 studies in sub-Saharan Africa and the USA found no evidence that intravaginal practices reduce the incidence of vaginal infections or HIV. There was inconclusive statistical evidence about the effect of intravaginal practices on women's risk of acquiring HIV infection; the direction of summary measures of associations suggested harm, but confidence intervals were wide and there were high levels of heterogeneity. There was evidence of an association between intravaginal washing and douching practices and bacterial vaginosis in unadjusted but not adjusted analyses. There was strong statistical evidence that bacterial vaginosis and trichomoniasis increase women's risk of acquiring HIV infection.The main strength of this study was the comprehensive search for studies examining all stages of a potential pathway linking intravaginal practices and HIV infection. Our systematic searches of multiple databases without language restrictions are unlikely to have missed important prospective studies of intravaginal practices and HIV infection. The specific keywords used to identify individual vaginal infections might, however, have failed to identify studies that were indexed only under the general term ‘sexually transmitted diseases’. We also attempted to distinguish the effects of different intravaginal practices using categories based on empirical research that describe practices according to the type of practice, method of application and product One previous review examined associations between intravaginal practices, vaginal infections and HIV Our review showed no evidence that the intravaginal practices investigated protect against the development of bacterial vaginosis, or acquisition of other vaginal infections or HIV, even though previous studies have found that women report that improving genital hygiene and treating symptoms are common motivations for these practices This study showed strong and consistent evidence that trichomoniasis increases women's risk of acquiring HIV in vitro activity and safety profiles did not prevent, and might have increased, the risk of HIV infection The findings of this study have implications for HIV prevention research, particularly in the field of vaginal microbicide development. A sub-group analysis of one trial found some evidence of benefit of 0.5% PRO 2000/5 gel Table S1Assessment of reporting of features related to the risk of bias. *** criterion well-covered; ** criterion adequately addressed; * criterion poorly addressed; - criterion not addressed or not reported; VI vaginal infection; VP intravaginal practice; HIV - human immunodeficiency virus infection; where more than one of our study objectives was addressed see , exposur(0.07 MB DOC)Click here for additional data file.Table S2Chlamydia trachomatis; HSV2, herpes simplex virus type 2; NG, Neisseria gonorrhoeae; NR, not reported; wy, woman years of follow up; WBC, white blood cells.Studies reporting associations between intravaginal practices and incident HIV infection (objective 1). BV, bacterial vaginosis; CT, (0.07 MB DOC)Click here for additional data file.Table S3Lactobacillus spp.; NR, not reported; TV, Trichomonas vaginalis; WBC, white blood cells.Studies reporting associations between intravaginal practices and vaginal infections (objective 2). BV, bacterial vaginosis; HSV2, herpes simplex virus type 2; H2O2, hydrogen peroxide producing (0.12 MB DOC)Click here for additional data file.


Our results demonstrate the feasibility of array-based approaches in studies of DNA methylation and highlight the vast differences between individual loci. The identification of CpG loci of which DNA methylation levels are under genetic control or are related to age or gender will facilitate further studies into the role of DNA methylation and disease.Cytosine-5 methylation within CpG dinucleotides is a potentially important mechanism of epigenetic influence on human traits and disease. In addition to influences of age and gender, genetic control of DNA methylation levels has recently been described. We used whole blood genomic DNA in a twin set  as well as healthy controls (N = 96) to investigate heritability and relationship with age and gender of selected DNA methylation profiles using readily commercially available GoldenGate bead array technology. Despite the inability to detect meaningful methylation differences in the majority of CpG loci due to tissue type and locus selection issues, we found replicable significant associations of DNA methylation with age and gender. We identified associations of genetically heritable single nucleotide polymorphisms with large differences in DNA methylation levels near the polymorphism ( Epigenetic factors have recently been found to play an important role in developmental plasticity AXIN1 (NC_000016) in peripheral blood COMT gene region (NC_000022), suggesting that DNA methylation may provide a mechanism through which birth weight affects the risk for schizophrenia IGF2/H19 locus (NC_000011) in twins revealed substantial heritability and cis genetic associations with single nucleotide polymorphisms (SNPs) Studies involving monozygotic and dizygotic twins have been used to investigate the influence of DNA methylation on phenotypic variation. A study using monozygotic twins discordant for caudal duplication anomaly revealed significant differences in DNA methylation patterns of the disease associated gene Based on these recent findings a picture is emerging in which DNA methylation levels at specific loci are to some degree under genetic control To investigate the heritability of DNA methylation as well as the effects of age and gender, we performed a pilot study of DNA methylation in healthy twin and control subjects using the Illumina GoldenGate Methylation assay. In this assay 1,505 CpG sites from more than 800 genes in the human genome are interrogated simultaneously. The aim of the study was two-fold. First, we were interested to see whether it was feasible to successfully measure DNA methylation levels in blood using commercially available arrays. Second, we determined whether previously reported effects of age, gender and genotype on DNA methylation levels were restricted to specific loci and whether the effects sizes were large enough to be detected.We generated DNA methylation data of MZ twins (N = 43), DZ twins (N = 43) and 96 healthy singletons using peripheral blood DNA samples and the GoldenGate Methylation assay. First, we used BeadStudio software to obtain intensity measures  for each sample. Using a threshold intensity value of 4,000, we eliminated eight subjects from five pairs (including replicate pairs) in the twin set from further analysis. We next excluded eight probes that had a detection p-value>0.05 in more than 5 % of the hybridizations. The failure rate in the remaining 1,497 probes was <0.01 %. We repeated the above steps for the 96 healthy control subjects included in the second set. In the latter series, 11 of the 1,505 DNA methylation probes and one subject failed quality control and were removed from further analysis.We performed two-dimensional unsupervised hierarchical clustering of autosomal loci in the filtered data set (Heatmap and histogram shown in Supplementary ACVR1) (NM_001105.2), interleukin 6 (IL6) (NM_000600.1), caspase recruitment domain-containing protein 15 (CARD15) (NM_022162.1), platelet-derived growth factor receptor alpha (PDGFRA) (NM_006206.3), nuclear factor kappa-B subunit 1 (NFKB1) (NM_003998.2), and ETS-domain protein (ELK) (NM_005229.2). The associations with age of all 251 DNA methylation probes are listed in Supplementary We investigated the association between DNA methylation levels of the 280 high-quality probes with age. We observed a strong correlation between the two experiments with an overall Spearman correlation for t-values of 0.77 (95% CI: 0.71–0.81). The combined analyses identified a total of 58 probes that were significantly related to age (21.5 percent). Six loci also showed a significant correlation with age in both samples series independently . The genWe next investigated the relationship of DNA methylation levels with gender. As expected, non-parametric analysis with Mann-Whitney U test showed that all X-chromosomal probes on the bead array were significantly more methylated in females. However, all but two X-linked probes failed the stringent normality test. For this reason we restricted the analysis of gender effects to the autosomal loci only. A total of 56 of the autosomal probes (20%) showed a significant effect of gender in the combined samples (see Supplementary AXL) (NM_001699.3), glutathione S-transferase M1 isoform 2 (GSTM1) (NM_000561.2), T-cell lymphoma invasion and metastasis 1 (TIAM1) (NM_003253.2) and TEK tyrosine kinase (TEK) (NM_000459.2) among others  yielded a significant heritability . The moscis and 5 trans effects after stringent multiple testing correction and confirmation by permutation testing . We here present genetic associations of three DNA methylation loci with nine SNPs that constituted a mean difference in DNA methylation level larger or equal than 0.17 to rule out the possibility of technical artifact and survived corrections for multiple testing both for the number of SNPs as well as the 512 phenotypes employed (ALOX12 (NM_000697.1), the gene locus with the most significant association of DNA methylation with genotype, is shown in GRPR , and PCTK1 ) with the SNPs rs12743401, rs3881953, rs12734338 rendered non-significant after including gender as a covariate. The association signals are most likely from a homolog area on the Y chromosome as the association of these SNPs with male gender (p<1E-8) confirms.Based on the high heritability estimate of DNA methylation profiles of multiple CpG sites, we performed association mapping using DNA methylation levels as quantitative traits. For 91 subjects of the second set, sample genotype data was available (Illumina 550K BeadChip), with a total of 437,968 SNPs genome-wide after quality control filtering. Genomic inflation factor as a measure of population stratification ranged from 0.96 to 1.05, the mean chi-square statistic ranged between 0.98 and 1.03 indicating an absence of population stratification. We observed significant associations between DNA methylation at 11 different CpG loci representing 35 employed . An examEVI2A (NM_001003927.1), which showed high heritability and strong associations with DNA methylation. We used a subset of subjects representing the upper and lower quartile of DNA methylation values and homozygosity of the associated alleles for four additional gene regions . We observed one novel sequence variation within the EVI2A_E420_F probe region in one subject. The remaining six loci did not show any genetic variation. (Detailed results of sequence data are available upon request.)To investigate whether sequence variation at the probe target regions may affect hybridization and cause spurious association findings, we performed a targeted sequencing effort of 15 gene regions. All subjects of the second set were included for sequencing This study surveys inter-individual DNA methylation in relation to genotype, age and gender effects simultaneously. Using genomic DNA extracted from peripheral blood, we analyzed DNA methylation patterns of a large number of CpG sites from more than 800 genes spread throughout the genome in 23 monozygotic and 23 dyzygotic healthy twin-pairs and 96 healthy singletons. We demonstrate that the application of readily available array based technology can provide meaningful results that open the opportunity of incorporating epigenetic information in genetic studies.cis effects constituted large effect sizes that are likely to be biologically relevant and survived strict correction for multiple testing. Other associations, although statistically significant, constituted smaller effect sizes and included trans effects of genetic variation on DNA methylation levels as well. Additional sequence analysis did not identify DNA variation within the targeted CpG probe regions that could have lead to spurious association findings Despite the fact that in the majority (86 percent) of CpG sites interrogated by this panel, no substantial DNA methylation variation was present due to tissue type and CpG selection, we did observe replicable, independent, significant effects of age and gender on DNA methylation status for some of the remaining sites. Furthermore, for a number of DNA methylation loci in the twin data, we observed high heritability values for their DNA methylation levels. Genome-wide mapping using DNA methylation as a quantitative trait in 91 healthy singletons with available SNP data yielded highly significant associations. Particularly some of the cis), within regions that are in linkage disequilibrium with the CpG sites assayed on the array. However, we also observed a number of significant genetic associations with DNA methylation status elsewhere in the genome on other chromosomes (i.e. trans effects) albeit with small effect sizes and with unknown biological relevance. Given the fact that trans effects were already observed in a relatively small sample size of 91 subjects with limited power in a small subset of CpG methylation sites, it is likely that larger studies will identify more trans effects and uncover new genetic regulation of epigenetic profiles. Further non-parametric analysis of association signals may implicate additional genetic control of methylation levels. It is noticeable that all significant trans effects are observed with common variants located within genes instead of gene deserts, suggesting biologically relevant interactions. However caution is warranted as some trans effects were identified as artifacts. For instance the genetic associations in trans from the X-chromsomal CpG sites were due to homologs of the associated SNPs on the Y-chromosome and the SNPs  listed at chromosome 6 but have homologs at chromosome 12 and therefore most likely constitute cis effects rather trans effects on methylation levels of the KRAS locus (NM_033360.2) on chromosome 12. However, another trans effect such as from rs3134269 in the Solute Carrier Family 25, member 32 gene  (involved in folate metabolism) that is associated with DNA methylation levels at TSC2  constitute a difference of 0.24 percent, well above detection sensitivity . These results support recent reports that changes in DNA methylation do not predominantly occur in promoter regions or CpG islands In addition to genetic control of DNA methylation levels, we found independent associations of DNA methylation with age and gender. Gender effects for methylation loci on the X chromosome are largely due to the X-inactivation dosage compensation mechanism in females PTHR1  gene that was found differentially methylated in females is associated with bone mineral density that shows substantial difference between males and females. Sequence variation of the IL6 gene (NM_000600.1) is related to metabolic syndrome in older males. Finally the AXIN1 locus (NM_003502.2) of which DNA methylation levels in peripheral blood has previously been associated with caudal duplication anomaly showed high heritability in our sample . Further studies into the phenotypic consequences of such methylation differences seem warranted.The DNA methylation loci with high heritability or with significant associations with age or gender identified in our study present some genes that have previously been implicated in disease. For example, the We realize that these findings are based on a relatively small sample size with limited statistical power. In addition, the Illumina GoldenGate Cancer Panel probes represent only a minute fraction (less than 0.005 percent) of all CpGs in the human genome. On estimate there are 32 million CpG dinucleotides constituting approximately 1 one percent of the genome et al who reported that the intraclass correlation between tissue type was significantly higher compared to intra-individual differences A remaining challenge will be the study of tissue-specificity of epigenetic control. We do not know what the meaning of differential DNA methylation in blood is for other tissues. Although several studies suggest an important role of DNA methylation in the specialization of tissues DNA methylation information can potentially be applied to the study of environmental influences, genetic mapping studies and the development of biomarkers. Further studies may unveil novel mechanisms by which the environment exerts influence on the susceptibility to diseases and may also be important for genetic linkage and association studies. It is possible that DNA methylation of disease-associated alleles may affect the relative risk of carriers to develop the trait, cause phenotypic heterogeneity or may explain some of the phenotypic differences in monozygous twins Written informed consent from all participants was obtained. This study was approved by the Medical Ethical Committee of the UMC Utrecht, The Netherlands.The twin sample included 23 healthy monochorionic or dichorionic monozygotic twin pairs (46 subjects) and 23 healthy dizygotic twin pairs  recruited via advertisements in local newspapers and media. Inclusion criteria included good general health and at least three Dutch grandparents. Zygosity of twin pairs was investigated previously by the Illumina DNA Test Panel consisting of 360 validated single nucleotide polymorphisms (SNPs). General Health was established using a general medical checklist; all subjects were free of medication. An independent second sample set consisted of 96 healthy controls, matched for age and gender to the original twin series. For this second sample set, genome-wide SNP data (Illumina 550K Infinium array) was available. The second sample was used to validate the age and gender effects observed in the twin sample and to perform genetic mapping using DNA methylation as quantitative traits. The mean age and gender information for both sample sets are listed in www.illumina.com/pages.ilmnID193).Genomic DNA extracted from peripheral blood was used for this study. Experiments of the twin data and singletons were performed independently. Genomic DNA samples (0.5 µg) from both sets were bisulfite converted using the EZ-96 DNA methylation kit  according to the manufacturer's protocol in two batches, first the twins and secondly the singletons. The twins and co-twins were randomly distributed across plates. DNA methylation profiles were obtained using the commercially available Illumina GoldenGate DNA methylation platform (DNA Methylation Cancer Panel I) as recommended by the manufacturer . With this assay, the DNA methylation levels of up to 1,505 CpG sites covering 807 gene regions are interrogated concurrently. The probe IDs are listed in Supplemental We used Illumina BeadStudio software for normalization and initial quality control. BeadStudio uses a background normalization that subtracts a background value derived by averaging the signals of built-in negative control bead types. The DNA Methylation status for each probe is presented as a Beta value, the ratio of signal from the methylated probe relative to the sum of both methylated and unmethylated probes. Beta values can range from 0 (unmethylated) to 1 (fully methylated). We tested for approximate normal distribution by means of a Lilliefors test for normality at the 0.1 level after FDR correction for multiple testing. Non-normal distributed probes were excluded from subsequent analysis. To ensure biological relevance of the methylation differences, we also excluded those probes that had a range smaller than 0.17 in the methylation levels. For further analysis, we used Z-scores of DNA methylation levels and age in a variance component analysis. Analyses of age and gender effects were conducted for the twins and singletons, both separately and jointly. The effects of age and gender on the level of methylation were tested in Mx Normalization steps were conducted in BeadStudio, variance component analysis was conducted using Mx 7) was performed for the associated SNPs to confirm our findings. These analyses were carried out on the Genetic Cluster Computer (http://www.geneticcluster.org). Association signals that were obtained from X-chromosomal loci were re-calculated using gender as covariate in a linear regression model.We generated SNP genotype data for 91 samples in the singletons using the Illumina HumanHapmapv1.1 550K platform at UCLA. Associations with the 512 DNA methylation levels that were normally distributed in this sample as quantitative phenotypes, were calculated using PLINK To investigate whether DNA sequence variation at probe regions affected the hybridization signals, we examined several candidate loci by standard sequencing. Selection of CpG sites for sequencing was based on the presence of highly significant association findings. All subjects from the second set were sequenced for PCR amplicons for the EVI2A_E420 locus; eight selected subjects were used to screen for sequence variation in the probe regions of ALOX12_P223_R, TDG_E129_F, CTSD_P726_F, TMPRSS4_P552_F. The latter subjects were selected by two criteria: DNA methylation levels in the upper or lower quintile, and homozygosity for the associated SNPs. The probes were selected from a range of strength of association signals based on the availability of suitable primers. Primer sequences are available upon request.Table S1Full age results(0.12 MB XLS)Click here for additional data file.Table S2Full gender results(0.13 MB DOC)Click here for additional data file.Table S3Full heritability result(0.10 MB DOC)Click here for additional data file.Table S4Full mapping results(0.06 MB XLS)Click here for additional data file.Table S5Probe IDs(1.09 MB XLS)Click here for additional data file.Figure S1Heatmap of twin sample(1.28 MB TIF)Click here for additional data file.Figure S2Heatmap of singleton sample(1.28 MB TIF)Click here for additional data file.


Trastuzumab (Herceptin), an antagonist to the human epidermal growth factor 2 (HER2) receptor significantly decreases the rates of breast cancer recurrence and mortality by 50%. Despite therapeutic benefits, the risk of cardiotoxicity with trastuzumab ranges from 10–15% when administered sequentially following anthraycline chemotherapy. Little is known about the utility of cardiac magnetic resonance (CMR) in the assessment of trastuzumab mediated cardiomyopathy.Between 2005–2006 inclusive, 160 breast cancer patients were identified at a single tertiary care oncology centre. Of the total population, 10 patients (mean age 40 ± 8 years) were identified with trastuzumab induced cardiomyopathy, based on a LVEF less than 40% on serial MUGA or echocardiography. CMR was performed in all patients to determine LV volumes, systolic function and evidence of late gadolinium enhancement (LGE). At the time of diagnosis of trastuzumab induced cardiomyopathy, the mean LVEF was 29 ± 4%. Subepicardial linear LGE was present in the lateral portion of the left ventricles in all 10 patients.LGE-CMR is a novel way of detecting early changes in the myocardium due to trastuzumab induced cardiotoxicity. Breast cancer is a major public health concern that affects 1 in 7 women in their lifetime . Recent Serial multiple gated acquisition scans (MUGA) are widely used to monitor cardiac dysfunction in breast cancer patients. However, with the improvement in both spatial and temporal resolution of cardiac magnetic resonance (CMR) over the past decade, it has now become the gold standard for the non-invasive assessment of left ventricular (LV) systolic dysfunction. Additionally, late gadolinium enhancement (LGE) can detect myocardial scarring. Although frequently used in the assessment of dilated cardiomyopathies secondary to ischemia or myocarditis , little Between 2005–2006 inclusive, 160 breast cancer patients who received trastuzamab in addition to anthracyline based adjuvant therapy were identified at a tertiary care oncology centre. All patients received FEC  for a total of 6 cycles. The mean duration between completion of chemotherapy and initiation of trastuzumab was 2 ± 1 months. Of the total population, 10 patients were identified with trastuzumab induced cardiomyopathy based on LV ejection fraction less than 40% on either serial MUGA or echocardiography. The medical records of all 10 patients were extensively reviewed for baseline demographic data. The retrospective study was approved by the local institutional review board.CMR was performed on all 10 patients using a 1.5 T scanner . Morphologic images in the cardiac short axis, 4 chamber long axis and 2 chamber long axis planes were acquired using IR-prepared dark blood HASTE sequences . In the same planes, cine-CMR was performed using a breath-hold balanced steady state free precession sequence . The cine-CMR short-axis images encompassed the entire LV from the base to the apex  to obtain a left ventricular ejection fraction (LVEF). Late gadolinium enhancement was performed after 10 minutes of 0.2 mmol/kg injection of Gadolinium  using a T1-weighted IR-prepared multislice TrueFISP sequence with magnitude and phase sensitive reconstruction. Images were acquired sequentially in the short axis, followed by horizontal and vertical long axis images .The total population included 10 patients  with normal LVEF at baseline and preserved systolic function following administration of anthracycline based chemotherapy using serial MUGA scans  is a relatively new chimerized monoclonal antibody that targets the extracellular portion of the HER2 membrane protein. Previous studies have demonstrated that trastuzumab provides considerable therapeutic benefits, when added to conventional anthracycline chemotherapy, in decreasing the rates of disease recurrence and death in HER2 overexpressed metastatic breast cancers -6. DespiVarious theories have been suggested regarding the possible pathogenetic origin of trastuzumab induced cardiomyopathy. These include potentiation of anthracycline induced cardiotoxicity and immune mediated destruction of cardiomyocytes . There iThe use of CMR for the non-invasive characterization of trastuzumab induced myocarditis is novel. Whereas CMR has become the standard diagnostic test in the evaluation of suspected myocarditis ,12, to dLate gadolinium enhancement using CMR is a novel way of detecting early changes in the myocardium due to trastuzumab induced cardiotoxicity. Future studies are required to validate identification of positive delayed enhancement using CMR as a subclinical marker for future LV dysfunction in this select population. Early detection of LV dysfunction using LGE-CMR may allow one to adjust treatment with trastuzumab prior to the development of irreversible heart failure.The author(s) declare that they have no competing interests.