Connective tissue diseases (CTDs) are a heterogeneous group of disorders that share certain clinical presentations and a disturbed immunoregulation, leading to autoantibody production. Subclinical or overt renal manifestations are frequently observed and complicate the clinical course of CTDs. Alterations of kidney function in Sjögren syndrome, systemic scleroderma (SSc), auto-immune myopathies (dermatomyositis and polymyositis), systemic lupus erythematosus (SLE), antiphospholipid syndrome nephropathy (APSN) as well as rheumatoid arthritis (RA) are frequently present and physicians should be aware of that.In SLE, renal prognosis significantly improved based on specific classification and treatment strategies adjusted to kidney biopsy findings. Patients with scleroderma renal crisis (SRC), which is usually characterized by severe hypertension, progressive decline of renal function and thrombotic microangiopathy, show a significant benefit of early angiotensin-converting-enzyme (ACE) inhibitor use in particular and strict blood pressure control in general. Treatment of the underlying autoimmune disorder or discontinuation of specific therapeutic agents improves kidney function in most patients with Sjögren syndrome, auto-immune myopathies, APSN and RA.In this review we focus on impairment of renal function in relation to underlying disease or adverse drug effects and implications on treatment decisions. Impairment of renal function is present to some extent in many connective tissue diseases (CTDs) with variable occurrence in Sjögren syndrome ,2, roughLymphocytic infiltration, leading to acute or chronic tubulointerstitial nephritis, is the predominant renal pathology in Sjögren syndrome ,6,7. SclLupus nephritis is one of the most severe organ manifestations of the disease and, depending on biopsy findings, needs aggressive immunosuppressive therapy. The histopathologic classification of lupus nephritis guides therapeutic interventions with the aim to reduce proteinuria and preserve kidney function. Renal manifestation in primary and secondary antiphospholipid syndrome (APS) is a well-described complication, frequently leading to arterial hypertension and occasionally impairment of renal function ,16. PatiPrimary Sjögren syndrome (PSS) is an autoimmune disorder of hitherto unknown origin which is characterized by polyclonal B-cell activation as well as lymphocytic infiltration of the exocrine glands, resulting in keratoconjunctivitis sicca (dry eyes disease) and/or xerostomia (dry mouth disease) . In addiAcute or chronic tubulointerstitial nephritis (TIN) with defects in tubular function is the predominant lesion in biopsy-proven renal involvement ,6. DistaTreatment with glucocorticoids should be initiated as first line therapy in patients with PSS and renal involvement since a good response to early treatment has been reported ,26. In aCorticosteroids are a mainstay in the treatment of TIN. Further histologic findings require specifically-tailored immunosuppression and most importantly, supplementation of bicarbonate and/or electrolytes, when indicated, should be commenced.SSc is a CTD characterized by deposition and overproduction of extracellular matrix proteins and collagen, resulting in tissue fibrosis and, subsequently, tissue dysfunction. Affected organs and tissues include the skin, gastrointestinal tract, heart, lungs and kidneys. Involvement of the vascular system generally results in the development of Raynaud’s phenomenon early in the disease course. Consecutively, severe clinical manifestations of vascular dysfunction can be observed in some patients leading to pulmonary fibrosis and pulmonary artery hypertension, esophageal motility dysfunction, watermelon stomach, cardiac involvement, as well as scleroderma renal crisis (SRC) -29. EpitDiagnosis of SRC is confirmed by renal biopsy, which shows a thrombotic microangiopathic process, particularly affecting small vessels. Vascular changes are accompanied by thrombosis, accumulation of myxoid material and later in the disease course, development of onion-skin lesions and/or fibrointimal sclerosis . FurtherEarly use of ACE inhibitors, on the basis of most experience in particular captopril, is undoubtedly a cornerstone in the management of hypertensive SRC. Immediate use with a progressive increase of ACE inhibitor dosage, even in the presence of deteriorating kidney function, is considered to prevent or even reverse renal failure ,37. AddiRenal involvement in SSc is often accompanied by progressive renal failure and rapid initiation of therapeutic interventions is mandatory. Blood pressure control, in particular with ACE-inhibitors and additional antihypertensive medication, is essential. If blood pressure is not adjustable or the patient shows signs of fluid overload, dialysis should be considered early in the disease course. Discontinuation of dialysis was reported in some patients. In patients with chronic hemodialysis and renal transplantation, a calcineurin-inhibitor free immunosuppressive regimen might be chosen.Auto-immune myopathies, namely dermatomyositis (DM) and polymyositis (PM), share common clinical features, such as proximal muscle weakness, muscle inflammation, presence of autoantibodies, elevated muscle enzymes, electromyographic alterations and extra muscular manifestations. Despite clinical similarities, both differ regarding muscle biopsy findings and DM is associated with cutaneous involvement. The presence of a heliotrope rash, which is characterized by a violaceous skin discoloration around the eyes, and Gottron’s sign (erythematous papules with involvement of joints) are pathognomonic for DM ,41. BothTwo types of renal involvement have been described in patients with PM/DM. First, rhabdomyolysis with release of myoglobin can lead to acute tubular necrosis with deterioration of renal function ,15. SecoHigh-dose oral corticosteroids are the cornerstone of DM/PM therapy. Moreover, the addition of immunosuppressive drugs, such as azathioprine (AZA) or cyclophosphamide (CYC), as well as anti-malaria medication in DM and methotrexate, CYC, intravenous immunoglobulins and CSA in PM has been reported to improve the renal outcome in DM/PM ,49,55-58Management of patients with auto-immune myopathies and renal involvement require special caution, because disease-related mortality due to rhabdomyolysis and hyperkalemia is greatly feared. Special therapeutic intervention with immunosuppression should be tailored to the underlying histology. In most cases, corticosteroids might be effective as one therapeutic component.SLE depicts a remarkable complex autoimmune disease with considerable heterogeneity in clinical manifestations and disease course. Classification of SLE was last edited by the American College of Rheumatology (ACR) in 1997  Table 22. EarlieAutoantibodies are directed against various nuclear antigens, in particular against chromatin components, such as nucleosomes, histones, anti-nuclear antibodies (ANA), double-stranded DNA antibodies (dsDNA) and ribonucleoproteins. Recently, it was suggested that the nucleosome might be the driving autoantigen in SLE. This hypothesis is supported by the finding that glomerular deposition of anti-dsDNA antibodies in lupus nephritis is mediated by nucleosomes ,67. The Involvement of the kidney in the natural history of disease is present in a majority of patients and is supposed to appear in almost 50% in the first year of diagnosis . Recent In general, use of ACE inhibitors significantly reduced the development of proteinuria and/or biopsy-proven lupus nephritis and was associated with a decreased risk of disease activity . Concomi2) in patients with class III through V lupus nephritis [Specific treatment follows the class of lupus nephritis, which is defined by the revised ISN criteria. Class I and class II require no therapy directed at the kidney in consequence of good long-term renal outcome . In contephritis . A furthephritis . Responsephritis . Patientephritis . In a smephritis .Following induction therapy, long-term immunosuppression is mandatory to avoid severe flares and to maintain stabilization of disease activity. Thus, immunosuppressants with a favorable safety profile and good efficacy are mandatory. MMF and AZA are deemed suitable and have shown efficacy in maintaining remission of lupus nephritis . EquivalB-cell depleting therapy with anti-CD20 antibody rituximab (RTX) proved efficient in patients with active SLE including patients with lupus nephritis, who were nonresponsive to standard immunosuppressive therapy . In prooNovel approaches with focus on targeted therapy have been developed and are currently being evaluated in clinical trials. Circulating B-lymphocyte stimulator (BLyS) is elevated in SLE, and titers correlate with increased disease activity and elevated dsDNA antibody concentrations . PatientPromising results have been obtained in a phase II trial for epratuzumab, a humanized anti-CD22 antibody ,109. AtaIn patients with highly active lupus nephritis with failure of conventional therapy, short-term as well as prolonged immuno-adsorption led to a significant reduction in proteinuria and to sustained remission rates . AutologThe histopathologic classification of lupus nephritis still guides the therapy. In proliferative lupus nephritis (III and IV), CYC and MMF have shown almost identical therapeutic responses as induction therapy in large trials. CSA might be an alternative to these immunosuppressive agents in pure class V lupus nephritis. In patients not responding to initial treatment, multi-target therapy might be an effective alternative. MMF seems to be superior to AZA in maintaining remission. The role of RTX in the treatment of lupus nephritis has to be further elucidated, as well as the significance of novel therapeutic approaches in the therapy of lupus nephritis.th week of gestation), and the presence of antiphospholipid antibodies (aPL), namely anticardiolipin antibodies (aCL) and lupus anticoagulant (LAC) [Antiphospholipid syndrome (APS) is defined by the association of vascular thrombosis potentially affecting all segments of the vascular bed, complications during pregnancy  . The APSnt (LAC) .et al. reported that all patients had severe hypertension and renal insufficiency in a small cohort of patients with secondary APS due to SLE [et al. examined kidney biopsies obtained from patients with SLE with or without presence of aPL. APSN was detected in almost 40% with aPL, compared with only 4.3% of patients without aPL [et al. examined 29 kidney biopsies of patients with APS [Renal manifestations in the context of APS may result from thrombosis occurring at any location in the renal vasculature. Renal artery stenosis (RAS) is a common complication of APS, leading to renovascular hypertension . In a ree to SLE . Thromboe to SLE , especiae to SLE . APSN ree to SLE ,123. Thre to SLE ,123. Tekhout aPL . Fakhourwith APS . In ninewith APS . Intereswith APS ,127.Blood pressure control is the key intervention in the treatment of APS-related renal involvement. Adequate anticoagulation (if evidence of microthrombi is present) has shown encouraging results in small cohorts and may prevent progression to end-stage renal disease . EvidencBlood pressure control is mandatory in patients with APSN. The role of anticoagulation with a target through INR above 3.0 in patients with APSN and microthrombi to prevent kidney function deterioration has to be elucidated in further, larger studies.RA is characterized by persistent synovial, systemic inflammation and autoantibodies (particularly to rheumatoid factor and citrullinated peptides). Genetic as well as environmental factors contribute to the risk of developing RA . Renal iA study of renal biopsy specimens indicated that mesangial glomerulonephritis is the predominant histopathologic finding in RA, followed by amyloidosis, membranous nephropathy, focal proliferative glomerulonephritis, minimal-change nephropathy and acute interstitial nephritis . DevelopImprovement of clinical and laboratory parameters was achieved in most cases after drug withdrawal and in case of necessary initiation of immunosuppression ,142,143.Therapy related deterioration of kidney function has to be excluded in patients with RA. In addition, the persisting inflammation can lead to deposition of amyloid. Thus, adequate therapy to reduce disease activity may be effective in preventing this late-onset complication. Specific therapeutic interventions should be tailored to the underlying histologic kidney involvement.Renal involvement is frequently present in CTDs and has variable phenotypes. Since there is a steady increase of knowledge regarding the pathophysiology behind auto-immune disorders, more specific therapeutic approaches have been developed and are currently in clinical trials.Acute or chronic TIN is the predominant kidney biopsy finding in Sjögren syndrome. Kidney function normalizes in most cases after corticosteroids are initiated ,6. In adResults from hematopoietic stem cell transplantation (HSCT) in SSc are promising. Current studies, namely the SCOT and ASTIS trials, have completed patient recruitment and the first results are expected soon . The ASSDiverse glomerular alterations and rhabdomyolysis have been reported in patients with auto-immune myopathies. Guided therapy with the aim to treat the underlying disease improves kidney function in most cases.post hoc analysis of the EXPLORER trial indicated that RTX-treated patients achieved lower disease activity without a subsequent severe disease flare when compared to those treated with placebo [In SLE, new therapeutic approaches have gained attention. One of these novel agents is belimumab, an inhibitor of serum BLyS, which was recently approved by the FDA for treatment of SLE with the exception of active lupus nephritis and central nervous system involvement. A randomized, controlled trial with inclusion of active lupus nephritis is currently being designed. Furthermore, BLyS inhibition may also be effective in the treatment of PSS, since patients with Sjögren syndrome exhibit increased BLyS levels . In pati placebo . Persist placebo . In addi placebo ,103. Des placebo -114, RTX placebo .APS-related renal manifestation potentially affects any segment of the vascular bed and is commonly accompanied by arterial hypertension. Blood pressure control is crucial, whereas the role and the target level of oral anticoagulation needs to be further elucidated. Chronic inflammation, as well as drug related adverse effects, is causative of kidney involvement in RA. Etanercept has shown encouraging results in reduction of serum amyloid A in amyloidosis and patients with a baseline serum creatinine below 2 mg/dl tended to show a benefit following TNF-alpha inhibition .Based on studies in non-diabetic nephropathy, patients with renal involvement in CTDs should receive RAAS blocking agents once proteinuria is >1 g/day ,150. RenIn summary, renal manifestations of CTDs are frequent. Renal biopsy to ensure diagnosis is necessary in most patients presenting with deterioration of renal function, increase of proteinuria or signs of nephritic syndrome (summarized in Table AA: Amyloid A; ACE: Angiotensin-converting-enzyme; aCL: Anticardiolipin antibodies; ACR: American college of rheumatology; ANA: Anti-nuclear antibodies; aPL: Antiphospholipid antibodies; APRIL: A proliferation-inducing ligand; APS: Antiphospholipid syndrome; APSN: Antiphospholipid syndrome nephropathy; AZA: Azathioprine; BLyS: B-lymphocyte stimulator; CSA: Cyclosporine A; CTD: Connective tissue disease; CTGF: Connective tissue growth factor; CYC: Cyclophosphamide; DM: Dermatomyositis; DMARD: Disease modifying antirheumatic drug; dsDNA: Double-stranded DNA antibodies; EMT: Epithelial to mesenchymal transition; FDA: Food and drug administration; FSGS: Focal segmental glomerulosclerosis; HSCT: Hematopoietic stem cell transplantation; INR: International normalized ratio; ISN: International society of nephrology; LAC: Lupus anticoagulant; LDL: Low-density lipoprotein; MMF: Mycophenolate mofetil; PM: Polymyositis; PSS: Primary sjögren syndrome; RA: Rheumatoid arthritis; RAAS: Renin-angiotensin-aldosterone system; RAS: Renal artery stenosis; RF: Rheumatoid factor; RPS: Renal pathology society; RTA: Renal tubular acidosis; RTX: Rituximab; SLE: Systemic lupus erythematosus; Sm: Smith; SRC: Scleroderma renal crisis; SOC: Standard of care; SSc: Systemic scleroderma; TGFß: Transforming growth factor ß; TIN: Tubulointerstitial nephritis; TNF: Tumor-necrosis factor; UNOS: United network of organ sharing.The authors declare that they have no competing interests.AK performed the literature search and wrote the manuscript. GM critically reviewed the manuscript. Both authors approved the final version of the manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1741-7015/11/95/prepub


A better understanding of phosphate regulation would allow for optimization of such processes.The phosphorus compounds serve as major building blocks of many biomolecules, and have important roles in signal transduction. The phosphate is involved in many biochemical reactions by the transfer of phosphoryl groups. All living cells sophisticatedly regulate the phosphate uptake, and survive even under phosphate-limiting condition, and thus phosphate metabolism is closely related to the diverse metabolism including energy and central carbon metabolism. In particular, phosphorylation may play important roles in the metabolic regulation at acidic condition and nitrogen limiting condition, which typically appears at the late growth phase in the batch culture. Moreover, phosphate starvation is a relatively inexpensive means of gene induction in practice, and the Escherichia coli was investigated in terms of fermentation characteristics and gene transcript levels for the aerobic continuous culture at the dilution rate of 0.2 h-1. The result indicates that the specific glucose consumption rate and the specific acetate production rate significantly increased, while the cell concentration decreased at low P concentration (10% of the M9 medium). The increase in the specific glucose uptake rate may be due to ATP demand caused by limited ATP production under P-limitation. The lower cell concentration was also caused by less ATP production. The less ATP production by H+-ATPase may have caused less cytochrome reaction affecting in quinone pool, and caused up-regulation of ArcA/B, which repressed TCA cycle genes and caused more acetate production. In the case of phoB mutant , the fermentation characteristics were less affected by P-limitation as compared to the wild type where the PhoB regulated genes were down-regulated, while phoR and phoU changed little. The phoR gene knockout caused phoB gene to be down-regulated as well as PhoB regulated genes, while phoU and phoM changed little. The effect of pH together with lower P concentration on the metabolic regulation was also investigated. In accordance with up-regulation of arcA gene expression, the expressions of the TCA cycle genes such as sdhC and mdh were down-regulated at acidic condition. The gene expression of rpoS was up-regulated, and the expression of gadA was up-regulated at pH 6.0. In accordance with this, PhoB regulated genes were up-regulated in the wild type under P-rich and P-limited conditions at pH 6.0 as compared to those at pH 7.0. Moreover, the effect of nitrogen limitation on the metabolic regulation was investigated, where the result indicates that phoB gene was up-regulated, and PhoB regulated genes were also up-regulated under N-limitation, as well as nitrogen-regulated genes.The effect of phosphate (P) concentration on the metabolism in The present result shows the complicated nature of the metabolic regulation for the fermentation characteristics upon phosphate limitation, acidic condition, and nitrogen limitation based on the transcript levels of selected genes. The result implies that the regulations under phosphate limitation, acidic condition, and nitrogen limitation, which occur typically at the late growth phase of the batch culture, are interconnected through RpoS and RpoD together with Pho genes. Escherichia coli contains about 15 mg of phosphate (P) per g (dry cell weight)  cluster per monomer under anoxic conditions. In the presence of oxygen, [4Fe-4S] FNR is converted to [2Fe-2S] FNR cluster and finally to apoFnr, which is no longer active in gene regulation : In a number of iron-sulfur proteins; GABA: α-Aminobutyric acid; GDH: Glutamate Dehydrogenase; Gln: Glutamine; GOGAT: Glutamate Syntheses; GS: Glutamine Synthetase; HdeA: Periplasmic protein; N: Nitrogen; OAA: Oxaloacetic Acid; P: Phosphate; PEP: Phospho-enol-pyruvate; ppGpp: Guanosine tetraphosphate; PYR: Pyruvate; TCA: Tri-carboxylic acid; PTS: Phosphotransferase System; UTase: Uridylyltransferase.The authors declare that they have no competing interests.LWM carried out fermentation experiments, assayed, made statistical analysis, and drafted the manuscript. KS considered the experimental design, analyzed the result, and prepared the manuscript together with LWM. All authors read and approved the final manuscript.E. coli (a) and its phoB mutant (b)Effect of phosphate concentration on the fermentation characteristics of wild type .Click here for fileGlobal regulators and their regulated genes.Click here for fileList of additional primers.Click here for file


Recently, we reported that low reward dependence, and to a lesser extent, low cooperativeness in the Temperature and Character Inventory (TCI) may be risk factors for treatment-resistant depression. Here, we analyzed additional psychological traits in these patients.We administered Costa and McCrae's five-factor model personality inventory, NEO Personality Inventory-Revised (NEO-PI-R), to antidepressant-treatment resistant depressed patients (n = 35), remitted depressed patients (n = 27), and healthy controls (n = 66). We also evaluated the relationships between scores on NEO and TCI, using the same cohort of patients with treatment-resistant depression, as our previous study.Patients with treatment-resistant depression showed high scores for neuroticism, low scores for extraversion, openness and conscientiousness, without changes in agreeableness, on the NEO. However, patients in remitted depression showed no significant scores on NEO. Patients with treatment-resistant depression and low openness on NEO showed positive relationships with reward dependence and cooperativeness on the TCI.Many studies have reported that depressed patients show high neuroticism, low extraversion and low conscientiousness on the NEO. Our study highlights low openness on the NEO, as a risk mediator in treatment-resistant depression. This newly identified trait should be included as a risk factor in treatment-resistant depression. It is well documented that 60 to 70 percent of depressed patients respond to first line antidepressant treatment at maximum dose, for at least two months. Between 80 and 90 percent of these patients respond to first or second choice prescribed antidepressant medication. The remaining 10 to 15 percent of patients who do not respond to therapy are deemed to have treatment-resistant depression Another personality inventory, the NEO Personality Inventory-Revised (NEO-PI-R) is also in common use Chronically depressed patients also reported higher levels of neuroticism and lower levels of extraversion, agreeableness, and conscientiousness, compared with those suffering acute forms of the disease The purpose of this study was to investigate in more depth, the presence of personality biases in patients with treatment-resistant depression, using the NEO-PI-R The study was approved by the ethics committee of Teikyo University Chiba Medical Center (study number 09-30) and performed in accordance with the Declaration of Helsinki. Written informed consent was obtained from all participants, after procedures had been fully explained.Sixty six healthy subjects, 27 depressed patients in remission, and 35 antidepressant treatment-resistant depressed patients were enrolled on this study Table 1.Personality was assessed using NEO PI-R. NEO PI-R utilized the five-factor model of personality: neuroticism, extraversion, openness, agreeableness, and conscientiousness TCI Scores in patients with treatment-resistant depression were taken from our recently reported study Data from five domains of the NEO were first analyzed using multiple analysis of variance (MANOVA), to check for the simultaneous existence of significant differences. Statistical differences among the three groups were determined by one-way factorial analysis of variance (ANOVA), followed by multiple comparison testing (Scheffe's test). Chi-square test was used for categorical variables. Statistical evaluation between the two groups was performed using a two-tailed Student's t-test. Coefficients among scores for NEO and TCI were estimated by Pearson coefficient. Differences were considered to be significant when p values were less than 0.01.MANOVA indicated a significant group effect . Subsequent one-way ANOVA demonstrated that patients with treatment-resistant depression showed significantly high scores for neuroticism and lower scores for extraversion, openness and conscientiousness on the NEO, compared with healthy controls or patients with remitted depression Figure 1The subscales of each domain on the NEO are shown in Neuroticism correlated significantly with HAM-D scores in all MDD patients including both remitted and treatment-resistant groups . In contrast, there was no correlation between NEO scores and the severity of depression in patients with treatment-resistant depression (data not shown).A significant negative correlation between neuroticism and extraversion was seen in healthy controls and remitted depression patients, but not in treatment-resistant depression patients Table 3.As shown in We found that patients with treatment-resistant depression showed significantly altered scores in neuroticism, extraversion, openness and conscientiousness, as measured by NEO. Previous studies using the NEO show that depressed patients scored highly for neuroticism, low extraversion and low conscientiousness We also detected a significant negative correlation between neuroticism and extraversion, in the healthy control and remitted depression groups, but not in the treatment-resistant depression group Table 3.Patients with treatment-resistant depression showed a negative relationship between neuroticism and agreeableness, which also was seen in healthy controls, but not in remitted depressed patients Table 3 Here, openness on the NEO showed a positive relationship with reward dependence and cooperativeness on the TCI, in the treatment-resistant depression group Table 4.We also found significant relationships between neuroticism on the NEO and harm avoidance and self-directedness on the TCI in treatment-resistant depression. Additionally, we detected association between extraversion on the NEO, and harm avoidance and reward dependence on the TCI, and between conscientiousness on the NEO and harm avoidance, persistence and self-directedness on the TCI in the same group of patients Table 4.Finally, this study failed to show any significant factors in remitted depression patients, using the NEO Figure 1We put forward that patients with treatment-resistant depression display lower levels of resilience, compared with healthy subjects and remitted depression patients. A previous study showed that resilient individuals exhibit lower levels of denial, avoidant coping, pessimism and behavioral disengagement In conclusion, patients with treatment-resistant depression demonstrated high scores for neuroticism, low scores for extraversion, openness and conscientiousness using the NEO. Previous studies report that depressed patients show high neuroticism, low extraversion and low conscientiousness on the NEO. This would strongly imply that the remaining factor, namely, low openness is a specific feature of treatment-resistant depression. Openness on the NEO has positive relationships with reward dependence and cooperativeness on the TCI, in treatment-resistant depression. Our results indicate that these three factors are important mediators in treatment-resistant depression.


The mild chronic inflammatory state associated with obesity may be an important link between adiposity and insulin resistance (IR). In a sample of 137 overweight and obese Chilean adolescents, we assessed associations between high-sensitivity C-reactive protein (hs-CRP), IR and adiposity; explored sex differences; and evaluated whether hs-CRP mediated the relationship between adiposity and IR. Positive relationships between hs-CRP, IR and 2 measures of adiposity were found. Hs-CRP was associated with waist circumference (WC) in boys and fat mass index (FMI) in girls. Using path analysis, we found that hs-CRP mediated the relationship between adiposity (WC and FMI) and the homeostatic model assessment of insulin resistance (HOMA-IR) (p < 0.05) in both sexes. Our novel finding is that inflammation statistically mediated the well described link between increased adiposity and IR. Adolescent obesity is a major public health problem associated with cardiovascular (CV) risk factors including abdominal obesity, insulin resistance (IR), dyslipidemia and hypertension ,2. While2]  . After aStatistical analyses were performed using SPSS  and SAS software . Bivariate associations were determined with Spearman correlation coefficients. We used the Mann-Whitney test to evaluate sex differences in CV risk variables (Table 2 = 0.13) and FMI for females . Hs-CRP was associated with HOMA-IR in males and females (p < 0.05), independent of adiposity. In our path analysis, hs-CRP partially mediated the relationship between adiposity and HOMA-IR, controlling for sex and Tanner stage (p < 0.05) . Future studies should include more direct measures of visceral and subcutaneous fat content (e.g. magnetic resonance or ultrasound imaging), and additional measures of inflammatory status  and insulin sensitivity. It would also be important to assess the magnitude of other factors that might be related to inflammatory status and metabolic performance, for example non-alcoholic steatohepatitis which often occurs with obesity and IR and can progress to inflammation and fibrosis ,20. The In conclusion, our sample of overweight and obese Chilean adolescents showed gender-specific associations between adiposity and systemic inflammation. Additionally, we found that hs-CRP statistically mediated the association between adiposity and IR. This suggests that a systemic inflammatory state is initiated by FM excess even during adolescence and that inflammation influences the metabolic consequences of overweight and obesity. Future research should include 1) detailed assessment of adiposity topography, 2) additional assessments of inflammation  and 3) studies in other populations.BMI: body mass index; CFI: comparative fit index; CV: cardiovascular; FM: fat mass; FMI: fat mass index; HOMA-IR: homeostatic model assessment of insulin resistance; Hs-CRP: high-sensitivity C-reactive protein; INTA: Institute of Nutrition and Food Technology; IR: insulin resistance; RMSEA: root mean square error of approximation; WC: waist circumference.The authors declare that they have no competing interests.MR participated in the design of the manuscript, data analysis, helped to draft the manuscript and significantly reviewed the manuscript before submission. SG participated in the design of the manuscript, data analysis, helped to draft the manuscript and significantly reviewed the manuscript before submission. ED participated in the data acquisition and interpretation and reviewed the manuscript before submission. EB participated in the design of the manuscript, data analysis, helped to draft the manuscript and significantly reviewed the manuscript before submission. LL participated in the data acquisition and analysis and reviewed the manuscript before submission. LL participated in the analysis and reviewed the manuscript before submission. RB participated in the data acquisition, analysis and interpretation, conceived the idea of the manuscript and reviewed the manuscript before submission. All authors read and approved the final manuscript.


DNA methylation, consisting of the addition of a methyl group at the fifth-position of cytosine in a CpG dinucleotide, is one of the most well-studied epigenetic mechanisms in mammals with important functions in normal and disease biology. Disease-specific aberrant DNA methylation is a well-recognized hallmark of many complex diseases. Accordingly, various studies have focused on characterizing unique DNA methylation marks associated with distinct stages of disease development as they may serve as useful biomarkers for diagnosis, prognosis, prediction of response to therapy, or disease monitoring. Recently, novel CpG dinucleotide modifications with potential regulatory roles such as 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine have been described. These potential epigenetic marks cannot be distinguished from 5-methylcytosine by many current strategies and may potentially compromise assessment and interpretation of methylation data. A large number of strategies have been described for the discovery and validation of DNA methylation-based biomarkers, each with its own advantages and limitations. These strategies can be classified into three main categories: restriction enzyme digestion, affinity-based analysis, and bisulfite modification. In general, candidate biomarkers are discovered using large-scale, genome-wide, methylation sequencing, and/or microarray-based profiling strategies. Following discovery, biomarker performance is validated in large independent cohorts using highly targeted locus-specific assays. There are still many challenges to the effective implementation of DNA methylation-based biomarkers. Emerging innovative methylation and hydroxymethylation detection strategies are focused on addressing these gaps in the field of epigenetics. The development of DNA methylation- and hydroxymethylation-based biomarkers is an exciting and rapidly evolving area of research that holds promise for potential applications in diverse clinical settings. Epigenetics is the study of reversible, heritable mechanisms that regulate gene expression without altering the DNA sequence , 2. DNA DNA methylation biomarkers offer several significant advantages over expression-based markers. For instance, they are readily amplifiable and easily detectable using polymerase chain reaction (PCR)-based approaches even if alterations are present only in a limited number of cells . DNA metAnother limitation of many current methodologies is the inability to distinguish between 5-mC and other novel structurally similar DNA modifications that have been recently discovered in mammalian DNA including 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC), and 5-carboxylcytosine (5-caC) . 5-hmC hThis review is divided into two parts that cover existing and emerging strategies applied to (A) discovery and (B) validation of DNA methylation-based biomarkers and describes their major advantages and limitations . ParticuOver the past few decades, there have been an increasing number of approaches devoted to generating genome-wide methylation profiles and aberrant methylation signatures, each with its own advantages, disadvantages, and areas of applicability. As DNA methylation information is lost during PCR amplification, the majority of techniques rely on methylation-dependent treatment of DNA prior to amplification. These assays can be classified into three main categories: restriction enzyme (RE) digestion, affinity-based analysis, and bisulfite modification. The combination of these three approaches with sequencing and microarray-based platforms has given rise to a wide range of techniques for global DNA methylation analysis.Global approaches to DNA methylation analysis are being widely used to generate genome-wide methylation profiles because they offer a number of advantages. In general, these approaches are high-throughput strategies with regard to the number of loci that can be analyzed at one time. In particular, sequencing platforms provide quantitative information about the methylation status of every CpG and allow for the analysis of methylation in repeat sequences and rare methylation variants, which is difficult to do using microarrays. Another advantage of sequencing approaches is that they can be used to analyze DNA methylation of regions with no prior knowledge of the sequence. The main weaknesses of sequencing strategies are library bias, cost, availability, and difficulties in data management and analysis, although the cost of massive sequencing technologies is rapidly decreasing. DNA methylation profiling using high-density microarrays is another commonly used method to identify broad differences between groups of samples. They are less time consuming, less labor intensive, and less costly than sequencing. In addition, microarrays allow for simultaneous analysis of a larger number of samples with a wider CGI coverage. Nevertheless, microarray analyses lack reliable quantitation and are limited by probe design, hybridization efficiency, and hybridization artifacts.HpaII tiny fragment enrichment by ligation-mediated PCR (HELP) which is based on digestion of high-molecular-weight genomic DNA with methylation-sensitive HpaII  and/or array-based platforms .–24.22–24ve HpaII  25]. In. InHpaIIlatforms . Other ey (LUMA)  27–29].–29.HpaIIquencing  30]. Th. ThHpaIIon (MCA) . This meed sites . Next, Dy (MCAM) , 32. Anoip (MAD)  33]. Mo. MoHpaII enzymes . Both te enzymes .McrBC. This enzyme recognizes closely spaced methylated cytosines and so has the capacity to digest densely methylated regions of DNA  glucose . E. E107].  samples . Given t samples , 108–110MSP is the most widely used locus-specific bisulfite-based DNA methylation analysis strategy that has been reliably applied to a large scale of clinical samples and has been previously reviewed in the literature , 111. BrGSTP1, APC, TGFβ2, HOXD3, MLH1, dickkopf homolog 1(DKK1), and secreted frizzled-related protein 1 (SFRP1), which has been shown to be detected in prostate and colon cancers [MethyLight utilizes methylation-specific primers and a TaqMan methylation-specific fluorescent reporter probe that anneals to the amplified region of interest . Anneali cancers , 119–122 cancers . HoweverCOL2A1) as well as to fully methylated and unmethylated standards. The limitations of all methods based on MCA are the use of dyes and the necessity of special equipment. Additionally, when heterogeneously methylated molecules are analyzed by MCA, the melting pattern becomes complex and difficult to interpret.MS-MCA is a method that employs an intercalating double-stranded DNA fluorescent dye such as SYBR green to monitor the melting properties of PCR products during MSP as temperatures rise . DNA melAn alternative approach called HeavyMethyl uses methylation-independent primers and oligonucleotide blockers that hybridize only to unmethylated DNA . Thus, oMS-SNuPE is another bisulfite modification-based strategy that has been previously reviewed , 125. ThBstUI, an enzyme that cuts unmodified cytosines [COBRA is a well-established bisulfite-based method that relies on methylation-independent DNA amplification and digestion with ytosines . Methylaytosines .MspI or HpaII, whereas in AIMS, it is digested with SmaI and XmaI. However, both techniques suffer from low-resolution and low-throughput, require high DNA quality and quantity, and utilize radioactive materials. Consequently, MS-AP-PCR and AIMS are rarely used for methylation analysis nowadays.The most well-known locus-specific DNA methylation analysis techniques based on methylation-sensitive RE are MS-AP-PCR and AIMS –133. In An alternative approach to enrich for methylated DNA is affinity-based enrichment. One class of affinity-based strategies, called MeDIP-PCR, utilize bead-immobilized anti-5-methylcytosine antibodies . Gene-spAluI and HpaII and MIRA to enrich for methylated DNA followed by quantitative real-time PCR (qPCR) for a more sensitive and specific methylation analysis than either approach alone [This method combines RE digestion with ch alone . HoweverThe development of DNA methylation-based biomarkers is an emerging and exciting area of research that holds promise for potential applications in diverse clinical settings. This review focuses on a large number of techniques that have been developed for methylation analysis at global and gene-specific levels for DNA methylation-based biomarkers discovery and validation. It is important to note that a key intermediate step between discovery and validation is the analysis of the heterogeneity of methylation in gene promoters and identification of contextually meaningful CpG sites that mediate gene transcription. DNA methylation changes may cause quantitative transcriptional changes and/or may lead to qualitative transcriptional silencing. Upstream regulatory regions of many genes are known to harbor more than one promoter. These promoters may serve to regulate expression of specific transcripts thereby leading to generation and/or expression of alternate transcripts. Differential methylation of such promoters may be context dependent – that is, certain promoters are preferentially regulated via methylation in certain tissues or cell types. Alternately, methylation signals of promoters may change in response to surrounding environmental milieu. Better understanding of these aspects will provide important clues underlying association of specific biomarkers with disease biology. However, there are still many challenges to the effective implementation of DNA methylation-based biomarkers. For example, many methylation studies published to date have not accounted for the presence of hydroxymethylated DNA. The role(s) of 5-hydroxymethylation is distinct from 5-mC and is being elucidated. Recent studies suggest that 5-hmC may serve as an intermediate in direct DNA demethylation , 137. ItEarlier methylation analyses relied exclusively on BS, but this approach has many challenges. Subsequently, array-based profiling approaches were leading the field of DNA methylation-based biomarker discovery, but NGS-based approaches have quickly caught up and are likely to become the platform of choice in the near future. If the $1000 personal genome becomes a reality in the future, personal epigenome will be a reality soon to follow. One can envision in the era of personalized medicine, individual methylation “signatures” will be tested in a variety of minimally invasive samples. Although currently the identification of methylation “signatures” is focused mostly on cancer, future focus will be on other diseases, beyond cancer.With respect to future frontiers in array-based platforms, the development of a triple microarray that will allow highly sensitive analysis of disease-related changes in DNA methylation, histone modifications, and microRNA expression simultaneously will provide new insights for more comprehensive epigenetic biomarker development.New advancements in epigenetic technologies in the future will most likely drive the development of easy, noninvasive, cost-effective, high-throughput, highly sensitive, and specific epigenetic tests in the clinic.


Gastroesophageal reflux disease (GERD) is one of the most common disorders in medical practice. It is the most common gastrointestinal diagnosis recorded during visits to outpatient clinics in the United States. Apart from the economic burden of the disease and its impact on quality of life, GERD is the most common predisposing factor for esophageal adenocarcinoma .Recently, many important issues have emerged regarding the classification, pathogenesis, natural history, and treatment of GERD. Although use of proton-pump inhibitor (PPI) is the treatment of choice for GERD, approximately, one-third of patients with GERD fail to response symptomatically to a standard-dose proton-pump inhibitor (PPI), either partially or completely . AdditioThe main focus of this special issue is on recent advances in the treatment of erosive esophagitis, NERD and Barrett's esophagus. In addition, the emerging diagnostic methods, pharmacological treatments, and endoscopic therapies for GERD are also discussed.The frequencies of gastroesophageal and extragastroesophageal symptoms in patients with mild erosive esophagitis, severe erosive esophagitis, and Barrett's esophagus, in Taiwan” is the first work simultaneously assessing the differences in reflux symptom profiles among the three different categories of GERD. The data showed that the frequencies of some esophageal and extraesophageal symptoms in patients with Los Angeles grade A/B erosive esophagitis were higher than those in patients with Los Angeles grade C/D erosive esophagitis and Barrett's esophagus. The paper entitled “Current pharmacological management of gastroesophageal reflux disease,” Y.-K. Wang et al. present the current and developing therapeutic agents for GERD treatment. The efficacies of PPIs and potassium-competitive acid blocker in GERD therapy are well reviewed. Additionally, the article summarizes the development of novel therapeutic agents focusing on the underlying mechanisms of GERD.In the paper entitled “Pharmacological therapy of gastroesophageal reflux in preterm infants,” L. Corvaglia et al. review the pathogenesis, presentation, diagnosis, and treatment of gastroesophageal reflux in preterm infants. A stepwise approach is advisable for the treatment of gastroesophageal reflux in preterm infants, firstly, promoting nonpharmacological interventions and secondly, limiting drugs to selected infants unresponsive to the conservative measures or who are suffering from severe gastroesophageal reflux with clinical complications.In the paper entitled “Stretta radiofrequency treatment for GERD: a safe and effective modality,” M. Franciosa et al. focus on the safety, efficacy, and durability of the Stretta radiofrequency treatment for GERD therapy. The novel endoscopic treatment reduces esophageal acid exposure, decreases the frequency of transient lower esophageal relaxation, decreases medication use and improves quality of life in GERD patients. In the paper entitled “Duodenal tube feeding: an alternative approach for effectively promoting weight gain in children with gastroesophageal reflux and congenital heart disease,” S. Kuwata et al. showed that duodenal tube feeding improves the weight gain of infants with gastroesophageal reflux who need treatment for congenital-heart-disease-associated heart failure.In the paper entitled “Changes in ghrelin-related factors in gastroesophageal reflux disease in rats,” M. Nahata et al. examined gastrointestinal hormone profiles and functional changes in rats with GERD. The results suggest that aberrantly increased secretion of peripheral ghrelin and decreased ghrelin responsiveness may occur in GERD rats.In the paper entitled “Surgical management of pediatric gastroesophageal reflux disease,” H. T. Jackson and T. D. Kane review the clinical presentation of GERD in pediatric population and discuss the options for surgical management and outcome in these patients.In the paper entitled “Current advances in the diagnosis and treatment of nonerosive reflux disease,” C. L. Chen and P. I. Hsu, review the literature about the pathogenesis, natural history, diagnosis and treatment of NERD. The authors suggest that a combination of 24-hour esophageal impedance and pH monitoring is indicated to differentiate acid-reflux-related NERD, weakly acid reflux-related NERD (hypersensitive esophagus), nonacid-reflux-related NERD, and functional heartburn in patients with poor response to appropriate PPI treatment.In the paper entitled “Antireflux endoluminal therapies: past and present,” K. C. Yew et al. and S.-K. Chuah review, highlight, and discuss three commonly employed antireflux endoluminal procedures: fundoplication or suturing techniques , intramural injection or implant techniques , and radiofrequency ablation of lower esophageal sphincter and cardia (the Stretta system). In the paper entitled “Ping-I HsuPing-I HsuNayoung KimNayoung KimKhean Lee GohKhean Lee GohDeng-Chyang WuDeng-Chyang Wu


Few cases of Epstein-Barr virus myelitis have been described in the literature. Multi-centric Castleman's disease is a lymphoproliferative disorder that is well known for its associations with the human immunodeficiency virus, human herpes virus 8, and Kaposi's sarcoma. The concurrent presentation of these two diseases in a patient at the same time is extremely unusual.We describe the case of a 43-year-old Caucasian man with acquired immune deficiency syndrome who presented with fever, weight loss and diffuse lymphadenopathy, and was diagnosed with multi-centric Castleman's disease. He presented three weeks later with lower extremity weakness and urinary retention, at which time cerebrospinal fluid contained lymphocytic pleocytosis and elevated protein. Magnetic resonance imaging demonstrated abnormal spinal cord signal intensity over several cervical and thoracic segments, suggesting the diagnosis of myelitis. Our patient was ultimately diagnosed with Epstein-Barr virus myelitis, as Epstein-Barr virus DNA was detected by polymerase chain reaction in the cerebrospinal fluid.To the best of our knowledge, this is the first case of multi-centric Castleman's disease followed by acute Epstein-Barr virus myelitis in a human immunodeficiency virus-infected patient. Clinicians caring for human immunodeficiency virus-infected patients should be vigilant about monitoring patients with increasing lymphadenopathy, prompting thorough diagnostic investigations when necessary. The Ebstein-Barr virus (EBV) is a member of the herpesviridae family. Even though most infections with EBV go unnoticed, in some cases it can be associated with the development of potentially serious conditions, including infectious mononucleosis. EBV has been associated with central nervous system (CNS) diseases , but rarely as a cause of myelitis ,2. The pMulti-centric Castleman's disease (MCD) is a systemic disorder characterized by significant peripheral lymphadenopathy and hepatosplenomegaly, as well as frequent fevers, night sweats, fatigue and weight loss . HIV-inf3, and a viral load of <50 copies/mL. He had been diagnosed with HIV-infection in 1990; his CD4 nadir was 4 (0.6%) in February 2004 with high viremia, 175,671 copies/mL. After initiation of potent anti-retroviral therapy the HIV viral load has been undetectable since October 2004.A 43-year-old Caucasian man on anti-retroviral therapy (lopinavir/ritonavi and emtricitabine/tenofovir) for HIV/AIDS presented in September 2008 with a three-month history of low grade fevers, night sweats, generalized fatigue, lethargy, unintentional weight loss, and bilateral lower extremity swelling. He had a CD4 count of 196 cells/mmμL, an albumin level of 2.5 mg/dL, and a Westergren sedimentation rate of 90 mm/hr. A lower extremity Doppler was negative for deep venous thrombosis.A physical exam of our patient revealed diffuse lymphadenopathy in his cervical, axillary and inguinal areas, splenomegaly, and pitting edema in both lower extremities. Computed tomography of his chest, abdomen and pelvis demonstrated widespread mediastinal, hilar, axillary, retro-peritoneal and pelvic lymphadenopathy, as well as the splenomegaly. A laboratory workup revealed a hemoglobin level of 8.5 mg/dL, a white blood cell count (WBC) of 4,600 cells/Histological examination of lymph node biopsy material revealed marked plasma cell infiltration, and follicles that were variable in appearance, from marked follicular hyperplasia to involution and dendritic cell hyperplasia Figure . ImmunohThree weeks later our patient presented with lower abdominal pain, urinary retention, and lower extremity weakness. Neurological examination revealed bilaterally reduced motor power (4/5) in all major muscle groups in his lower extremities, along with a positive Babinski sign on the right. Deep tendon reflexes were normal bilaterally. An ultrasound of his pelvis demonstrated bladder distention. A Foley catheter was inserted and 2 liters of urine was evacuated, with complete relief of pain.A Gadolinium magnetic resonance imaging (MRI) of his spine was done to rule out spinal cord compression; it revealed abnormal spinal cord signal intensity involving several cervical and thoracic segments, associated with expansion of the cord and mild enhancement of the areas of abnormal T2 signal Figure . MRI of 3, with 90% lymphocytes, and a high protein level of 243 mg/dL; cytological analysis identified mature reactive lymphocytes with no evidence of lymphoma on flow cytometry. Polymerase chain reaction (PCR) was positive for EBV DNA in the CSF, but negative for HHV-8, cytomegalovirus (CMV), herpes simplex virus (HSV) and Varicella-Zoster virus (VZV). Treatment with high-dose dexamethasone was initiated. Our patient was then transferred to the National Institute of Health (NIH) and started on a treatment protocol of high-dose zidovudine (AZT) and valganciclovir for MCD . Subsequent laboratory follow-up revealed marked improvement over the first seven days of treatment, including a marked decrease in WBC and protein in his CSF. A follow-up MRI demonstrated resolution of all spinal cord lesions. He is now doing well, his last CD4 was 322 (15%), viral load < 50 copies/mL in August 2010.A lumbar puncture was performed: cerebrospinal fluid (CSF) contained an elevated WBC of 50 cells/mmThis case is notable for two reasons. First, a 43-year-old man with AIDS initially suspected to have lymphoma due to his clinical features at presentation, was subsequently diagnosed with MCD. Second, within three weeks he presented with EBV myelitis.With respect to the former condition, this case brings to light the importance of obtaining definitive histological diagnoses in HIV-infected patients who present with lymphadenopathy and systemic symptoms. MCD is relatively uncommon cause for such a presentation. Though clinically similar to lymphoma, MCD is an entity that is distinct from malignant lymphoproliferative disorders in both histology and prognosis. It is characterized by significant peripheral lymphadenopathy and hepatosplenomegaly, as well as by frequent fevers, night sweats, fatigue and weight loss . AbnormaAlthough no standard treatment has been established, MCD often is treated systemically  with sucEBV is a γ-herpes virus that is detectable in over 90 percent of the general population. Even though most infections with EBV go unnoticed, in some cases it can be associated with the development of serious conditions, including infectious mononucleosis. EBV has been associated with CNS diseases, including meningitis and encephalitis ,2. EBV mPatients with AIDS have 10 to 20 times as many circulating EBV-infected B-cells as those who are healthy. T-cells from patients with AIDS suppress EBV-infected B-cells less effectively than do cells from normal controls . A decliIn our patient, both the clinical and radiological findings were characteristic of myeloradiculopathy. The CSF abnormalities further indicated that the disease was inflammatory. Extensive virological analysis of serum and CSF revealed the presence of EBV DNA in the CSF, as well as the absence of CMV, HSV, VZV and HHV-8. The clinical features of myelitis, and the increased signal in the cervical and thoracic spinal cord in this patient on MRI, were similar to those described in previously reported EBV myelitis cases ,15-17.The International Herpes Management Forum recommends that the diagnosis of EBV infections of the CNS can include evidence of EBV DNA in CSF by PCR. Unfortunately, there is no definitive treatment for EBV infection of the nervous system. Steroids and immunoglobulin have been used empirically, but their effects on disease progression are unknown. Anti-viral therapy has not demonstrated clinical efficacy in the treatment of EBV-related CNS disorders .Our patient presented with two rare disorders in rapid succession, first MCD, followed by EBV myelitis. Histological evaluation revealed the first diagnosis, and CSF PCR analysis the second. Clinicians caring for HIV-infected patients should be vigilant about monitoring patients with increasing lymphadenopathy, prompting thorough diagnostic investigations when necessary. Treatment for MCD can be complex, and referral to dedicated programs with unique treatment protocols should be considered. EBV myelitis is too rare for there to be published therapeutic trials; nonetheless, systemic steroids may be beneficial in its treatment.AIDS: acquired immunodeficiency syndrome; CNS: central nervous system; CSF: cerebrospinal fluid; CMV: cytomegalovirus: EBV: Ebstein-Barr virus; HHV-8: human herpes virus 8; HSV: human simplex virus; KS: Kaposi's sarcoma; MCD: multi-centric Castleman's disease; MRI: magnetic resonance imaging; PCR: polymerase chain reaction; VZV: Varicella-Zoster virus; WBC: white blood cell count.Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.CA, GP and VS analyzed and interpreted the patient data and wrote the manuscript. JB performed the histopathologic and immunohistochemical analyses of the biopsy, and was a major contributor in writing the manuscript. All authors read and approved the final manuscript.


Unintentional non-adherence has been characterized as passively inconsistent medication-taking behavior (forgetfulness or carelessness). Our objectives were to: (1) study the prevalence and predictors of unintentional non-adherence; and (2) explore the interrelationship between intentional and unintentional non-adherence in relation to patients’ medication beliefs.We conducted a cross-sectional survey of adults with asthma, hypertension, diabetes, hyperlipidemia, osteoporosis, or depression from the Harris Interactive Chronic Illness Panel. The analytic sample for this study included 24,017 adults who self-identified themselves as persistent to prescription medications for their index disease. They answered three questions on unintentional non-adherence , 11 questions on intentional non-adherence, and three multi-item scales assessing perceived need for medication (k = 10), perceived medication concerns (k = 6), and perceived medication affordability (k = 4). Logistic regression was used to model predictors of each unintentional non-adherence behavior. Baron and Kenny’s regression approach was used to test the mediational effect of unintentional non-adherence on the relationship between medication beliefs and intentional non-adherence. Bootstrapping was employed to confirm the statistical significance of these results.For the index disease, 62% forgot to take a medication, 37% had run out of the medication, and 23% were careless about taking the medication. Common multivariate predictors (p < .001) of the three behaviors were: (1) lower perceived need for medications; (2) more medication affordability problems; (3) worse self-rated health; (4) diabetes or osteoporosis (relative to hypertension); and (5) younger age. Unique statistically-significant predictors of the three behaviors were: (a) ‘forgot to take medications’ - greater concerns about the index medication and male gender; (b) ‘run out of medications’ - non-white race, asthma, and higher number of total prescription medications; (c) ‘being careless’ - greater medication concerns. Mediational tests confirmed the hypothesis that the effect of medication beliefs  on intentional non-adherence is mediated through unintentional non-adherence.For our study sample, unintentional non-adherence does not appear to be random and is predicted by medication beliefs, chronic disease, and sociodemographics. The data suggests that the importance of unintentional non-adherence may lie in its potential prognostic significance for future intentional non-adherence. Health care providers may consider routinely inquiring about unintentional non-adherence in order to proactively address patients’ suboptimal medication beliefs before they choose to discontinue therapy all together. Medication adherence refers to the extent of conformity to treatment recommendations with respect to the timing, dosage, frequency, and duration of a prescribed medication . Non-adhResearch over the past 20 years has consistently shown that intentional non-adherence is driven by patient beliefs about their treatment, disease, and prognosis as well as their objective experiences with medications -27. SomeAlthough recent studies have begun to clear the veil on unintentional non-adherence, the generalizability of the evidence to date is limited by small samples in niche populations. A majority of these results were reported in samples of about 150 patients or less ,15,20,30yes if they agreed to participate. The study was approved by the George Mason University Institutional Review Board.A cross-sectional survey of U.S. adults with self-reported chronic disease was conducted using the Harris Interactive Chronic Illness Panel (CIP). CIP participants are recruited through a number of avenues, including postal-mail invitations, television advertisements, telephone recruitment for under-represented populations, targeted email solicitations, and text-banner placements on websites . In February and March of 2009, randomly-selected members of the CIP were sent an e-mail invitation to participate in the survey. Panel members responding to the e-mail invitation were instructed to read the informed consent form and click on Panel members were eligible for participation if they were aged 40 and older, resided in the U.S., and reported one of six chronic diseases . Only persons age 40 and older were sampled because they bear a greater burden of chronic disease compared to adults aged 18–39. The six chronic diseases reflect a mix of symptomatic, asymptomatic, and mental-health conditions. They are some of the most highly prevalent conditions in the U.S.  and are Requests for survey participation were sent to 204,266 randomly-selected CIP members. Of these invitations, there were 11,945 invalid e-mail addresses. Of the 192,321 invitations with valid e-mail addresses, 17,121 were ineligible due to age less than 40. Of the 175,200 age-eligible persons with a valid e-mail address, 51,774 completed the screener (29.5% survey contact rate per standards recommended by The American Association for Public Opinion Research ).During the screening portion of the survey, panel members’ chronic disease status was reconfirmed and their medication-taking behavior for the index condition was solicited. The screener solicited the number of medications respondents currently took for each disease as well as the length of time they reported continuously taking the medication. These items were used to classify respondents as currently persistent to their medication. To identify respondents as non-persisters, the survey asked if, in the last year, they had stopped taking a prescription medication for one of the six conditions without their providers telling them to do so. To identify respondents as non-fulfillers, the survey asked if, in the last year, they had received, but did not fill, a new prescription from their provider for one of the six target conditions. Only subjects who self-identified themselves as persistent to  prescription medications for their index disease form the analytic sample for this study. The index disease was the disease for which respondents completed the medication-belief items. If self-reported persisters reported more than one of the six target conditions, one was randomly selected as the index disease. Because non-fulfillers never filled the index medication, they were not asked questions about unintentional non-adherence. Self-reported non-persisters were excluded from the analytic sample because their complete discontinuation of therapy cannot, logically, be unintentional.six months: (1) did you ever forget to take the prescription medication; (2) did you ever run out of the prescription medication; and (3) were you careless at times about taking the prescription medication? Respondents also answered 11 questions on intentional non-adherence in reference to their medication-taking behavior in the past six months for their index disease. These questions surveyed the respondents on the following behaviors: took less medication than instructed because they felt better/worse; skipped taking medication because they felt better/worse; altered dose of medication to suit own needs; stopped taking medication because they felt better/worse; skipped doses to make medication last longer; took smaller doses to make medication last longer; skipped doses of medication because they did not think it was helping them; and stopped medication because they did not think it was helping them. Both sets of measures of unintentional and intentional non-adherence were adapted from validated measures published in the peer-review literature  and others an inverse effect similar to that reported herein ,10. UnliPatient’s medication beliefs, especially perceived need for medication and perceived medication affordability, were strong predictors of unintentional non-adherence. Patients in the lowest quartile of perceived need for medications were 1.9 times more likely to report being careless at times with medications. Patients in the lowest quartile of perceived medication affordability were 4.65 times more likely to report running out of medications at times. Other research has also reported medication beliefs to be associated with unintentional non-adherence ,30. OverThis study has several strengths compared to previous empirical work on unintentional non-adherence. A large, internet-based panel of adults with chronic disease was accessed. The obtained sample represents persons aged 40 and older with one of six chronic diseases from all 50 U.S. states. Our sample is by far the largest in which predictors of unintentional non-adherence has been assessed. The multi-item scales assessing perceived need for medications, perceived medication concerns, and perceived medication affordability have been previously tested and found to be reliable and valid . Unlike Despite these strengths, several limitations must be taken in to account when interpreting the results. This study was conducted over the Internet with 29.5% survey contact rate. The CIP recruits panel members through multiple avenues in an attempt to provide a representative sample of the adult, chronically-ill population and to reach under-represented and hard-to-reach populations of interest. However, some biases were observed in the sample. Specifically, compared to the U.S. adult population, the obtained sample was under-represented by adults with income less than $25,000 annually , under-rreasons for forgetfulness or carelessness. The analyses reported herein suggest that unintentional non-adherence is influenced by the same drivers as intentional non-adherence—patients’ beliefs about the medications. While unintentional non-adherence may appear to be a rather benign behavior, its importance lies in its potential prognostic significance. Qualitative research has depicted how patients test or experiment with their medications [Numerous measures assessing unintentional non-adherence exist. For example, the four-item Morisky Scale  containsications ,65,66. Iications , having ications , or haviications ,69. Our Non-persistence most commonly occurs in the first six months of therapy -77. It iFor our study sample, unintentional non-adherence does not appear to be random and is predicted by medication beliefs, chronic disease, and sociodemographics. Our data suggests that the importance of unintentional non-adherence may lie in its potential prognostic significance for future intentional non-adherence. Health care providers may consider routinely inquiring about unintentional non-adherence in order to proactively address patients’ suboptimal medication beliefs before they choose to discontinue therapy all together.Drs. Gadkari and McHorney are full-time employees of and own stock in Merck & Co., Inc.Both AG and CM contributed to the conception of the manuscript idea. CM led the instrument development and data collection process. AG led the statistical analysis and interpretation of data for the manuscript. Both AG and CM contributed to the original draft of the manuscript as well as to the critical revision of the manuscript for important intellectual content. Both AG and CM have given final approval for the current version of the manuscript to be published.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6963/12/98/prepub


The clinical scenario of a new or worsening pleural effusion following the initiation of antituberculous therapy has been classically referred to as a 'paradoxical' pleural response, presumably explained by an immunological rebound phenomenon. Emerging evidence suggests that there also may be a role for a lupus-related reaction in the pathophysiology of this disorder.An 84-year-old Asian man treated with isoniazid, along with rifampin, pyrazinamide and ethambutol for suspected extrapulmonary tuberculosis, presented with a recurrent pleural effusion, his third episode since the initiation of this therapy. The first effusion occurred one month after the start of treatment, without any prior evidence of pulmonary tuberculosis involvement. Follow-up testing, including thoracoscopic pleural biopsies, never confirmed tuberculosis infection. Further evaluation yielded serological evidence suggesting drug-induced lupus. No effusions recurred following the discontinuation of isoniazid, although other antituberculosis medications were continued.The immunological rebound construct is inconsistent with the evolution of this case, which indicates rather that drug-induced lupus may explain at least some cases of new pleural effusions following the initiation of isoniazid. Mycobacterium tuberculosis infection (TB), occurring in up to 30% of cases [Pleural effusions are frequently observed in pulmonary of cases . New onsof cases ,3. Publiof cases . While tAn 84-year-old Asian man was admitted to hospital for dyspnea and was found to have a large, recurrent right-sided pleural effusion. This was the third time our patient presented with an effusion since the initiation of rifampin, isoniazid, pyrazinamide and ethambutol (RIPE) therapy for presumed extrapulmonary TB. Nine months prior to this presentation, our patient underwent a screening colonoscopy with a biopsy that reportedly yielded a single granulomatous, acid-fast stain-positive, but TB culture-negative lesion. No anti-TB treatment was initiated at that time. The purportedly acid-fast stain-positive specimen could not be located subsequently for re-review. A repeat colonoscopy with biopsy at six months demonstrated no granulomas or other evidence of TB. There was no radiographic evidence of pulmonary TB and interval sputum cultures were negative, as was a serum QuantiFERON-TB Gold test. Nevertheless, our patient was started on RIPE combination therapy.Four weeks after the initiation of this regimen (two months prior to the episode described in this report), our patient complained of dyspnea, cough and weakness. He was admitted to hospital with the initial right-sided pleural effusion presentation Figure . ThoracoTwo weeks later, a re-accumulated pleural effusion led to the admission we report Figure . Our patThe application of immunological rebound as an explanation for this case, in which the anti-TB therapy was empiric and without a convincingly demonstrated infection, is not consistent with the specific clinical data in hand. Moreover, the adenosine deaminase value we observed was below the cut-off point <35U/L) associated with a greater than 90% negative predictive value for pleural TB, even if the pre-test likelihood of disease was as high as 70% U/L assoc,3. GivenThe case we describe, in conjunction with other cases in the medical literature, underscores the likely pivotal role for DIL in at least some new or worsening effusions during isoniazid treatment. Isoniazid-induced lupus should be considered in the differential diagnosis of a treatment emergent effusion and appropriate diagnostic tests should be pursued before such an effusion is attributed to a paradoxical pleural response.Mycobacterium tuberculosis infection.ANA: anti-nuclear antibody; DIL: drug-induced lupus; RIPE: rifampin, isoniazid, pyrazinamide, ethambutol; TB: Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.Both VV and PB were directly involved in the care of the patient during the admission described in this report. VV was a major contributor in writing the manuscript with significant overall guidance and editorial assistance from PB. Both authors read and approved the final manuscript.


Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by chronic synovitis that progresses to destruction of cartilage and bone. Bone marrow (BM) cells have been shown to contribute to this pathogenesis. In this study, we compared differentially expressed molecules in BM cells from RA and osteoarthritis (OA) patients and analyzed abnormal regulatory networks to identify the role of BM cells in RA.Gene expression profiles (GEPs) in BM-derived mononuclear cells from 9 RA and 10 OA patients were obtained by DNA microarray. Up- and down-regulated genes were identified by comparing the GEPs from the two patient groups. Bioinformatics was performed by Expression Analysis Systemic Explorer (EASE) 2.0 based on gene ontology, followed by network pathway analysis with Ingenuity Pathways Analysis (IPA) 7.5.The BM mononuclear cells showed 764 up-regulated and 1,910 down-regulated genes in RA patients relative to the OA group. EASE revealed that the gene category response to external stimulus, which included the gene category immune response, was overrepresented by the up-regulated genes. So too were the gene categories signal transduction and phosphate metabolism. Down-regulated genes were dominantly classified in three gene categories: cell proliferation, which included mitotic cell cycle, DNA replication and chromosome cycle, and DNA metabolism. Most genes in these categories overlapped with each other. IPA analysis showed that the up-regulated genes in immune response were highly relevant to the antigen presentation pathway and to interferon signaling. The major histocompatibility complex (MHC) class I molecules, human leukocyte antigen (HLA)-E, HLA-F, and HLA-G, tapasin (TAP) and TAP binding protein, both of which are involved in peptide antigen binding and presentation via MHC class I molecules, are depicted in the immune response molecule networks. Interferon gamma and interleukin 8 were overexpressed and found to play central roles in these networks.Abnormal regulatory networks in the immune response and cell cycle categories were identified in BM mononuclear cells from RA patients, indicating that the BM is pathologically involved in RA. Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by chronic synovitis that is often pathogenic and destructive to articular cartilage and bone. To understand the complex pathogenesis and heterogeneous manifestations of autoimmune diseases including RA, DNA microarray has emerged as a powerful tool -4. We haIn RA pathology, fibroblast-like synoviocyte (FLS) has been shown to play an essential role in the chronic inflammation of RA joints . TherefoThe BM contains three types of stem cells: hematopoietic stem cells (HSCs), which produce all the mature blood lineages for leukocytes, erythrocytes, and platelets; mesenchymal stem cells, which can differentiate into osteoblasts, chondrocytes, and adipocytes; and endothelial stem cells. The proliferations and differentiations of these heterogeneous cell populations are dependent on the BM microenvironment and are regulated by highly sophisticated networks, either through cell-cell interactions or cytokine networks. Indeed, a remarkable elevation in IL6 and IL8 levels in the BM serum from RA patients has been reported to relate to the synovial proliferation seen in multiple joints . TherefoIn this study, we identify aberrant regulatory networks in BM cells from RA patients by analyzing differentially expressed genes based on their gene expression profiles with those of osteoarthritis (OA) patients. OA patients were chosen because the OA pathology is relatively well understood and the BM cells from these patients are far more readily available than those from healthy subjects.Nine patients  with RA satisfying the 1987 revised diagnostic criteria of the American College of Rheumatology  and 10 pPatient BM were collected and kept at 4°C. All BM-derived mononuclear cells (BMMC) were isolated by using Ficoll-Paque™ Plus  gradient centrifugation according to the manufacturer's recommendations. Total RNA from the BMMCs was extracted by using the RNeasy Mini Kit . A 3 μg sample of total RNA was used for DNA microarray analysis by using GeneChip Human Genome U133 Plus 2.0 Array . Signal values were obtained according to the manufacturer's instructions and normalized by eliminating the highest and lowest 2% of the data, respectively. Only data with present or marginal detection calls were selected for further analysis. Microarray data have been deposited in NCBIs Gene Expression Omnibus (GEO) and are accessible through GEO series accession number [GSE27390].Genes were identified as differentially expressed if their mean signal values were at least 50% different between the RA and OA groups. These genes were functionally categorized using Expression Analysis Systematic Explorer (EASE) version 2.0 bioinformatics software . InteracThe false-discovery rate was used to determine statistically significant differences in the mRNA expression levels between the RA and OA groups. The criterion for the statistical significance was q < 0.001.DNA microarray analysis revealed that 2,674 genes were differentially expressed in BMMC from patients with RA compared with those from patients with OA: 764 out of the 2,674 genes were up-regulated and the remaining 1,910 genes were down-regulated.To identify any aberrant biological function in the BMMC of RA patients, EASE based on the Gene Ontology (GO) database, which can classify large gene lists into functionally related gene groups and rank their importance, was performed. EASE classified the gene categories into three GO systems: biological process, cellular component, and molecular function. Up-regulated and down-regulated genes for the GO system biological process based on EASE are shown in Tables EASE of the up-regulated genes identified four major gene categories: response to external stimulus, signal transduction, phosphate metabolism, and RNA splicing, via transesterification reactions Table . Based oEASE for the down-regulated genes identified three major gene categories: cell proliferation, DNA replication and chromosome cycle, and DNA metabolism Table . The dowThe gene category immune response for up-regulated genes and mitotic cell cycle for down-regulated genes had the lowest EASE scores, respectively. The relations among the 56 up-regulated genes and the 97 down-regulated genes in these two gene categories were further analyzed by IPA.IPA analysis revealed that the up-regulated genes in immune response were highly relevant to the antigen presentation pathway and to interferon (IFN) signaling. There were four networks represented by the 56 up-regulated genes Figure . The firIPA found down-regulated molecules to significantly affect the role of polo-like kinase in mitosis, the role of CHK protein in cell cycle checkpoint control, and affect pyrimidine metabolism, and ataxia telangiectasia mutated (ATM) signaling. There were four networks constructed by the 97 down-regulated genes in the mitotic cell cycle Figure . SeveralIt is commonly known that autoimmunity plays a pivotal role in the pathology of RA. However, the exact etiology and pathogenesis are poorly understood. Our work, comparing the gene expression profiles of BMMC between RA patients and OA patients by microarray technology and gene ontology analysis, found abnormal immune responses in BMMC. This agrees with accumulating evidence indicating that abnormalities in BM cells may contribute to the pathogenesis of RA . To our Several reports have shown that peripheral blood from SLE patients has remarkably homogenous gene expression patterns and an overexpression of IFI genes ,15-17. T-/- embryos [Meanwhile, our finding that the MHC class I molecules HLA-E, HLA-F, and HLA-G, TAP, and TAPBP were all overexpressed in the BM cells of RA patients is also novel. All these genes relate to the antigen presentation pathway. For example, up-regulation of HLA-E is considered a potential marker for cancer. Additionally, its expression can confer resistance to NK cell-mediated lysis ,20. HLA- embryos . Kawane  embryos . Althougde novo synthesis of the nucleoside thymidine required for DNA synthesis. However, subsequent analysis showed MTX treatment does not correlate with the down-regulated gene expressions (data not shown). Alternatively, we considered the fact the BMMC samples in this study were isolated by using Ficoll-Paque, which may cause nucleated erythroblasts to be miscible in mononuclear cell proportions and thus affect cell cycle. Finally, a high concentration of serum IL6 in BM has been reported in RA patients [Another interesting observation is that underexpressed genes were dominantly related to cell cycle and DNA metabolism. We are the first to report the suppression of cell cycle and DNA metabolism in BM cells from RA patients. Initially, there appear to be several possible mechanisms that can explain this result. One is a therapeutic effect caused by MTX, as MTX acts by inhibiting the metabolism of folic acid, which is needed for the patients . This ispatients ,29. Hepcpatients . They fupatients . TogetheAccording to our unpublished data, the genes expressed in the peripheral blood cells of RA patients that correspond to cell cycle and DNA metabolism were not down-regulated as observed in BM cells, but the down-regulation for those in RNA metabolism- or translation-related genes were found. As all mature blood lineages in peripheral blood are produced from HSCs in the BM, the s abnormality in immune response and suppression of cell cycle in BM may contribute to the pathogenesis of RA.BM cells from RA patients had abnormal functional networks in immune response and cell cycle when compared with the BM cells from OA patients. Our results suggest that the overexpression of genes that take part in the antigen presentation pathway and IFN signaling contribute to the pathogenesis of RA. Our results also suggest that the underexpression of genes relating to cell cycle in the BM may be a potential pathogenic factor for RA.ATM: ataxia telangiectasia mutated; BM: bone marrow; BMMC: BM-derived mononuclear cells; CCN: cyclin; CDC: cell division cycle; CDK: cyclin-dependent kinase; CRP: C-reactive protein; DMARDs: disease-modifying antirheumatic drugs; EASE: expression analysis systematic explorer; FLS: fibroblast-like synoviocyte; GEO: Gene Expression Omnibus; GO: gene ontology; HLA: human leukocyte antigen; HNF: hepatocyte nuclear factor; HSCs: hematopoietic stem cells; IFI: IFN-inducible; IFN: interferon; IL: interleukin; IPA: ingenuity pathway analysis; JIA: juvenile idiopathic arthritis; MAPK: mitogen-activated protein kinase; MHC: major histocompatibility complex; MyD88: myeloid differentiation primary response gene 88; NFκB: nuclear factor of kappa light polypeptide; OA: osteoarthritis; PBMC: peripheral blood mononuclear cells; POL: polymerase; RA: rheumatoid arthritis; RF: rheumatoid factor; SLE: systemic lupus erythematosus; TAP: tapasin; TAPBP: TAP binding protein; TCR: T cell receptor; TLRs: Toll-like receptors.The authors declare that they have no competing interests.H-ML performed the data and statistical analysis, and drafted and revised the manuscript. HS and CA assisted with the acquisition of data and analysis. RS performed mRNA expression analysis with microarrays. YS and KO treated and recruited the patients for this study, and analyzed the clinical data of the patients. TO and NN made substantial contributions to the conception and design of the experiments, and analysis and interpretation of the data. All authors read and approved the final manuscript.


Motivation: The interaction between drugs and their targets, often proteins, and between antibodies and their targets, is important for planning and analyzing investigational and therapeutic interventions in many biological systems. Although drug-target and antibody-target datasets are available in separate databases, they are not publicly available in an integrated bioinformatics resource. As medical therapeutics, especially in cancer, increasingly uses targeted drugs and measures their effects on biomolecular profiles, there is an unmet need for a user-friendly toolset that allows researchers to comprehensively and conveniently access and query information about drugs, antibodies and their targets.Summary: The PiHelper framework integrates human drug-target and antibody-target associations from publicly available resources to help meet the needs of researchers in systems pharmacology, perturbation biology and proteomics. PiHelper has utilities to (i) import drug- and antibody-target information; (ii) search the associations either programmatically or through a web user interface (UI); (iii) visualize the data interactively in a network; and (iv) export relationships for use in publications or other analysis tools.Availability: PiHelper is a free software under the GNU Lesser General Public License (LGPL) v3.0. Source code and documentation are at http://bit.ly/pihelper. We plan to coordinate contributions from the community by managing future releases.Contact:pihelper@cbio.mskcc.org In cancer biology, systems pharmacology and perturbation biology, researchers designing targeted drug experiments often need to choose targeted drugs and antibodies of interest for their experimental studies. For such studies, drug- and antibody-target databases are valuable resources and are increasingly publicly available in computable formats. Unfortunately, this information is in separate databases that use mostly incompatible formats, making it difficult to integrate data across different resources. This, coupled with strict constraints on distribution of the data, hinders access to up-to-date integrated data.Here we describe an open-source framework, PiHelper, for easy aggregation, integration and visualization of drug- and antibody-target data from multiple sources. PiHelper provides a platform-independent command-line tool to help users, with minimal configuration, import and export drug- and antibody-target information in a human- and gene-centric manner; a Java application programming interface and a REST-ful  web service to facilitate programmatic access to the aggregated data; and a web-based UI to help users query data in a gene-centric manner and export the results as an image or undirected binary network.We believe PiHelper will facilitate hypothesis generation and design of new experiments by enabling researchers to access and query integrated drug-target and antibody-target data from multiple resources in an automatic way.importer component supports automatic fetching of background gene information, gene sets, gene-centric drug-target and antibody-target annotations from multiple resources . These files can then be used for further analysis tools, e.g. by importing the data into Cytoscape as a binary network and running graph-based queries or visualizing larger networks (Once the database is populated through the admin tool, the The web-based UI distributed as a part of PiHelper enables users to query antibodies and drugs in a gene-centric manner .Fig. 1.Drug, Gene and DrugTarget, which capture drug– and antibody–gene relationships. These elements, together with their querying methods, help developers build custom applications or analysis tools that depend on drug or antibody annotation data.The core module provides the model Java classes and basic finder methods. The model classes consist of basic elements, such as Besides the Java application programming interface, the core module also includes a web service component that provides basic querying methods through REST protocols. The web service supports obtaining the results in either JavaScript Object Notation or HyperText Markup Language. The former provides flexibility for developers who prefer other programming languages than Java; the latter enables users to interact with the database via their web browser of choice.