Plasmodium vivax has been interrupted by epidemic expansions and imported cases. The impact of these changes on the local parasite population, and concurrent risks of future resurgence, was assessed.In Central China the declining incidence of Plasmodium vivax isolates collected from Anhui and Jiangsu provinces, Central China between 2007 and 2010 were genotyped using capillary electrophoresis at seven polymorphic short tandem repeat markers. Spatial and temporal analyses of within-host and population diversity, population structure, and relatedness were conducted on these isolates.HE = 0.8 – 0.85). Differentiation between Anhui and Jiangsu was modest (F’ST = 0.1). Several clusters of isolates with identical multi-locus haplotypes were observed across both Anhui and Jiangsu. Linkage disequilibrium was strong in both populations and in each year tested (IAS = 0.2 – 0.4), but declined two- to four-fold when identical haplotypes were accounted for, indicative of occasional epidemic transmission dynamics. None of five imported isolates shared identical haplotypes to any of the central Chinese isolates.Polyclonal infections were infrequent in the 94 isolates from Anhui (4%) and 25 from Jiangsu (12%), with a trend for increasing frequency from 2008 to 2010 (2 to 19%) when combined. Population diversity was high in both provinces and across the years tested (P. vivax in Central China highlights unstable transmission, with limited barriers to gene flow between the central provinces. Despite low endemicity, population diversity remained high, but the reservoirs sustaining this diversity remain unclear. The challenge of imported cases and risks of resurgence emphasize the need for continued surveillance to detect early warning signals. Although parasite genotyping has potential to inform the management of outbreaks, further studies are required to identify suitable marker panels for resolving local from imported P. vivax isolates.The population genetic structure of  China has made considerable progress in reducing the burden of malaria, since the launch of the National Malaria Control Programme in 1955. Despite two major outbreaks in the 1960s and 1970s, the incidence of malaria cases has dropped steadily from an estimated 6.79 million cases in 1954 to less than 15,000 in 2009 [1-Σpi2], where n is the number of isolates analysed and pi is the frequency of the ith allele in the given population. HE was averaged across the 7 loci in each population to provide a measure of population diversity.Population-level genetic diversity was characterized using a measure of the expected heterozygosity  . STRUCTURE results were displayed in barplots using Distruct software version 1.1 [STRUCTURE software version 2.3.3  was usedlsewhere  for eachsion 1.1 .IAS) using the web-based LIAN 3.5 software [IAS estimates was assessed using 10,000 random permutations of the data. For each population, LD was assessed in 1) the full sample set, and 2) a curtailed sample set with each unique haplotype represented once only.After exclusion of loci with more than 15% missing data, multi-locus haplotypes (or infection haplotypes) were reconstructed from the predominant allele at each locus in isolates with no missing data at the remaining loci. Using these haplotypes, multi-locus linkage disequilibrium (LD) was measured by the standardized index of association (software . Under tps), and using (1-ps) as a measure of genetic distance [pca function in the Modular toolkit for Data Processing framework in Python [The genetic relatedness between sample pairs was assessed by a simple measure of the proportion of alleles shared between haplotype pairs  could be genotyped successfully at a minimum of six of the seven loci investigated. Subsequent analysis was restricted to these 128 “pass” isolates, which included 94 isolates from Anhui, 25 from Jiangsu, two from Henan, two from Yunnan, with five internationally imported .A summary of the number of isolates included in the study by site and year of collection is presented in Table Insufficient sample sizes were available for temporal analyses within Jiangsu Province . However, as detailed further in the results section on “Population Structure and Differentiation”, allele sharing between Anhui and Jiangsu was sufficient to enable pooling of the two provinces for both spatial and temporal investigations in “Central China”. After pooling, sufficient sample sizes were available for comparisons between 2008, 2009 and 2010. As detailed in Table The genotyping assays exhibited less than 2% failure in the pass samples, apart from the MS10 and MS5 loci, which exhibited a 21% and 11% genotyping failure rate respectively. With the exception of the MS10 locus, 84% (108/128) of the pass samples exhibited a full set of genotype calls at all loci.P = 0.160). The frequency of polyclonal infection increased over time in Central China, from 1.9% (1/54) in 2008, to 5.1% (2/39) in 2009, and 19% (4/21) in 2010 .Table Measures of the MOI corresponded with the patterns observed in the frequency of polyclonal infections Table . The MOIHE) ranging from 0.809 in Jiangsu and 0.816 in Anhui, with an overall HE = 0.824 across both sites (Table HE = 0.849) compared to 2008 (HE = 0.816) or 2009 (HE = 0.796).Population diversity was high across all isolates, heterozygosity (FST between Anhui and Jiangsu was low (FST = 0.018). Comparably low levels of differentiation were observed between 2008, 2009 and 2010 (unadjusted FST range = 0.000 - 0.039). Only the differentiation between 2009 and 2010 approached significance . Adjustment for the marker diversity increased the levels of differentiation between Anhui and Jiangsu and in the annual pair-wise comparisons, but the overall levels remained low to moderate (standardized FST range = 0.000 - 0.214).As detailed in Table ΔK method, STRUCTURE analysis suggested evidence of population sub-structure, with K = 4 identified as the most likely number of sub-populations , Jiangsu , and across Central China as a whole  . After accounting for outbreak samples by analysing unique haplotypes only in each test group, the IAS dropped two to three-fold in each of Anhui and Jiangsu, and across Central China. In the temporal analyses, the largest reduction in IAS (four-fold) was observed in 2008, with two-fold and three-fold reductions observed in 2009 and 2010, respectively.Moderately high linkage disequilibrium (LD) was observed in Anhui  and four suspected imported cases  with no missing data Figure . A totalP = 0.01). Further visual representation of haplotype dynamics over time is presented in Figure A weak but significant correlation was found between genetic distance (estimated from the number of genotype differences) and temporal distance classified by annual quarter . However, the number of isolates from Jiangsu was small, reducing the power of the analysis. Nonetheless, in accordance with the F’ST results, limited evidence for differentiation between Anhui and Jiangsu was observed with STRUCTURE analysis, with any sub-populations rather being shared amongst the two provinces. The level of differentiation observed with the F’ST analysis was lower than levels observed in intra-country comparisons in Sabah (F’ST = 0.5 - 0.6) [F’ST = 0.4 - 0.7) [P. vivax endemicity is comparably low. Rather, the differentiation is more comparable to levels observed between sites in Papua New Guinea (PNG) (F’ST = 0.14 - 0.16) [P. vivax strains endemic to Central China may have contributed to the persistence and spread of infections both temporally and geographically. Indeed, the majority of the multi-locus haplotypes observed three or more times in the data set (10/11), were observed in two or three different years of collection. Hence the current evidence suggests that the recently malaria-free province of Jiangsu is at moderately high risk of re-introduced malaria from Anhui.Parasite isolates were sourced from two neighbouring provinces in Central China (Anhui and Jiangsu). Anhui exhibits the highest incidence of malaria in Central China (API 5.0 - 0.28 between 2007 and 2010), significantly greater than Jiangsu (API 0.05 - 0.09 between 2007 and 2010). The latter has not experienced any indigenous malaria cases in 2012 and 2013. Only moderate genetic differentiation was observed between the two provinces and this was apparent even after adjusting for the extensive marker diversity (4 - 0.7) , where P - 0.16) . The mecP. vivax surface antigens on isolates from Anhui Province demonstrated a low frequency of polyclonal infections in 2004 (6%) [The current analysis of seven STR markers demonstrated polyclonal infections to be infrequent in both Anhui (4%) and Jiangsu (12%). These levels of polyclonal infection are generally lower than those observed in tropical and sub-tropical endemic populations across the globe ,27,45-49004 (6%) , and mod004 (6%) . Across P. vivax transmission in Central China. As observed in P. vivax populations from multiple endemic settings across the globe [HE = 0.8). The reservoirs sustaining this diversity in the face of aggressive containment efforts remain unclear. Sub-patent and asymptomatic infections may present a largely hidden reservoir, with undetected imported infections in particular enabling maintained diversity. Indeed, the propensity of P. vivax to relapse from its dormant liver stage weeks to months after the primary infection may greatly aid the introduction of imported strains. A recent study investigating temporal trends in the diversity of P. vivax isolates in Sri Lanka demonstrated that despite rapidly declining endemicity between 2003-2004 and 2006-2007, population diversity remained unexpectedly high in the latter years [P. vivax (HE = 0.85) relative to P. falciparum (HE = 0.54), frequent relapse may have facilitated polyclonal infection and outcrossing in the P. vivax population, with moderate contribution to the observed diversity [The results of the analyses of population diversity and linkage disequilibrium (LD) demonstrate evidence of moderate instability in he globe ,27,45-49er years . The autiversity . Howeveriversity , the narIAS = 0.2 – 0.4), suggestive of low recombination, as might be anticipated with largely clonal transmission and limited opportunity for transmission. However, when each distinct haplotype was represented just once, the strength of LD (IAS) declined two- to four-fold, with the greatest decline observed in 2008 . The strengthening of LD by the expansion of a few haplotypes in an otherwise moderately panmictic population is suggestive of occasional outbreaks/epidemic transmission. Indeed, multiple small outbreak clusters of identical haplotypes were observed in the neighbour-joining plot. In Sabah, similar outbreak dynamics were observed, but with the majority of the identical haplotypes observed within short temporal windows and moderately confined geographic space [Despite the high population diversity, LD remained high in both Anhui and Jiangsu and in each year tested  has been made available for enhanced comparison of STR-based P. vivax data between studies [P. vivax endemic regions across the globe, so that the utility of the APMEN marker panel as well as new markers can be investigated comprehensively. Indeed, as recently demonstrated in a global study of variation in the P. vivax mitochondrial genome, SNPs in this organelle may offer some assistance in resolving the geographic origin of isolates at least at a regional level [P. falciparum isolates [In settings such as Central China, where the endemicity of local infections is declining rapidly but the relative proportion of imported cases rising, geographic markers enabling confirmation of imported versus local  studies . This mual level . Furtherisolates .P. vivax resurgence in Central China appear to have been successful in reducing the parasite incidence, genotyping analysis confirms complex parasite dynamics during the suppression phase. The ability of certain parasite lineages to expand locally demonstrates the instability of transmission and risk of future resurgence. With limited barriers to gene flow between Anhui and Jiangsu, the risks of resurgence in the latter demand continued dedication to surveillance to detect early warning signals. Parasite genotyping may facilitate early detection of outbreaks. Further studies utilising the consensus marker sets or barcodes informed by nuclear and organellar genomic data will be required to discriminate local from imported P. vivax isolates in Central China and other vivax endemic regions. Such studies will inform the main reservoirs of infection, the degree to which imported cases may affect the local transmission, and in doing so help to focus appropriate malaria control resources to ensure the ultimate elimination of the parasite.Although interventions to suppress the recent The authors declare that they have no competing interests.YL, QG, QC, RH, SA, JC and RNP conceived and designed the study. YL, JC, and KG performed the laboratory assays. HT, SA, TGC, YL and JC performed the data analysis. JC, YL, QG and HYZ provided essential contribution to the sample collections. YL, SA, QC, RNP, HT, JC, RH and QG wrote the manuscript and facilitated data interpretation. All authors read and approved the final manuscript.Assay details.Click here for fileInference of most likely STRUCTURE-defined K in Anhui and Jiangsu. The top panel presents ΔK against K , with weak evidence for the most likely K of 4 [K (from 1 - 10), with weak evidence for the most likely K at 1.y K of 4 . The botClick here for filePopulation structure in Anhui and Jiangsu inferred by STRUCTURE at K = 4. Each vertical bar represents a sample, with colour-coding reflecting the predicted ancestry to each of the 4 (K) sub-populations. K1 = light green, K2 = dark green, K3 = red, and K4 = orange.Click here for filePrincipal component analysis of the genetic variation between local and imported P. vivax cases in Central China. The PCA was generated using data from 104 Chinese and 4 imported isolates which exhibited full genotype profiles at the loci investigated (excluding MS10). As with the neighbour-joining tree (Figure Click here for file


We determined the expression and quantity of IC subsets in the three study groups and performed correlation analyses with demographic and clinical parameters. The quantity of several IC subsets differed between the AIS/TIA, CCD, and HV groups. Several clinical and demographic variables independently predicted the quantity of IC subsets in patients with AIS/TIA. No significant changes in the quantity of IC subsets occurred within the first three days after AIS/TIA. Overall, these findings strengthen the evidence for a pathophysiologic role of IC in human ischemic stroke and the potential use of IC-based biomarkers for the prediction of stroke risk. A comprehensive description of IC kinetics is crucial to enable the design of targeted treatment strategies.Immune cells (IC) play a crucial role in murine stroke pathophysiology. However, data are limited on the role of these cells in ischemic stroke in humans. We therefore aimed to characterize and compare peripheral IC subsets in patients with acute ischemic stroke/transient ischemic attack (AIS/TIA), chronic cerebrovascular disease (CCD) and healthy volunteers (HV). We conducted a case-control study of patients with AIS/TIA ( Peripheral immunodepression is a common observation after acute ischemic stroke ,2,3 and The stroke-induced immunodeficiency  has impoExperimental stroke studies in rodents described the pathophysiologic relevance of immune cell subsets ,14,15,16While the importance of neuroimmunologic interactions after ischemic stroke—including the role of distinct immune cell subsets—is being increasingly recognized ,27,28,29This case-control study has been conducted to evaluate whether: (i) peripheral immune cell subsets differ between healthy volunteers (HV), patients with acute cerebrovascular disease (AIS/transient ischemic attack [TIA]), and those with CCD; and (ii) to identify demographic and clinical predictors of the numbers of distinct peripheral immune cells in patients with AIS/TIA.Overall, the study included 116 patients with AIS/TIA. Patients had a mean age of 70 ± 12 years, 53% were male and 58% of patients presented with an AIS. Baseline clinical severity, measured using the National Institutes of Health Stroke Scale (NIHSS) and Barthel Index, was 4.8 ± 6.0 and 74 ± 30, respectively. The demographic and clinical characteristics of patients presenting with an AIS or TIA are summarized in p < 0.001; neutrophils, 4.2 ± 1.4/nL, p < 0.001) and HV . In contrast, lymphocytes were higher in HV (2.0 ± 0.6/nL) compared with patients with CCD 1.8 ± 0.6/nL, p < 0.05) and those with AIS/TIA . Also FoxP3+ regulatory T cells (Treg), a subset of lymphocytes, were decreased in patients with AIS/TIA (2.4% ± 1.2%) compared with patients with CCD  and HV . There was no difference in the number of monocytes as well as the fraction of CD4+CD8− or CD8+CD4− T cells between the groups.The numbers or fractions of immune cell subsets in patients with AIS/TIA and CCD and in HV are shown in p < 0.001) and neutrophil numbers (p < 0.001) as well as the fraction of FoxP3+ Treg (p = 0.02) were higher in patients with AIS compared with patients with TIA. In contrast, the number of monocytes was lower in patients with AIS than in patients with TIA (p = 0.02). Older patients showed a lower quantity of lymphocytes (p = 0.02) and a smaller fraction of CD4+CD8− T cells (p = 0.03). Severity of stroke at admission was associated with different immune cell subsets . Again, leukocytes (p = 0.01) and neutrophils (p = 0.003) were associated with thrombolysis. Interestingly, sex and pretreatment with platelet inhibitors did not influence the number or fraction of immune cell subsets.Results from univariate analysis of the association between the number or fraction of immune cells subsets and key demographic and clinical characteristics are summarized in vs. TIA) as an independent predictor of leukocyte (p = 0.006), neutrophil (p = 0.005), and monocyte count (p = 0.04). Sex was only associated with the fraction of CD8+CD4− T cells (p = 0.03). Age only accounted for CD4+CD8− T cell regulation (p = 0.012). NIHSS scores were independent predictors of the CD4+CD8− as well as the CD8+CD4− fractions . We found no independent predictor of FoxP3 Treg. In addition, none of the measured variables  were influenced by the time of blood withdrawal .Multivariate analysis (adjusted for age and sex)  identifiIn this case-control study, we analyzed peripheral immune responses in different cerebrovascular disease settings and showed that the number or fraction of distinct immune cell subsets is differentially regulated between patients with AIS/TIA and CCD, compared with HV. Moreover, within the AIS/TIA group, several clinical  or demographic  parameters predicted the number or fraction of immune cells even after adjustment for age and sex.+CD8− T cells and a high percentage of CD8+CD4− T cells were independently associated with high clinical severity of patients with AIS/TIA at admission. This observation can be confirmed by a study showing that subjects with a high fraction of CD8+ cells often have comorbidities that include insulin resistance and an increased risk of cardiovascular events [+ T cells in AIS [+ cells have detrimental properties in cardiovascular disease. FoxP3 Treg were associated with clinical severity in univariate analysis but not after adjustment for age and sex.Most of our findings in patients with AIS/TIA are consistent with the results of previously published studies regarding ischemic stroke ,33,34,35r events . Therefos in AIS , it can et al. [reg over time in patients with intracerebral hemorrhage and Sarrafzadeh et al. [+ and CD8+ T cells in a subpopulation of patients in the first days after aneurysmal subarachnoid hemorrhage.Very importantly, other diseases of the CNS—such as like aneurysmal subarachnoid hemorrhage  or acuteet al.  reportedh et al.  found an+ Treg were even higher in patients with CCD compared with those with AIS/TIA, suggesting a pathophysiologic role of Treg in CCD. Atherosclerosis is currently understood as a systemic disease that might also be influenced by pro- and anti-inflammatory cytokines. A recent report suggested that the level of detrimental cytokines could be decreased by physical exercise [Despite clear evidence that inflammatory mechanisms and immune cells play an important part in the pathophysiology of atherosclerosis—including plaque progression and instability ,37—to thexercise . Furthervs. TIA, age, sex, NIHSS). It seems that the number of immune cells depends on various non-modifiable clinical and demographic variables, making it difficult to develop universal treatment strategies.As immune cells are not only biomarkers after ischemic stroke, but also potential therapeutic targets , a detaiThere are several limitations to this study that should be considered. First, it should be remembered that the potential for reverse causation as a result of blood withdrawal following a cerebrovascular event cannot be disregarded. Accordingly, the current study describes the magnitude and significance of associations between immune cell subsets and demographic/clinical parameters without attributing causality. Further prospective studies are required to formally elucidate causality. Second, all patients were required to provide informed consent before participating in the study. However, this may have resulted in patients who have suffered a severe stroke and/or aphasia being underrepresented in this study because neurological deficits related to their condition may have prevented them from being capable of providing informed consent. Third, it was not possible to completely rule out a non-vascular origin for symptoms in 42% of the TIA patient population, meaning that the possibility of the aforementioned factors influencing the regulation of immune cell subsets remains.i.e., an acute ischemic lesion on brain imaging) and TIA (no acute ischemic lesion on brain imaging), blood samples must have been drawn within 24 hours of symptom onset; in the CCD group, patients must have presented with extracranial and/or intracranial stenosis of the large cerebral arteries with (n = 66) or without (n = 51) a history of AIS or TIA; and for the control HV subjects, aged ≥50 years with no history of stroke, myocardial infarction, or peripheral arterial disease. Patients with AIS, TIA or CCD were excluded from the study if they presented with intracerebral hemorrhage, were aged <18 years, had a known plasmatic coagulation disorder, or a detailed medical history indicated the presence of platelet dysfunction.Patients with acute cerebrovascular disease (AIS/TIA) and CCD were included in this study, while control subjects were HV from the local population. All study participants were required to meet the following inclusion criteria: for patients presenting with an AIS  and written informed consent was provided by all participants. In total, 337 patients were eligible to participate in the study, including 116 patients with AIS or TIA, 117 patients with CCD, and 104 HV. Patient treatment and care remained at physician discretion and was not affected by participation in this study.vs. no thrombolysis) were recorded, as well as NIHSS [An adapted version of the TOAST  criteria  was applas NIHSS  and Bartas NIHSS .+CD8−, CD8+CD4−, and FoxP3+ Treg was performed using peripheral blood mononuclear cells (PBMCs), with density gradient centrifugation used to isolate PBMCs from peripheral blood. Cells were analyzed on a BD FACSCalibur flow cytometer . The following primary antibodies were used: FoxP3-APC , CD4− FITC , and CD8− PE . The respective isotype controls were purchased from BD Biosciences. The gating strategy is illustrated in Blood samples were drawn from an antecubital vein using a 21-gauge butterfly needle between 08.00 and 12.00 h on Days 0, 1, and 3 in patients with acute cerebrovascular disease. Blood samples were only drawn once in patients with CCD and HV. Pre-analytic preparations for blood collection were carried out according to specific standard operating procedures and only non-hemolyzed blood samples were analyzed. Differential hematology—including the absolute number of leukocytes, lymphocytes, neutrophils and monocytes—has been analyzed at the Division of Laboratory Medicine of the University Hospital Würzburg. Flow cytometric analysis of the fractions of CD4p values derived, as appropriate. Coefficients and corresponding 95% confidence intervals for potential predictors of the numbers of distinct immune cells were estimated using a linear regression model that included all variables without collinearity in a multivariate model that was adjusted for age and sex. Immune cell subsets were compared between the different patient groups , and distributions analyzed using the Kolmogorov-Smirnov test. It was assumed that the immune cell numbers were normally distributed and the groups were compared using ANOVA with a Bonferroni post-hoc test. These comparisons were additionally adjusted for age and sex. All reported p values are derived from two-sided tests, with a p value <0.05 considered to be statistically significant. Analyses were performed using SPSS Version 21 and SAS software version 9.1 .Continuous variables are presented as mean ± standard deviation or median with interquartile range, as appropriate. Categorical variables are expressed as percentages. Analysis of variance (ANOVA) and chi-square tests were used to investigate the association between the absolute number or fraction of immune cell subsets and demographic and clinical characteristics , TOAST criteria, duration between symptom onset and blood withdrawal, NIHSS score, Barthel Index score, treatment modality (intravenous thrombolysis or not), and treatment with platelet inhibitors in the days before blood withdrawal) and Changes in peripheral immune cell numbers are a well-known signature after ischemic stroke. At the same time, immune cell subsets play major roles in the pathophysiology of murine ischemic stroke and might also become future targets of novel therapeutic approaches in humans. We provide here an overview of the regulation of distinct immune cell subsets after AIS/TIA in comparison with CCD and HV. The description of independent predictors of immune cells raises new questions, which might be valuable for the understanding of pathophysiologic mechanisms, and could finally help to enable focused treatment strategies.


Objective. The aim of this study is to analyze the high-frequency hearing levels in patients with rheumatoid arthritis and to determine the relationship between hearing loss, disease duration, and immunological parameters. Materials and Methods. A descriptive cross-sectional study including fifty-three patients with rheumatoid arthritis was performed. The control group consisted of 71 age- and sex-matched patients from the study population . Both a pure tone audiometry and an extended-high-frequency audiometry were performed. Results. Extended-high-frequency audiometry diagnosed sensorineural hearing loss in 69.8% of the patients which exceeded the results obtained with pure tone audiometry (43% of the patients). This study found significant correlations in patients with sensorineural hearing loss related to age, sex, and serum anti-cardiolipin (aCL) antibody levels. Conclusion. Sensorineural hearing loss must be considered within the clinical context of rheumatoid arthritis. Our results demonstrated that an extended-high-frequency audiometry is a useful audiological test that must be performed within the diagnostic and follow-up testing of patients with rheumatoid arthritis, providing further insight into a disease-modifying treatment or a hearing loss preventive treatment. Autoimmune hearing loss was reported for the first time by McCabe in 1979.  A descriRheumatoid arthritis (RA) is a connective tissue disease that has a disseminated erosive arthropathy associated with systemic manifestations. This immune-mediated disease may entail mainly SNHL –11, whicThe pathogenesis of SNHL in RA still remains unclear, although it is potentially related to vasculitis , neuritiThis study aimed to assess the real prevalence of SNHL in RA using advanced audiometric analysis based on extended-high-frequency audiometry (HFA), thus considering its effectiveness and clinical utility for inclusion in the routine diagnostic hearing tests. Furthermore, we analyzed the immunological parameters to obtain an indicator of the inner ear impairment in these patients.A cross-sectional descriptive study was carried out with comparative cases and controls matched for age and sex.Inclusion Criteria. Fifty-three patients with RA (106 ears), both sexes, aged between 20 and 60, were diagnosed and treated in the office of the Rheumatology and Nephrology Departments of “Severo Ochoa” Hospital . The diagnostic criteria and their respective scoring used for a definitive RA diagnosis were as follows: (A) joint involvement: 1 large joint = 0; 2–10 large joints = 1; 1–3 small joints (with or without involvement of large joints) = 2; 4–10 small joints (with or without involvement of large joints) = 3; >10 joints  = 5; (B) serology (at least 1 test result is needed for classification): Negative Rheumatoid Factor (RF) and negative anti-citrullinated protein antibody (ACPA) = 0; low-positive RF or low-positive ACPA = 2; high-positive RF or high-positive ACPA = 3; (C) acute phase reactants (at least 1 test result is needed for classification): normal C-reactive protein (CRP) and normal Erythrocyte Sedimentation Rate (ESR) = 0; abnormal CRP or abnormal ESR = 1; (D) duration of symptoms: <6 weeks = 0; >6 weeks = 1. The classification criteria from the American College of Rheumatology (ACR) for RA  consider that a score of > or = 6/10 is needed for classification of a patient as having a definitive RA. inclusion criteria: patients who suffered from rheumatoid arthritis, both sexes, aged between 20 and 60.Both a medical history and an ENT examination were performed, using the followingExclusion Criteria. Exclusion criteria are patients who suffered from another immune-mediated disease, inner ear disease, traumatic brain injury, Menière's disease, metabolic diseases, and cardiovascular disease, exposure to noise, and the use of ototoxic medication different from that used in the treatment of RA.A control group of 71 patients (142 ears) was consecutively recruited in “Area 9” of Madrid, from January 2010 to February 2011.The control group was composed of patients with a normal state of health, free from all signs or symptoms of ear disease and from obstructing wax in the ear canal, and with no history of exposure to noise, potentially ototoxic drugs, or familial hearing loss that attended our ENT department for head and neck or rhinologic disorders.If the control population presented hearing thresholds in the limits of the normal range, according to the range of age and sex, assessed by pure tone audiometry (PTA) using frequencies of 125 to 8000 Hz, consequently an extended-high-frequency audiometry (HFA) of 8000 to 18000 Hz was also performed to determine the normal hearing parameters and use those parameters as references for the study.A number of variables have been studied, such as demographic variables (age and sex), hearing variables (hearing thresholds with PTA and HFA), and variables related to the rheumatologic disease . Disease activity is measured as active or nonactive.The first otological test included an initial otoscopy and a hearing screening with PTA and HFA.All the subjects suitable for inclusion were evaluated based on a questionnaire (Annex A in ISO 389-9: 2009) according to a written protocol based on ISO 389-9 recommendations on the determination of reference hearing threshold levels.Audiometric tests were conducted by experienced audiometric technicians. For each test frequency, the signal was manually increased by steps of 5 dB until the test person responded, after which the signal was decreased by 10 dB and increased by 5 dB until response. The intensity to which the listener responded three out of five times was recorded as threshold (masking was used). The pure tone hearing thresholds (125–8000 Hz) were measured with a manual audiometer  and equipped with TDH-39 supra-aural earphones . Bone and air conduction audiometry were performed.HFA thresholds  were determined using a Madsen clinical audiometer  with a Sennheiser HDA 200 closed circumaural earphone . All testing equipment for audiometry was calibrated according to ISO 389-5  and the manufacturer's recommendations. The threshold was defined as the lowest decibel hearing level at which responses occurred in at least 50% of a series of ascending trials  , 27.The limit for the hearing loss to be considered was established when one or more frequencies have higher threshold than the frequency previously identified as normal in the control population .An immunological analysis of all the parameters, including immunological RA activity (CRP and ESR), subgroup markers of RA (RF and antinuclear antibodies), acute phase reactants (APR), serum cryoglobulins, and anti-cardiolipin (aCL) and antithyroid antibodies, was performed.All the procedures used in this study and practiced on each patient were in agreement and regulated with approval from the “Severo Ochoa” Hospital (Madrid) Institutional Review Board and ethical code identified by the MINUTE: 11/09, 25/11/2009, IEC (Independent Ethics Committee) Internal code: 431-A (65/09), and the Declaration of Helsinki (1983). Informed consent was obtained from each participant. The same person performed all interviews. The subjects received no monetary compensation. Informed consent was obtained from all subjects, after being informed of details about the purpose of the study, according to the recommendations from the Ethics Committee in our centre.Statistical Package for the Social Sciences) for Windows. This study used quantitative and qualitative descriptive statistics, Chi-square test, Student's t-test, and Mann-Whitney U test. Logistic regression analysis was achieved by adjusting the estimate for some risk factors, including independent variables such as risk factors related to the patient, subjective hearing or rheumatic disease, with a dependent variable represented by the presence of sensorineural hearing loss. The estimate was established with 95% confidence intervals of the hearing loss prevalence in the PTA (threshold > 30 dB HL) and in the HFA (according to the hearing threshold of the control population for each frequency), adjusted for age  and sex. A p value of <0.05 was considered significant.Statistical analyses were performed using SPSS 15 (The average age of the patients with RA was 50.5 years (interquartile range: 38–59 years), and the patients included 14 males and 39 females with a 26.4%/73.6% male/female ratio. The control population included 71 subjects (142 ears) belonging to Madrid Area 9; the patients were consecutively enrolled, with an average age of 38 years .p = 0.001) and 8000 Hz (p = 0.001) frequencies between patients with RA and the control population.In this study, 43% of the study population with PTA had hearing loss (46 ears). The type of hearing loss was sensorineural, with high-frequency loss predominant. High frequencies were affected in 56.6%, medium frequencies in 17%, and low frequencies in 13.2% of the population. There was a significant difference in hearing thresholds (arithmetic mean) at 6000 Hz (p = 0.038), 6000 (p = 0.004), and 8000 Hz (p = 0.009) frequencies; there were lower auditory hearing thresholds in females compared to males. We observed that in patients with RA there was a statistically significant relationship between the hearing loss diagnosed by PTA and sex (OR = 3.46 (1.29–9.27)) and the age at which it was diagnosed (OR = 1.09 (1.04–1.15)).SNHL was bilateral and symmetric in 74% of the cases. There were no cases of conductive or mixed hearing loss. Our results showed that patients with RA had greater high-frequency loss than the control population (according to age range) . Regardip < 0.0001)  . These r 0.0001) .p = 0.009) and the 10000 Hz (p = 0.026) threshold frequencies, between male and female patients with RA, which demonstrated a greater hearing loss in males than in females.Regarding sex, we obtained a significant difference between the means of the 8000 Hz ).The diagnosis of SNHL in RA reached 69.8% with HFA and 43.4% with PTA. The chance of diagnosing hearing loss with HFA is 33.6 times higher compared to PTA OR = 29 (4.8–1184.43).RA may cause SNHL , similarNevertheless, nowadays the treatment used in RA includes immune-modulating drugs that could control the progression of the SNHL in some patients. Thereby an early diagnosis of a possible subclinical SNHL by means of HFA  in order to allow a prior treatment (transtympanic or systemic corticosteroids) is of paramount importance.(1) PTA in RA. The prevalence of SNHL with PTA in our study (43.4%) is within the same range as described in previous literature, which has been estimated between 25.2 to 60% [2 to 60% , 12, 13.2 to 60% , 31, 32.2 to 60% , who cla2 to 60% , who repA comparison between the study and control groups using PTA revealed a significant hearing loss in patients with RA at frequencies of 6000 and 8000 Hz. Our results differ from other authors, who did not find significant differences between the two populations , 13.Moreover, 73.9% of our patients with RA had bilateral, symmetric SNHL, which was a slightly lower result compared to the results obtained by Özcan et al.: 84.6% with bilateral SNHL , 21. In In our study, we observed a significant relationship between SNHL and sex (OR > 1) with males demonstrating a higher risk of hearing loss and significant differences in high frequencies at approximately 4000, 6000, and 8000 Hz. These findings were consistent with those observed by Dikici et al. . This heThe persistent findings of high-frequency thresholds in female subjects might be explained due to the fact that the female ear canal has smaller average length and volume. Nevertheless, there could be other explanations for the gender difference, such as differences in exposure to workplace or extracurricular activity noise or different hearing levels as a result of a different phylogenetic origin. Moreover, the aging process may be different in males and females. There are many health factors that influence hearing status  which may also influence the differences observed between males and females .(2) HFA in RA. In most of the studies, the hearing loss diagnosis in patients with RA was performed using pure tone audiometry. In few studies a hearing screening was performed using frequencies higher than 8000 Hz [ 8000 Hz , 34, 35. 8000 Hz , 35 and,Furthermore, 69.8% of our patients with RA were diagnosed with SNHL using HFA, which is a higher percentage compared to those obtained using PTA. Some studies showed that there were no references regarding the index of SNHL using HFA, because previous results were global (125–16000 Hz) , 36.Our results do not show a significant correlation between SNHL diagnosed using HFA and age. This finding takes into consideration that normal age-related hearing loss in patients older than 50 from the control population is expected to have higher thresholds than those with RA since hearing loss is generally observed since early stages of the disease . RegardiIn our study there were statistically significant differences in all of the frequencies studied between the hearing threshold in both the control population and patients with RA. The hearing loss is greater starting at 40 years of age with thresholds higher than 60 dB.Öztürk et al. obtained similar results and found significant differences between the control population and the patients with RA, from a frequency 220 to 16000 Hz . In our We found no correlation between SNHL diagnosed using PTA and HFA and the duration of the disease, which was consistent with other studies , 32, 33.p = 0.029) between SNHL diagnosed using HFA and anti-cardiolipin (aCL) positivity (OR < 1) was observed. This finding might be due to the potential for inner ear thrombosis to be developed, which is found to be associated with high levels of aCL (risk factor for SNHL). In our study, 43.3% of the patients with RA had aCL positivity, which may cause confusion since it is usually associated with SLE [The analysis of immunological parameters using specific laboratory tests was performed without obtaining conclusive results , 38, 39.with SLE –42.No correlation between positivity to anti-nuclear antibodies (ANA), rheumatoid factor (RF), Sjögren's syndrome (SS), aCL, ESR, and SNHL has been reported , 12, 14.There was a higher rate of patients diagnosed with SNHL using HFA  compared to studies previously reported. SNHL in RA was bilateral and predominantly symmetric, affecting mainly high frequencies. Males over 40 had a greater risk of suffering from SNHL.There were more cases of subclinical hearing loss diagnosed using HFA compared to PTA. These results lead us to consider HFA as a necessary test to diagnose subclinical hearing loss in patients with RA. An early intervention in this subgroup of patients may cease the development of hearing loss. Thus, we propose that HFA should be performed as a routine screening not only for the study of RA but also for all of the inner ear disorders , 28, 43.


Emergence of colonization and infection with vancomycin-resistant enterococci (VRE) has become a worldwide challenge. To investigate whether the increasing incidence of VRE isolation can be correlated with use of glycopeptides in the hospital setting, we conducted a hospital-wide two-year study in the university hospital of Vienna.Within the period from January 2011 through December 2012 all patients with isolation of invasive or non-invasive VRE were retrospectively included. Specialty-specific data concerning the consumption of vancomycin and teicoplanin, fluoroquinolones and third generation cephalosporins in defined daily doses (DDDs) from June 2010 through May 2012 were extracted from the hospital pharmacy computer system. To assess the relationship between the usage of those antibiotics and the incidence of VRE (VRE-rate per 10 000 patients) a Poisson regression was performed.p < 0.001). The results of the Poisson regression for vancomycin (p = 0.0018) and teicoplanin (p < 0.0001) separately were both highly significant. Spearman’s correlation coefficient indicated a strong correlation between the two variables (rho = 0.8).In the study period 266 patients were colonized or infected with VRE. Specialty-specific VRE isolation was as follows: general surgical units (44 patients), bone marrow transplant unit (35 patients), general medical units (33 patients), cardiothoracic surgery (27 patients), nephrology (26 patients), haematooncology (22 patients), gastroenterology (17 patients), urology (17 patients), and the infectious diseases unit (11 patients). Hospital-wide consumption of glycopeptides was higher for teicoplanin than for vancomycin (26 242 versus 8677 DDDs). Specialty-specific VRE incidence significantly increased with the use of glycopeptides, fluoroquinolones or third generation cephalosporins (Overall usage of glycopeptides, fluoroquinolones or third generation cephalosporins contributed to the emergence of VRE in the hospital setting. Enterococci are part of the gastrointestinal bacterial flora and urogenital mucosa in healthy humans, but important nosocomial pathogens in a variety of invasive infections including endocarditis, bloodstream infections, wound infections, and meningitis , 2.Enterococcus faecium, are emerging worldwide [E. faecium increased from 1.8 % in 2008 to 5.9 % in 2013 [E. faecium [Colonization and infection with vancomycin-resistant enterococci (VRE), predominantly but not exclusively with orldwide –5. The corldwide , 7. AccoThe aim of the present study was to investigate whether this increasing incidence of VRE isolation in our patients can be correlated with use of glycopeptides in the hospital setting.The General Hospital of Vienna, Austria, is a 2133–bed central hospital and the seat of the clinics of the Medical University of Vienna. Each year more than 100 000 patients receive inpatient treatment and a total of 1.25 million patients attend the outpatient clinics . The uniEnterococcus faecalis was identified by the formation of black colonies on tellurit agar plates, while other Enterococcus species were identified by VITEK 2 GP (bioMérieux) or MALDI-TOF mass spectrometry . If the vancomycin and teicoplanin disc diffusion test according to the current version of the EUCAST methodology [In case of VRE screening selective enrichment media  were inoculated, while clinical samples for non-targeted culture were processed following standard procedures dependent on the type of specimen, i.e. non-selective enrichment broth (brain-heart infusion) was included only in case of material from primary sterile body sites. hodology  displayehodology .For avoidance of nosocomial transmissions, a targeted risk adjusted screening policy with subsequent isolation  of patients colonized or infected with VRE is implemented at our institution.Glycopeptides, fluoroquinolones and third generation cephalosporins were classified according to the anatomical therapeutic chemical (ATC) system. In our institution the following agents were used: vancomycin, teicoplanin, ciprofloxacin, levofloxacin, moxifloxacin, ceftriaxone, cefotaxime, ceftazidime and cefixime. Hospital-wide and specialty-specific data on consumption of those antibiotics were derived from the hospital pharmacy for the period from June 1, 2010 through and including May 31, 2012. Antibiotic usage was measured in defined daily doses (DDDs). In accordance with the ATC classification/DDD index 2013, the glycopeptide DDDs used were vancomycin 2 g and teicoplanin 0.4 g, the fluoroquinolone DDDs used were levofloxacin 0.5 g, moxifloxacin 0.4 g and ciprofloxacin 1 g for oral administration or 0.5 g for parenteral administration, the third generation cephalosporin DDDs used were ceftriaxone 2 g, cefotaxime 4 g, ceftazidime 4 g and cefixime 0.4 g [In order to assess the relationship between the antibiotic usage and the incidence of VRE (VRE-rate per 10 000 patients), a Poisson regression model was fitted with the number of VRE-positive patients as the dependent variable, logarithm of the mean usage of glycopeptides, fluoroquinolones or third generation cephalosporins as independent variable and the total number of patients in each unit as an offset.et al. a dynamic relationship between antimicrobial use of glycopeptides and an increase in the VRE incidence was identified with average delays between 2 and 6 months [As in a previous surveillance study by Kritsotakis 6 months , we seleP-values of ≤0.05 were considered to be statistically significant. All calculations were performed using R 3.0.2.Model diagnostics revealed that the non-transformed mean usage did not fit the data well due to a high leverage point . Further to control for possible violations of the distribution assumptions, White’s robust covariance estimator was calculated . Z-testsIn the pre-study period from 2004 through 2010, a continuous increase in the number of VRE-positive patients was detected in the hospital, from a single documented case of VRE in 2004 (corresponding to 0.001 patients per 10 000 admissions), 67 cases of VRE in 2007 (corresponding to 0.066 patients per 10 000 admissions) to a peak of 129 VRE-positive patients in 2010 (corresponding to 0.125 patients per 10 000 admissions).E. faecium, n = 258; E. faecalis, n = 3), and five patients were colonized with 2 VRE species . The VanA phenotype was detected in the VRE isolates of 234 (88 %) patients and VanB phenotype in 33 (12 %) patients.During the subsequent two-year study period, a total of 99 635 patients were admitted to the hospital in 2011 and 102 083 in 2012. The overall VRE incidence was 0.131 per 10 000 admissions in the first year and 0.156 per 10 000 admissions in the second year. Among these patients, 261 patients were colonized or infected with a single VRE species , followed by the bone marrow transplantation (BMT) unit (35 patients), general medicine (33 patients), cardiothoracic surgery (27 patients), nephrology (26 patients), haematooncology (22 patients), gastroenterology (17 patients), urology (17 patients), and the infectious diseases unit (11 patients) . Figure p = 0.0018) and teicoplanin separately (p < 0.0001) were both highly significant. Spearman’s correlation coefficient indicated a strong correlation between the two variables (rho = 0.8).Specialty-specific VRE incidence significantly increased with the use of glycopeptides, fluoroquinolones or third generation cephalosporins , management of invasive VRE infections is challenging because of limited treatment options. Furthermore, enterococcal bacteraemia was associated with increased risk of death in allogeneic haematopoietic stem cell transplant recipients [E. faecium [In the present study we identified a positive non-linear relationship between the specialty-specific isolation of VRE and total glycopeptide, fluoroquinolone or third generation cephalosporin consumption. Most patients with VRE-positive cultures were simply colonized, but 6.8 % experienced bloodstream infection caused by vancomycin-resistant cipients . During cipients . In contcipients  but havecipients . In a recipients . But, in faecium . However faecium . PatientVancomycin and teicoplanin are used therapeutically in our hospital to treat invasive infections with gram-positive multi-drug resistant microorganisms or as empirical therapy in severe sepsis or septic shock in intensive care and immunosuppressed patients. During the period 2000 through 2011, vancomycin was used most frequently in our institution as first-line treatment in patients with MRSA bacteraemia, followed by teicoplanin .Clostridium difficile, oral glycopeptides can promote acquisition of gastrointestinal VRE [C. difficile, such as fidaxomicin, decrease the risk of VRE acquisition [Furthermore, in patients with moderate to severe colitis caused by inal VRE , whereasuisition .A limitation of our study was that we could not assess the impact of oral versus intravenous use of glycopeptides on the incidence of VRE separately. During the observation period, only the intravenous formulations of vancomycin and teicoplanin were provided for oral use, because no oral formulations were available.Enterococcus species in the hospital setting, the implementation of a program for prudent use of glycopeptides, fluoroquinolones and third generation cephalosporins is warranted.We demonstrated a positive non-linear relationship between the specialty-specific isolation of VRE and glycopeptide, fluoroquinolone or third generation cephalosporin usage in the University Hospital of Vienna. Although VRE incidence is increasing in our institution, the increase remains moderate, and is probably controlled by infection control measures. Nevertheless, to minimize the spread of glycopeptide-resistant


P < 0.005,  r2 = 0.4958). In contrast, Gremlin expression showed no correlation with tumor subtype or Knosp score. The high level of expression of Gremlin in pituitary adenoma tissue with many CD34-positive vessels and the strong coherence of these regions indicate that Gremlin is associated with angiogenesis in pituitary adenoma cells.Gremlin is an antagonist of bone morphogenetic protein (BMP) and a major driving force in skeletal modeling in the fetal stage. Several recent reports have shown that Gremlin is also involved in angiogenesis of lung cancer and diabetic retinopathy. The purpose of this study was to investigate the role of Gremlin in tumor angiogenesis in pituitary adenoma. Double fluorescence immunohistochemistry of Gremlin and CD34 was performed in pituitary adenoma tissues obtained during transsphenoidal surgery in 45 cases . Gremlin and microvascular density (MVD) were detected by double-immunofluorescence microscopy in CD34-positive vessels from tissue microarray analysis of 60 cases of pituitary adenomas . In tissue microarray analysis, MVD was significantly correlated with an increased Gremlin level (linear regression:  Angiogenesis is a complex multistep process that has a crucial role in tumor growth, invasion, and metastasis. Improved understanding of angiogenesis will provide insights into tumor stage and the response of tumor vessels to antiangiogenic therapy and may lead to more personalized cancer therapy , 2. CurrMicrovascular density (MVD) has been studied in a number of neoplasia and generally there is a close relationship between angiogenesis and tumor progression. Thus, MVD may be a predictive factor for disease progression and response to treatment. Zhang et al. found that cervical cancer progression is correlated with MVD and VEGF , and Zhaβ1 may regulate angiogenesis in pituitary adenomas by initially increasing levels of proangiogenic VEGF-A and then stimulating antiangiogenic molecules in a human pituitary cell line (HP75). Zatelli et al. [VEGF is likely to have a role in tumor angiogenesis in pituitary adenomas that is similar to that in other neoplasms, and VEGF also regulates the growth of pituitary tumor cells through its receptors VEGFR-1, VEGFR-8, and VEGFR-9. Onofri et al.  showed ti et al.  showed ti et al. . A noveli et al. , 14; howi et al. . Thus, iβ and VEGF. Gremlin is expressed in the endothelium and stimulates migration and invasion of endothelial cells in fibrin and collagen gels, binds with high affinity to various endothelial cells, and triggers tyrosine phosphorylation of intracellular signaling proteins [ in vitro and in vivo [Gremlin is an antagonist of bone morphogenic protein (BMP) and is expressed during embryonic development and under different pathologic conditions, including cancer. Gremlin was identified as a novel proangiogenic factor belonging to the cysteine-knot superfamily that includes TGF-proteins . Similar in vivo , 18. Thu in vivo .To the best of our knowledge, expression of Gremlin has not been examined in pituitary adenomas. Therefore, the aim of this study was to investigate the role of Gremlin in tumor angiogenesis in pituitary adenomas. Our results show a close relationship of Gremlin with tumor angiogenesis and proliferation in human pituitary adenoma tissues.n = 2), GHoma (n = 17), NFoma (n = 17), PRLoma (n = 7), and TSHoma (n = 2). No patients with acromegaly received octreotide and none with prolactinoma received preoperative dopamine antagonists.Forty-five pituitary adenoma tissues samples were selected from operative specimens obtained during transsphenoidal surgery in the Department of Neurosurgery at Nippon Medical School from April 2010 to August 2011. The subjects included 28 women (17–76 years old) and 17 men (22–75 years old). Based on previous immunohistochemical staining data, tumors were classified as ACTHoma  and boiled for 5 min in a microwave oven at 600 W to retrieve antigen. Slides were cooled at room temperature for 30 min, rinsed in phosphate-buffered saline (PBS), and blocked by incubation with 1% nonfat milk  for 30 min. Slides were then incubated with mouse anti-human CD34 monoclonal antibody  for 30 min followed by incubation with Texas Red- (TR-) conjugated goat anti-mouse IgG  for 30 min. Thereafter, sections were incubated with rabbit anti-human Gremlin polyclonal IgG antibody  for 60 min followed by incubation with FITC-conjugated bovine anti-rabbit IgG  for 60 min. After counterstaining with Meyer's hematoxylin, each section was mounted with mounting medium . Expression in each section was evaluated in six randomly selected visual fields by fluorescence microscopy  at 40x magnification and analyzed using a computerized image analysis system . Bitmap analysis was used to examine the distribution of the luminance of regions stained in fluorescence immunohistochemistry using Image-Pro Plus, with quantification based on numerical brightness values.All specimens were promptly fixed in 10% buffered formalin, embedded in paraffin, and stored. After characterization for pituitary hormones, 4 n = 5), GHoma (n = 23), NFoma (n = 22), PRLoma (n = 6), and TSHoma (n = 4). Samples were paraffin embedded and used to build tissue microarrays that were analyzed immunohistochemically using a protocol available online (http://genome-www.stanford.edu/TMA/). Tissue microarrays were incubated with rabbit anti-human Gremlin polyclonal antibody (1 : 100 dilution), rabbit anti-β-actin monoclonal antibody , or normal rabbit IgG , followed by incubation with a secondary antibody . Expression was examined by fluorescence microscopy . The fluorescence intensity of the negative control was subtracted and the level of Gremlin relative to β-actin was calculated as a percentage using image analysis software .Pituitary adenoma tissues samples were selected from 60 subjects, including 28 women (17–76 years old) and 32 men (22–75 years old) with ACTHoma (β-actin) in microarray analyses were analyzed by Spearman rank correlation test. Comparisons between microadenomas (<1 cm) and macroadenomas (>1 cm) in preoperative images (MRI enhanced with Gd-DTPA) and tissue microarray analysis were analyzed by Mann-Whitney test. A Kruskal-Wallis test followed by a post hoc Dunn multiple comparison test was used for group comparisons of Gremlin among pituitary adenoma subtypes. The relationship between CD34-positive vessels and Gremlin expression determined by tissue microarray analysis was evaluated using linear regression analysis. All statistical analyses were performed using GraphPad Prism ver. 5.0 . P ≤ 0.05 was considered significant. All data are shown as means ± standard deviation (SD).Relationships between CD34-positive vessels and Gremlin expression in pituitary adenoma tissues were evaluated using quadratic regression analysis. Correlations between the Knosp score, a measure of pituitary tumor invasiveness, and Gremlin expression , the area of the region of overlapping fluorescent probe was calculated by pixel. The rate of colocalization with Gremlin is in CD34-positive cells in the range of 0.169 to 0.998; the average is 0.644 (64.4%) (SEM 0.049) . Gremlinβ-actin as a positive control and normal rabbit IgG as a negative control   . NeitherIn the current study, Gremlin was shown to be expressed abundantly in pituitary adenoma tissues. This expression was significantly related to CD34-positive vessels, but not to the tumor invasion grade or age, sex, or tumor size. These results strongly indicate that Gremlin may regulate tumor angiogenesis.Gremlin is a glycoprotein that is expressed during embryonic development and acts as a BMP agonist by binding to BMPs 2, 4, and 7. Gremlin is expressed in osteoblasts and opposes BMP effects on osteoblastic differentiation and function. Head muscle formation is locally repressed by Wnt and BMP signaling and induced by antagonists of these signaling pathways secreted by adjacent tissues . Gremlinβ and VEGF. Gremlin activates VEGFR2 in endothelial cells, leading to VEGFR2-dependent angiogenic responses with implications in vascular development, angiogenesis-dependent diseases, and tumor neovascularization [Gremlin is also expressed under pathological conditions, including in cancer, and is a proangiogenic protein in the cysteine-knot superfamily that also includes TGF-rization . Gremlinrization .β and is directly correlated with renal dysfunction [Abundant Gremlin expression also occurs in diabetic nephropathy , 31, occfunction . BMP-7 afunction , 34. Dolfunction . This fuβ pathways, all of which have a role in diabetic fibrotic disease. This implies a role for Gremlin in the pathogenesis of diabetic retinopathy [Gremlin expression is also induced in bovine renal pericytes in response to elevated glucose and in the retina of the streptozotocin-induced diabetic mouse. This expression is modulated by hyperglycemic induction of MAPK, reactive oxygen species, and TGF-inopathy . Gremlininopathy . Thus, Ginopathy .β-catenin-independent signaling pathways, and conclusively showed Wnt signaling in estrogen-induced lactotroph proliferation [One of the few studies on Wnt signaling in normal pituitary or adenoma cells revealed a critical function of the Wnt pathway in control of the progenitor/stem cell pool in the pituitary and, in particular, in craniopharyngioma . Giles eferation . A revieferation .In the current study, we found that Gremlin is strongly expressed in pituitary adenoma tissues and that the expression level was significantly associated with CD34-positive vessels. These data suggest the possibility that Gremlin plays an important role by the tumor angiogenesis in pituitary adenomas. Signaling cascades mediated by Gremlin should be further investigated since Gremlin may be a novel candidate for molecular targeted therapy for pituitary adenoma.


ΔSUA) and change in eGFR (ΔeGFR) during the same period. Multivariate regression analysis of ΔeGFR indicated that the increase in SUA over time were a negative predictor of the change in eGFR. Our result indicates that the decline of eGFR over years is larger in subjects with an increased SUA level, which helps to underline the importance of SUA level management in the context of kidney function preservation.Serum uric acid (SUA) level has been proposed to have important connections with chronic kidney disease (CKD), while the impact of SUA level on the natural history of glomerular filtration rate (GFR) decline remains unknown. The present study aims to study the association of the SUA level with the GFR decline in a general population. Two thousand, seven hundred and eighty-nine subjects who visited the Health Checkup Clinic both at 2008 and 2013 were identified. A significant inverse correlation was observed between change in SUA from 2008–2013 ( Uric acid (UA) is a poorly soluble end product of purine nucleotides degradation, and hyperuricemia is defined as serum uric acid (SUA) level >420 umol/L (7 mg/dl) in males and >360 umol/L (>6 mg/dl) in females . Recent 2)  has attracted a lot of nephrologists and there are many studies published. It has been established that in patients with gout and acute UA nephropathy, the treatment of hyperuricemia is of clinical benefits. For patients without gout and acute nephropathy, the first aspect is the relationship between hyperuricemia and CKD progression. Whether SUA is an independent risk factor or only a marker of the progression of CKD is still a matter of debate. In the past, hyperuricemia was thought to be simply a reflection of kidney damage because it was assumed that a decrease in kidney blood flow causes the decrease in UA clearance. However, high uric acid levels was reported to be associated with increased rates of glomerular filtration rate (GFR) decline in multiple cross sectional studies in CKD patients . On the 2) .While the conclusion is still uncertain, most studies were done study the relationship of hyperuricemia and CKD incidence/progression, it also remains unknown whether the SUA level is associated with the natural decline of GFR with aging. It is important to study the strategies that could prevent the incidence of CKD and preserve the GFR in general population. The present study aims to study the association of the SUA level with the GFR decline in a general population who visited the Health Checkup Clinic regularly, and our results suggested a role of SUA level in the GFR decline.The protocol of this study was approved by the Clinical Investigation Ethics Committee of Shandong Provincial Hospital Affiliated Shandong Provincial Hospital (Reference No. 2015-040) and was conducted in strict adherence with the principles of the Declaration of Helsinki.This is a retrospective study. Computer-based data of Health Checkup Clinic, Shandong Provincial Hospital Affiliated to Shandong University  were available from Jan 1st, 2008–Dec 31st, 2013. Subjects who visited the Heath Checkup at 2008 and 2013 were identified using ID numbers, birthdates and other identifiers. All subjects are >18 years old. This study excludes outpatient or clinical patients. Patients with acute kidney injury, amputation, heart failure, severe liver disease, infection disease, malignant disease and pregnancy were excluded. To confirm the accuracy, all the matches were visually inspected. Thus, we identified 2,789 adults, of which 205 subjects were excluded for incomplete records. This left 2,584 subjects  for analysis. All subjects gave the oral consent that the Health Checkup Clinic of Shandong Provincial Hospital could use the anonymous data for research purpose only when they visited at the first time.In 2008 and 2013, all subjects completed a detailed questionnaire administered by trained interviewers on life style factors such as smoking, consumption of alcohol and physical activity. Weight and height were measured with light indoor clothes and without shoes. An automated sphygmomanometer was used to measure systolic and diastolic blood pressures (SBP/DBP) with subjects in the seated position in triplicate. A blood sample was taken after an overnight fast. Biomedical parameters including serum uric acid, serum creatinine (Scr), fasting plasma glucose (FPG) and lipids were measured using a biochemical auto-analyzer in the Clinical Laboratory of the Shandong Provincial Hospital affiliated to Shandong University using standardized procedures.ΔeGFR) were calculated as the eGFR at 2013 minus the eGFR at 2008 (eGFR13 − eGFR08). Hyperuricemia is defined as serum uric acid level >420 umol/L (7 mg/dl) in males and >360 umol/L (>6 mg/dl) females  divided by the square of the height (m). Estimated Glomerular Filtration Rate (eGFR) was calculated using Modification of Diet in Renal Disease (MDRD) formula for Chinese . Accordi females .p < 0.05 were considered to be statistically significant. Kolmogorov-Smirnov test was used for normality distribution. Descriptive statistics are presented as means with standard deviations for normally distributed data, median with interquartile range  for non-normally distributed data, and frequencies with percentage for categorical data. Paired t-test were used for normally distributed continuous variables, while Wilcoxon Signed-Rank test were used for non-normally distributed data in comparison between data in year of 2008 and that in 2013. Univariate and multivariate linear regression analysis was employed to estimate the relationship between SUA level and eGFR change over 5 years.Statistical analysis was conducted by using SAS, version 9.4 (SAS Institute Inc). The graph was drafted using SPSS, version 22. Values of p < 0.01). While the decline of eGFR across the 5-year period was statistically significant –101.19  mL/min/1.73 m2, p = 0.08) but in a very small manner. There were no differences in the BMI, systolic blood pressure (BP). Fasting plasma glucose (FPG), serum total cholesterol (TC) and serum triglycerides (TG) were slightly increased in 2013 compared to 2008, and the diastolic BP was marginally decreased. The ratio of subjects with dipstick-positive proteinuria at 2008 and 2013 was 3.4% and 2.8% respectively.ΔeGFR) . In contrast, a significant inverse correlation was observed between change in SUA from 2008–2013 (ΔSUV) and ΔeGFR during the same period as shown in r = −0.37, p < 0.01).Univariate linear regression analysis did not indicate a significant correlation between baseline SUA at 2008 and the change in eGFR from 2008–2013 . Drinking alcohol (p = 0.95) or smoking (p = 0.98), BMI (p = 0.42), systolic blood pressure (SBP) (p = 0.87), and diastolic blood pressure (DBP) (p = 0.52) were not associated with ΔeGFR.As listed in p-value >0.05). The baseline SUA is lower in the Increased SUA group. Together with SUA level, BMI, DBP, FPG and TG, were lower in the Increased SUA group compared with the other two groups. eGFR of the Increased SUA group was the highest. This indicates that the Increased SUA was at a “healthier” state at 2008. However, after 5 years, the eGFR of the Increased SUA group declined by 5.26  mL/min/1.73 m2, and the eGFR of the Decreased SUA group incremented by 3.23  mL/min/1.73 m2 with a p-value <0.01.Secondary analyses included a trichotomized variable defined by subject’s SUA increase versus decline from year 2008–2013. For a changed SUA level <10 umol/L, either increased or decreased, it is not clinically meaningful since it could be the experiment error. Therefore, we divided the subjects into three groups: subjects with increased SUA (ΔSUA >10 umol/L), those with unchanged SUA (−10 umol/L < ΔSUA < 10 umol/L), and those with decreased SUA (ΔSUA < −10 umol/L). The demographic characteristics of threes groups were shown in p of univariate linear regression analysis <0.1, and second, there were differences among three groups. Together with the trichotomized variable defined by subject’s SUA change, baseline SUA, FPG, TC, TG, and baseline eGFR were fitted in the multiple linear regression model. The collinearity diagnostics show that vif was close to 0, and condition index was >10, indicating that multicollinearity exists. However, there were no closely related variables detected. Using forward stepwise selection with inclusion criteria of p < 0.05 and exclusion criteria of p > 0.10, we get the multivariate regression analysis results as shown in 2 of 0.17.Based on the result of univariate linear regression analysis and the comparison between the three groups, we identified the independent variables that were put into multivariate linear regression model. They meet both criteria, first, The present study demonstrates that the change in SUA during a 5-year period is inversely associated with the decline of eGFR during the same period.Chronic kidney disease (CKD) has become a global public health problem because of the high prevalence and the accompanying increase in the risk of end-stage renal disease (ESRD), cardiovascular disease and premature death . There a2, allopurinol or control was randomly assigned. Those patients with lower serum uric acid levels by the use of allopurinol had an increase in eGFR of 3.3 ± 1.2 mL/min/1.73 m2 per year, whereas eGFR decreased 1.3 ± 0.6 mL/min/1.73 m2 in the control group during a 12-month follow-up (p < 0.04) (2 at 6 months. Placebo group showed a significant decrease of eGFR from 32.6 ± 11.6–28.2 ± 11.5 mL/min/1.73 m2 (p < 0.003)  . In anot< 0.003) . Thus, lIn contrast with studies that were done in patients who already have chronic kidney disease, our results show that in health check-up individuals, the increase in SUA level is associated with a greater decline in GFR in a 5-year period. This is in consistent with previous studies that show lowering uric acid level also could preserve GFR in individuals with normal kidney function. Serum uric acid level >480 umol/L (8 mg/dL) or <300 umol/L (5 mg/dL) was found to be linked with a subsequent increased risk for kidney failure within 2 years (2.9-fold in men and 10.0-fold in women) in a study of 6,400 individuals with normal kidney function . AnotherIn our study, we focused on the relationship between SUA level changes and eGFR changes, while the predictive value of hyperuricemia on the incidence of CKD or Cardiovascular Disease (CVD) was not explored. SUA as a risk factor for the incidence of CVD was clearly shown by two recent meta analysis . In one The mechanisms that uric acid leads to CKD progression are not fully understood. Uric acid crystals are able to adhere to the renal epithelial cells, which could induce an acute inflammatory response . Besides2 from 2008–2013 (There are limitations in our study. The major limitation of the present study is that our study subjects were not randomly sampled. Thus, some selection bias cannot be excluded in the cohort. This might explain that some continuous variables in our study are not normally distributed. Second, there are no information on medications used, such as uricosuric drugs, xanthine oxidase, and anti-hypertension drugs. So there are maybe some other confounding factors that could not be detected by our analysis. Third, some subjects showed increased or decreased eGFR that is greater than 50 mL/min/1.73 m008–2013 , which mIn conclusion, in this retrospective study, we observed that larger decline of eGFR over years is associated with the increased SUA level in general population. Together with more causal mechanisms to be discovered, our findings help to underline the importance of SUA level management in the context of kidney function preservation.10.7717/peerj.1859/supp-1Supplemental Information 1Click here for additional data file.


Salviae Miltiorrhizae Bunge. Previous studies have demonstrated that SAA is a multi-target agent and has various pharmacological activities. The pleiotropic properties of SAA predict its potential in the treatment of NS. The study investigated the effect of SAA on doxorubicin-induced nephropathy. The kidney function related-biochemical changes, hemorheological parameters and oxidative stress status were determined, and histological examination using light and transmission electron microcopies and western blot analysis were also performed. Results revealed that treatment with SAA alleviated histological damages, relieved proteinuria, hypoalbuminemia and hyperlipidemia, reduced oxidative stress, as well as improving hemorheology. Furthermore, SAA restored podocin expression, down-regulated the expression of NF-κB p65 and p-IκBα while up-regulating IκBα protein expression. Overall, as a multifunctional agent, SAA has a favorable renoprotection in doxorubicin-induced nephropathy. The anti-inflammation, antioxidant, amelioration of podocyte injury, improvement of hemorheology and hypolipidemic properties may constituent an important part of its therapeutic effects. All these indicate that SAA is likely to be a promising agent for NS.Nephrotic syndrome (NS) is still a therapeutic challenge. To date there is no ideal treatment. Evidence suggest that multidrug therapy has more effect than monotherapy in amelioration of renal injury. Salvianolic acid A (SAA) is the major active component of  Nephrotic syndrome (NS) is a common and multiple disease in the section of nephrology. Its main clinical symptoms include heavy proteinuria, albuminuria, hypoalbuminemia and hyperlipidaemiaTo date there is no ideal treatment for NS. Hormone and immunosuppressors are the major treatments52The pathogenesis of NS is complex and not entirely clear, which involves multisystem disturbances, namely immunologic, circulatory and metabolic systems. Evidence suggest that oxidative stress, immune injury and inflammation play key roles in the development and progression of NS2710111215Panax Pseudoginseng, Radix Astragali and Salviae Miltiorrhizae Bunge have showed favorable renoprotection1718Most traditional Chinese medicine and natural product both have pleiotropic properties, and may have particular therapeutic advantages in the process of NS treatment. As reported previously, the Chinese herbs Salviae Miltiorrhizae Bunge , a versatile Chinese herbal drug. Several pharmacological studies have demonstrated that SAA is a multi-target agent and possesses a variety of pharmacological activities, such as antiplatelet and anti-thrombosis23Salvianolic acid A (SAA) is one of the major active components of Based on the above description, the pharmacological effects of SAA is closely related to the pathophysiology of NS. The pleiotropic properties of SAA predict its potential in the treatment of NS. Thus, we evaluated the effect of SAA in an established model of doxorubicin-induced nephropathy and initially explored its molecular mechanisms.P = 0.016). Beginning on day 14, the urinary protein of DOX-treated rats rapidly increased (P = 0.001). Treatment with prednisone acetate significantly decreased urinary protein at 2, 3, and 4 weeks . SAA also decreased urinary protein. The effect was significant at doses of 5 and 10 mg/kg at 3 and 4 weeks . SAA 5 mg/kg markedly decreased urinary protein only at 3 weeks (P = 0.000), and had no notable effect at 2 and 4 weeks.As shown in As indicated in P = 0.026 and P = 0.002, respectively), and 10 mg/kg for MDA level (P = 0.009). Prednisone acetate had no notable effect on SOD activity and MDA level   increased SOD activity while decreasing MDA level. The effect was significant at doses of 5 and 10 mg/kg for SOD activity (ctively) .Whole blood viscosity, plasma viscosity and hematocrit considerably elevated in DOX nephropathy rats. SAA substantially improved hemorheologic parameters through decrease of blood viscosity, plasma viscosity and hematocrit .P = 0.000). Fortunately, prednisone acetate and SAA with different doses could attenuate the severity of pathological damages in a certain degree, in the form of reduction of inflammatory cells infiltrations and protein cast formation. The pathological scores were correspondingly decreased, and the effect was significant in the groups of SAA 5 and 10 mg/kg and prednisone acetate .Transmission electron microscopic examination was performed to analyze the ultrastructural changes in renal tissue. There were no apparent damages as evaluated by intactness of glomerulus and tubules in renal tissues of control rats. The foot processes were clearly observed, and there was no swelling and hypertrophy. In DOX-treated rats, the basal structure of glomerulus was damaged, which manifested as extensive fusion and effacement of foot processes and mild thickening of glomerular basement membrane (GBM). In renal tissues of rats treated with prednisone acetate and SAA, only focal segmental podocyte foot process fusion and effacement were observed. These results suggested that SAA could protect podocyte from damage caused by DOX .P = 0.046).As shown in In the current study, we used a single doxorubicin injection method to successfully induce an experimental nephrotic syndrome model, which is characterized by large proteinuria, hyperlipidemia and hypoalbuminemia, increased levels of BUN and SCr , associated with hyperviscous status and increased oxidative stress. In addition, extensive effacement of podocyte foot processes were also observed in nephrotic rats. But all the features of nephropathy could be lightened by administration of SAA as evidenced by reduction of proteinuria, hypoalbuminemia and glomerular damage, amelioration of renal damage, and improvement of lipid metabolism and hemorheology, as well as reduction of oxidative stress. These results confirm that SAA has a beneficial effect on DOX nephropathy. Furthermore, the effect of SAA appeared to be superior to that of prednisone acetate in terms of lipid-lowering and improvement of hemorheology.Venous and arterial thromboembolism are considered to be the most severe complications of NS, which affect the treatment and outcome of NS. Hypercoagulability status was considered to contribute to this thrombophilic phenomenon27281719Oxidative stress is one of the significant mechanisms in the pathogenesis of renal injury, which is produced by excessive generation of reactive oxygen species (ROS) or inefficient antioxidant defenses7732333637The beneficial effect of SAA was further illustrated by histological evaluation using light and transmission electron microcopies. Histopathologic examination showed that glomerular epithelial hyperplasia, tubular dilatation, abundant protein exudation in renal tubular lumen and numerous inflammatory cells infiltrations were present in renal tissue of nephrotic rats. In addition, extensive fusion and effacement of podocyte foot processes could be also observed. Treatment with SAA decreased the severity of pathologic damage, reduced extent of podocyte foot effacement. It is known that podocytes are critical in maintaining the normal filtration barrier of glomerulus and podocyte slit diaphragm (SD) plays a major role within it. Nephrin and podocin are protein components of SD, involving in regulating normal renal function14As noted earlier, NF-κB as a transcription factor plays a central role in the regulation of a large number of genes involved in the progression of renal disease. NF-κB is activated in podocytes and persistent proteinuria and peroxides are stimulants of NF-κB. The activation of NF-κB is regulated by IκB protein and IκB kinases (IKKs). When inactive, the NF-κB dimer associates with IκB proteins, the best-studied and most important of which is IκBα913In conclusion, our results suggest that SAA has multiple therapeutic activities and can partially protect against DOX-induced nephropathy in rats. The anti-inflammation, antioxidant, inhibition of podocyte injury, improvement of hemorheology and hypolipidemic properties may constituent an important part of its therapeutic effects. The molecular mechanism may involve the inhibition of NF-κB activation and regulation of podocin expression. These novel findings provide the pharmacological foundation for the further development of SAA for the treatment of NS.Male Sprague-Dawley (SD) rats  were purchased from Vital River Laboratory Animal Technology Co., Ltd. (certificate No.11400700039133). All animals were allowed to acclimate for at least 1 week at a temperature of 24 ± 1 °C and humidity of 55% ± 5%. All rats were housed in cages with food and tap water ad libitum. All experiments were performed in accordance with relevant guidelines and regulations approved by the Experimental Animal Research Committee of Yantai University.SAA was provided by Shandong Target Drug Research Co. Ltd . Prednisone acetate was the product of Zhejiang Xianju Pharmaceutical Co. Ltd. . Doxorubicin hydrochloride for injection was produced by Shenzhen Main Luck Pharmaceuticals Inc. . The antibodies used in this study were anti-NF-κB p65 , anti-inhibitor of NF-κB (IκB) α , anti-phosphorylation-IκBα  and anti-podocin . Goat anti-rabbit IgG was also the product of Abcam. BCA protein assay kit, anti-β-actin antibody, HRP-labeled goat anti-mouse IgG (H+L) and lipid peroxidation malonaldehyde (MDA) assay kit were obtained from Beyotime Institute of Biotechnology . Superoxide dismutase (SOD) detection kit was purchased from Nanjing Jiancheng Bioengineering Institute .Thirty male Sprague-Dawley (SD) rats were randomly divided into six groups of five animals each: control, doxorubicin (DOX) alone, DOX plus prednisone acetate , DOX plus SAA . Doxorubicin (7.5 mg/kg) were given intravenously once to rats to induced kidney injury as previously describedOn days 3, 7, 14, 21 and 28 after DOX injection, rats in each group were put into metabolic cages, and 24-hour urine samples were collected for measurement of urinary protein. On days 29, these rats were sacrificed with intraperitoneal injection of chloral hydrate (350 mg/kg). Blood were collected from aorta abdominalis, and four milliliters of blood were anti-coagulated with heparin. Hemorheological parameters of hematocrit (Hct), plasma viscosity (PV) and whole blood viscosity (WBC) including high shear rate, middle shear rate and low shear rate were determined by routine laboratory assays. The rest of the blood were centrifuged for 15 min at 3000 rpm. The serum was isolated and the levels of serum albumin (ALB), total cholesterol (TC), triglyceride (TG), serum creatinine (Scr) and blood urea nitrogen (BUN) were measured by Automatic Biochemical Analyzer. Twenty-four hours of urinary protein excretion and serum total protein were measured by BCA protein assay kit.Before collection of blood, the two kidneys were rapidly removed. The left kidney was cut into two parts. One part of the kidney was fixed in 10% formalin, embedded in paraffin and stained with hematoxylin and eosin (H and E), and assessed under light microscopy. Sections were examined by a qualified and blinded pathologist to evaluate the degree of pathological changes based on glomerular epithelial hyperplasia, tubular dilatation and protein cast formation, and inflammatory cells infiltrations. These were scored from 0 to 3 . The total score for each renal sample was calculated. Another part of the kidney was used for electron microscopic examination. Small blocks of kidneys were fixed in 3% glutaraldehyde, postfixed in 2% osmium tetroxide, dehydrated in graded ethanol and acetone, and then embedded in epoxy resin. Ultrathin section were stained with lead citrate and uranyl acetate and examined using an electron microscopy . The renal cortex from right kidney was stored at −80 °C and thawed just before use.The renal cortex was homogenized on ice in PBS buffer. The supernatants were prepared by centrifugation at 12000 rpm for 10 min and collected, and then total protein concentration was measured using a BCA kit. The supernatants were then used for the determination of activity of SOD and MDA level. The process of assay was performed according to the manufacturers’ instructions. The final results were expressed as U/mg protein for SOD activity and μmol/mg protein for MDA level.Nuclear and cytoplasmic extracts were prepared using a nuclear and cytoplasmic protein extraction kit . Briefly, renal cortex (approximately 80 mg) were homogenized in cytoplasmic extraction buffer. Tissue homogenates were rapidly lysed by vortexing. The homogenate was centrifuged at 12000 rpm for 10 min at 4 °C and proteins were collected from the supernatant. The pellets were re-suspended in 50 μL of nuclear protein extraction buffer, lysed by ultrasound in an ice water bath, and then centrifuged to yield the nuclear fraction. Protein concentration was measured with a BCA protein assay kit. Protein samples (80–100 μg) were separated using 10% SDS–PAGE gels and transferred onto PVDF membranes. The membranes were blocked with 5% skim milk in Tris-buffered saline with Tween 20 for 2 h at room temperature and then incubated with primary antibodies to anti-podocin (1:200 dilution), anti-NF-κB p65, anti-IκBα, or anti-p-IκBα (1:4000–1:10000 dilution) overnight at 4 °C. Then, the membrane was washed 4 times for 10 min each and incubated with a horseradish peroxidase-conjugated anti-rabbit IgG antibody. Protein bands were detected using an enhanced chemiluminescence detection kit (Beyotime Institute of Biotechnology) and visualized by exposure to photographic film. The protein bands were visualized by exposure to photographic film. Densitometric analysis of protein bands was performed using Quantity One v4.4.0.36 analyzer software (BIO-RAD).U test. P < 0.05 was considered significant.Statistical analyses were performed using SPSS software 17.0 for Windows. All results are expressed as mean ± standard deviation (SD). Quantitative data were tested for homogeneity of variance. If the variance was homogeneous, one-way ANOVA followed by the least significant difference (LSD) test was used. Comparisons between groups of nonparametric data were made using the Kruskal-Wallis test followed by the Mann-Whitney How to cite this article: Fan, H.-Y. et al. Salvianolic acid A as a multifunctional agent ameliorates doxorubicin-induced nephropathy in rats. Sci. Rep.5, 12273; doi: 10.1038/srep12273 (2015).


Objective. To investigate the association of thyroid function and diabetic nephropathy (DN) in euthyroid patients with type 2 diabetes. Methods. A total of 421 patients were included in this cross-sectional study. The following parameters were assessed: anthropometric measurements, fast plasma glucose, serum creatinine, lipid profile, HbA1c, free triiodothyronine (FT3), free thyroxine, thyroid-stimulating hormone levels, and urinary albumin-to-creatinine ratio (UACR). Patients with UACR of ≥30 mg/g were defined as those suffering from DN. Results. Of the 421 patients, 203 (48.2%) suffered from DN, and no difference was found between males and females. The patients with DN yielded significantly lower FT3 levels than those without DN (P < 0.01). The prevalence of DN showed a significantly decreasing trend across the three tertiles based on FT3 levels . After adjustment for gender and age, FT3 levels were found to correlate positively with estimated glomerular filtration rate (P = 0.03) and negatively with UACR (P < 0.01). Multiple linear regression analysis showed that FT3 level was independently associated with UACR . Conclusion. Serum FT3 levels are inversely associated with DN in euthyroid patients with type 2 diabetes, independent of traditional risk factors.  Diabetic nephropathy (DN) is one of the most common microvascular complications of diabetes mellitus and leading causes of end-stage renal disease worldwide and accounts for a significant increase in morbidity and mortality in diabetic patients. Although DN is alleviated through conventional treatments, such as strict glycemic control, limited protein intake, blood pressure control, and renin-angiotensin-aldosterone system inhibition, these treatments cannot completely prevent the progression of DN in diabetic patients .Several clinical studies show that thyroid dysfunction is related to renal disease. Overt and subclinical hypothyroidism are associated with a low estimated glomerular filtration rate (eGFR) and an increased risk of chronic kidney disease (CKD) –5. ThesePrevious studies have shown a close interrelationship between thyroid hormone and DN. Type 2 diabetic patients with subclinical hypothyroidism (SCH) have been found to be associated with the high prevalence of DN in several studies , 11. In A total of 632 patients with type 2 diabetes treated at the Shanghai Jiao Tong University Affiliated Shanghai First People's Hospital from 2009 to 2012 were enrolled in this study. The inclusion criteria were patients with normal thyroid function  and negative for thyroid autoantibodies, such as thyroid peroxidase antibody (TPOAb) and thyrotropin receptor antibody (TRAb). The following exclusion criteria were considered: current malignancy, pregnancy, acute intercurrent illness, nondiabetic renal problems, chronic liver disease, urinary tract infection, history of thyroid disease, or any thyroid medication (levothyroxine or antithyroid drugs). A total of 421 patients  were included in the final analysis. The study protocol was approved by the Ethics Committee of the Shanghai First People's Hospital.Patient data, including demographic characteristics, lifestyle habits (smoking and drinking), medical history, and medication use, were obtained via a standard questionnaire. Smoking habit was defined as the consumption of more than five cigarettes daily for at least one year. Height and body weight were measured using a digital scale, and body mass index (BMI) was calculated by dividing body weight (in kilograms) by height (in square meters). Blood pressure (BP) was measured twice using a mercury sphygmomanometer, with participants in a seated position after 5 min of rest. Two BP readings were obtained 1 min apart, and the mean was calculated. Hypertension was defined as systolic blood pressure (SBP) of ≥140 mmHg and/or diastolic blood pressure (DBP) of ≥90 mmHg, or treatment with antihypertensive drugs.2) = 186 × (Scr/88.4)−1.154 × (age)−0.203 ×  [Blood samples were collected after the patients fasted overnight. Biochemical parameters, including the levels of fasting plasma glucose (FPG), serum creatinine (SCr), serum total cholesterol (TC), serum triglyceride (TG), serum high-density lipoprotein cholesterol (HDL-C), and serum low-density lipoprotein cholesterol (LDL-C), were measured via routine laboratory methods by using a HITACHI 7600 instrument . HbA1c was detected through high-performance liquid chromatography . eGFR was calculated using the equation of the Modification of Diet in Renal Disease: eGFR  .Urinary albumin-to-creatinine ratio (UACR) was evaluated by obtaining the average of three UACR measurements. Before examination was conducted, the patients were instructed to avoid exercise for 1 h. Urinary albumin concentration was measured using immunoturbidimetry , and urinary creatinine concentration was determined by a modified Jaffe method on an automatic analyzer . UACR was calculated as urinary albumin concentration divided by creatinine concentration and expressed in mg/g. DN was defined by an increased UACR of ≥30 mg/g in the absence of urinary tract infection or other renal abnormalities.All of the patients were assessed by an experienced ophthalmologist using a direct ophthalmoscope and a digital retinal camera . Diabetic retinopathy (DR) was diagnosed according to the diagnostic code of the American Academy of Ophthalmology. The minimum criterion for the DR diagnosis was the presence of at least one microaneurysm in any photographed field .Serums FT3, FT4, TSH, TPOAb, and TRAb levels were measured using an electrochemiluminescence analyzer  with a mating reagent .t-test or Mann-Whitney U test, and categorical data were compared via chi-square test. Non-normally distributed variables, such as HbA1c, TG, TSH, and UACR, were natural log-transformed or arctan-transformed into approximately normal distributed data before correlation and regression analysis were conducted. The correlation between serum thyroid hormone levels and related clinical variables was determined through partial correlation analysis. The independent determinants of UACR were identified through multiple linear regression analysis. A two-tailed P value of <0.05 was considered statistically significant. Data were analyzed using SPSS version 16.0 for Windows .Continuous variables were expressed as mean ± standard deviation or median (interquartile range), and categorical variables were expressed as percentages. Continuous data were compared via Student's P > 0.05). The comparison results of the clinical characteristics between patients with and without DN are shown in Among the 421 patients, 203 (48.2%) were diagnosed with DN, and no difference was found between males and females . The first FT3 quartile group showed a significantly higher prevalence of DN than the second and third groups , FT4 , or TSH . The prevalence of DN showed a significantly decreasing trend across the three tertiles based on FT3 levels ; by contrast, FT3 levels were correlated negatively with FPG, HbA1c, and UACR . FT4 levels were also correlated negatively with HbA1c (P = 0.03). TSH levels were correlated positively with BMI and TG (P < 0.01 for both); conversely, TSH levels were correlated negatively with diabetic duration and HDL-C (P < 0.01 for both)  .β = −0.18, t = −3.70, and P < 0.01). Other risk factors included diabetic duration, hypertension, DBP, presence or absence of DR, and FPG and eGFR levels  expression [κB and JNK pathways are also involved in the development of DN [The mechanism of the association between FT3 and DN can be explained by the following. First, thyroid hormones elicit crucial effects on vascular and endothelial functions. Patients with SCH experience endothelial dysfunction characterized by a reduction in nitric oxide (NO) availability; this alteration is partially independent of dyslipidemia and can be reversed through levothyroxine supplementation . Völzke gression . Third, pression . NF-κB ant of DN . a relevant parameter in assessing the thyroid function in diabetes.Interestingly, our study fails to show a significant association between TSH levels and albuminuria in diabetic patients, which is different from the results of previous studies. The possible explanations are as follows: Firstly, patients with positive TPOAb are not excluded from most previous studies. In general, patients with positive TPOAb have higher TSH levels than those without, even in the euthyroid state. Moreover, TPOAb is also found to be associated with renal disease. Xiang et al.  reportedβ-cell growth and proliferation in human and rodent insulinoma cell lines [Similar to other studies , 36, thell lines . A clinill lines . These oAs a limitation, a cross-sectional design was implemented in our study. This cross-sectional study investigated patients with type 2 diabetes treated with various medications. Further prospective and longitudinal studies should be conducted to confirm a causal relationship between FT3 levels and DN in diabetic patients, who are stratified with and without medications.In summary, our study suggests that serum FT3 levels are inversely associated with DN in euthyroid patients with type 2 diabetes, which is independent of other risk factors. The results of our study may help predict the risk of DN development. Our study may also improve our understanding of this disease. Furthermore, our study can be used as a basis to establish effective prevention strategies.