Cytogenetic aberrations and gene mutations have long been regarded as independent prognostic markers in AML, both of which can lead to misexpression of some key genes related to hematopoiesis. It is believed that the expression level of the key genes is associated with the treatment outcome of AML.CEBPA, NPM1, FLT3, C-KIT, NRAS, WT1, DNMT3A, MLL-PTD and IDH1/2, as well as expression levels of MECOM, ERG, GATA2, WT1, BAALC, MEIS1 and SPI1.In this study, we analyzed the clinical features and molecular aberrations of 560 newly diagnosed non-M3 AML patients, including mutational status of MECOM, MEIS1 and BAALC. FLT3, C-KIT and NRAS mutations contained conversed expression profile regarding MEIS1, WT1, GATA2 and BAALC expression, respectively. FLT3, DNMT3A, NPM1 and biallelic CEBPA represented the mutations associated with the prognosis of AML in our group. Higher MECOM and MEIS1 gene expression levels showed a significant impact on complete remission (CR) rate, disease free survival (DFS) and overall survival (OS) both in univariate and multivariate analysis, respectively; and an additive effect could be observed. By systematically integrating gene mutational status results and gene expression profile, we could establish a more refined system to precisely subdivide AML patients into distinct prognostic groups.Certain gene expression levels were associated with the cytogenetic aberration of the disease, especially for Gene expression abnormalities contained important biological and clinical informations, and could be integrated into current AML stratification system.The online version of this article (doi:10.1186/s12967-017-1279-4) contains supplementary material, which is available to authorized users. C-KIT, FLT3 and NRAS; and Class II, mutations affecting transcription factors (TF) or co-factors and causing impaired differentiation such as point mutation of CEBPA, AML1 and gene fusion of AML1-ETO [DNMT3A, IDH1, IDH2, and TET2, which shared a common feature of aggressive diseases, old age and poor prognosis [NPM1, activated signaling, myeloid TFs, TF fusions, DNA methylation, chromatin modifier, tumor suppressors, cohesin complex, and spliceosome. However, their function and potential clinical translation in guiding treatment and judging prognosis should be further confirmed in future clinical trials [Acute myeloid leukemia (AML) is a group of hematological malignancies, arising from stem cells, whose leukemogenesis and clinical behavior was deeply affected by the underlying cytogenetic and molecular abnormalities –3. ClassAML1-ETO . In our rognosis . With thl trials .FLT3 and CEBPA mutations being the most common gene mutations in Western and Chinese populations respectively; represent poor and favorable indicators in AML  [MLL, DNMT3A, TET2, and ASXL1 mutations [NPM1 is regarded as a favorable one [TP53 and WT1 in cytogenetic normal AML (CN-AML) [C-KIT in core binding factor AML (CBF-AML) [TP53 mutations, biallelic CEBPA mutations, MLL fusion gene, GATA2, MECOM, IDH2, and t/DEK-NUP214, with each of the subtypes presenting distinct clinical behaviors [Until now, the roles of a sizable portion of gene mutations are addressed and integrated in clinical practice. For instance, r CEBPA) , 14–16. utations , 17, 18,able one , 19, 20.(CN-AML) –24 and CCBF-AML) . MetalloCBF-AML) . Similarehaviors .MECOM , BAALC, ERG and WT1. A remote GATA2 hematopoietic enhancer alteration in inv(3) by activating EVI1 expression was reported [BAALC and ERG [MEIS1, which is up-regulated by MLL abnormalities, still needs to be addressed in AML [How these gene mutations are involved in the leukemogenesis needs further investigation. It is believed that normal hematopoiesis and cellular differentiation is highly dependent on the transcriptional regulation systems. The expression of lineage-determining transcription factor is in strict time order. Gene alterations, including fusions and mutations, could lead to the abnormal expression of key genes, and these kinds of misexpression disrupt the TF-dependent genetic network. In recent years, the expression level of several genes became research interest, exemplified as reported . It is breported –33; howe and ERG , 35. Thed in AML , 37.Hence, we performed this study to systemically investigate the role of a series of gene expression in AML, including previously known ones and newly established ones. Moreover, we intent to integrate these new markers into current established gene mutation profile to provide a more precise stratification of AML.The newly diagnosed non-M3 patients were selected from Shanghai Institute of Hematology (SIH). Patients with leukemia either transformed from myelodysplasia syndrome (MDS) or secondary to other malignancies were excluded from this study. Cytogenetic analysis was performed centrally in SIH in every patient. The bone marrow (BM) samples of de novo AML patients were studied mostly by R- and/or G-banding analysis, and were confirmed in most cases with relevant molecular markers .This study was approved by the ethic board of Ruijin hospital. All patients had given informed consent for both treatment and cryopreservation of BM and peripheral blood (PB) according to the Declaration of Helsinki.2, D1-3; and Ara-C 100 mg/m2, D1-7. In the consolidation therapy, they were treated with high-dose cytarabine based chemotherapy for 4 cycles. For old patients , the treatment was mainly decided by the physician: fit patients underwent a regimen similar to younger patients, but with a reduced consolidation cycles of high dose Ara-C to 2 cycles; unfit patients, underwent either low dose treatment, demethylation treatment or palliative care [Younger AML patients (age ≤ 60) received standard first line treatment of DA like regimen, which consisted of daunorubicin 45–60 mg/mive care .WT1 [ABL1 control gene [MECOM, ERG, GATA2, BAALC, MEIS1 and SPI1 expression levels were quantified by using the TaqMan Gene Expression Assay assays, according to the manufacturer’s instructions . These seven genes’ transcripts were normalized to ABL1 by using the respective plasmid standards to generate normalized copy numbers. Reactions were performed using ABI ViiA™7 . Each sample was analyzed in duplicate. Data were reported using a common threshold of 0.1. Positive and negative controls were included in all assays.Gene mutations/fusions were detected as previously reported . The WT1WT1  and ABL1rol gene  RQ-PCR aFisher’s exact P test was used to compare the gene expression levels in different subgroups, as well as the difference of CR rates. One way Anova test was used to compare the clinical features such as age and WBC count in different groups. OS was measured from the date of disease diagnosis to death (failure) or alive at last follow-up (censored). DFS was defined as the duration from the documentation of CR to treatment failure such as relapse, refractory disease, death, or alive in CR at last follow-up (censored). Kaplan–Meier analysis was used to calculate the distribution of OS and DFS. Hazard ratio analysis was performed to compare the difference of survivals. Binary logistic regression and COX model was used for the multivariate analysis of associations between mutational status and the achievement of CR and OS and DFS, respectively. A limited backward selection procedure was used to exclude redundant variates. All above statistical procedures were performed with the SPSS statistical software package, version 16.0.MECOM, ERG, WT1, GATA2, BAALC, MEIS1 and SPI1 in bone marrow (BM) of de novo AML patients were shown in Additional file Gene expression levels of WT1, BAALC and ERG into high and low group by their median values in patients according to the previous reports [MECOM, MEIS1, GATA2 and SPI1 were also chosen at their median values. Survival analysis showed that median value as cut-off value could separate patients with different prognosis in MECOM, MEISI and SPI1 expression group , while using cut-off value at quartile 1 (Q1) or Q3 would lose the power to separate the patients  and 86 elderly patients (age > 60 years old). The patients were classified into 3 groups: Group 1, Core Binding Factor AML (CBF-AML), which includes 89 patients, Group 2, Cytogenetic intermediate risk AML, which includes 401 patients with normal karyotype  or cytogenetic aberration without prognostic significance, and Group 3, Cytogenetic high risk patients (55 patients) group. Among group 2, young patients with normal karyotype (265 patients) or insignificant cytogenetic aberration accounted for 84% (336 patients), while elderly patients accounted for 16% (65 patients) including 55 CN-AML patients. Cytogenetic results were failed or unavailable in 15 patients. The distribution of the patients in FAB classification and cytogenetic abnormalities of enrolled patients was listed in Additional file MEISI expression had the higher median age (P = 0.026). Similarly, in old patients (age > 60), more patients harbored higher MEIS1 and SPI1 expression levels . Female patients tended to have higher expression of MECOM, MEIS1 and WT1 . The high SPI1 expression group manifested the feature of high WBC count at disease presenting (P = 0.003). The detailed clinical features were shown in Table Age was strongly associated with several gene expression levels. The patients with higher MECOM, MEIS1, SPI1, ERG, WT1, GATA2, and BAALC in different cytogenetic risk groups. In CBF-AML, MEIS1 and WT1 (both P < 0.001) were lower in CBFα-AML ), but higher in CBFβ-AML (inv(16)) ; while BAALC was higher in both groups (both P < 0.001). And SPI1 was high in CBFβ-AML (P < 0.001) as previously reported [MECOM and BAALC were lower (both P < 0.001), while GATA2 was higher (P = 0.014). Particularly, MECOM tended to have a higher level in poor risk factors both in 11q23 rearrangement (P = 0.001) and others (P = 0.015). 11q23 abnormalities were associated with higher expressions of MECOM (P = 0.001), MEIS1 (P = 0.003), SPI1 (P = 0.031), WT1 (P = 0.032), and interestingly, a lower BAALC expression (P = 0.003) in our group. Figure There was a difference of gene expression levels including those of reported . In CN-AFLT3-ITD/TKD mutations, 45 (8.0%) were with NRAS mutations, 59 (10.5%) were with C-KIT mutations, 100 (17.9%) were with NPM1 mutations, 38 (6.8%) were with WT1 mutations, 118 (21.1%) were with CEBPA mutations, 62 (11.1%) were with DNMT3A mutations, 79 (14.1%) were with IDH1/2 mutations, and 27 (4.8%) were with MLL-PTD mutations, respectively. The distribution of gene mutations was accordant to previous reports. The detailed distribution of gene mutations in different cytogenetic risk group was shown in Additional file Out of 560 AML patients, 116 (20.7%) patients were with FLT3 mutations were associated with high MEIS1 (P < 0.001), WT1 (P < 0.001), and GATA2 (P = 0.004) and lower BAALC (P = 0.007) expressions, respectively; while C-KIT was associated with lower MEIS1 (P < 0.001), WT1 (P < 0.001) and GATA2 (P = 0.008) and higher BAALC (P < 0.001) expressions, respectively. At the same time, another class I mutation, NRAS was not associated with any gene expression level. The mutual exclusion of Class II mutations was also reflected in gene expression level. CEBPA was associated with lower MECOM (P < 0.001), MEISI (P < 0.001), SPI1 (P < 0.001), and WT1 (P < 0.001) expressions, respectively. NPM1 was related with lower ERG (P < 0.001) and BAALC (P < 0.001), but higher MEIS1 (P < 0.001), WT1 (P < 0.001) and GATA2 (P = 0.001) expression levels, respectively. Other potential mutual relationship with gene expression level was also observed in epigenetic modifier gene mutations, such as DNMT3A and IDH1/2, respectively. The detailed data were shown in Fig. The potential relationship of mutual co-existence and co-exclusion was observed between gene mutation group and investigated gene expression level. FLT3 (P = 0.036) and biallelic CEBPA (P = 0.009) mutations were associated with lower and higher CR rate, respectively. In different gene expression groups, higher MECOM, MEIS1 and SPI1 expression was associated with a lower CR rate , while other factors were not associated with the induction outcome of the cytogenetic intermediate risk patients  and high MECOM and MEIS1 expression , respectively. A multivariate analysis was also performed in cytogenetic intermediate risk group AML patients. The results were similar in terms of gene expression level . In our group, the median OS and DFS were observed at 17 ± 2.3 and 27.5 ± 4.2 months (24 ± 3.1 and 30 ± 5.0 months for young patients), respectively , respectively).CEBPA mutations were associated with favorable OS  and DFS , respectively, as well as NPM1-mut/DNMT3A-wt , which was accordant to previous study. In contrast, FLT3-ITD/TKD and DNMT3A mutations were associated with poor prognosis , high MEIS1  and high SPI1  were associated with the survival. The two parameters, MECOM and MEIS1, even had an additive effect, high MECOM and high MEIS1 group showed worst prognosis, low MECOM and low MEIS1 group presented most favorable treatment outcome, while either high MECOM or MEIS1 group was in the middle  and DFS .Similarly, according to gene expression level, in univariate analysis, high CEBPA mutations; (2) intermediate risk I: low MECOM and MEIS1 expression without biallelic CEBPA mutations or NPM1-mut/DNMT3A-wt; (3) intermediate risk II: others; (4) high risk: FLT3-ITD/TKD with the absence of NPM1-mut/DNMT3A-wt or DNMT3A mutations or high MECOM and MEIS1 expression  low risk: biallelic FLT3 mutations (for both OS and DFS), biallelic CEBPA mutations (for OS), NPM1-mut/DNMT3A-wt (for OS), as well as high MECOM (both for OS and DFS) and high MEIS1 (for OS) expressions were independent prognostic factors for AML (q21q26.2), which has lower incidence in AML [MLL-AF9 pediatric AML, which was associated with poor prognosis [MLL rearrangement in another Japanese series with 130 pediatric AML patients [MECOM, several groups use WT1 expression level to monitor the minimal residual diseases (MRD) in BM and PB in AML, whose increasing strongly indicates poor prognosis and relapse [MECOM and WT1 over-expression are poor indicators, while clinical value of BAALC and ERG are controversial. Some genes of myeloid transcriptional factors are also drawn of attraction, such as GATA2 and SPI1; especially for MEIS1, which is proved to be regulated by MLL mutation in previous reports [MECOM, WT1, ERG, BAALC, and 3 new ones with potential value, GATA2, SPI1 and MEIS1, to try to address this question.However, in addition to gene mutations, some gene expression levels, which are caused by the regulation of a certain gene, or even several genes, are also involved in leukemogenesis. Among them, e in AML , 49. Therognosis ; as wellpatients . In addi relapse . When co reports . AlthougMECOM expression was identified to be low in CN-AML group, but high in 11q23 aberration group. MEIS1 expression was low in t group, but high in the group with poor cytogenetic makers. High BAALC expression was associated with CBF-AML, but less distributed in CN- and 11q23 AML. Moreover, gene mutational status was also associated with the gene expression level. FLT3 was associated with a high MEIS1, WT1, and GATA2 and lower BAALC expression, respectively; while C-KIT was associated with a lower MEIS1, lower WT1 and GATA2 and higher BAALC expression, respectively. And interestingly, NRAS was not associated with any gene expression level. Above results seemed to be mutually exclusive. CEBPA, as the most common mutation type in Chinese population, seemed to have favorable gene expression profile: lower MECOM (P < 0.001), MEISI (P < 0.001), SPI1 (P < 0.001), and WT1 (P < 0.001) expression, respectively. Mutant NPM1 was related with lower ERG (P < 0.001), BAALC (P < 0.001), but higher MEIS1 (P < 0.001), WT1 (P < 0.001) and GATA2 (P = 0.001), respectively. As we know, FLT3, C-KIT, CEBPA and NPM1 mutations were considered to have prognostic value in predicting the prognosis of AML, and their related gene expression profile might be the reason. We believe that certain mutations elicit numerous expressional changes in other genes which are associated with leukemogenesis, leading to different clinical behaviors of the diseases. Such kind of mutual relationship with gene expression level was also observed in epigenetic modifier gene mutations.Firstly, we identified that certain gene expression levels were associated with the cytogenetic aberrations of the disease. In this study, FLT3 and higher MECOM, MEIS1 and SPI1 expressions were associated with a lower CR rate , while CEBPA (P = 0.009) mutations were associated with a higher CR rate. Higher MECOM and MEIS1 expressions remained significant in multivariate analysis, while NPM1-mut/DNMT3A-wt appeared to be an independent factor (P = 0.003). In survival, biallelic CEBPA mutations and NPM1-mut/DNMT3A-wt were associated with favorable OS  and DFS , respective, while FLT3-ITD/TKD  and DNMT3A  were associated with poor prognosis. As for gene expression profile, high MECOM  and high MEIS1  levels were associated with the survival. An additive effect could be observed when we combined the two gene expression levels together, high MECOM and high MEIS1 group showed worst prognosis, low MECOM and low MEIS1 group presented most favorable treatment outcome. Furthermore, WT1 could help to separate the low risk group of low MECOM and MEIS1 group into a more refined subgroup, the high WT1/low MECOM and MEIS1 expression was associated with relative poor OS  and DFS .In treatment outcome analysis, univariate analysis showed that DNMT3A, MLL, NPM1, CEBPA, FLT3 mutations etc., and more importantly gene expression profile, especially for MECOM, MEIS1 and WT1, one could discriminate the AML patients with different clinical behaviors.Finally, we have established a new system to stratify the AML integrating cytogenetic risk, gene mutational status and gene expression profile. Through examination of traditional cytogenetic markers, gene mutations, exemplified as Gene expression aberrations are associated with the cytogenetic abnormalities and gene mutations in AML, as well as the clinical behavior of the patients. Of note, their value in predicting the prognosis of AML was demonstrated in this study.Additional file 1: Figure S1. Gene expression of MECOM, ERG, WT1, GATA2, BAALC, MEIS1 and SPI1 in bone marrow (BM) of de novo AML patients was normalized to ABL expression. Each median value after a log-transformation is indicated by a horizontal line.Additional file 2: Figure S2. Kaplan–Meier survival analysis for overall survival (OS) using different cut-offs of relative gene expression. Log-rank P value was portrayed for each survival analysis.Additional file 3: Table S1. FAB subgroups and cytogenetic abnormalities in AML patients.Additional file 4: Table S2. The distribution of gene mutations in different cytogenetic risk groups.Additional file 5: Table S3. The relationship of gene mutational status and gene expression level.Additional file 6: Table S4. The relationship of gene mutational status and gene expression level (intermediate risk).Additional file 7: Table S5. Multivariate analysis of intermediate risk group.Additional file 8: Table S6. CR rate of different gene mutation and expression groups in intermediate risk group (young AML patients).Additional file 9: Table S7. Multivariate analysis of prognostic value of young AML patients.Additional file 10: Table S8. Multivariate analysis of intermediate risk group (young AML patients).Additional file 11: Figure S3. Kaplan–Meier curves for OS and DFS according to genotypes.Additional file 12: Figure S4. Kaplan–Meier curves of young AML patients for OS and DFS according to genotypes.Additional file 13: Figure S5. Comparison of OS and DFS between young AML patients with low or high gene expression with statistical significance in univariate analysis.Additional file 14: Figure S6. Risk stratification of young AML according to gene mutations and gene expression levels.


Background/Aims. Subclinical atherosclerosis and long-term glycemic variability have been reported to predict incident chronic kidney disease (CKD) in the general population. However, these associations have not been investigated in patients with type 2 diabetes with preserved kidney function. Methods. We prospectively followed up 162 patients with type 2 diabetes  and assessed whether carotid intima-media thickness (IMT) measured by B-mode ultrasound and visit-to-visit HbA1c variability are associated with deterioration of CKD (incident CKD defined as estimated GFR [eGFR] < 60 mL/min/1.73 m2 and progression of CKD stages) over a median follow-up of 6.0 years. At baseline, 25 patients (15.4%) had CKD. Cox proportional hazards regression models were used for identifying associated factors of CKD deterioration. Results. Estimated GFR decreased from 75.8 ± 16.3 to 67.4 ± 18.2 mL/min/1.73 m2 (p < 0.01). Of 162 patients, 32 developed CKD and 8 made a progression of CKD stages. Multivariate Cox regression analysis revealed that carotid IMT  and coefficient of variation of HbA1c  were predictors of deterioration of CKD independently of age, mean HbA1c, urinary albumin/creatinine ratio, baseline eGFR, uric acid, and leucocyte count. Conclusions. Subclinical atherosclerosis and long-term glycemic variability predict deterioration of chronic kidney disease (as defined by incident or worsening CKD) in type 2 diabetic patients with preserved kidney function. Chronic kidney disease (CKD) has been consistently and independently associated with longitudinal risk for cardiovascular disease and heart failure. Progression towards end-stage renal disease exposes CKD patients to an increased risk of development of vascular disease and cardiovascular morbidity and mortality , 2. EstaThese overlapping risk factor patterns lead us to hypothesize that atherosclerosis may be an important mechanism leading to declines in kidney function. Prospective studies have shown a positive correlation between increased carotid artery intima-media thickness (IMT), a subclinical marker of atherosclerosis and cardiovascular disease, and the risk for kidney function decline and incident CKD in the elderly , 10. In The setting for this observational study was the same as previously reported . We repoFor each subject on each monthly visit, waist circumference, weight, and BP were measured by registered nurses. As previously reported in detail , blood wIntrapersonal mean and coefficient of variation (CV) of HbA1c, fasting and postmeal plasma glucose , and serum TG  taken during the first 12 months after enrollment were calculated and their means ± SD were shown in 2 after 6.0 years in participants with an eGFR > 60 mL/min/1.73 m2 at baseline. Linear regression was used to estimate changes in eGFR using a median of 52 creatinine measurements (interquartile range: 31–60) over 6.0 years of follow-up in each patient. Patients were staged according to the level of eGFR in mL/min/1.73 m2: G1: >90, G2: 60–89, G3a: 45–59, G3b: 30–44, and G4: 15–29 [Urinary albumin was measured once in random urine samples obtained during the first 3-4 months after enrollment using a turbidimetric immunoassay and expressed as albumin/creatinine ratio (ACR). Normoalbuminuria, microalbuminuria, and macroalbuminuria were defined as ACR < 30 mg/g, ACR between 30 and 299 mg/g, and ACR ≥ 300 mg/g, respectively . Serum aIMT was measured by a well-trained medical technologist of Sadamitsu Hospital using ultrasonic diagnosis equipment  that was programmed with IMT software  as previously described . Carotidt-test and frequencies of conditions by chi-square tests. Bivariate and multivariable Cox proportional hazards regression models were used for identifying associated factors of deterioration of CKD. Results were expressed as hazard ratio (HR) with their 95% confidence intervals (CI). A two-tailed p < 0.05 was considered statistically significant. All calculations were performed with SPSS system 15.0 .Data were presented as means ± SD unless otherwise stated. Differences between 2 groups were analyzed by The baseline characteristics of 162 participants are shown in p = 0.001) there was no difference in the proportion of patients with microalbuminuria . Consequently, patients with CKD tended to have higher proportion of ACR ≥ 30 mg/g (p = 0.06). There was no difference in glycemic variables and carotid IMT between the 2 groups.As compared to patients without CKD, those with CKD at baseline were older and had longer duration of diabetes and higher proportion of patients on BP-lowering agents . Urinary 30 mg/g . Diastol2 at baseline to 67.4 ± 18.2 mL/min/1.73 m2 after 6.0 years of follow-up (p < 0.01) with an annual decline of 1.11 ± 2.94 mL/min/1.73 m2. Proportion of patients with CKD (eGFR < 60 mL/min/1.73 m2) increased from 15.5% (n = 26) at baseline to 32.7% (n = 55) after follow-up whereas proportion of patients with stage G2 decreased from 63.6% (n = 107) to 54.7% (n = 92) and proportion with G1 decreased from 20.9% (n = 35) to 12.5% (n = 21).The level of eGFR decreased from 75.8 ± 16.3 mL/min/1.73 m2) occurred in 32 patients. Progression from stage 3A to stage 3B and stage 4 occurred in 7 patients and 1 patient, respectively. In total deterioration of CKD occurred in 40 patients . Maximum IMT and CV HbA1c were significant predictors of CKD deterioration. Leucocyte count and baseline eGFR were associated with CKD deterioration, but the relations were of borderline significance. In a model which included mean IMT instead of maximum IMT, CV-HbA1c , mean IMT , baseline eGFR , and leucocyte count  were independent predictors of CKD deterioration.Cox proportional hazard regression models were used for identifying associated factors of deterioration of CKD . IndepenThis study demonstrates in type 2 diabetic patients with preserved kidney function and relatively good glycemic, lipid, and BP control at baseline that the presence of CVD measured as subclinical carotid atherosclerosis was a predictor for deterioration of CKD. In addition, a nontraditional risk factor, namely, visit-to-visit variation in HbA1c, was found to be a risk factor of CKD deterioration.Cross-sectional associations between reduced kidney function and IMT thickening are reported in patients with type 2 diabetes , 18, hypLong-term glycemic variability has been suspected to be a major factor of diabetic complications . We haveThere were no differences in BP between patients whose kidney function deteriorated and those whose kidney function did not in the present study. This may be in line with observation that BP control of 138/86 or 132/78 mmHg appeared to stabilize renal function in hypertensive type 2 diabetic patients without overt albuminuria over a 5-year period , as progThe strength of the current study is that mean and CV of HbA1c and other traditional and nontraditional risk factors shown in Measurements of carotid IMT and visit-to-visit HbA1c variability retain predictive power with respect to CKD progression even after traditional and nontraditional risk factors for kidney disease progression have been taken into consideration. These results suggest that type 2 diabetic patients with subclinical carotid atherosclerosis should be recognized as a group at high risk for CKD progression. In addition, these findings also raise the issue of whether aggressive treatment of traditional risk factors for subclinical carotid atherosclerosis might, in fact, decrease progression to renal replacement therapy. Further studies are needed to confirm the association in other ethnic groups with more patients.


Mycobacterium tuberculosis infection can potentially induce an immune response against the mycobacteria. To examine the role of dendritic cells in this response, macrophage apoptosis was induced by incubating the macrophage with cell wall extracts of mycobacteria expressing LpqH. The apoptogenic proteins of the cell wall extracts were engulfed by the macrophage and then were translocated from the cytosol to the nuclei of the dying cells. Dendritic cells that engulfed the apoptotic macrophages acquired an immunogenic phenotype that included upregulation of MHC-I, increased expression of the costimulatory molecules, CD40, CD80, and CD86, and increased production of IL-12, IL-10, TNF-α, and TGF-β. In addition, the dendritic cells triggered a proliferative response of CD8+ T cells with IFN-γ production via cross-presentation. Taken together, these findings support a model in which phagocytosis of whole apoptotic cells carrying mycobacterial antigens promotes a potentially protective immune response.While homeostatic apoptosis is immunologically silent, macrophage apoptosis during  Mycobacterium tuberculosis (Mtb) involves both innate and adaptive immune mechanisms  and disrupted with a glass homogenizer. Released nuclei were passed through a 30% saccharose gradient (0.88 M) in nuclear buffer (500 ml) and separated in 15% PAGE-SDS gels. After the proteins were transferred to PVDF membranes, they were incubated with a rabbit antiserum to MsmegLpqH cell-wall proteins (diluted 1:200), or with a mAb recognizing LpqH (diluted 1:200), at 4°C overnight. After rinsing, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies diluted 1:1000. After 1 h, reactive bands were visualized with a SuperSignal West Dura kit  or by DAB/H2O2 staining.To study the intracellular localization of apoptosis-inducing proteins by immunofluorescence, 5 x 10The method proposed by Inaba et al. was followed with minor modifications . Briefly5 cells) labeled with PKH-67; 50 μg protein of apoptotic MØs correspond to 200,000 cells. To synchronize phagocytosis, apoptotic MØs were pelleted by centrifugation  onto DC slides. After 1 h, 4 h, and 24 h, the cells were examined by flow cytometry and the percent of phagocytosis was determined. In addition, in assays with PKH-26 labeled apoptotic MØs, phagocytosis was analyzed setting a geometric mean fluorescence index (MFI). For fluorescence microscopy a sample of each set of cells was rinsed with PBS, fixed with 1% paraformaldehyde, and mounted with an anti-fade reagent and DAPI onto prepared cytospin slides. The cells were examined with an Olympus BX51 immunofluorescence microscope and with a LSM 5 Pascal Zeiss laser scanning confocal microscope with Software 2.8.Since the uptake of antigenic material is a prerequisite for antigen processing and presentation to T cells , the cap5 DCs were incubated in RPMI 1640 supplemented with 5% FBS, for 24 h with 50 μg MsmegLpqH or UV-induced apoptotic MØs. As a positive activation control, DCs were treated with 1 μg of LPS. Each set of cells was stained with mAbs recognizing CD11c, antigen presentation molecules, MHC-I and MHC-II, and costimulatory molecules, CD40, CD80, and CD86. The CD11c+ cells were gated and a geometric mean fluorescence index (MFI) of the markers was set. Cytokine release in the supernatant of the DCs cocultured with apoptotic MØs for 24 h was also assayed. According to manufacturers’ instructions, a sandwich enzyme-linked immunosorbent assay (ELISA) method with mAbs recognizing IL-12, TNF-α, TGF-β, and IL-10 was used. The levels of cytokines released into the culture medium were calculated based on absorbance values measured at 450 nm with a microplate reader and standard curves that were generated with recombinant cytokines. The results are expressed in picograms per milliliter.Immature DCs were obtained from bone marrow precursor cells as described above. Briefly, 5 x 10+ and CD8+ populations were gated and the percentage of cells within each gate was recorded. We measured the cytokines released in the supernatant with an ELISA assay for IL-12, TNF-α, IL-10, and IFN-γ as described above. In addition, proliferation assays were performed with purified CD4 and CD8 T cells obtained from mice spleens by cell sorting. Purified cells were labeled with CFSE (1 μM) in serum-free PBS for 5 min at room temperature and cocultured for 3 days with DCs activated with MsmegLpqH or UV apoptotic MØs at a 10:1 ratio. Flow cytometry CFSE dilution assays were performed to assess cell proliferation.Mononuclear spleen cell suspensions were obtained from naïve 6-week-old BALB/c mice. Briefly, spleens were resected, placed in Petri dishes containing RPMI 1640, and physically disrupted with a cell strainer. The resulting cell suspension was rinsed with RPMI 1640 and centrifuged for 5 min at 652 x g. To isolate T cells, erythrocyte lysis was performed. Then, the spleen mononuclear cells were labeled with a mAb to the pan-T cell marker Thy-1.2 (CD90.2)  that was5 DCs were incubated with 50 μg MsmegLpqH or UV-induced apoptotic MØs protein for 24 h. After rinsing, the DCs were cocultured with CFSE-labeled, cell sorted CD8+ or CD4+ T cells at a 1:10 ratio. After 3 d, cell proliferation was assessed by CFSE dilution flow cytometry. Supernatants were collected and levels of IFN-γ were quantitated with a BioLegend ELISA kit.Cross presentation assays with purified CD4 and CD8 T cells were carried out. DCs were treated for 1 h with the proteasome inhibitor, MG-132 (0.2 μm), and with the proton pump inhibitor bafilomycin (0.05 μm) . After 1Statistical analyses were performed with GraphPad PRISM software . Data expressed as the mean ± standard deviation were analyzed with non parametric Kruskal Wallis with Anova, Student´s t-test, Wilcoxon signed test and Mann Whitney test.M. smegmatis strain was transformed to express LpqH. The cell wall fraction from this strain was isolated by sonication and separated by SDS-PAGE. A subsequent immunoblot with a monoclonal antibody recognizing LpqH identified a 19-kDa band , and with an inhibitor of vacuolar H+ ATPase, bafilomycin (0.05 μm). Similar treatments were used with purified CD4+ T cells. It was found that both MG-132 and bafilomycin similarly decreased the proliferation of CD8+ T cells (+ T cell proliferation (+ and CD4+ T cells was measured in the supernatant. It was observed MG-132 and bafilomycin inhibited markedly the release of IFN-γ by CD8 T cells (+ T cells (To confirm that the DCs that engulfed MsmegLpqH apoptotic MØs were cross-presenting antigen to CD8 T cells . In contferation . IFN-γ r T cells . MG-132  T cells .Given the central role that DCs play in the adaptive T cell response , the aimDuring apoptosis, cells undergo dramatic changes . In the To activate DCs, we obtained late phase apoptotic MØs that were free of blebs. Apoptosis was induced by exposing MØ to extracts of mycobacterial cell wall proteins, particularly LpqH, an apoptogenic Mtb lipoprotein known to induce T cell immunity . IntraceIt has been demonstrated that immature DCs endocytose apoptotic MØs with great efficiency . In the Following the phagocytosis of antigenic material, DCs process and present antigen to T cells via MHC-II, MHC-I, and CD1 antigen presentation molecules , 36. In + T cells. These effects may be due to the presence of mycobacterial antigens in the apoptotic bodies, since the DCs that engulfed the UV-induced apoptotic MØs were free of exogenous antigens and they exhibited a distinct phenotype that did not include upregulation of costimulatory and antigen presentation molecules or the production of proinflammatory cytokines. Furthermore, instead of activating CD8 T cells, the UV apoptotic bodies induced the proliferation of activated CD4 T cells.The results of the present study demonstrate that DCs that have phagocytosed MØs carrying mycobacterial antigens, acquire a mature phenotype which is characterized by increased expression of the antigen presentation molecule, MHC-I, and the costimulatory molecules, CD40 and CD86. Additionally, it was observed that the marked increase in production of the anti-inflammatory cytokine, IL-10, did not affect the ability of the DCs to cross-present antigen to CD8


Pheochromocytoma is an endocrine tumor that causes hypertension, facial pallor, and headache. Pheochromocytoma patients rarely present with acute heart failure or cardiogenic shock.We discuss the case of a female patient with Takotsubo-pattern cardiomyopathy who presented with acute heart failure caused by pheochromocytoma.Treatment was adjusted based on the data of the pulse contour cardiac output system. After intensive hydration and medication for heart failure, the condition of the patient stabilized.Before confirming the diagnosis, pulse contour cardiac output data could provide a direction for diagnosis and treatment. Therefore, confirming the diagnosis before laboratory data are available is a challenge.Pheochromocytoma is a rare endocrine tumor that secretes catecholamine and presents with a classic triad of headache, sweating, and palpitation.21 through V4  154/96 mm Hg; body temperature (BT) 36°C; respiratory rate (RR) 30 breaths/min; and oxygen saturation 98% in 3 L of oxygen administered through a nasal cannula  was 40,300 pg/mL. Other data included the following values: troponin I, 33.4 μg/L (<0.5 μg/L); creatinine kinase (CK), 738 U/L (30–223 U/L); and CK-MB, 123.6 ng/mL (0.6–6.3 ng/mL).2, a systemic vascular resistance index (SVRI) of 5177 dyn.sec/cm5.m2, an intrathoracic blood volume index (ITBVI) of 812 mL/m2 (850–1000 mL/m2), and a global end-diastolic volume index (GEDVI) of 650 mL/m2 (680–800 mL/m2). Echocardiography revealed apical akinesis with relatively preserved LV basal wall motion, and the LV ejection fraction (LVEF) was 11%.Four hours later, her BP suddenly decreased to 72/50 mm Hg, and she developed acute shortness of breath, diaphoresis, and a cold appearance. Endotracheal intubation was performed because of respiratory failure, and profound shock was observed. The pulse contour cardiac output (PICCO) system  was used to evaluate her hemodynamic condition. Evaluation revealed a cardiac index (CI) of 1.42 L/min/m2, an SVRI of 1929 dyn.sec/cm5.m2, and an ITBVI of 701 mL/m2. Echocardiography revealed a reduction in abnormal contraction in the apical wall of the LV, and the LVEF was 64%.Owing to the decreased ITBVI and CI, intensive hydration with normal saline and inotropic agents was prescribed. Antibiotics and steroids were administered because of the possibility of myocarditis. After the treatment, the PICCO data showed a CI of 3.69 L/min/mOn the fifth hospital day, the urine catecholamine levels  revealed the following values: epinephrine, 320.1 μg/d (0–27 μg/d); norepinephrine, 305.5 μg/d (0–97 μg/d); dopamine, 80.4 μg/d (0–500 μg/d); and vanillyl mandelic acid (VMA), 25.06 mg/d (1–7.5 mg/d). The alpha blocker was prescribed under the suspicion of adrenal pheochromocytoma. Later, the patient was transferred to the ordinary ward in a relatively stable condition.A contrast-enhanced computed tomography scan of the abdomen showed a left adrenal heterogeneous enhanced mass measuring 3.9 × 3.4 cm -α and interleukin (IL)-6. These inflammatory factors down-regulated the expression of the sarcoplasmic reticulum Ca2+-ATPase gene and delayed diastolic calcium reuptake. Whereas the diastolic function improved, the levels of these cytokines decreased. Among the Chinese population, risk-conferring genetic variants of the angiotensin II type 1 receptor gene for diastolic heart failure were found, which were previously identified.Patients with myocarditis may have a viral prodrome, and various clinical scenarios can occur, such as acute coronary syndrome, acute dilated cardiomyopathy, and heart failure.Based on clinical presentation alone, diagnosis is difficult to make, and collection of urine data of catecholamine and VMA takes time. Before the urinalysis results become available, PICCO data could provide an indication of the diagnosis. The hemodynamic status showed low CI and high SVRI among the 3 conditions. However, the intravascular volumes of Takotsubo cardiomyopathy and myocarditis were increased, whereas the intravascular volume of pheochromocytoma with acute heart failure was depleted. The low intravascular volume was attributable to the catecholamine-induced volume contraction. Therefore, volume expansion should be performed with caution in patients with pheochromocytoma. Hence, PICCO data could provide a direction for diagnosis and treatment before the data of urine VMA and catecholamine become available.Our case indicated that a diagnosis of pheochromocytoma should be considered when echocardiography or angiography reveals Takotsubo-pattern cardiomyopathy. Moreover, PICCO data should be evaluated when patients have acute LV systolic dysfunction with heart failure and no coronary artery stenosis. Finally, for patients presenting with acute heart failure, pheochromocytoma should always be considered.


Purpose. This study aimed to evaluate the characteristics of the HVGGSSV peptide, exploring radiation-guided delivery in a mouse model of nasopharyngeal carcinoma. Methods. Mice with CNE-1 nasopharyngeal carcinoma were assigned to two different groups treated with Cy7-NHS and Cy7-HVGGSSV, respectively. Meanwhile, each mouse received a single dose of 3 Gy radiation. Biological distribution of the recombinant peptide was assessed on an in vivo small animal imaging system. Results. The experimental group showed maximum fluorescence intensity in irradiated tumors treated with Cy7-labeled HVGGSSV, while untreated (0 Gy) control tumors showed lower intensity levels. Fluorescence intensities of tumors in the right hind limbs of experimental animals were 7.84 × 107 ± 1.13 × 107, 1.35 × 108 ± 2.66 × 107, 4.05 × 108 ± 1.75 × 107, 5.57 × 108 ± 3.47 × 107, and 9.26 × 107 ± 1.73 × 107 photons/s/cm2 higher compared with left hind limb values at 1, 2, 15, 24, and 48 h, respectively. Fluorescence intensities of tumor in the right hind limbs of the experimental group were 1.66 × 108 ± 1.71 × 107, 1.51 × 108 ± 3.23 × 107, 5.38 × 108 ± 1.96 × 107, 5.89 × 108 ± 3.57 × 107, and 1.62 × 108 ± 1.69 × 107 photons/s/cm2 higher compared with control group values at 1, 2, 15, 24, and 48 h, respectively. Fluorescence was not specifically distributed in the control group. Compared with low fluorescence intensity in the heart, lungs, and tumors, high fluorescence distribution was found in the liver and kidney at 48 h. Conclusions. HVGGSSV was selectively bound to irradiated nasopharyngeal carcinoma, acting as a targeting transport carrier for radiation-guided drugs that are mainly metabolized in the kidney and liver. Although concurrent chemoradiotherapy has become an important treatment modality for cancer , nonspecPeptides are widely used in targeted drug carrier research due to high affinity and specificity, low molecular weight, lack of immunogenicity, and absorbability by the tumor cells –8. LowerAs proposed by Hariri et al. , we synt2, in a humid environment. Five-week-old male nude mice weighing 18–20 g were anesthetized by intraperitoneal injection of chloral hydrate. A 0.1 mL cell suspension (1 × 107/mL) was inoculated subcutaneously in bilateral hind limbs of each animal. Once the tumor had reached approximately 1 cm in diameter (after 1-2 weeks), the mice were subjected to in vivo experiments.Human nasopharyngeal carcinoma CNE-1 cells were provided by Tumor Radiation Biological Laboratory of Fujian Provincial Tumor Hospital and cultured in complete RPMI 1640 at 37°C and 5% COThe fluorescent molecule Cy7 mono NHS ester was conjugated to the peptide to yield the Cy7-HVGGSSV fluorescence polypeptide, which was purified by SephadexG-15. After buffer elution, two bands were identified: the first outflow was the labeled conjugate Cy7-HVGGSSV while the band atop the column was free Cy7. The samples were freeze-dried for 66 h, and absorption spectra of the Cy7-HVGGSSV compound were measured on a spectrophotometer.n = 8) and received 3 Gy radiation to the right hind limb tumor. 4 h after irradiation, control group mice were administered with 0.1 mL of free Cy7 solution (1 mg/L) to tail vein while the mice in the experimental group were injected with 0.1 mL of Cy7-HVGGSSV solution (1 mg/L) similarly.Mice were randomly divided into two groups (Mice were anesthetized and fastened in a thermoplastic membrane. Right hind limb tumors and outer margins (0.5 cm around) were considered the radiation field, while left hind limb tumor was avoided. The mice were subjected to 3 Gy dose on a 6 MV X-ray linear accelerator. in vivo. The mice were fasted for 6 h to reduce interference of the gastrointestinal content. Subsequently, the animals were anesthetized by chloral hydrate and subjected to imaging after drug injections at 1, 2, 15, 24, and 48 h, respectively. Maximum excitation wavelength was 747 nm, for emission at 775 nm. Bilateral limb tumors were selected in fluorescent images as the region of interest (ROI). Then, fluorescence quantum in the ROI was measured, and fluorescence intensity of the tumor area was analyzed and compared in bilateral limbs. Changes in fluorescence quantum at different time points in the same ROI were monitored.Fluorescence distribution in bilateral hind limb tumors was assessed on a small animal imaging system in vivo.Because of the complicated mouse structure and composition, hair, necrotic tumor tissue, and food in the gastrointestinal tract may produce spontaneous fluorescence that increases the background noise, affecting the recognition of specific fluorescent substances. To reduce such interference, the mice were sacrificed at 48 h after drug administration, and hearts, livers, lungs, kidneys, and tumors were excised and assessed on a small animal imaging system in vivo [ in vivo distribution of the fluorescence polypeptide. Orange indicated the strongest fluorescence intensity at 750 nm excitation, with highest content of the fluorescent molecule Cy7, whereas purple reflected a very low fluorescence intensity. The color spectrum of orange, red, yellow, green, blue, and purple indicated high to low fluorescence intensity in that order.Pseudocolor and scanning images for each mouse were overlaid and fused by the small animal imaging system in vivo , 21. Thet-test. P < 0.05 was considered statistically significant.Data are mean ± standard deviation (SD) and were analyzed by the SPSS 19.0 software. Differences between groups were assessed by Maximum excitation wavelength was 747 nm, for a maximum emission wavelength of 775 nm. Spectroscopic results are shown in  in vivo small animal imaging system (Cy7-HVGGSSV and free Cy7 fluorescent molecules (control) were administered by tail vein injection. Then, distribution and metabolism of the polypeptide were assessed on ang system .8 ± 1.75 × 107 and 5.57 × 108 ± 3.47 × 107 photons/s/cm2 higher compared with left hind limb values. No fluorescence distribution was found in experimental mice after 48 h. Figures According to the linear distribution theory of fluorescence intensity, fluorescence intensities in other body areas were significantly different from values in right hind limb tumors, which were remarkably lower compared with those in tumors treated by radiation injury. Fluorescence signal data in tumors of the Cy7-HVGGSSV group are shown in P ≥ 0.05). Mean fluorescence intensities of right hind limb tumors of the Cy7 group were 1.66 × 108 ± 1.71 × 107, 1.51 × 108 ± 3.23 × 107, 5.38 × 108 ± 1.96 × 107, 5.89 × 108 ± 3.57 × 107, and 1.62 × 108 ± 1.69 × 107 photons/s/cm2 lower compared with Cy7-HVGGSSV group values at 1, 2, 15, 24, and 48 h, respectively.Cy7 group results are shown in  in vivo. Fluorescence signals and fluorescence image of separated organ in mice of the Cy7-HVGGSSV group are shown in Mice were sacrificed at 48 h after drug administration, and hearts, livers, lungs, kidneys, and tumors were excised for assessment on a small animal imaging system in vivo and its binding characteristics to tumors after radiation injury were evaluated. On fluorescence images of tumor-bearing nude mice treated with Cy7-labeled HVGGSSV, fluorescent peptides were accumulated in irradiated right hind limb tumors, while less fluorescence was observed in nonirradiated left hind tumors. Substantially higher fluorescence signals were obtained in the liver and kidney compared with heart and lung values 48 hours after injection, suggesting that the polypeptide might be degraded in the liver and kidney, thereby avoiding side effects on other tissues. According to the instructions of small animal imaging system in vivo, fluorescence intensities of different regions could only be compared in the same background. To compare fluorescence signals at different times, both fluorescence intensity and quantum should be taken into consideration. Additionally, to highlight the differences among various regions, with this peptide mainly metabolized in the kidney, different orders of magnitude were, respectively, used in Figures 9 versus 108). Our results showed that the targeting carrier could accumulate in irradiated tumors and might be metabolized in the kidney and liver.This study assessed the biological features and drug loading properties of HVGGSSV, a new nanometer-sized targeting carrier. Using a small animal imaging system, distribution and metabolism of Cy7-labeled HVGGSSV were observed in vivo and in vitro differences in polypeptide metabolism or differences in affinity and clearance between polypeptides and targeted proteins. The small animal imaging system can directly assess the labeled experimental subjects in real time and in situ, rapidly yielding quantitative information in a completely in vivo environment.The phage display technology described here allowed us to examine polypeptides that specifically bind to tumors. It could rapidly screen bioactive peptides, proteins, receptors, and other novel or lead compounds with high stability and specificity in the body , 16. HowThe molecular weights of polypeptides are lower than antibody values (1-2 kDa). Therefore, polypeptides have better bioavailability and permeability, with higher safety in the tumor tissue. Moreover, they also exhibit accelerated metabolism in the body. Their limitations include instability on the tumor cell surface and alterations according to the environment , 18. SugHerein, we synthesized HVGGSSV according to Lowery et al. , with thFluorescence distribution patterns in bilateral hind limb tumors between the Cy7-HVGGSSV and Cy7 groups at 1 or 2 h were similar. At 15 and 24 h after treatment, red signals were clearly visible in right hind limb tumors in Cy7-HVGGSSV group mice, while no signal was visualized in the corresponding right hind limb tumors of Cy7 group animals. These results suggested that the distribution of free fluorescent Cy7 molecule is not restricted. Fluorescent signals in the left hind limb tumor area were lower and dissipated rapidly compared to those in right hind limbs in Cy7-HVGGSSV group animals, suggesting that distribution of the HVGGSSV polypeptide was higher in radiation-damaged tumors.No signals were observed in Cy7-HVGGSSV or Cy7 group mice at 48 h. In addition, Lowery et al.  found fl in vivo could not be quantitatively evaluated due to interference by objective factors. Mice were sacrificed after 48 h of in vivo observation; the organs were then excised to assess polypeptide distribution. Our results showed the following order for polypeptide distribution: kidney > liver > right hind limb tumor > left hind limb tumor > lung > heart. Fluorescence distribution in the kidney was markedly persistent, suggesting that this carrier was primarily metabolized in the kidney, largely damaging this organ. Fluorescence distribution in the liver was lower than that in the kidney; meanwhile, right hind limb tumor signals were higher than left limb values. These findings suggested that the fluorescence carrier persisted in right hind limb tumors. Since the fluorescence carrier was cleared from the body, it could not be definitely claimed that HVGGSSV was targeted to radiation-injured tumors. The current data showed that this peptide might be metabolized in kidney, followed by liver; therefore, hepatotoxicity and nephrotoxicity constitute the focus of future research in our laboratory.Unlike the current study, Lowery et al.  did not The current study reported the successful building of HVGGSSV, a targeting carrier with adequate biological compatibility and high binding capacity with irradiated nasopharyngeal carcinoma. HVGGSSV is anticipated to become an efficient transport carrier of targeted therapeutics for cancer, thus contributing towards an advanced approach for an ideal combination model of chemotherapy and radiotherapy.


Updated population-based data on the frequency and distribution of risk factors for pneumococcal disease is scarce. This study investigated the prevalence of distinct comorbidities and underlying risk conditions related to an increasing risk of pneumococcal disease among Catalonian middle-aged and older adults.Information System for the Development of Research in Primary Care (SIDIAP database) was used to identify high-risk (asplenia and/or immunocompromising conditions) and other increased-risk conditions  among study subjects.Cross-sectional population-based study including 2,033,465 individuals aged 50 years or older registered at 01/01/2015 in the Catalonian Health Institute . The clinical research database of the p < 0.001; 41.7% in people 50–64 years vs 54.7% in persons 65 years or older, p < 0.001). An amount of 176,600 individuals (8.7%) had high-risk conditions . On the other hand, 803,710 persons (39.5%) had one or more other risk conditions. In fact, 212,255 (10.4%) had chronic pulmonary diseases, 248,377 (12.2%) cardiac disease, 41,734 (2.1%) liver disease, 341,535 (16.8%) diabetes mellitus, 58,781 (2.9%) alcoholism and 317,558 (15.6%) were smokers.Globally, 980,310 (48.2%) of the 2,033,465 study population had at least one risk condition of suffering pneumococcal disease . Streptococcus pneumoniae is a Gram-positive bacterium that typically colonises the respiratory tract in humans, being the cause of serious illness and death in some individuals. After colonisation, possible outcomes are the clearance, the asymptomatic persistence (carrier state), or the bacterium can progress to the disease, causing mucosal infections  or resulting in invasive pneumococcal disease (IPD) such as bacteremic pneumonias, meningitis or sepsis. The outcome depends on interactions between factors related to the host, therapy and microorganism. [rganism. , 2.At present, pneumococcal infections remain a major cause of morbidity and mortality around the world, being one of the ten leading causes of death worldwide.  The riskSeveral studies have investigated the prevalence of distinct risk conditions to suffer IPD among adult populations and have reported incidence of IPD among healthy adults as compared with those with underlying conditions. –11 UsualAt present, two pneumococcal vaccines are available for use in adults: the “classical” 23-valent pneumococcal polysaccharide vaccine (PPV23) and the “new” 13-valent protein-polysaccharide conjugate vaccine (PCV13). The PPV23 was marketed in 1983  whereas In Catalonia and Spain, since the 2000s, the PPV23 is recommended and publicly funded for all people aged 65 years or older (with or without risk conditions) as well as for those persons 18–64 years with certain underlying risk conditions such as asplenia, immunocompromising conditions, chronic pulmonary, cardiac, renal or liver disease, diabetes, alcoholism and smoking. , 14 The In 2015, the reported pneumococcal vaccination coverage in Catalonia was 39% among adults ≥50 years-old (4.8% in 50–59 years vs 35.5% in 60–69 years vs 71.9% in 70–79 years vs 79.5% in 80 years or older).  AccordinAs mentioned above, several studies have reported the prevalence of chronic illnesses and underlying conditions among hospitalised patients with IPD or pneumonia, –11 but tThe present study investigated the prevalence of distinct comorbidities and underlying conditions related to increasing risk  for pneumococcal infections among Catalonian middle-aged and older adults. High-risk persons in the same population have been previously described elsewhere. .Catalonian Health Institute on January 1, 2015 .This is a cross-sectional population-based study involving all individuals aged 50 years or older, who were registered in the Primary Health Care Centres (PHCCs) of the Catalonian Health Institute manages 274 PHCCs, serving a population of approximately five million people. Doctors and nurses systematically use electronic medical records to record medical diagnoses, underlying conditions, prescriptions, and other clinical patient management activities coded according to the International Classification of Diseases, 10th Revision (ICD-10).In Catalonia, a region in North Eastern Spain with seven million people, there are 358 PHCCs  which are distributed by geographical area and are responsible for the health care of the population in their areas. The Catalonian Health Institute Information System for the Development of Research in Primary Care (“SIDIAP” database) compiles coded clinical information from the Electronic PHCC’s records, [The records,  and it hrecords,  The SIDIrecords, .SIDIAP database was complete, so a condition was considered absent if it was not recorded.The “SIDIAP” research database was used to identify chronic comorbidities and underlying medical conditions among study subjects, who were classified into three risk strata on the basis of immunocompromise degree and current recommendations for pneumococcal vaccination. , 13, 14 p < 0.05 (two-tailed). Data were analyzed using the SPSS version 18.0 .The statistical differences between prevalence for distinct risk conditions according to distinct population subgroups were evaluated using the Chi-squared tests, or Fisher’s exact test as appropriate. Odds Ratios (ORs) with 95% confidence intervals (CIs) were estimated to compare prevalence between gender and age groups. Statistical significance was set at Of the total 2,033,465 study population, 935,705 (46%) were men and 1,097,760 (54%) were women, being 1,021,648 (50.2%) aged 50–64 years and 1,011,817 (49.8%) aged 65 years or older.p < 0.001; 41.7% in people 50–64 years vs 54.7% in persons 65 years or older, p < 0.001). Among the 50–64 years age subgroup, prevalence of persons with any high-risk condition (stratum 1) was 4.7% in men and 5.4% in women, whereas prevalence of persons with any medium-risk condition (risk stratum 2) was 42.9% in men and 30.7% in women. Among the 65 years or older age subgroup, prevalence of persons with any high-risk condition (stratum 1) was 14.4% in men and 10.7% in women, whereas prevalence of persons with any medium-risk condition (risk stratum 2) was 50% in men and 36.7% in women  of the 2,033,465 study population had at least one risk condition to suffer pneumococcal disease  of the total 2,033,465 study subjects had only one risk condition, 243,127 (12.0%) had two risk conditions, 69,308  had four or more risk conditions.If we consider those conditions associated with highest risk for pneumococcal infections (risk stratum 1), 3274 (<0.1%) had primary immunodeficiency, 589 (0.2%) HIV infection, 1480 (0.1%) nephrotic syndrome, 15,468 (0.8%) had severe renal failure, 5034 (0.2%) had received bone marrow transplantation, 103,948 persons (5.1%) had recent cancer diagnosis and 72,040 (3.5%) received immunosuppressive treatment/radiotherapy in last 12 months. An amount of 294 (<0.1%) had asplenia, 76 (<0.1%) cochlear implants and 41 (<0.1%) CSF leaks.p < 0.001; 5.1% in persons 50–64 years vs 12.3% in persons 65 years or older, p < 0.001).Considering that some persons had more than one high-risk condition, only 176,600 individuals (8.7%) of the overall study population were classified in risk stratum 1  had chronic pulmonary/respiratory diseases, 248,377 persons (12.2%) had chronic heart diseases, 41,734 (2.1%) chronic liver diseases, 341,535 (16.8%) diabetes mellitus, 58,781 (2.9%) alcoholism and 317,558 (15.6%) were smokers.p < 0.001; 36.7% in persons 50–64 years vs 42.4% in persons 65 years or older, p < 0.001).Overall, 803,710 individuals (39.5%) of the 2,033,465 study subjects were classified in risk stratum 2 because they had one of the above mentioned conditions but had no concomitantly conditions mentioned in risk stratum 1  of the overall study population were classified as risk stratum 3 (lowest risk) because they did not present any risk condition mentioned in stratum 1 and/or 2. The prevalence for each one of the distinct risk factor/condition, according to gender and age strata, is shown in Tables Pneumococcal disease is recognized as a major cause of morbidity and mortality around the world.  However,As main findings, approximately 10 % (8.7%) of the overall study population have some underlying conditions related with highest risk for pneumococcal disease  and 40 % (39.5%) have other conditions related to increasing risk . Therefore, almost 50 % (48.2%) of the overall population may be considered at-risk for pneumococcal infections in our study. In the present study, most underlying conditions were more common in men than in women. Substantial differences  were observed for smoking, alcoholism and HIV infection. Only prevalence of primary immunodeficiency, immunosuppressive treatment, severe renal failure, CSF leaks and cochlear implant were more frequent in women. Recent literature suggests that pneumococcal vaccine effectiveness may be influenced by sex,  which unSeveral studies have estimated the incidence of IPD among persons with high-risk conditions such as sickle-cell anemia, HIV infection, chronic pulmonary diseases, cardiac diseases, alcoholism and/or smoking , 25, 26 Advisory Committee on Immunization Practices (ACIP) of the Centers for Diseases Control and Prevention , a sequential pneumococcal vaccination using PCV13 + PPV23 is recommended for persons aged 65 years or older (with or without underlying conditions) and persons 19–64 years with CSF leaks, cochlear implants, functional or anatomic asplenia and/or immunocompromising conditions . [Most studies assessing the presence of risk conditions for pneumococcal diseases were hospital based studies involving hospitalized patients with IPD, but there is limited population-based data about the prevalence of some of these conditions. –11 The ayeloma).  At preseyeloma).  however,yeloma). , 28 In Cyeloma). .In the present study, risk strata were defined on the basis of immunocompromise degree and presence of other underlying conditions generally considered to establish pneumococcal vaccination recommendations. According to the current ACIP’s recommendations , –20 all pOur data provides important information for policy makers in order to better determine the size of different target population considering distinct pneumococcal vaccination strategies . According to our data, approximately 5% of people 50–64 years are at highest-risk for pneumococcal infections and consequently dual vaccination could be recommended. Within this 50–64 years age subgroup, approximately 37% of persons have other risk conditions and therefore should be vaccinated with a unique pneumococcal vaccine (PPV23 according to current ACIP/CDC recommendations).  If we coIn England, a study focused on adults who had received PPV23 estimated that 12.7% of population in 2009 had at least one of the risk conditions included in the recommendations for pneumococcal vaccination there .  In SpainOur study has several strengths. Study design was population-based and large enough to assess the prevalence of main underlying risk conditions related to invasive pneumococcal infections. In the study setting, similar to the rest of Spain, all inhabitants are covered by the National Health Service by a compulsory health assurance system, all inhabitants are assigned to a PHCC, medical assistance is free and most treatments are publicly funded; so a bias related to people who are not seeking care or have not been entered into the SIDIAP database is unlikely. As limitation, although the validity of clinical data source was previously checked,  informatConsidering the epidemiology of pneumococcal disease most studies report specific data on elderly people  who experience substantial morbidity and mortality. However, data focused on younger and/or middle-aged adults is uncommon. In the present study, together with elderly people, we also included middle aged persons  because they have a non-insignificant incidence of invasive pneumococcal disease , –11 and tThis large population-based study involving more than 2 million people investigating the prevalence of main risk conditions for pneumococcal infections among the general population 50 years or older in Catalonia, Spain, shows that approximately 50 % of them may be considered at-risk population for pneumococcal disease. Of this 50 % nearly 10 % have high-risk conditions and 40 % have medium-risk conditions. Updated epidemiological studies focused on distinct populations and geographical settings are necessary to know the prevalence of different risk conditions for pneumococcal disease, to clarify the true size of target populations for vaccination, and better allocate health care resources considering distinct vaccination strategies in adults.


Solanum tuberosum L.) is the third most widely consumed plant food by humans. Its tubers are rich in starch and vitamin C, but have low or null levels of essential nutrients such as provitamin A and vitamin E. Transformation of potato with a bacterial mini-pathway for β-carotene in a tuber-specific manner results in a “golden” potato (GP) tuber phenotype resulting from accumulation of provitamin A carotenoids (α- and β-carotene) and xanthophylls. Here, we investigated the bioaccessibility of carotenoids and vitamin E as α-tocopherol (αTC) in boiled wild type and golden tubers using in vitro digestion. Golden tubers contained up to 91 μg provitamin A carotenes (PAC)/g D, increased levels of xanthophylls, phytoene and phytofluene, as well as up to 78 μg vitamin E/g DW. Cubes from wild type and GP tubers were boiled and subjected to simulated digestion to estimate bioaccessibility of carotenoids and αTC. Retention in boiled GPs exceeded 80% for β-carotene (βC), α-carotene (αC), lutein, phytoene ± and αTC, but less than 50% for phytofluene. The efficiency of partitioning of total βC, αC, E-lutein, phytoene, phytofluene and αTC in the mixed micelle fraction during small intestinal digestion was influenced by genotype, tuber content and hydrophobicity. Apical uptake of the compounds that partitioned in mixed micelles by monolayers of human intestinal Caco-2 cells during incubation for 4h was 14–20% for provitamin A and xanthophylls, 43–45% for phytoene, 23–27% for phytofluene, and 53% for αTC. These results suggest that a 150 g serving of boiled golden potatoes has the potential to contribute 42% and 23% of the daily requirement of retinol activity equivalents (RAE), as well as 34 and 17% of the daily vitamin E requirement for children and women of reproductive age, respectively.Potato ( Vitamin A deficiency (VAD) remains the major cause of blindness in children and increases the probability of morbidity and mortality due to infectious diseases. β-carotene has the highest provitamin A activity and accumulates in plant foods such as carrots, apricots and peaches, but not in major staples such as rice, wheat, maize, cassava and potatoes that are often the primary source of energy for those residing in developing regions . The CopOR gene [Erwinia herbicola. The resulting “golden” tubers display a deep yellow to orange fleshed tuber phenotype, largely resulting from the accumulation of β-carotene , lutein (30 fold), β-β-xanthophylls (9 fold) and α-carotene [Use of the actual potato ancestors likely began about 13,000 years ago by Mesolithic populations living in Chilean forest-lands and the Peruvian Andean Mountains . Domesticarotene .In order to mediate the essential functions of vitamin A, dietary provitamin A (αC and βC) and its retinyl ester metabolites must be absorbed and transferred to target tissues. This requires carotenoid release from the food matrix, solubilization in triglyceride-rich oil droplets, and transfer to mixed micelles along with products of lipid digestion. Subsequently, the carotenoids are delivered to enterocytes and incorporated intact or after cleavage and conversion to retinyl esters into chylomicrons that are secreted into lymph for distribution to peripheral tissues . Partitiin vitro digestion/Caco-2 human intestinal cell model [The primary objectives of this study were to investigate the retention of provitamin A carotenoids and vitamin E in boiled golden potato (GP) tubers, and their bioaccessibility using the coupled ll model . Furthertrans-β-carotene , αC (98% pure) and αTC were purchased from Sigma-Aldrich . Phytoene and phytofluene were kindly provided by Dr. Ken Riedl, Nutrient & Phytochemical Analytic Shared Resource (NPASR) at The Ohio State University. All other reagents and supplies were purchased from ThermoFisher Scientific unless otherwise specified.Reagents for extraction and chromatographic grade solvents were purchased from Thermo Fisher Scientific . Fetal bovine serum (FBS) was purchased from Gemini Bio-Products . Fungizone, penicillin-streptomycin, L-glutamine and non-essential amino acids were purchased from Life Technologies Corporation . DMEM medium with 25 mmol/L glucose, HEPES buffer, bile extract, porcine amylase, pepsin, lipase and pancreatin, apo-8’-carotenal, all-3), pooled and transferred to 50 mL polypropylene tubes, blanketed with argon, sealed and immediately frozen in liquid nitrogen and stored at -80°C. Frozen tubes were shipped on dry ice to The Ohio State University, Columbus, OH, USA. Upon thawing, cubes for each genotype were divided into two portions. The first was homogenized, transferred to 50 mL polypropylene tubes, blanketed with nitrogen gas (99% purity), sealed and re-frozen at -80°C as raw samples. Six replicate samples of thawed homogenate of raw potato of each genotype were used to determine moisture and carotenoid content. The second set of thawed pooled cubes was boiled at 95–100°C for 10 min to a soft texture as determined by insertion of a metal probe. After cooling in an ice bath, boiled potatoes were homogenized, transferred to 50 mL polypropylene tubes, blanketed with nitrogen gas and stored at -80°C. Six replicate samples of thawed homogenate of boiled potato of each genotype were analyzed to determine moisture and carotenoid content, and digested in vitro to determine bioaccessibility and uptake by Caco-2 human intestinal cells as described below.Wild-type (cv Desiree) and golden potato transgenic lines 17 and 30 were grown in the ENEA transgenic greenhouse as described previously . At the Simulated oral, gastric and small intestinal digestion was performed as previously described . BrieflyCaco-2 human intestinal cells  were grown and maintained in T-75 flasks. Medium contained 15% fetal bovine serum (FBS) during the cell replication phase and 7.5% once the monolayer was confluent. Fresh medium was added to confluent monolayers every second day and monolayers were used for experiments 11 days after achieving confluence . Freshlyet al. [™ C30 column  protected with a C18 guard column with a Waters 2695 HPLC system coupled with a Waters model 2996 photodiode array detector. βC, αC, lutein, zeaxanthin, violaxanthin, phytoene, phytofluene and αTC were identified by comparison of retention times and absorption spectra with pure standards. Quantities were estimated using five-point standard curves of each pure compound. Z-βC and Z-lutein were identified by their absorption spectra and quantities were estimated using the standard curve of the respective all E-isomers. Lutein esters in golden potato were identified as described elsewhere [1%1cm, were as follows: 2,550 at 445 nm for lutein in ethanol; 2,540 at 450nm for zeaxanthin in ethanol; 2,550 at 440 nm for violaxanthin in ethanol; 2,710 at 445nm for αC in hexane; 2,592 at 450nm for βC in petroleum ether; 1,250 at 286nm for phytoene in petroleum ether; 1,350 at 350nm for phytofluene in petroleum ether, and 75.8 at 292nm for αTC in ethanol [Carotenoids were extracted from raw and boiled potato tubers according to Kimura et al. . Extractet al. . Quantitlsewhere . Extinct ethanol . Apo-8’-Moisture content of homogenized raw and boiled golden potato was determined by drying 2–3 g of homogenized sample in tarred crucibles at 105°C to constant weight (± 2%). Cell protein content (11.8 ± 0.2 mg per flask) was determined by bicinchoninic acid assay  with bovine serum albumin as standard.in vitro digestion of the homogenates; and six monolayers of Caco-2 cells exposed to the mixed micelle fraction. Statistically significant differences (p < 0.05) were determined by one-way analysis of variance with Newman-Keuls as the post-hoc test.Results (means ± SD) were analyzed using GraphPad Prism, version 6  for each genotype as follows: six technical replicates of homogenates of raw and boiled potatoes from pooled cubes prepared from at least two potatoes from five plants for each genotype; six replicates of chyme and mixed micelle fraction generated during E- and Z-lutein, lutein esters, and violaxanthin, along with trace quantities of E-βC, Z-βC and zeaxanthin esters were present in WT potatoes. In contrast, βC and αC were the most abundant carotenoids in GP17 and GP30 with total concentrations of provitamin A carotenoids being more than 100-fold greater than that in WT. GP potato also contained 2–3 times higher quantities of total lutein than WT. The concentration of the epoxy-carotenoid violaxanthin in WT and GP potato was similar. Furthermore, while phytoene and phytofluene were not detected in WT potato, the concentrations of these acyclic carotenoids were similar to that of total lutein in GP17 and GP30. Phytoene, but not phytofluene, was previously reported in GP tubers [Moisture content of raw WT potato (86.1 ± 0.9%) was slightly, but significantly, greater than that of raw GP17 and GP30 potato (82.4 ± 0.9% and 82.4 ± 1.1% respectively). Total carotenoid content in raw GP17 and GP30 were 6 and 8 times, respectively, greater than in raw WT potato , S1 Fig.in vitro digestion of potatoes varied according to genotype and chemical structure, but the relative extent of micellarization of αTC was independent of genotype  of micellarization of carotenes, lutein, phytoene and phytofluene during genotype . Althouggenotype . The quaE-lutein (20%), Z-βC (18%) > Z-lutein (15–17%), E-βC (15–16%), and αC (13%)  .et al. [Arabidopsis thaliana [Potatoes are an important food crop in many Asian, African and South American countries with significant VAD prevalence –8. For eet al.  transforet al. ), cassavet al. , and soret al. ). GP17 aet al. –29, and et al. . The amoet al.  or 15-fothaliana . CollectCarotenoids are secondary metabolites that are susceptible to isomerization and degradation during the thermal processing of several plant foods including potato tubers , 33. TheIn vitro digestion is a cost-effective, qualitative predictor of the relative bioavailability of nutrients and other dietary compounds [E-βC and E-lutein after digestion of WT potato likely resulted from less competition for transfer from lipid droplets to mixed micelles as their concentrations were very low compared to those in GPs. The efficiency of micellarization of E-βC from GP17 and GP30  exceeded that reported for transgenic sorghum porridges  was also lower than the mean bioaccessibility of lutein and zeaxanthin (approx. 50% each) for seven accessions of yellow flesh boiled potatoes [Quantitation of the absorption of ingested provitamin A carotenoids and the retinyl esters generated by β-carotene 15,15’oxygenase requires feeding the food of interest to human subjects. The amounts of ingested provitamin A and its cleavage products possessing vitamin A activity is dependent on various factors, including the quantity and physicochemical properties of the carotenoids in the food matrix, post-harvest processing, style of preparing the food for consumption, other components of the meal, gastrointestinal health and the vitamin A status of the host . In vitrompounds . The pre.9–4.9%; ) and ora.9–4.9%; ), but le.9–4.9%; ). The repotatoes . The apppotatoes . Transpopotatoes . The ext approx. % each foBarua and Olson  reportedet al. [et al. [14C-phytoene in Mongolian gerbils was 23%. The efficiency of micellarization of phytoene and phytofluene during simulated small intestinal digestion of GP17 and GP30 exceeded that of carotenes as did their uptake from mixed micelles by Caco-2 cells. Future studies using Caco-2 cells are expected to yield insights on the mechanisms of apical uptake, possible intracellular metabolism, and the transfer of these more saturated, colorless C40 carotenoids across the basolateral membrane.Phytoene and phytofluene are colorless, acyclic precursors of lycopene, that are present in carotenoid rich foods such as tomatoes, carrots, red grapefruit and some peppers –29. Theset al.  digested [et al.  reportedet al. [Che et al.  recentlyet al.  and metaet al. . These rWe investigated the retention during boiling and bioaccessibility of provitamin A carotenes, xanthophylls, phytoene, phytofluene and α-tocopherol in two lines of GP. The data highlighted several nutritionally interesting features of GP tubers. These include the following: (i) increased amounts of provitamin A carotenoids, αTC (vitamin E), phytoene and phytofluene; (ii) greater than 82% retention of αTC and carotenoids other than phytofluene during boiling; (iii) relative bioaccessibilities of provitamin A, xanthophylls, phytoene, phytofluene and αTC similar to that reported for other vegetables; and (iv) uptake across the apical surface of Caco-2 cells from micelles generated during simulated digestion of GP ranging from 14–20% for provitamin A carotenoids and xanthophylls, 53% for αTC, 43–45% for phytoene and 23–27% for phytofluene. These data suggest GP has the potential to contribute to the provitamin A and αTC nutritional requirements of populations at risk of vitamin A and E deficiency, especially in countries where potato is an important staple food. Their use for dietary interventions should, therefore, be considered by national and international programs aimed at preventing vitamins A and E deficiency.S1 Fig(TIF)Click here for additional data file.S2 Figp < 0.05).Data are mean ± SD for 6 replicate samples from a pooled sample prepared from 10 tubers from 5 plants for each genotype. Different letters above error bars for each compound indicate that mean retention significantly differed for the indicated genotype ((TIF)Click here for additional data file.S3 Figp < 0.05).Data are mean ± SD for n = 6 replicate samples from a pooled sample prepared from 10 tubes from 5 plants of each genotypes. Different letters above error bars for each compound indicate that mean retention significantly differed for the indicated genotype ((TIF)Click here for additional data file.


The clinical characteristics of Takayasu arteritis (TAK) developing in individuals older than 40 years (TAK >40) are little-known.We retrospectively analyzed 43 patients with TAK treated at three hospitals in Japan from April 2000 to March 2016. From medical records we collected baseline variables at diagnosis including clinical symptoms, laboratory data, and subsequent relapses. We compared these indices in the patients with TAK onset at >40 years of age (TAK >40) to those with TAK onset ≤40 years (TAK ≤40). Multiplex cytokine/chemokine bead assays were performed using preserved serum supernatants from 24 patients with TAK and 40 healthy donors.p = 0.019), carotid bruit (7 (35%) vs. 16 (70%), p = 0.034), and chest pain (0 (0%) vs. 6 (26%), p = 0.023) compared to patients with TAK ≤40 (n = 23). The initial prednisolone dose was significantly lower in TAK >40 . Assessed by the log-rank test, the relapse-free survival rate after remission was significantly higher in the patients with TAK >40 (p = 0.029). The interleukin 17 levels were significantly lower in patients with TAK >40 compared to patients with TAK ≤40 and healthy donors.Of the 43 patients, 20 had TAK >40; this group had significantly fewer instances of orthostatic hypotension (2 (10%) vs. 10 (43%), Compared to TAK ≤40, TAK >40 could be treated by lower initial doses of prednisolone to achieve remission, and with fewer relapses. These differences might be due to the difference of T helper 17 (Th17) activity suggested by the cytokine profiles. Takayasu arteritis (TAK) is a type of large-vessel vasculitis that predominantly affects young women . In geneThe patient’s age at diagnosis is important in autoimmune diseases. For example, the clinical characteristics of patients with elderly-onset rheumatoid arthritis (RA) are different from those of younger patients with RA . SimilarOhigashi et al. reported the clinical characteristics of patients with TAK >40; these patients had significantly more coronary artery disease and hypertension, and significantly fewer instances of aortic regurgitation . HoweverHere we attempted to identify the clinical differences, including relapse and prognosis, in patients with TAK >40 and patients with TAK ≤40. In addition, we evaluated differences in the profile of serum cytokines and chemokines at diagnosis between TAK >40 and TAK ≤40.We retrospectively analyzed all patients with TAK who were registered and followed by the Nagasaki University Hospital, Sasebo Chuo Hospital, and NHO Nagasaki Medical Center between April 2000 and March 2016. We included patients with diagnosis based on the ACR 1990 criteria for the classification of TAK .Fig. 1FlTo compare the characteristics of TAK among patients of different age at disease onset, we divided the 43 patients by age at disease onset: >40 years old (the TAK >40 group) and ≤40 years old at disease onset (the TAK ≤40 group). None of the TAK >40 patients had history of symptoms of TAK 40 years of age. TAK >40 fulfilled three or more items in the ACR criteria for TAK without the definition of onset of age. TAK ≤40 also fulfilled three or more items, including age.Relapse was determined by Japan College of Rheumatology-certified rheumatologists by confirmation of the recurrence of active disease as described . The criWe collected baseline information at diagnosis, including clinical characteristics and laboratory data and subsequent relapses, by accessing the patients’ medical records. The baseline clinical characteristics at diagnosis included age, sex, symptoms, body mass index (BMI), history of smoking, angiographic classification types, and complications including diabetes mellitus (DM) and chronic kidney disease (CKD) (these complications influence vascular conditions), inflammatory bowel diseases (IBD), and pyoderma gangrenosum (PG) (TAK is frequently complicated with IBD or PG).The initial laboratory data included human leukocyte antigen (HLA) typing, white blood cell count, hemoglobin, platelet count, C reactive protein (CRP), erythrocyte sedimentation rate (ESR), albumin (Alb), and immunoglobulin G (IgG). Baseline information at diagnosis was collected within 2 weeks before treatment started, except for the HLA typing.We analyzed the corticosteroid doses received in both the initial treatment and on relapse in the 43 patients with TAK who had been followed up for ≥6 months. Information on the use of pulsed methylprednisolone and immunosuppressants including methotrexate, cyclosporine, and tacrolimus was also collected, as was information on the use of biologics including infliximab, adalimumab, and tocilizumab. The corticosteroid doses were calculated as daily prednisolone equivalents. The rate of reduction in the prednisolone dose (mg/month) was calculated using the reduction dose during the 6 months after starting corticosteroid treatment. In particular, we subtracted the prednisolone dose at 6 months after remission from the initial prednisolone dose and we divided the difference by 6 months.Multiplex cytokine/chemokine bead assays were performed using diluted serum supernatants and Milliplex MAP Human Cytokine/Chemokine Panel 1 Pre-mixed 41Plex  analyzed with a Bio-Plex® MAGPIX™ Multiplex Reader  according to the manufacturers’ instructions. The cytokines/chemokines measured by the Milliplex MAP Human Cytokine/Chemokine Panel 1 Pre-mixed 41Plex included vascular endothelial growth factor (VEGF), TNF-β, TNF-α, transforming growth factor (TGF)-α, regulated and normal T cell expressed and secreted (RANTES), platelet-derived growth factor (PDGF)-AB/BB, PDGF-AA, macrophage inflammatory protein (MIP)-1β, MIP-1α, myeloid dendritic cells (MDC) (CCL22), monocyte chemotactic protein (MCP)-3, MCP-1, IP-10, IL-17, IL-15, IL-13, IL-12 (p70), IL-12 (p40), IL-10, IL-9, IL-8, IL-7, IL-6, IL-5, IL-4, IL-3, IL-2, IL-1ra, IL-1β, IL-1α, interferon (IFN)-γ, IFN-α2, growth-related cytokine (GRO), granulocyte macrophage-colony stimulating factor (GM-CSF), granulocyte-colony stimulating factor (G-CSF), fractalkine, Flt-3 ligand, fibroblast growth factor (FGF)-2, eotaxin, epidermal growth factor (EGF), and sCD40L.Serum obtained from patients with TAK at diagnosis and from HD was centrifuged within 30 minutes at 1500 rpm at 4 °C for 5 minutes, and the liquid phase serum was stored at −80 °C until use. Serum from HD was divided into two analysis groups of HD older than 40 years (HD >40) and HD who were ≤40 years old (HD ≤40). We compared serum from HD with serum from patients with TAK >40 and TAK ≤40. Serum was obtained from HD in 2013–2015. The earliest obtained serum from a patient at diagnosis was 2009. Multiplex cytokine/chemokine bead assays using serum from patients and HD were performed in 2015–2016.p value <0.05 was considered significant. The Steel-Dwass test was performed for comparisons of multiplex cytokine/chemokine bead assay results in the four groups, i.e., TAK >40, TAK ≤40, HD >40, and HD ≤40. All statistical analyses were performed using JMP Statistical Software, ver. 11  and GraphPad Prism ver. 7.0 .We compared the baseline characteristics, including demographic data, hematologic data, serum markers, treatments, and follow-up duration between the TAK >40 and TAK ≤40 groups. Variables are described using frequencies for categorical variables and with the median and interquartile range (IQR) for quantitative variables. We assessed the association between variables using Fisher’s exact test for categorical variables and Wilcoxon’s rank sum test for quantitative variables. The relapse-free survival rate after remission was estimated by the Kaplan-Meier method. The association between the relapse-free survival rate and TAK >40 or TAK ≤40 was evaluated by the log-rank test. All tests were two-sided and a n = 23) groups. Median age in the TAK >40 and TAK ≤40 groups was 56 and 23 years, respectively. The positive rate of HLA-B52, the rate of complications, and angiographic classification were not significantly different between the two groups.The study population was 43 patients who were identified over the 16-year period. Twenty of the patients with TAK were classified as TAK >40. Table p = 0.019), carotid bruit (7 (35%) vs. 16 (70%), p = 0.034) and chest pain (0 (0%) vs. 6 (26%), p = 0.023) at diagnosis compared to the TAK ≤40 group. There were no significant between-group differences in CRP or ESR. Furthermore, because we could not eliminate the potential bias in the diagnosis of TAK by using the criterion of age at disease onset, we extracted TAK ≤40 who fulfilled at least three of the five criteria excluding the criterion of age at disease onset from the whole TAK ≤40 group and then compared these patients with the TAK >40 group  vs. 10 (43%), 0% vs. 10p = 0.024). The initial prednisolone dose per kg of body weight was also significantly lower in the TAK >40 compared to the TAK ≤40 group . There were no significant between-group differences in the other immunosuppressants. Five patients in the TAK ≤40 group were treated with infliximab compared to none of the patients in the TAK >40 group. There were no significant between-group differences in the rate of reduction in the dose of prednisolone or follow-up duration.Table p = 0.029) using the Kaplan-Meier method , and only one patient died, due to pancreatic cancer.We evaluated the relapse-free survival rates in the TAK >40 and TAK ≤40 groups, and observed that the TAK >40 patients had significantly fewer relapses as assessed by the log-rank test p = 0.09 using tWe performed a multiplex cytokine/chemokine bead assay using serum from patients with TAK >40 and patients with TAK ≤40 obtained at diagnosis Fig. . We obtaThe TAK >40 group had significantly lower IL-17 compared to the TAK ≤40 and the HD >40 groups (median (IQR) 1.1 (0.2–2.5) pg/mL for TAK >40; 8.4 (2.6–17.8) pg/mL for TAK ≤40; 5.1 3.5–9.0) pg/mL for HD >40; and 9.4 (6.0–39.5) for HD ≤40). In the complete series of 24 patients with TAK, IL-6 was significantly higher in the HD >40 and the HD ≤40 groups (median (IQR) 26.9 (8.1–65.2) pg/mL; 23.4 (8.2–56.1) pg/mL; 0.6 (0–2.1) pg/mL; and 1.4 (0–5.5) pg/mL, respectively). TNF-α in the TAK >40 group was significantly higher than that in the HD >40 group (16.3 (11.2–22.1) pg/mL for TAK >40; 13.5 (10.0–21.0) pg/mL for TAK ≤40; 7.1 (5.3–9.4) pg/mL for HD >40; and 8.3 (6.9–13.0) pg/mL for HD ≤40 ). However, there were no significant differences in IL-6 or TNF-α between the TAK >40 and TAK ≤40 groups. There were no significant differences between the TAK >40 and TAK ≤40 groups in IFN-γ, IL-2, IL-4, IL-5, IL-8, IL-10, IL-13, GM-CSF, or RANTES 1 and Th17 pathways in patients with TAK contributes to the systemic and vascular manifestations , 11. In We observed that IL-6 and TNF-α were higher in patients with TAK compared to the HD, which is compatible with previous studies using ELISA –13. Our We observed no significant differences in Th1-related cytokines, including IFN-γ and IL-2 and Th2-related cytokines, including IL-4, IL-5, and IL-13, between the TAK >40 and TAK ≤40 groups. Glucocorticoids are a standard part of the treatment of TAK, and a previous study demonstrated a decrease in Th1-derived cytokines  produced from PBMCs after glucocorticoid treatment, whereas the production of Th17-derived cytokines  was not inhibited . Misra eBy contrast, elevated serum IL-17 in patients with TAK compared to HD have been reported previously . In addiOur study has some limitations. First, we could not eliminate potential bias in the diagnosis of TAK by using the criterion of age at disease onset. Because TAK classification criteria include the criterion of age at disease onset ≤40 years, a prospective study is absolutely imperative to eliminate bias in the diagnosis of TAK. To partially address this problem, we also performed comparison of patients with TAK ≤40 who fulfilled at least three criteria excluding the age criterion and those with TAK >40. Second, more cases are needed to confirm these results, particularly in assessing the results of multiplex cytokine/chemokine bead assays. However, this study is valuable as a hypothesis-generating study. Third, we could not assess the T cell subpopulations using fluorescence-activated cell sorting because this was a retrospective study. We plan to analyze T cell subpopulations in patients with TAK >40 and TAK ≤40 in the future. Fourth, we used the ACR criteria to differentiate TAK from GCA and to exclude GCA. TAK and GCA appear more similar than different, based on clinical and pathological findings . In contPatients with TAK >40 had fewer relapses after remission compared to patients with TAK ≤40. The levels of IL-17 in the TAK >40 group at diagnosis were lower compared to those in the TAK ≤40 group. Our findings clarified the relationships between age, clinical characteristics, and profiles of cytokines and chemokines in patients with TAK.