Prognostic impact of cortactin in patients with hypopharyngeal cancer and its role for tegafur-uracil maintenance after adjuvant chemoradiotherapy.

The prognosis of patients with hypopharyngeal cancer (HPC) remains poor. Our study aims to investigate the prognostic impact of cortactin in patients with HPC and its role for tegafur-uracil (UFUR) maintenance after adjuvant chemoradiotherapy (CRT). Patients who were diagnosed to have HPC and underwent laryngopharyngectomy followed by adjuvant CRT were enrolled into our study. Immunohistochemical staining was performed for cortactin evaluation. Kaplan-Meier curves were depicted for recurrence-free survival (RFS) and overall survival (OS). A total of 157 patients were enrolled into our study. After stratified by cortactin, 53 patients were cortactin (+) and 104 patients were cortactin (-). The median RFS was 86.7 months in cortactin (-) and 10.2 months in cortactin (+) (P < 0.001). The median OS was 93.4 months in cortactin (-) and 16.9 months in cortactin (+) (P < 0.001). Patients were further classified according to UFUR maintenance or not after adjuvant CRT. In cortactin (+) patients, the median RFS and OS were 13.6 months versus 7.0 months (P = 0.006) and 24.0 months versus 10.0 months (P < 0.001) in UFUR (+) and UFUR (-), respectively. In cortactin (-) patients, the median RFS and OS were 96.0 months versus 72.2 months (P = 0.262) and 98.5 months versus 105.0 months (P = 0.665) in UFUR (+) and UFUR (-), respectively. Cortactin has a significantly impact in HPC patients. UFUR maintenance provided survival benefits in patients with cortactin (+) after adjuvant CRT.

The use of a low-flux hemo-dialyzer is associated with impaired platelet aggregation in patients undergoing chronic hemodialysis.

In patients with chronic hemodialysis (HD), both abnormal thrombotic and bleeding events are commonly observed. Uremic platelet dysfunction is one of the important attributing factors. Moreover, HD may also result in aggregation dysfunction of platelets during the therapeutic procedure. However, how the HD process affects platelet and coagulation function is unknown and dialyzer membrane flux could have an impact on it. We aimed to compare the impacts of low-flux and high-flux HD on the platelet function of patients undergoing chronic HD. This was a cross-sectional study conducted in the HD unit of E-Da hospital in Taiwan. A total of 78 patients with maintenance HD three times per week for more than one year, including 40 with high- and 38 with low-flux hemodialysis, were recruited. Their platelet functions were evaluated using an in vitro platelet function analyzer (PFA-100) before and after the HD session. Of the 78 patients undergoing HD, 60 (76%) had prolonged pre-dialysis collagen/epinephrine (CEPI) and collagen/adenosine diphosphate closure times. Those receiving low-flux dialyzer had a significant increase in CEPI closure time (pre-dialysis 212.3 ±â€…62.1 seconds. post-dialysis 241.5 ±â€…64.3 seconds, P = .01), but not collagen/adenosine diphosphate closure time, after HD. After adjusting confounding factors, only the low-flux dialyzer demonstrated an independent association with the prolonged CEPI closure time after HD therapy (odds ratio = 23.31, 95% CI: 1.94-280.61, P = .01). We observed that impaired platelet aggregation is prevalent in patients undergoing chronic HD. Therefore, the use of low-flux dialyzers may further worsen platelet aggregation after dialysis. Patients with uremic bleeding diathesis should take precautions. We suggest that further studies using flow cytometry should be conducted to explore the mechanism of dialysis flux and platelet activity during HD.

Influence of RBC Indices on HbA1c Measurement by Capillary Electrophoresis and HPLC Methods.

BACKGROUND: HbA1c is the gold standard of diabetic surveys to monitor the long-term glycemic control. Anemia is cited as a major confounder to HbA1c analysis; however, the effect of RBC indices influences on HbA1c analysis is not known. The aim of this study is to compare ion-exchange high-performance liquid chromatography, and capillary electrophoresis to evaluate the influence of RBC parameters on HbA1c values in anemia patients. METHODS: Erythrocyte parameters were collected from the 307 randomly selected specimens from the Hematology division. HbA1c was measured on the same specimen using Tosoh G8 and Capillarys 2 Flex Piercing on the same day. RESULTS: There is acceptable concordance between the results of capillary electrophoresis and HPLC methods (R2 = 0.953, p < 0.001). However, significant differences in HbA1c value between the two assay methods were obtained in the patients with abnormal RBC indices (p < 0.001). CONCLUSIONS: Our results demonstrated HbA1c differences were significantly different in the patients with low Hb (≤ 8 g/dL) and high RDW-CV (≥ 13.7%). It is suggested that in the analysis of HbA1c level in anemia patients, simultaneous testing for hemoglobin level is needed. In addition, development of a new reference value of HBA1c for patients with severe anemia should be considered.

The role of conserved arginine and proline residues in enterovirus VP1 protein.

BACKGROUND: High diversity of VP1 protein among enteroviruses has been a barrier in developing universally effective antiviral drugs. To maintain structure stability during evolution, several residues of VP1 protein of enteroviruses are conserved. Therefore, investigation of highly conserved residues in VP1 protein may provide information for antiviral drug candidates against enteroviruses. METHODS: To identify highly conserved amino acid sequences of the VP1 in enterovirus genus, the Consurf and CABS-flex 2.0 web software were applied. Through the combination with secondary structure information, we focused on conserved amino acids of VP1 property analysis. RESULTS: Most conserved residues of VP1 were in the interior and interacted with VP2, VP3 and VP4 capsid proteins. Structure of EV-A71 (PDB code 4AED) showed conserved residues were at hydrophobic pocket and close to the junction between the loop and ß-barrel. Interestingly, arginine was the most common conserved residue of VP1. Proline was the second most common conserved residue and was found in the loop and ß-barrel intersection areas. VP1 protein flexibility was associated with the secondary structure. Conserved residues of VP1 in ß-barrel showed significantly low flexibility. CONCLUSION: Through large scale sequence analysis, we identified the amino acid distribution and location of conserved residues in VP1. This knowledge can be extrapolated for the Enterovirus genus and may contribute to developing the potential compound as an anti-enteroviral agent.

Association between ADAMTS13 deficiency and cardiovascular events in chronic hemodialysis patients.

A mild decrease of ADAMTS13 (a disintegrin and metalloprotease with thrombospodin type 1 motif 13) could attribute to stroke and coronary heart disease in general population. However, the role of ADAMTS13 in hemodialysis (HD) patients remains to be explored. This cross-sectional and observational cohort study enrolled 98 chronic HD patients and 100 normal subjects with the aims to compare the ADAMTS13 activity between chronic HD patients and normal subjects, and to discover the role of ADAMTS13 on the newly developed cardiovascular events for HD patients in a 2-year follow-up. Our HD patients had a significantly lower ADAMTS13 activity than normal subjects, 41.0 ± 22.8% versus 102.3 ± 17.7%, p < 0.001. ADAMTS13 activity was positively correlated with diabetes, triglyceride and hemoglobin A1c, and negatively with high-density lipoprotein cholesterol levels in HD patients. With a follow-up of 20.3 ± 7.3 months, the Cox proportional hazards model revealed that low ADAMTS13, comorbid diabetes, and coronary heart diseases have independent correlations with the development of cardiovascular events. Our study demonstrated that chronic HD patients have a markedly decreased ADAMTS13 activity than normal subjects. Although ADAMTS13 seems to correlate well with diabetes, high triglyceride and low high-density lipoprotein cholesterol levels, ADAMTS13 deficiency still carries an independent risk for cardiovascular events in chronic HD patients.

The Association of Urinary Sclerostin and Renal Magnesium Handling in Type 2 Diabetic Patients with Chronic Kidney Disease.

INTRODUCTION: Sclerostin could enhance renal excretion of calcium (Ca) and phosphate (P). The association between sclerostin and magnesium (Mg) has not yet discovered. In patients with type 2 diabetes mellitus (T2DM) or chronic kidney disease (CKD), higher serum sclerostin and altered renal excretion of Ca, P, and Mg were detected. Therefore, we tried to evaluate if there was any association between sclerostin and fractional excretion of Ca, P, and Mg (FeCa, FeP, and FeMg) in T2DM with CKD. METHODS: In this prospective cohort study, 43 T2DM patients without CKD or with CKD stage 1-5 were enrolled. Values of parameters, including serum and urine sclerostin, were collected at baseline and 6 months later. For baseline data, the Mann-Whitney U test, χ2 test, or Spearman's correlation were used. For multivariate repeated measurement analysis, generalized estimating equation (GEE) model was utilized. RESULTS: Patients with lower estimated glomerular filtration rate had higher serum sclerostin, FeP, FeMg, and lower FeCa. By correlation analysis, serum sclerostin was negatively associated with FeCa (p = 0.02) and positively associated with FeP (p = 0.002). The urine sclerostin to creatinine ratio (Uscl/Ucre) was positively correlated with FeP (p = 0.007) and FeMg (p = 0.005). After multivariate analyses by GEE model, serum sclerostin was still inversely associated with FeCa, while Uscl/Ucre was significantly associated with FeMg. On the other hand, FeP lost its associations with serum sclerostin or Uscl/Ucre. CONCLUSION: In our study population of T2DM patients with or without CKD, the inverse correlation between serum sclerostin and FeCa could not be explained by the calciuric effect of sclerostin. In addition, a newly discovered positive association between urinary sclerostin and FeMg indicated a possible role of urinary sclerostin in regulating renal Mg handling especially over distal convoluted tubules.

Novel Application of Magnetite Nanoparticle-Mediated Vitamin D3 Delivery for Peritoneal Dialysis-Related Peritoneal Damage.

PURPOSE: Vitamin D3 is useful for the treatment of peritoneal dialysis (PD)-related peritoneal damage, but its side effects, such as hypercalcemia and vascular calcification, limit its applicability. Thus, we developed vitamin D-loaded magnetic nanoparticles (MNPs) and determined their therapeutic efficacy and side effects in vivo. MATERIALS AND METHODS: Alginate-modified MNPs were combined with 1α, 25 (OH)2D3 to generate vitamin D-loaded nanoparticles. The particles were conjugated with an antibody against peritoneum-glycoprotein M6A (GPM6A). The particles' ability to target the peritoneum was examined following intraperitoneal administration to mice and by monitoring their bio-distribution. We also established a PD animal model to determine the therapeutic and side effects of vitamin D-loaded MNPs in vivo. RESULTS: Vitamin D-loaded MNPs targeted the peritoneum better than vitamin D3, and had the same therapeutic effect as vitamin D3 in ameliorating peritoneal fibrosis and functional deterioration in a PD animal model. Most importantly, the particles reduced the side effects of vitamin D3, such as hypercalcemia and body weight loss, in mice. CONCLUSION: Vitamin D-loaded MNPs could be an ideal future therapeutic option to treat PD-related peritoneal damage.

Indoxyl Sulfate Mediates the Low Inducibility of the NLRP3 Inflammasome in Hemodialysis Patients.

The NLRP3 inflammasome is responsible for the maturation of caspase-1 and interleukin-1ß (IL-1ß). Despite the study about basal activity of the NLRP3 inflammasome in hemodialysis (HD) patients, little is known about its inducibility in the milieu of uremia. Peripheral blood mononuclear cells (PBMCs) isolated from 11 HD patients and 14 volunteers without a history of chronic kidney disease, as well as macrophages with or without the uremic toxin indoxyl sulfate (IS) pretreatment, underwent canonical NLRP3 inflammasome induction. Despite the high plasma levels of IL-1ß in HD patients, caspase-1 and IL-1ß in the PBMCs of HD patients remained predominantly immature and were not secreted in response to the canonical stimulus. In addition, while IS alone facilitated the inflammasome-independent secretion of IL-1ß from macrophages, IS exposure before induction reduced the inducibility of the NLRP3 inflammasome, characterized by insufficient maturation of caspase-1. The low expression of inflammasome components, which was observed in both IS-pretreated cells and the PBMCs of HD patients, was probably responsible for the low inducibility.

Frequent transfusion and specific HLA-DRB1 alleles correlate independently with Miltenberger blood group alloimmunization in dialysis patients.

Anti-"Mia " is the most frequent irregular RBC antibody in Taiwan due to high prevalence of Miltenberger antigens. Dialysis patients, a special patient group that needs frequent transfusions of RBCs, may have the greatest risk for developing anti-"Mia " antibodies. The aim of this study was to investigate the association between specific HLA-DRB1 alleles and Mi alloimmunization among dialysis patients. A cohort of 267 maintenance dialysis patients who had ever received at least one RBCs transfusion was enrolled. Anti-"Mia " was identified in patients' serum using the manual polybrene technique and HLA-DRB1 genotyping was carried out using polymerase chain reaction and sequence-specific oligonucleotide probe nonradioactive hybridization. Twenty-one (7.9%) of patients had positive anti-"Mia " tests and had received significantly more units of RBC transfusions than those without anti-"Mia " antibodies (11.3 ± 14.5 U vs 4.5 ± 10.1 U, P = .005). DRB1*04, *07, and *09 alleles were also more prevalent in patients with anti-"Mia " compared to those without Mi III alloimmunization. The multivariate logistic regression analysis showed that the number of RBC transfusions and the presence of DRB1*04, *07, and *09 phenotypes correlated independently with Mi III immunization (Odds ratios [OR] 1.05 (P = .001) for each unit of RBCs transfused; ORs 4.80 (P = .006), 12.29 (P = .005), and 5.42 (P = .003) for presence of DRB1*04, *07, and *09, respectively). This study is the first to demonstrate markedly high prevalence of Mi III alloimmunization in dialysis patients of Taiwan. Extending Mi III matching for RBC transfusions in susceptible dialysis patients may help to reduce the risk of Mi III alloimmunization in this patient population.

Extensively drug-resistant Haemophilus influenzae - emergence, epidemiology, risk factors, and regimen.

BACKGROUND: Concern about Haemophilus influenzae infection has been increasing over recent decades. Given the emergence of H. influenzae with severe drug resistance, we assessed the prevalence of as well as risk factors and potential therapies for extensively drug-resistant (XDR) H. influenzae infection in Taiwan. RESULTS: In total, 2091 H. influenzae isolates with disk diffusion-based antibiotic susceptibility testing from 2007 to 2018 were enrolled. H. influenzae strains resistant to ampicillin, chloramphenicol, levofloxacin, and trimethoprim-sulfamethoxazole tended to be isolated from patient wards (≧41%), whereas those resistant to amoxicillin-clavulanate, cefotaxime, and cefuroxime were more likely to be isolated from intensive care units (approximately 50%). XDR H. influenzae was first identified in 2007, and its incidence did not significantly change thereafter. Overall prevalence of single, multiple, and extensively drug-resistant H. influenzae over 2007-2018 was 21.5% (n = 450), 26.6% (n = 557), and 2.5% (n = 52), respectively. A stepwise logistic regression analysis revealed that blood culture (odds ratio: 4.069, 95% confidence intervals: 1.339-12.365, P = 0.013) was an independent risk factor for XDR H. influenzae infection. No nosocomial transmission of XDR H. influenzae observed. Antibiotic susceptibility testing results demonstrated that cefotaxime was effective against 78.8% (n = 41) of the XDR strains. CONCLUSIONS: The presence of XDR H. influenzae strains was identified in Taiwan, and cefotaxime was efficacious against most of these strains.

High correlation between human rhinovirus type C and children with asthma exacerbations in Taiwan.

BACKGROUND/PURPOSES: Human rhinovirus type C (HRV-C) has been associated with asthma exacerbation (AE) in children in several countries. However, in Taiwan the association between HRV, especially HRV-C, and AE in children has yet to be elucidated. We sought to investigate the prevalence of respiratory viruses in children with acute lower respiratory tract infection (ALRTI) in Taiwan and the association between different types of HRV and AE in children. METHODS: This prospective study was conducted from 2011 to 2013, and enrolled children with ALRTI, including an asthma exacerbation group (AE; n = 28) and a Non-asthma group (n = 66). Viruses were detected by culture, reverse transcription-polymerase chain reaction, and molecular sequencing of nasopharyngeal swabs. RESULTS: The prevalence of identified respiratory viruses was 78.6% in the AE group and 65.2% in the Non-asthma group. The prevalence rates of HRV and HRV-C were significantly higher in the AE group than in the Non-asthma group (67.9% vs. 33.3% in HRV, p = 0.002; and 50% vs. 15.2% in HRV-C, p < 0.001). Among the children with HRV, the prevalence of HRV-C (68.4%) was higher than that of the other types of HRV (31.6%, including HRV-A 26.3%, and HRV-B 5.3%) in the AE group but not in the Non-asthma group (40.9% vs. 59.1%). CONCLUSIONS: HRV is the most predominant viral infection responsible for pediatric AE in Taiwan, and HRV-C is responsible for more of these exacerbations than HRV-A or HRV-B.

The Clinical Implication of Vitamin D Nanomedicine for Peritoneal Dialysis-Related Peritoneal Damage.

PURPOSE: Vitamin D is a novel potential therapeutic agent for peritoneal dialysis (PD)-related peritoneal fibrosis, but it can induce hypercalcemia and vascular calcification, which limits its applicability. In this study, we create nanotechnology-based drug delivery systems to investigate its therapeutics and side effects. MATERIALS AND METHODS: 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [amino-(polyethylene glycol)2000] (DSPE-PEG) and L-α-phosphatidylcholine (PC), which packages with 1α,25(OH)2D3, were used to construct vitamin D nanoliposomes. To confirm the function and safety of vitamin D nanoliposomes, peritoneal mesothelial cells were treated with TGF-ß1 and the reverse was attempted using vitamin D nanoliposomes. Antibodies (Ab) against the peritoneum-glycoprotein M6A (GPM6A) Ab were conjugated with vitamin D nanoliposomes. These particles were implanted into mice by intraperitoneal injection and the animals were monitored for the distribution and side effects induced by vitamin D. RESULTS: Vitamin D nanoliposomes were taken up by the mesothelial cells over time without cell toxicity and it also provided the same therapeutic effect in vitro. In vivo study, fluorescent imaging showed vitamin D nanoliposomes allow specific peritoneum target effect and also ameliorate vitamin D side effect. CONCLUSION: Nanoliposomes vitamin D delivery systems for the prevention of PD-related peritoneal damage may be a potential clinical strategy in the future.

Zoledronic acid blocks the interaction between breast cancer cells and regulatory T-cells.

BACKGROUND: Zoledronic acid (ZA), a nitrogen-containing bisphosphonate, inhibits osteoclastogenesis. Emerging evidence suggests that ZA has anti-tumor and anti-metastatic properties for breast cancer cells. In a mouse model of ZA-related osteonecrosis of the jaw, ZA administration was found to suppress regulatory T-cells (Tregs) function. Our previous reports also demonstrated ZA acted as an immune modulator to block Tregs. Manipulation of Tregs represents a new strategy for cancer treatment. However, the relationship among ZA, Tregs, and cancer cells remains unclear. In this study, we investigated the effects of ZA on the interaction of breast cancer cells and Tregs. METHODS: The anti-tumor effect of ZA on triple negative breast cancer cell lines were validated by XTT, wound healing and apoptosis analysis. A flow cytometry-based assay was used to analyze the immunosuppressive effect of Tregs treated with media conditioned by breast cancer cells, and a transwell assay was used to evaluate the chemotactic migration of Tregs. Differential gene expression profile on MDA-MB-231 treated with ZA (25 µM) was analyzed by. microarrays to describe the molecular basis of actions of ZA for possible direct anti-tumor effects. Enzyme-linked immunosorbent assays and quantitative real-time PCR were used to investigate the effect of ZA on the expression of cytokines/factors by breast cancer cells. RESULTS: ZA was found to inhibit the proliferation and migration of breast cancer cells. Media conditioned by the MDA-MB-231 cells promoted the expansion, chemotactic migration, and immunosuppressive activity of Tregs, and these effects were attenuated in a dose-dependent manner by ZA treatment, and the attenuation was due to reduced expression of selected breast cancer cell factors (CCL2, CCL5, and IDO). CONCLUSIONS: ZA can significantly affect the interaction between breast cancer cells and Tregs. Our findings indicate that ZA is a potential therapeutic agent that can be used to reduce cancer aggressiveness by abolishing the supportive role of Tregs.

Cortactin as a potential predictor of second esophageal neoplasia in hypopharyngeal carcinoma.

OBJECTIVE: Hypopharyngeal carcinoma has a very poor prognosis. The high incidence of second esophageal neoplasia is one of the major causes. To establish an efficient follow-up scheme for increasing the diagnostic yield and reducing the adverse impact of second esophageal neoplasia on survival, the purpose of this study was to explore a biomarker to predict second esophageal neoplasia. METHODS: In this retrospective cohort study, consecutive tissue specimens from those patients who underwent tumor resection between September 2007 and October 2015 were collected. Gene amplification was performed by real-time PCR. The expression of cortactin was evaluated by immunohistochemistry. The predictive risk factors of developing second esophageal neoplasia and prognostic factors related to survival were analyzed. RESULTS: A total of 187 patients were included with a mean follow-up of 48months (12-118months). Second esophageal tumors were found in 53 (28.3%), including 41 (21.9%) esophageal squamous cell carcinoma and 12 severe dysplasia. The results of multivariate analyses revealed that age (OR 2.81, 95% CI 1.16-6.78), cortactin overexpression (OR 2.49, 95% CI 1.17-5.33), and stage IV versus I (OR 6.49, 95% CI 1.68-25.18) were independent predictors of second esophageal neoplasia, and second esophageal neoplasia (HR 1.78, 95% CI 1.05-3.01) was an independent predictor of overall survival. CONCLUSION: This is the first report to identify a potential biomarker for predicting second esophageal neoplasia in patients with hypopharyngeal carcinoma. In those patients with cortactin overexpression and younger age (≤60years old), close surveillance for second esophageal neoplasia is required. In addition, the real effect of cortactin overexpression on development of primary esophageal carcinoma is required to be validated in a large cohort study.

Differences in TLR7/8 activation between monocytes and macrophages.

The recognition of single-stranded RNA by TLR7/8 leads to the production of NF-κB-mediated cytokines and type I IFNs. However, the role of TLR7/8 activation in monocytes and macrophages is still unclear. The aim of this study was to investigate the differences in the activation of TLR7/8 between these two cell types. Microarray analysis, qRT-PCR and flow cytometry were used to analyse TLR7/8 signalling pathways in monocytes and macrophages after stimulation with agonists. Our data indicated that TLR8 agonists activated the NF-κB- and IRF-mediated pathways in THP-1 cells, whereas TLR7 agonists did not. However, silent TLR8 and enhanced TLR7 expression could increase TLR7-induced NF-κB activation in monocytes. TLR7 and TLR8 agonists induced NF-κB activation but no ISG response in PMA-differentiated THP-1 cells. The mRNA levels of pro-inflammatory cytokine were elevated upon CL075 stimulation in macrophages compared to monocytes. Thus, TLR7 and TLR8 might modulate different immune responses in monocytes and macrophages.

Bacterial colonization patterns in daily chlorhexidine care at the exit site in peritoneal dialysis patients-A prospective, randomized controlled trial.

Bacterial colonization patterns in daily chlorhexidine care at the exit site in peritoneal dialysis (PD) patients were not known. We performed a prospective, randomized controlled trial enrolling 89 PD patients. After stratification by initial Staphylococcus aureus (SA) carrier status, patients were randomly assigned to receive daily 4% chlorhexidine care (intervention group) or normal saline (control group) at the exit site. Monthly, we cultured bacteria from the exit site and nasal swabs for 1 year. The SA colonization rates at exit site at 6 and 12 months were significantly lower in the intervention group than the control group (5.0% vs. 22.9%, p = 0.023 and 8.6% vs. 28.1%, p = 0.037 for 6 and 12 months, respectively). The Methicillin-resistant SA (MRSA) colonization rate at exit site at 6 months was similar (5.7% vs. 2.5%,p = 0.596) in control and intervention group, but significantly lower in the intervention group than the control group at exit site at 12months (0% vs. 12.5%, p = 0.047). The gram-negative bacilli (GNB) colonization rates were similar between the intervention and control groups at 6 and 12 months. Genotyping of all MRSA isolates showed ST (sequence type) 59 was the most predominant clone. In conclusion, chlorhexidine care at the exit site in PD patients may be a good strategy for SA and MRSA decolonization. TRIAL REGISTRATION: ClinicalTrials.gov NCT02446158.

Immune modulation of CD4+CD25+ regulatory T cells by zoledronic acid.

BACKGROUND: CD4+CD25+ regulatory T (Treg) cells suppress tumor immunity by inhibiting immune cells. Manipulation of Treg cells represents a new strategy for cancer treatment. Zoledronic acid (ZA), a nitrogen-containing bisphosphonate, inhibits the expression of receptor activator of nuclear factor kappa-B ligand (RANKL) on osteoblasts to inhibit osteoclastogenesis. In a mouse model of bisphosphonate-related osteonecrosis of the jaw, administration of ZA suppressed Treg-cell activity and activated inflammatory Th17 cells. However, the interaction between ZA and Treg cells remained unclear. This study investigated the immune modulation of Treg cells by ZA. METHODS: Flow cytometry was used to analyze the phenotypic and immunosuppressive characteristics of Treg cells treated with ZA. Chemotactic migration was evaluated using transwell assays. Quantitative real-time PCR (qRT-PCR) was used to investigate the effect of ZA on the expression of suppressive molecules by Treg cells. RESULTS: Proliferation of isolated Treg cells in culture was inhibited by ZA, although ZA did not induce apoptosis. qRT-PCR and flow cytometry showed that ZA significantly downregulated the expression of CCR4, CTLA4, PD-1 and RANKL on Treg cells. Chemotactic migration and immunosuppressive functions were also significantly attenuated in Treg cells pretreated with ZA, and these effects were dose-dependent. Co-culture with Treg cells significantly increased the migration rate of breast cancer cells, while pretreatment of Treg cells with ZA attenuated this effect. CONCLUSIONS: Our findings demonstrated that ZA acted as an immune modulator by significantly inhibiting the expansion, migration, immunosuppressive function and pro-metastatic ability of Treg cells. Immunomodulation of Treg cells by ZA represents a new strategy for cancer therapy.

Functional polymorphisms of the TLR7 and TLR8 genes contribute to Mycobacterium tuberculosis infection.

Tuberculosis (TB) has recently re-emerged as a major global public health threat and Mycobacterium tuberculosis (MTB) is a highly successful pathogen that evolved remarkable strategies to establish persistent infection. There is strong evidence that host genetic factors influence individual susceptibility to TB. In this study, we evaluated the associations between the TLR7 and TLR8 genetic polymorphisms and TB susceptibility in Chinese individuals. The results demonstrated that the frequency of the TLR8-129C allele was higher in male patients with pulmonary TB than in healthy controls (22.9% vs. 6.8%, p < 0.001). Based on haplotype analysis, the frequency of the TLR7 IVS2-151A/TLR8 -129C haplotype increased the risk for TB infection compared to the wild-type allele (TLR7 IVS2-151A/TLR8 -129G), with OR = 3.23 (95% CI = 1.58-6.61; p = 0.001). An ex vivo phagocytosis assay that examined the functional effects of these polymorphisms on the defense against MTB revealed higher phagocytosis in monocytes from males with the TLR7 IVS2-151A/TLR8 -129C genotype than in those with the wild-type allele (73.0 ± 20.3% versus 34.6 ± 8.1%; p = 0.03). In addition, mRNA expression and cytokine production were analyzed in the whole blood of male healthy volunteers stimulated with inactivated MTB ex vivo. TNFα production was lower in TLR7 IVS2-151A/TLR8 -129C subjects than in those with the wild-type allele (578.4 ± 90.3 pg/ml versus 1043 ± 136 pg/ml; p = 0.03), and the expression of TLR7 was significantly impaired (0.8 ± 0.1 folds, p = 0.05) after MTB stimulation. In conclusion, these findings provide evidence that TLR7 and TLR8 genetic polymorphisms are associated with susceptibility to MTB infection, and the link is shaped by less effective MTB phagocytosis and impaired TLR signaling.

Impaired responsiveness of platelets to epinephrine due to α2A adrenoreceptor deficiency in Male Chinese.

Epinephrine is known as a weak, but important, agonist for platelet activation. It has been reported that the responsiveness of platelets to epinephrine was markedly impaired in 6% of Caucasians and in 16% of Japanese. The purpose of this study was to screen and characterize this abnormality in healthy Taiwanese Chinese volunteers. We used aggregometry, flow cytometry and platelet function analyzer (PFA)-100 system to assess in 50 healthy male volunteers the responsiveness of platelets to epinephrine stimulation. Using α2A adrenoceptor antagonist BRL44408 maleate competition and a [(3)H]yohimbin binding assay, we evaluated α2A adrenoceptors on platelets. The aggregation of platelets after stimulation with 10 µM of epinephrine indicated two distinct groups of study participants: 24 (48.0%) good- and 26 (52.0%) impaired-responders to epinephrine. Flow cytometric analysis of platelets after stimulated with 1 µM epinephrine showed that glycoprotein (GP) IIb/IIIa and P-selectin expression of epinephrine good- and impaired-responders were 27.1 ± 11.0% vs. 9.9 ± 5.4% (p = 0.003) and 12.2 ± 6.2% vs. 3.6 ± 3.5% (p < 0.001), respectively. The PFA-100 system showed that epinephrine-impaired-responders had a longer collagen-epinephrine induced closure time. Good-responder platelets incubated with BRL44408 maleate had an impaired response to epinephrine stimulation. [(3)H]yohimbine binding studies showed fewer α2A adrenoreceptors on the platelets of epinephrine-impaired-responders than on those of good-responders. The prevalence of impaired responsiveness to epinephrine was high and probably due to α2A adrenoreceptor deficiency in male Taiwanese Chinese.

Vitamin D can ameliorate chlorhexidine gluconate-induced peritoneal fibrosis and functional deterioration through the inhibition of epithelial-to-mesenchymal transition of mesothelial cells.

BACKGROUND: Peritoneal dialysis (PD) can induce fibrosis and functional alterations in PD patients' peritoneal membranes, due to long-term unphysiological dialysate exposure, partially occurring via triggering of epithelial-to-mesenchymal transition (EMT) in peritoneal mesothelial cells (MCs). Vitamin D can ameliorate these negative effects; however, the mechanism remains unexplored. Therefore, we investigated its possible links to MCs EMT inhibition. METHODS: Peritoneal fibrosis was established in Sprague-Dawley rats by chlorhexidine gluconate (CG) intraperitoneal injection for 21 days, with and without 1α,25(OH)2D3 treatment. Morphological and functional evaluation and western blot analysis of EMT marker were performed upon peritoneum tissue. In vitro study was also performed in a primary human peritoneal MC culture system; MCs were incubated with transforming growth factor-ß1 (TGF-ß1) in the absence or presence of 1α,25(OH)2D3. EMT marker expression, migration activities, and cytoskeleton redistribution of MCs were determined. RESULTS: 1α,25(OH)2D3 ameliorated CG-induced morphological and functional deterioration in animal model, along with CG-induced upregulation of α-SMA and downregulation of E-cadherin expression. Meanwhile, 1α,25(OH)2D3 also ameliorated TGF-ß1-induced decrease in E-cadherin expression, increase in Snai1 and α-SMA expression, intracellular F-actin redistribution, and migration activity in vitro. CONCLUSION: 1α,25(OH)2D3 can ameliorate CG-induced peritoneal fibrosis and attenuate functional deterioration through inhibiting MC EMT.