[Determination of 22 mycotoxins in milk by liquid chromatography-quadrupole/orbitrap mass spectrometry].

Mycotoxins have carcinogenic, mutagenic, hepatotoxic, nephrotoxic, immunotoxic, neurotoxic, and teratogenic properties. Thus, these substances have attracted significant attention because they pose a threat to human health. As research on mycotoxins deepens, new structural analogues of mycotoxins are constantly being discovered. In this study, a method based on high performance liquid chromatography-quadrupole/orbitrap mass spectrometry was established for the simultaneous determination of 22 mycotoxins in milk. A simple, effective, and rapid pretreatment method was optimized by focusing on the solvent type, extractant volume, and extracting salt based on the characteristics of the mycotoxins and sample matrix. The analytes were extracted using 0.5% formic acid acetonitrile solution and added with sodium chloride to separate fats from water. The samples were centrifuged at 8000 r/min (4 ℃) for 5 min using a centrifuge and then concentrated using nitrogen. The dry residue was dissolved with 50% methanol aqueous solution. Twenty-two mycotoxins were separated on a ZORBAX Eclipse Plus C18 chromatographic column (100 mm×2.1 mm, 1.7 µm), and quantitative analysis was performed using the isotope internal standard method. The analytes were determined by liquid chromatography-quadrupole/orbitrap mass spectrometry in positive electrospray ionization mode. Qualitative analyses of the compounds were performed in full mass spectrometry/data-dependent tandem mass spectrometry (MS/dd-MS2) mode. Good linearities in the range of 0.5-100.0 µg/L were observed for the 22 mycotoxins, and the correlation coefficients (R2) were greater than 0.999. The limits of detection (S/N=3) and quantification (S/N=10) ranged from 0.3 to 0.5 µg/kg and from 1.0 to 1.5 µg/kg, respectively. The average recoveries of the 22 mycotoxins at three spiked levels of 1.5, 5.0, and 15 µg/kg were between 84.7% and 100.8%, with relative standard deviations (RSDs) of 1.2%-9.9%. These findings indicate that the method has high sensitivity and accuracy as well as good precision. Finally, the method was applied to the detection and analysis of mycotoxins in 25 actual commercial milk samples. The results revealed that the selected samples were not contaminated with any of the mycotoxins analyzed. Thus, the proposed method is useful as a quick preprocessing and confirmatory method for the simultaneous determination of mycotoxins in milk.

Lipoprotein a Combined with Fibrinogen as an Independent Predictor of Long-Term Prognosis in Patients with Acute Coronary Syndrome: A Multi-Center Retrospective Study.

Background: Patients with acute coronary syndrome (ACS) still have a high risk of recurrence of major adverse cardiovascular and cerebrovascular events (MACCE). However, there are rare studies on the prediction of MACCE in patients with ACS using lipoprotein a [Lp(a)] combined with fibrinogen. The aim of this study was to analyze the predictive value of Lp(a) combined with fibrinogen for the long-term prognosis of patients with ACS. Methods: 804 patients with ACS admitted to 11 tertiary general hospitals in Chengdu from January 2017 to June 2019 were included in the study. According to the Lp(a) 300 mg/L, patients were assigned to the non-high Lp(a) group and high Lp(a) group. Patients were assigned to the non-high or high fibrinogen groups using the fibrinogen level of 3.08 g/L. Subsequently, patients were divided into group A, B, or C by Lp(a) combined with fibrinogen. The study endpoints were MACCE, including all-cause death, non-fatal myocardial infarction, non-fatal stroke, and revascularization. The incidences of MACCE among groups were compared. Lp(a), fibrinogen, Lp(a) combined with fibrinogen classifications were each added into the basic model to construct three new models. The C-index, net reclassification index (NRI) and integrated discrimination improvement (IDI) of the three new models were then compared. Results: The median follow-up was 16 months. During follow-up, the cumulative incidence of MACCE in group C was significantly higher than that measured in group A and B (p < 0.001). The results of the multivariate Cox regression analysis of MACCE showed that Lp(a) ≥300 mg/L with fibrinogen ≥3.08 g/L was an independent predictor of MACCE. According to the GRACE score and the statistical analyses, the basic model was constructed, which had a C-index of 0.694. The C-index, NRI, and IDI of the new model constructed using the basic model + Lp(a) combined with fibrinogen classification were 0.736, 0.095, and 0.094 respectively. Conclusions: Single Lp(a), single fibrinogen and Lp(a) combined with fibrinogen were independent predictors of MACCE in patients with ACS. The predictive value of Lp(a) combined with fibrinogen in patients with ACS was better than that of single Lp(a) and single fibrinogen.

Handheld versus conventional ultrasound for assessing carotid artery in routine volunteers.

AIM: Carotid ultrasound is a key tool for the diagnosis and evaluation of cardio disease, and the measurement of carotid intima-media thickness (CIMT) and hemodynamic parameters is of paramount importance for the imaging method. The aim of this study was to evaluate the feasibility and accuracy of handheld ultrasound devices for measuring carotid parameters. METHODS: We performed a carotid ultrasound on 25 participants using a handheld ultrasound device and a conventional ultrasound machine. For each participant, max and mean CIMT of common carotid artery (CCA) and peak systolic velocity (PSV), end diastolic velocity (EDV) and resistive index (RI) of CCA, bilateral external carotid artery (ECA), internal carotid artery (ICA) and the vertebral artery were measured. Agreement and repeatability were evaluated by linear regression and Bland-Altman analysis. RESULTS: We found a good repeatability and consistent of handheld ultrasound device in measuring mean CIMT (r = 0.68, P < 0.01). Furthermore, there was a moderate to good agreement between handheld and conventional ultrasound systems in measuring max IMT, mean IMT, PSV, EDV and RI of CCA (0.73, 0.79, 0.52, 0.58 and 0.84, respectively). CONCLUSION: Handheld ultrasound devices were able to provide carotid IMT and hemodynamic parameters measurements similar to those of conventional ultrasound. Such capabilities of handheld ultrasound devices might be useful for the primary assessment of carotid in clinical work.

The Impact of COVID-19 on Postdischarge Outcomes for Dialysis Patients in the United States: Evidence from Medicare Claims Data.

Background: Recent investigations have shown that, on average, patients hospitalized with coronavirus disease 2019 (COVID-19) have a poorer postdischarge prognosis than those hospitalized without COVID-19, but this effect remains unclear among patients with end-stage kidney disease (ESKD) who are on dialysis. Methods: Leveraging a national ESKD patient claims database administered by the US Centers for Medicare and Medicaid Services, we conducted a retrospective cohort study that characterized the effects of in-hospital COVID-19 on all-cause unplanned readmission and death within 30 days of discharge for patients on dialysis. Included in this study were 436,745 live acute-care hospital discharges of 222,154 Medicare beneficiaries on dialysis from 7871 Medicare-certified dialysis facilities between January 1 and October 31, 2020. Adjusting for patient demographics, clinical characteristics, and prevalent comorbidities, we fit facility-stratified Cox cause-specific hazard models with two interval-specific (1-7 and 8-30 days after hospital discharge) effects of in-hospital COVID-19 and effects of prehospitalization COVID-19. Results: The hazard ratios due to in-hospital COVID-19 over the first 7 days after discharge were 95% CI, 1.53 to 1.65 for readmission and 95% CI, 1.38 to 1.70 for death, both with P<0.001. For the remaining 23 days, the hazard ratios were 95% CI, 0.89 to 0.96 and 95% CI, 0.86 to 1.07, with P<0.001 and P=0.50, respectively. Effects of prehospitalization COVID-19 were mostly nonsignificant. Conclusions: In-hospital COVID-19 had an adverse effect on both postdischarge readmission and death over the first week. With the surviving patients having COVID-19 substantially selected from those hospitalized, in-hospital COVID-19 was associated with lower rates of readmission and death starting from the second week.

Understanding the Tradeoffs Between Travel Burden and Quality of Care for In-center Hemodialysis Patients.

BACKGROUND: Renal dialysis is a lifesaving but demanding therapy, requiring 3 weekly treatments of multiple-hour durations. Though travel times and quality of care vary across facilities, the extent to which patients are willing and able to engage in weighing tradeoffs is not known. Since 2015, Medicare has summarized and reported quality data for dialysis facilities using a star rating system. We estimate choice models to assess the relative roles of travel distance and quality of care in explaining patient choice of facility. RESEARCH DESIGN: Using national data on 2 million patient-years from 7198 dialysis facilities and 4-star rating releases, we estimated travel distance to patients' closest facilities, incremental travel distance to the next closest facility with a higher star rating, and the difference in ratings between these 2 facilities. We fit mixed effects logistic regression models predicting whether patients dialyzed at their closest facilities. RESULTS: Median travel distance was 4 times that in rural (10.9 miles) versus urban areas (2.6 miles). Higher differences in rating [odds ratios (OR): 0.56; 95% confidence interval (CI): 0.50-0.62] and greater area deprivation (OR: 0.50; 95% CI: 0.48-0.53) were associated with lower odds of attending one's closest facility. Stratified models were also fit based on urbanicity. For rural patients, excess travel was associated with higher odds of attending the closer facility (per 10 miles; OR: 1.05; 95% CI: 1.04-1.06). Star rating differences were associated with lower odds of receiving care from the closest facility among urban (OR: 0.57; 95% CI: 0.51-0.63) and rural patients (OR: 0.18; 95% CI: 0.08-0.44). CONCLUSIONS: Most dialysis patients have higher rated facilities located not much further than their closest facility, suggesting many patients could evaluate tradeoffs between distance and quality of care in where they receive dialysis. Our results show that such tradeoffs likely occur. Therefore, quality ratings such as the Dialysis Facility Compare (DFC) Star Rating may provide actionable information to patients and caregivers. However, we were not able to assess whether these associations reflect a causal effect of the Star Ratings on patient choice, as the Star Ratings served only as a marker of quality of care.

COVID-19 Risk Factors and Mortality Outcomes Among Medicare Patients Receiving Long-term Dialysis.

Importance: There is a need for studies to evaluate the risk factors for COVID-19 and mortality among the entire Medicare long-term dialysis population using Medicare claims data. Objective: To identify risk factors associated with COVID-19 and mortality in Medicare patients undergoing long-term dialysis. Design, Setting, and Participants: This retrospective, claims-based cohort study compared mortality trends of patients receiving long-term dialysis in 2020 with previous years (2013-2019) and fit Cox regression models to identify risk factors for contracting COVID-19 and postdiagnosis mortality. The cohort included the national population of Medicare patients receiving long-term dialysis in 2020, derived from clinical and administrative databases. COVID-19 was identified through Medicare claims sources. Data were analyzed on May 17, 2021. Main Outcomes and Measures: The 2 main outcomes were COVID-19 and all-cause mortality. Associations of claims-based risk factors with COVID-19 and mortality were investigated prediagnosis and postdiagnosis. Results: Among a total of 498 169 Medicare patients undergoing dialysis (median [IQR] age, 66 [56-74] years; 215 935 [43.1%] women and 283 227 [56.9%] men), 60 090 (12.1%) had COVID-19, among whom 15 612 patients (26.0%) died. COVID-19 rates were significantly higher among Black (21 787 of 165 830 patients [13.1%]) and Hispanic (13 530 of 86 871 patients [15.6%]) patients compared with non-Black patients (38 303 of 332 339 [11.5%]), as well as patients with short (ie, 1-89 days; 7738 of 55 184 patients [14.0%]) and extended (ie, ≥90 days; 10 737 of 30 196 patients [35.6%]) nursing home stays in the prior year. Adjusting for all other risk factors, residing in a nursing home 1 to 89 days in the prior year was associated with a higher hazard for COVID-19 (hazard ratio [HR] vs 0 days, 1.60; 95% CI 1.56-1.65) and for postdiagnosis mortality (HR, 1.31; 95% CI, 1.25-1.37), as was residing in a nursing home for an extended stay (COVID-19: HR, 4.48; 95% CI, 4.37-4.59; mortality: HR, 1.12; 95% CI, 1.07-1.16). Black race (HR vs non-Black: HR, 1.25; 95% CI, 1.23-1.28) and Hispanic ethnicity (HR vs non-Hispanic: HR, 1.68; 95% CI, 1.64-1.72) were associated with significantly higher hazards of COVID-19. Although home dialysis was associated with lower COVID-19 rates (HR, 0.77; 95% CI, 0.75-0.80), it was associated with higher mortality (HR, 1.18; 95% CI, 1.11-1.25). Conclusions and Relevance: These results shed light on COVID-19 risk factors and outcomes among Medicare patients receiving long-term chronic dialysis and could inform policy decisions to mitigate the significant extra burden of COVID-19 and death in this population.

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Vascular smooth muscle cell (VSMC) is one of the main cell types in blood vessel wall, and the VSMC apoptosis is involved in the development of atherosclerosis (AS). In early AS, the apoptosis of VSMC is regarded as a compensatory mechanism for neointimal hyperplasia and lumen stenosis. However, this process will also accelerate the rupture of atherosclerotic plaques, resulting in serious cardiovascular complications. More studies on the related genes, RNA, and inducing factors of VSMCs apoptosis in occurrence and development of AS can provide scientific strategies for prevention and treatment of AS in different periods.

Evaluating national trends in outcomes after implementation of a star rating system: Results from dialysis facility compare.

OBJECTIVE: To examine which factors are driving improvement in the Dialysis Facility Compare (DFC) star ratings and to test whether nonclinical facility characteristics are associated with observed longitudinal changes in the star ratings. DATA SOURCES: Data were collected from eligible patients in over 6,000 Medicare-certified dialysis facilities from three annual star rating and individual measure updates, publicly released on DFC in October 2015, October 2016, and April 2018. STUDY DESIGN: Changes in the star rating and individual quality measures were investigated across three public data releases. Year-to-year changes in the star ratings were linked to facility characteristics, adjusting for baseline differences in quality measure performance. DATA COLLECTION: Data from publicly reported quality measures, including standardized mortality, hospitalization, and transfusion ratios, dialysis adequacy, type of vascular access for dialysis, and management of mineral and bone disease, were extracted from annual DFC data releases. PRINCIPAL FINDINGS: The proportion of four- and five-star facilities increased from 30.0% to 53.4% between October 2015 and April 2018. Quality improvement was driven by the domain of care containing the dialysis adequacy and hypercalcemia measures. Additionally, independently owned facilities and facilities belonging to smaller dialysis organizations had significantly lower odds of year-to-year improvement than facilities belonging to either of the two large dialysis organizations (Odds Ratio [OR]: 0.736, 95% Confidence Interval [CI]: 0.631-0.856 and OR: 0.797, 95% CI: 0.723-0.879, respectively). CONCLUSIONS: The percentage of four- and five-star facilities has increased markedly over a three-year time period. These changes were driven by improvement in the specific quality measures that may be most directly under the control of the dialysis facility.

Delay optimization of multipoint pacing increases the cardiac index and narrows the QRS width.

INTRODUCTION: The benefits of MPP delay optimization on hemodynamics and ventricular contraction synchronicity can be quantified with cardiac index (CI) and QRS width. A delay with the maximum CI and minimum QRS width may be the optimized settings for multipoint pacing (MPP). METHODS: Twelve patients with advanced heart failure who received cardiac resynchronization therapy defibrillation with MPP at the Third People's Hospital of Chengdu from March 2016 to April 2019 were included. Interventricular and intraventricular delays were optimized through noninvasive cardiac output monitoring and a 12 lead ECG. RESULTS: According to CI, the optimized left ventricular- left ventricular - right ventricular delay setting was mainly 25 ms-25 ms and 40 ms-40 ms. And the delay with the minimum QRS width was mainly in 5 ms-5 ms, 25 ms-25 ms, and 40 ms-25 ms. The optimal MPP configuration increased CI compared to the MPP setting that produced the minimum CI (4.5 ± 1.3 vs. 2.8 ± 1.0 L/min/m2, P < 0.001). The QRS width of the optimized MPP was narrower than the MPP setting that produced the maximum QRS width (127 ± 20 vs. 160 ± 29 ms, P < 0.001). CONCLUSION: Delay optimization improves hemodynamic response and ventricular contraction synchronicity. The delay of 25 ms-25 ms may be the optimal setting for most MPP patients.

Ultrasonographic optic nerve sheath diameter monitoring of elevated intracranial pressure: two case reports.

Ultrasonography assessments of optic nerve sheath diameter (ONSD) is a non-invasive method that may help identify elevated intracranial pressure (ICP). However, this technique was used to evaluate the elevated ICP caused by traumatic brain injury. The objective of this study was to examine clinical cases of the changes in ICP with venous sinus stenosis and venous sinus thrombosis found the advantage of this technique in the application. And we dynamically monitor ONSD and ICP as a lens for understanding the dynamic assessment for ICP. The first case of venous sinus stenosis with elevated ICP identified in real-time by changes in ONSD, which are correlated with ICP before and after stenting. Another case of venous sinus thrombosis with elevated ICP. And after treatment, the patient underwent an ultrasound ONSD examination and lumbar puncture (LP) at the 1st, the 2nd and 3rd month of follow-up. The previously enlarged ONSDs retracted and LP opening pressure gradually returned to normal. These cases indicate that ONSD examination may help dynamically assess ICP changes and evaluate the efficacy of ICP treatment. These results provide utile, evidence based, preliminary clinical recommendations and indicate that ONSD examination might be a useful method of evaluating ICP, especially if repeated evaluations are needed.

Anti-N-methyl-D-aspartate receptor encephalitis induced by bilateral ovarian teratomas with distinct histopathologic types: A case report and brief literature review.

RATIONALE: Anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis is an autoimmune disorder that is most frequently induced by ovarian teratoma in young females. The condition can be controlled and reversed via ovarian tumor resection and immunotherapy. However, anti-NMDAR encephalitis induced by bilateral ovarian teratomas with distinct histopathologic types is rarely reported in the literature. PATIENT CONCERNS: A 23-year-old woman presented with seizures. DIAGNOSES: The diagnosis was anti-NMDAR encephalitis associated with ovarian teratomas based on positive anti-NMDAR antibody tests in both the cerebrospinal fluid and serum, and the detection of bilateral ovarian lesions on pelvic computed tomography. The postoperative histopathologic examination confirmed that the left lesion was an immature teratoma, and the right lesion was a mature teratoma. INTERVENTIONS: We performed surgical resection of the ovarian teratomas and administered immunotherapy for the control of anti-NMDAR encephalitis. Chemotherapy was administered for the immature teratoma. OUTCOMES: The patient recovered without any postoperative complications. She has been confirmed to be in complete clinical remission, and has not had a recurrence during 18 months of follow-up. LESSONS: Anti-NMDAR encephalitis induced by bilateral ovarian teratomas of differing histopathologic types (1 immature and 1 mature) is rare. Early diagnosis and treatment with tumor resection, immunotherapy, and chemotherapy are critical for a good prognosis.

Functional analysis of gene expression profiling-based prediction in bladder cancer.

The present study aimed to analyze the modification of gene expression in bladder cancer (BC) by identifying significant differentially expressed genes (DEGs) and functionally assess them using bioinformatics analysis. To achieve this, two microarray datasets, GSE24152 (which included 10 fresh tumor tissue samples from urothelial bladder carcinoma patients and 7 benign mucosa samples from the bladder), and GSE42089 (which included 10 tissues samples from urothelial cell carcinoma patients and 8 tissues samples from the normal bladder), were downloaded from the Gene Expression Omnibus database for further analysis. Differentially expressed genes (DEGs) were screened between benign the mucosa and control groups in GSE24152 and GSE42089 datasets. Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) analysis were performed on overlapping DEGs identified in GSE24152 and GSE42089. Protein-protein interaction (PPI) networks and sub-networks were then constructed to identify key genes and main pathways. GO terms analysis was also performed for the selected clusters. In total, 1,325 DEGs in GSE24152 and 647 DEGs in GSE42089 were screened, in which 619 common DEGs were identified. The DEGs were mainly enriched in pathways and GO terms associated with mitotic and chromosome assembly, including nucleosome assembly, spindle checkpoint and DNA replication. In the interaction network, progesterone receptor (PGR), MAF bZIP transcription factor G (MAFG), cell division cycle 6 (CDC6) and members of the minichromosome maintenance family (MCMs) were identified as key genes. Histones were also considered to be significant factors in BC. Nucleosome assembly and sequence-specific DNA binding were the most significant clustered GO terms. In conclusion, the DEGs, including PGR, MAFG, CDC6 and MCMs, and those encoding the core histone family were closely associated with the development of BC via pathways associated with mitotic and chromosome assembly.

[Impact of Lung Cancer Deaths on Life Expectancy and Years of Potential Life Lost from 1981 to 2015 in Kunshan, Jiangsu Province, China].

BACKGROUND: Previous reports have shown that lung cancer incidence and mortality were increasing both in male and female; however, the temporal trends in lung cancer eliminated life expectancy and potential years of life lost (PYLL) are very rare. Thus, we examine the temporal trends in lung cancer eliminated life expectancy and PYLL in Kunshan city, Jiangsu province, 1981-2015. METHODS: Data were collected from vital registry of Kunshan city. Lung cancer eliminated life expectancy and the PYLL were calculated by sex. The Chinese population in 2000 was used to calculate age-standardized PYLL. Estimate annual percentage changes (eAPC) and 95% confidence interval (CI) were used to examine the temporal trendss. RESULTS: During 1981 to 2015, substantially increasing trend was observed for the lung cancer eliminated life expectancy, which increased by 0.34 years in 1981 to 0.86 in 2015 (APC=3.2%, 95%CI: 2.8%-3.6%), and a significant increasing trend was found for male (APC=3.0%, 95%CI: 2.5%-3.5%) and female (APC=3.6%, 95%CI: 3.0%-4.2%). Moreover, the age-standardized PYLL among both sex (APC=-0.1%, 95%CI: -0.6%-0.4%) and male (APC=-0.5%, 95%CI: -1.1%-0.1%) were stable, but increasing trend was observed in females (APC=1.5%, 95%CI: 0.3%-2.7%). CONCLUSIONS: Although there was no significant change over the past 3 decades regarding the effect of premature deaths due to lung cancer, a substantial increasing trend was observed in lung cancer eliminated life expectancy, which suggested that targeted lung cancer prevention and control measures are urgently need.

[Association between cigarette smoking and hypertension in men: a dose response relationship analysis].

OBJECTIVE: To estimate the dose response relationship between cigarette smoking and hypertension in men based on restricted cubic spline method. METHODS: Under the proportion to the population size, 38 520 subjects were randomly selected from May to August 2012 with cluster sampling method in urban and rural areas of Kunshan, China.Each participant received face-to-face interview with the standardized questionnaire, and physical examination.Restricted cubic spline was employed to estimate the dose response relation of cigarette smoking on the risk of hypertension. RESULTS: The overall prevalence of cigarette smoking was 22.6% (8 691/38 520), prevalence of cigarette smoking in men was 46.1% (8 499/18 454). Multiple logistic regression analysis showed that current smoking (OR = 1.16, 95%CI:1.05-1.28) and previous cigarette smoking (OR = 1.32, 95%CI:1.07-1.63) were associated with hypertension after adjusted confounding factors (age, sex, body mass index, education, family income per month, urban or rural areas, physical activity and physical exercise) in men. After further adjusting drinking status, only previous cigarette smoking was associated with hypertension (OR = 1.28, 95%CI:1.04-1.58). The restricted cubic spline model indicated a linear dose-response relation between hypertension and cigarette smoking per day in men (non-linearity test P = 0.604 1).However, a non-linear dose response relation was found between duration of smoking (non-linearity test P < 0.000 1), smoking index (non-linearity test P = 0.009 9) and hypertension. CONCLUSION: Long-term and heavy cigarette smoking is associated with hypertension in men.

Clinical and biochemical profiles suggest fibromuscular dysplasia is a systemic disease with altered TGF-ß expression and connective tissue features.

Fibromuscular dysplasia (FMD) is a rare, nonatherosclerotic arterial disease for which the molecular basis is unknown. We comprehensively studied 47 subjects with FMD, including physical examination, spine magnetic resonance imaging, bone densitometry, and brain magnetic resonance angiography. Inflammatory biomarkers in plasma and transforming growth factor ß (TGF-ß) cytokines in patient-derived dermal fibroblasts were measured by ELISA. Arterial pathology other than medial fibrodysplasia with multifocal stenosis included cerebral aneurysm, found in 12.8% of subjects. Extra-arterial pathology included low bone density (P<0.001); early onset degenerative spine disease (95.7%); increased incidence of Chiari I malformation (6.4%) and dural ectasia (42.6%); and physical examination findings of a mild connective tissue dysplasia (95.7%). Screening for mutations causing known genetically mediated arteriopathies was unrevealing. We found elevated plasma TGF-ß1 (P=0.009), TGF-ß2 (P=0.004) and additional inflammatory markers, and increased TGF-ß1 (P=0.0009) and TGF-ß2 (P=0.0001) secretion in dermal fibroblast cell lines from subjects with FMD compared to age- and gender-matched controls. Detailed phenotyping of patients with FMD allowed us to demonstrate that FMD is a systemic disease with alterations in common with the spectrum of genetic syndromes that involve altered TGF-ß signaling and offers TGF-ß as a marker of FMD.

Virus-specific peptide dependent NK cell cytotoxicity.

NK cells do not express recombination-dependent antigen-specific receptors and are traditionally defined as cells of the innate immune response. The activation of NK cells was believed to be controlled by the net balance of signals from a multitude of activating and inhibitory receptors irrespectively of antigen specificity. However, murine antigen-specific memory NK cells in liver have been described to mediate hapten or viral specific recall response and are capable of infiltrating to the site of infection. The mechanisms by which NK cells recognize target cells in an antigen-specific manner are largely unclear. Using a novel multiplex killing assay, we screened the NK cell (human) cytotoxic activity of 35 different donors against different virus peptide pools loaded autologous B cells. We have found that human NK cells from some CMV and EBV positive donors can recognize peptide loaded autologous B cells as targets and perform antigen-specific cytotoxic killing. This may provide evidence that NK cells are able to scan the peptide repertoire on the target cell surface and virus-derived peptides may influence the NK cell activation-inhibition balance.

Multiplex and functional detection of antigen-specific human T cells by ITRA--indirect T cell recognition assay.

The identification and functional characterization of pathogen-specific T cells plays a critical role in immunological research and diagnostics. In addition to the present standard technologies such as intracellular cytokine staining (ICS), enzyme-linked immunospot (ELISPOT) and peptide-major-histocompatibility-complex (MHC) multimer staining, we aimed to develop a multiplex detection assay, which provides fast in vitro functional data for both human CD4 and CD8 T cells with different antigen specificities in one sample. In this study, we have exploited the expression of CD83 on B cells to develop the cell array-based indirect T cell recognition assay (ITRA). In detail, B cells are pulsed with different pathogen peptide pools and fluorescently barcoded. Thereafter the B cells are pooled and co-cultured with autologous T cells. Subsequently each B cell population is analyzed via flow cytometry for CD83 expression, which indicates antigen-specific interaction with CD4 T cells. Moreover, we revealed donor dependent variations of cytotoxic activity of pathogen-specific CD4 T cells and CD8 T cells, evidenced by specific lysis of peptide-pulsed B cells. Taken together, ITRA is a novel antigen presenting cell (APC) array based method to analyze the presence and function of various antigen-specific T cells in one sample. It has the potential to be used in the future for epitope/antigen screening in research and for analysis of anti-tumor, anti-pathogen or autoimmune T cell responses in patient samples.

Outcomes of adult living donor liver transplantation: comparison of the Adult-to-adult Living Donor Liver Transplantation Cohort Study and the national experience.

The study objectives were to determine whether the findings of the Adult-to-Adult Living Donor Liver Transplantation Cohort Study (A2ALL) reflect the U.S. national experience and to define risk factors for patient mortality and graft loss in living donor liver transplantation (LDLT). A2ALL previously identified risk factors for mortality after LDLT, which included early center experience, older recipient age, and longer cold ischemia time. LDLT procedures at 9 A2ALL centers (n = 702) and 67 non-A2ALL centers (n = 1664) from January 1998 through December 2007 in the Scientific Registry of Transplant Recipients database were analyzed. Potential predictors of time from transplantation to death or graft failure were tested using Cox regression. No significant difference in overall mortality between A2ALL and non-A2ALL centers was found. Higher hazard ratios (HRs) were associated with donor age (HR = 1.13 per 10 years, P = 0.0002), recipient age (HR = 1.20 per 10 years, P = 0.0003), serum creatinine levels (HR = 1.52 per loge unit increase, P < 0.0001), hepatocellular carcinoma (HR = 2.12, P<0.0001) or hepatitis C virus (HR = 1.18, P = 0.026), intensive care unit stay (HR = 2.52, P< 0.0001) or hospitalization (HR = 1.62, P < 0.0001) versus home, earlier center experience (LDLT case number 15: HR = 1.61, P < 0.0001, and a cold ischemia time >4.5 hours (HR = 1.79, P = 0.0006). Except for center experience, risk factor effects between A2ALL and non-A2ALL centers were not significantly different. Variables associated with graft loss were identified and showed similar trends. In conclusion, mortality and graft loss risk factors were similar in A2ALL and non-A2ALL centers. These analyses demonstrate that findings from the A2ALL consortium are relevant to other centers in the U.S. performing LDLT, and conclusions and recommendations from A2ALL may help to guide clinical decision making.

Phosphorylation of mouse immunity-related GTPase (IRG) resistance proteins is an evasion strategy for virulent Toxoplasma gondii.

Virulence of complex pathogens in mammals is generally determined by multiple components of the pathogen interacting with the functional complexity and multiple layering of the mammalian immune system. It is most unusual for the resistance of a mammalian host to be overcome by the defeat of a single defence mechanism. In this study we uncover and analyse just such a case at the molecular level, involving the widespread intracellular protozoan pathogen Toxoplasma gondii and one of its most important natural hosts, the house mouse (Mus musculus). Natural polymorphism in virulence of Eurasian T. gondii strains for mice has been correlated in genetic screens with the expression of polymorphic rhoptry kinases (ROP kinases) secreted into the host cell during infection. We show that the molecular targets of the virulent allelic form of ROP18 kinase are members of a family of cellular GTPases, the interferon-inducible IRG (immunity-related GTPase) proteins, known from earlier work to be essential resistance factors in mice against avirulent strains of T. gondii. Virulent T. gondii strain ROP18 kinase phosphorylates several mouse IRG proteins. We show that the parasite kinase phosphorylates host Irga6 at two threonines in the nucleotide-binding domain, biochemically inactivating the GTPase and inhibiting its accumulation and action at the T. gondii parasitophorous vacuole membrane. Our analysis identifies the conformationally active switch I region of the GTP-binding site as an Achilles' heel of the IRG protein pathogen-resistance mechanism. The polymorphism of ROP18 in natural T. gondii populations indicates the existence of a dynamic, rapidly evolving ecological relationship between parasite virulence factors and host resistance factors. This system should be unusually fruitful for analysis at both ecological and molecular levels since both T. gondii and the mouse are widespread and abundant in the wild and are well-established model species with excellent analytical tools available.