TIA-1 cytotoxic granule-associated RNA binding protein improves the prognostic performance of CD8 in mismatch repair-proficient colorectal cancer

Background

Evidence suggests a confounding effect of mismatch repair (MMR) status on immune response in colorectal cancer. The identification of innate and adaptive immune cells, that can complement the established prognostic effect of CD8 in MMR-proficient colorectal cancers patients, representing 85% of all cases, has not been performed.

Methodology/Principal Findings

Colorectal cancers from a test (n = 1197) and external validation (n = 209) cohort of MMR-proficient colorectal cancers were mounted onto single and multiple punch tissue microarrays. Immunohistochemical quantification (score 0-3) was performed for CD3, CD4, CD8, CD45RO, CD68, CD163, FoxP3, GranzymeB, iNOS, mast cell tryptase, MUM1, PD1 and TIA-1 tumor-infiltrating (TILs) reactive cells. Coexpression experiments on fresh colorectal cancer specimens using specific cell population markers were performed. In the test group, higher numbers of CD3+ (p<0.001), CD4+ (p = 0.029), CD8+ (p<0.001), CD45RO+ (p = 0.048), FoxP3+ (p<0.001), GranzymeB+ (p<0.001), iNOS+ (p = 0.035), MUM1+ (p = 0.014), PD1+ (p = 0.034) and TIA-1+ TILs (p<0.001) were linked to favourable outcome. Adjusting for age, gender, TNM stage and post-operative therapy, higher CD8+ (p<0.001; HR (95%CI): 0.66 (0.64-0.68)) and TIA-1+ (p<0.001; HR (95%CI): 0.56 (0.5-0.6)) were independent prognostic factors. Moreover, among patients with CD8+ infiltrates, TIA-1 further stratified 355 (35.6%) patients into prognostic subgroups (p<0.001; HR (95%CI): 0.89 (95%CI: 0.8-0.9)). Results were confirmed on the validation cohort (p = 0.006). TIA-1+ cells were mostly CD8+ (57%), but also stained for TCRγδ (22%), CD66b (13%) and only rarely for CD4+, macrophage and NK cell markers.

Conclusions

TIA-1 adds prognostic information to TNM stage and adjuvant therapy in MMR-proficient colorectal cancer patients. The prognostic effect of CD8+ TILs is confounded by the presence of TIA-1+ which translates into improved risk stratification for approximately 35% of all patients with MMR-proficient colorectal cancers.     

Genetic association study identifies HSPB7 as a risk gene for idiopathic dilated cardiomyopathy

Dilated cardiomyopathy (DCM) is a structural heart disease with strong genetic background. Monogenic forms of DCM are observed in families with mutations located mostly in genes encoding structural and sarcomeric proteins. However, strong evidence suggests that genetic factors also affect the susceptibility to idiopathic DCM. To identify risk alleles for non-familial forms of DCM, we carried out a case-control association study, genotyping 664 DCM cases and 1,874 population-based healthy controls from Germany using a 50K human cardiovascular disease bead chip covering more than 2,000 genes pre-selected for cardiovascular relevance. After quality control, 30,920 single nucleotide polymorphisms (SNP) were tested for association with the disease by logistic regression adjusted for gender, and results were genomic-control corrected. The analysis revealed a significant association between a SNP in HSPB7 gene (rs1739843, minor allele frequency 39%) and idiopathic DCM (p = 1.06×10⁻⁶, OR = 0.67 [95% CI 0.57–0.79] for the minor allele T). Three more SNPs showed p < 2.21×10⁻⁵. De novo genotyping of these four SNPs was done in three independent case-control studies of idiopathic DCM. Association between SNP rs1739843 and DCM was significant in all replication samples: Germany (n = 564, n = 981 controls, p = 2.07×10⁻³, OR = 0.79 [95% CI 0.67–0.92]), France 1 (n = 433 cases, n = 395 controls, p = 3.73×10⁻³, OR = 0.74 [95% CI 0.60–0.91]), and France 2 (n = 249 cases, n = 380 controls, p = 2.26×10⁻⁴, OR = 0.63 [95% CI 0.50–0.81]). The combined analysis of all four studies including a total of n = 1,910 cases and n = 3,630 controls showed highly significant evidence for association between rs1739843 and idiopathic DCM (p = 5.28×10⁻¹³, OR = 0.72 [95% CI 0.65–0.78]). None of the other three SNPs showed significant results in the replication stage.This finding of the HSPB7 gene from a genetic search for idiopathic DCM using a large SNP panel underscores the influence of common polymorphisms on DCM susceptibility.     

Differential expression of NK receptors CD94 and NKG2A by T cells in rheumatoid arthritis patients in remission compared to active disease

Objective

TNF inhibitors (TNFi) have revolutionised the treatment of rheumatoid arthritis (RA). Natural killer (NK) cells and Natural Killer Cell Receptor⁺ T (NKT) cells comprise important effector lymphocytes whose activity is tightly regulated through surface NK receptors (NKRs). Dysregulation of NKRs in patients with autoimmune diseases has been shown, however little is known regarding NKRs expression in patients with TNFi-induced remission and in those who maintain remission vs disease flare following TNFi withdrawal.

Methods

Patients with RA were recruited for this study, (i) RA patients in clinical remission following a minimum of one year of TNFi therapy (n = −15); (2) Active RA patients, not currently or ever receiving TNFi (n = 18); and healthy control volunteers (n = 15). Patients in remission were divided into two groups: those who were maintained on TNFi and those who withdrew from TNFi and maintained on DMARDS. All patients underwent full clinical assessment. Peripheral blood mononuclear cells were isolated and NKR (CD94, NKG2A, CD161, CD69, CD57, CD158a, CD158b) expression on T-(CD3⁺CD56⁻), NK-(CD3⁻CD56⁺) and NKT-(CD3⁺CD56⁺) cells was determined by flow cytometry.

Results

Following TNFi withdrawal, percentages and numbers of circulating T cells, NK cells or NKT cell populations were unchanged in patients in remission versus active RA or HCs. Expression of the NKRs CD161, CD57, CD94 and NKG2A was significantly increased on CD3⁺CD56-T cells from patients in remission compared to active RA (p<0.05). CD3⁺CD56-T cell expression of CD94 and NKG2A was significantly increased in patients who remained in remission compared with patients whose disease flared (p<0.05), with no differences observed for CD161 and CD57. CD3⁺CD56⁻ cell expression of NKG2A was inversely related to DAS28 (r = −0.612, p<0.005).

Conclusion

High CD94/NKG2A expression by T cells was demonstrated in remission patients following TNFi therapy compared to active RA, while low CD94/NKG2A were associated with disease flare following withdrawal of therapy.     

Metal ionophore treatment restores dendritic spine density and synaptic protein levels in a mouse model of Alzheimer's disease

We have previously demonstrated that brief treatment of APP transgenic mice with metal ionophores (PBT2, Prana Biotechnology) rapidly and markedly improves learning and memory. To understand the potential mechanisms of action underlying this phenomenon we examined hippocampal dendritic spine density, and the levels of key proteins involved in learning and memory, in young (4 months) and old (14 months) female Tg2576 mice following brief (11 days) oral treatment with PBT2 (30 mg/kg/d). Transgenic mice exhibited deficits in spine density compared to littermate controls that were significantly rescued by PBT2 treatment in both the young (+17%, p<0.001) and old (+32%, p<0.001) animals. There was no effect of PBT2 on spine density in the control animals. In the transgenic animals, PBT2 treatment also resulted in significant increases in brain levels of CamKII (+57%, p = 0.005), spinophilin (+37%, p = 0.04), NMDAR1A (+126%, p = 0.02), NMDAR2A (+70%, p = 0.05), pro-BDNF (+19%, p = 0.02) and BDNF (+19%, p = 0.04). While PBT2-treatment did not significantly alter neurite-length in vivo, it did increase neurite outgrowth (+200%, p = 0.006) in cultured cells, and this was abolished by co-incubation with the transition metal chelator, diamsar. These data suggest that PBT2 may affect multiple aspects of snaptic health/efficacy. In Alzheimer''s disease therefore, PBT2 may restore the uptake of physiological metal ions trapped within extracellular β-amyloid aggregates that then induce biochemical and anatomical changes to improve cognitive function.     

The sodium iodide symporter (NIS) and potential regulators in normal, benign and malignant human breast tissue

Introduction

The presence, relevance and regulation of the Sodium Iodide Symporter (NIS) in human mammary tissue remains poorly understood. This study aimed to quantify relative expression of NIS and putative regulators in human breast tissue, with relationships observed further investigated in vitro.

Methods

Human breast tissue specimens (malignant n = 75, normal n = 15, fibroadenoma n = 10) were analysed by RQ-PCR targeting NIS, receptors for retinoic acid (RARα, RARβ), oestrogen (ERα), thyroid hormones (THRα, THRβ), and also phosphoinositide-3-kinase (PI3K). Breast cancer cells were treated with Retinoic acid (ATRA), Estradiol and Thyroxine individually and in combination followed by analysis of changes in NIS expression.

Results

The lowest levels of NIS were detected in normal tissue (Mean(SEM) 0.70(0.12) Log₁₀ Relative Quantity (RQ)) with significantly higher levels observed in fibroadenoma (1.69(0.21) Log₁₀RQ, p<0.005) and malignant breast tissue (1.18(0.07) Log₁₀RQ, p<0.05). Significant positive correlations were observed between human NIS and ERα (r = 0.22, p<0.05) and RARα (r = 0.29, p<0.005), with the strongest relationship observed between NIS and RARβ (r = 0.38, p<0.0001). An inverse relationship between NIS and PI3K expression was also observed (r = −0.21, p<0.05). In vitro, ATRA, Estradiol and Thyroxine individually stimulated significant increases in NIS expression (range 6–16 fold), while ATRA and Thyroxine combined caused the greatest increase (range 16–26 fold).

Conclusion

Although NIS expression is significantly higher in malignant compared to normal breast tissue, the highest level was detected in fibroadenoma. The data presented supports a role for retinoic acid and estradiol in mammary NIS regulation in vivo, and also highlights potential thyroidal regulation of mammary NIS mediated by thyroid hormones.     

Urinary podocyte-associated mRNA profile in various stages of diabetic nephropathy

Background

Podocyte injury and subsequent excretion in urine play a crucial role in the pathogenesis and progression of diabetic nephropathy (DN). Quantification of messenger RNA (mRNA) expression in urinary sediment by real-time PCR is emerging as a noninvasive method of screening DN-associated biomarkers. We hypothesized that the urinary mRNA profile of podocyte-associated molecules may provide important clinical insight into the different stages of diabetic nephropathy.

Methods

DN patients (N = 51) and healthy controls (N = 13) were enrolled in this study. DN patients were divided into a normoalbuminuria group (UAE<30 mg/g, n = 17), a microalbuminuria group (UAE 30∼300 mg/g, n = 15), and a macroalbuminuria group (UAE>300 mg/g, n = 19), according to their urinary albumin excretion (UAE). Relative mRNA abundance of synaptopodin, podocalyxin, CD2-AP, α-actin4, and podocin were quantified, and correlations between target mRNAs and clinical parameters were examined.

Results

The urinary mRNA levels of all genes studied were significantly higher in the DN group compared with controls (p<0.05), and mRNA levels increased with DN progression. Urinary mRNA levels of all target genes positively correlated with both UAE and BUN. The expression of podocalyxin, CD2-AP, α-actin4, and podocin mRNA correlated with serum creatinine (r = 0.457, p = 0.001; r = 0.329, p = 0.01; r = 0.286, p = 0.021; r = 0.357, p = 0.006, respectively). Furthermore, podocalyxin mRNA was found to negatively correlate with eGFR (r = −0.349, p = 0.01).

Conclusion

The urinary mRNA profiles of synaptopodin, podocalyxin, CD2-AP, α-actin4, and podocin were found to increase with the progression of DN, which suggested that quantification of podocyte-associated molecules will be useful biomarkers of DN.     

Brown adipose tissue in morbidly obese subjects

Background

Cold-stimulated adaptive thermogenesis in brown adipose tissue (BAT) to increase energy expenditure is suggested as a possible therapeutic target for the treatment of obesity. We have recently shown high prevalence of BAT in adult humans, which was inversely related to body mass index (BMI) and body fat percentage (BF%), suggesting that obesity is associated with lower BAT activity. Here, we examined BAT activity in morbidly obese subjects and its role in cold-induced thermogenesis (CIT) after applying a personalized cooling protocol. We hypothesize that morbidly obese subjects show reduced BAT activity upon cold exposure.

Methods and Findings

After applying a personalized cooling protocol for maximal non-shivering conditions, BAT activity was determined using positron-emission tomography and computed tomography (PET-CT). Cold-induced BAT activity was detected in three out of 15 morbidly obese subjects. Combined with results from lean to morbidly obese subjects (n = 39) from previous study, the collective data show a highly significant correlation between BAT activity and body composition (P<0.001), respectively explaining 64% and 60% of the variance in BMI (r = 0.8; P<0.001) and BF% (r = 0.75; P<0.001). Obese individuals demonstrate a blunted CIT combined with low BAT activity. Only in BAT-positive subjects (n = 26) mean energy expenditure was increased significantly upon cold exposure (51.5±6.7 J/s versus 44.0±5.1 J/s, P = 0.001), and the increase was significantly higher compared to BAT-negative subjects (+15.5±8.9% versus +3.6±8.9%, P = 0.001), indicating a role for BAT in CIT in humans.

Conclusions

This study shows that in an extremely large range of body compositions, BAT activity is highly correlated with BMI and BF%. BAT-positive subjects showed higher CIT, indicating that BAT is also in humans involved in adaptive thermogenesis. Increasing BAT activity could be a therapeutic target in (morbid) obesity.     

Predictors of health-related quality of life in patients at risk for cardiovascular disease in European primary care

Background

Cardiovascular risk management plays an important role in primary care. In patients at high risk for cardiovascular diseases (CVD) lifestyle and, where appropriate, medical interventions are recommended in guidelines. Health-related quality of life (HRQoL) is an important outcome in clinical practice. This study aimed to assess the HRQoL of this patient group and to investigate the impact of both patients'' characteristics and practice quality scores on their assessments of HRQoL.

Methods and Findings

An observational study in 218 general practices from 8 European countries was conducted. 2142 patients at risk for CVD (33.5% female) with a mean age of 66.3 (SD 9.1) years completed a questionnaire including the EQ-5D instrument and provided data from medical record. Validated quality indicators of general practices were assessed using practice questionnaires and face-to-face interviews. A hierarchical multilevel analysis was performed to identify predictors of EQ-5D scores at patient and practice level. The mean EQ-5D score was 0.78 (SD 0.19). Female gender (r = −0.03, p<0.0016), age (r = −0.01, p = 0.0387) and lower educational level (r = −0.03, p<0.0001) were correlated negatively with EQ-5D scores. Clinically more important was the correlation of HRQoL with the frequency of practice contacts (r = −0.12, p<0.0001) and the number of uncontrolled risk factors (r = −0.01, p<0.0039). Medication adherence (r = 0.032, p<0.0001), and physical activity (r = 0.02, p<0.0001) were identified as positive predictors of HRQoL. The EUPROPEP-score category ‘organization’ (r = 0.02, p<0.0001) was positively related to EQ-5D scores, whereas other practice scores were not correlated to EQ-5D-scores.

Conclusions

In patients at risk for CVD, good medication adherence, regular physical activity, controlling of biomedical risk factor levels and patient-centered practice organization have been shown to be positively correlated to HRQoL and should therefore be targeted in interventions not only to reduce morbidity but also to sustain or even to ameliorate HRQoL.     

Skeletal muscle phosphodiester content relates to body mass and glycemic control

Background

Aging and insulin resistance have been related to reduced mitochondrial function and oxidative stress. Muscular phosphodiesters (PDE) are comprised of metabolites of phospholipid breakdown and may reflect membrane damage. We aimed to test the hypothesis that myocellular PDE are increased in patients with type 2 diabetes (T2D) and correlate inversely with mitochondrial ATP turnover.

Methods

A Cross-sectional study in the Clinical Research Facility of an University hospital was performed. 10 nonobese middle-aged patients with T2D, 10 healthy humans matched for sex, age and physical activity index (CONm) and 18 young healthy humans (CONy) were included. Myocellular PDE and unidirectional flux through ATP synthase (fATP) were measured with ³¹P magnetic resonance spectroscopy (MRS). Intramyocellular (IMCL) and hepatocellular lipid deposition (HCL) were quantified with ¹H MRS. Insulin sensitivity (Rd) was assessed from hyperinsulinemic-euglycemic clamp tests in 10 T2D, 10 CONm and 11 CONy.

Results

During fasting, T2D and CONm had 1.5 fold greater PDE than CONy (2.8±0.2, 2.5±0.2, 1.7±0.1 mmol/l, P = 0.004). Stimulation by insulin did not affect PDE in any group. PDE correlated negatively with Rd (r = −0.552, p<0.005) and fATP (r = −0.396, p<0.05) and positively with age (r = 0.656, p<0.001) and body mass (r = 0.597, p<0.001). PDE also related positively to HbA1c (r = 0.674, p<0.001) and fasting plasma glucose (r = 0.629, p<0.001) within T2D and across all participants.

Conclusions

Muscular PDE concentrations associate with age, lower resting mitochondrial activity and insulin resistance, which is determined mainly by body mass and glycemia.     

Hypertension is associated with marked alterations in sphingolipid biology: a potential role for ceramide

Background

Hypertension is, amongst others, characterized by endothelial dysfunction and vascular remodeling. As sphingolipids have been implicated in both the regulation of vascular contractility and growth, we investigated whether sphingolipid biology is altered in hypertension and whether this is reflected in altered vascular function.

Methods and Findings

In isolated carotid arteries from spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats, shifting the ceramide/S1P ratio towards ceramide dominance by administration of a sphingosine kinase inhibitor (dimethylsphingosine) or exogenous application of sphingomyelinase, induced marked endothelium-dependent contractions in SHR vessels (DMS: 1.4±0.4 and SMase: 2.1±0.1 mN/mm; n = 10), that were virtually absent in WKY vessels (DMS: 0.0±0.0 and SMase: 0.6±0.1 mN/mm; n = 9, p<0.05). Imaging mass spectrometry and immunohistochemistry indicated that these contractions were most likely mediated by ceramide and dependent on iPLA₂, cyclooxygenase-1 and thromboxane synthase. Expression levels of these enzymes were higher in SHR vessels. In concurrence, infusion of dimethylsphingosine caused a marked rise in blood pressure in anesthetized SHR (42±4%; n = 7), but not in WKY (−12±10%; n = 6). Lipidomics analysis by mass spectrometry, revealed elevated levels of ceramide in arterial tissue of SHR compared to WKY (691±42 vs. 419±27 pmol, n = 3–5 respectively, p<0.05). These pronounced alterations in SHR sphingolipid biology are also reflected in increased plasma ceramide levels (513±19 pmol WKY vs. 645±25 pmol SHR, n = 6–12, p<0.05). Interestingly, we observed similar increases in ceramide levels (correlating with hypertension grade) in plasma from humans with essential hypertension (185±8 pmol vs. 252±23 pmol; n = 18 normotensive vs. n = 19 hypertensive patients, p<0.05).

Conclusions

Hypertension is associated with marked alterations in vascular sphingolipid biology such as elevated ceramide levels and signaling, that contribute to increased vascular tone.     

Functional energetics of CD4+-cellular immunity in monoclonal antibody-associated progressive multifocal leukoencephalopathy in autoimmune disorders

Background

Progressive multifocal leukoencephalopathy (PML) is an opportunistic central nervous system- (CNS-) infection that typically occurs in a subset of immunocompromised individuals. An increasing incidence of PML has recently been reported in patients receiving monoclonal antibody (mAb) therapy for the treatment of autoimmune diseases, particularly those treated with natalizumab, efalizumab and rituximab. Intracellular CD4⁺-ATP-concentration (iATP) functionally reflects cellular immunocompetence and inversely correlates with risk of infections during immunosuppressive therapy. We investigated whether iATP may assist in individualized risk stratification for opportunistic infections during mAb-treatment.

Methodology/Principal Findings

iATP in PHA-stimulated, immunoselected CD4⁺-cells was analyzed using an FDA-approved assay. iATP of mAb-associated PML (natalizumab (n = 8), rituximab (n = 2), efalizumab (n = 1)), or other cases of opportunistic CNS-infections (HIV-associated PML (n = 2), spontaneous PML, PML in a psoriasis patient under fumaric acids, natalizumab-associated herpes simplex encephalitis (n = 1 each)) was reduced by 59% (194.5±29 ng/ml, mean±SEM) in comparison to healthy controls (HC, 479.9±19.8 ng/ml, p<0.0001). iATP in 14 of these 16 patients was at or below 3^rd percentile of healthy controls, similar to HIV-patients (n = 18). In contrast, CD4⁺-cell numbers were reduced in only 7 of 15 patients, for whom cell counts were available. iATP correlated with mitochondrial transmembrane potential (ΔΨ_m) (iATP/ΔΨ_m−correlation:tau = 0.49, p = 0.03). Whereas mean iATP of cross-sectionally analysed natalizumab-treated patients was unaltered (448.7±12 ng/ml, n = 150), iATP was moderately decreased (316.2±26.1 ng/ml, p = 0.04) in patients (n = 7) who had been treated already during the pivotal phase III trials and had received natalizumab for more than 6 years. 2/92 (2%) patients with less than 24 months natalizumab treatment revealed very low iATP at or below the 3^rd percentile of HC, whereas 10/58 (17%) of the patients treated for more than 24 months had such low iATP-concentrations.

Conclusion

Our results suggest that bioenergetic parameters such as iATP may assist in risk stratification under mAb-immunotherapy of autoimmune disorders.     

Epidemiology of concomitant infection due to Loa loa and Mansonella perstans in Gabon

Background

The filarial parasites Loa loa and Mansonnella perstans are endemic in the central and western African forest block. Loa loa is pathogenic and represents a major obstacle to the control of co-endemic filariae because its treatment can cause fatal complications such as encephalitis.

Methodology/Principal Findings

4392 individuals aged over 15 years were studied both by direct examination and a concentration technique. The overall prevalence rates were 22.4% for Loa loa microfilaremia, 10.2% for M. perstans microfilaremia, and 3.2% for mixed infection. The prevalence of both filariae was higher in the forest ecosystem than in savannah and lakeland (p<0.0001). The intensity of microfilariae (mf) was also higher in the forest ecosystem for both parasites. The prevalence and intensity of microfilaria were both influenced by age and gender. Correlations were found between the prevalence and intensity of Loa loa microfilariae (r = 0.215 p = 0.036), and between the prevalence of Loa loa and the prevalence of individuals with microfilaria >8000 mf/ml (r = 0.624; p<0.0001) and microfilariae >30 000 mf/ml (r = 0.319, p = 0.002). In contrast, the prevalence of pruritis and Calabar swellings correlated negatively with the prevalence of Loa loa microfilaria (r = −0.219, p = 0.032; r = −0.220; p = 0.031, respectively). Pruritis, Calabar swellings and eye worm were not associated with L. loa mf intensity (r = −0.144, p = 0.162; r–0.061, p = 0.558; and r = 0.051, p = 0.624, respectively), or with the prevalence or intensity of M. perstans microfilariae.

Conclusions/Significance

This map of the distribution of filariae in Gabon should prove helpful for control programs. Our findings confirm the spatial uniformity of the relationship between parasitological indices. Clinical manifestations point to a relationship between filariae and allergy.     

IL-17A expression is localised to both mononuclear and polymorphonuclear synovial cell infiltrates

Introduction

This study examines the expression of IL-17A-secreting cells within the inflamed synovium and the relationship to in vivo joint hypoxia measurements.

Methods

IL-17A expression was quantified in synovial tissue (ST), serum and synovial fluid (SF) by immunohistochemistry and MSD-plex assays. IL-6 SF and serum levels were measured by MSD-plex assays. Dual immunofluorescence for IL-17A was quantified in ST CD15+ cells (neutrophils), Tryptase+ (mast cells) and CD4+ (T cells). Synovial tissue oxygen (tpO₂) levels were measured under direct visualisation at arthroscopy. Synovial infiltration was assessed using immunohistochemistry for cell specific markers. Peripheral blood mononuclear and polymorphonuclear cells were isolated and exposed to normoxic or 3% hypoxic conditions. IL-17A and IL-6 were quantified as above in culture supernatants.

Results

IL-17A expression was localised to mononuclear and polymorphonuclear (PMN) cells in inflamed ST. Dual immunoflourescent staining co-localised IL-17A expression with CD15+ neutrophils Tryptase+ mast cells and CD4+T cells. % IL-17A positivity was highest on CD15+ neutrophils, followed by mast cells and then CD4+T-cells. The number of IL-17A-secreting PMN cells significantly correlated with sublining CD68 expression (r = 0.618, p<0.01). IL-17A SF levels correlated with IL-6 SF levels (r = 0.675, p<0.01). Patients categorized according to tp0₂< or >20mmHg, showed those with low tp0₂<20mmHg had significantly higher IL-17A+ mononuclear cells with no difference observed for PMNs. Exposure of mononuclear and polymorphonuclear cells to 3% hypoxia, significantly induced IL-6 in mononuclear cells, but had no effect on IL-17A expression in mononuclear and polymorphonuclear cells.

Conclusion

This study demonstrates IL-17A expression is localised to several immune cell subtypes within the inflamed synovial tissue, further supporting the concept that IL-17A is a key mediator in inflammatory arthritis. The association of hypoxia with Il-17A expression appears to be indirect, probably through hypoxia-induced pro-inflammatory pathways and leukocyte influx within the joint microenvironment.     

HLA-A*02:07 is a protective allele for EBV negative and a susceptibility allele for EBV positive classical Hodgkin lymphoma in China

HLA-A2 protects from EBV+ classical Hodgkin lymphoma (cHL) in Western Europe, but it is unknown whether this protective effect also exists in the Chinese population. We investigated the association of HLA-A2 and specific common and well documented HLA-A2 subtypes with EBV stratified cHL patients (n = 161) from the northern part of China. Quantitative-PCR and sequence-based subtyping was performed to identify HLA-A2 positive samples and their subtypes. 67 (42%) of the cHL patients were EBV+. There were no significant differences in percentages of HLA-A2 positivity between cHL and controls (65% vs 66%) and between EBV+ and EBV− cHL patients (70% vs 61%). The frequency distribution of HLA-A2 subtypes was significantly different between EBV stratified cHL subgroups and controls. This difference was most striking for the HLA-A*02:07 type with a frequency of 38% in EBV+ cHL, 8% in EBV− cHL and 20% in controls. Significant differences were also observed for the HLA-A*02:07, HLA-A2 (non-02:07) and the A2-negative typings between EBV+ cHL vs controls (p = 0.028), EBV− cHL vs controls (p = 0.045) and EBV+ vs EBV− cHL cases (p = 2×10⁻⁵). In conclusion, HLA-A*02:07 is a predisposing allele for EBV+ cHL and a protective allele for EBV− cHL in the northern Chinese population.     

Prediction of emergent heart failure death by semi-quantitative triage risk stratification

Objectives

Generic triage risk assessments are widely used in the emergency department (ED), but have not been validated for prediction of short-term risk among patients with acute heart failure (HF). Our objective was to evaluate the Canadian Triage Acuity Scale (CTAS) for prediction of early death among HF patients.

Methods

We included patients presenting with HF to an ED in Ontario from Apr 2003 to Mar 2007. We used the National Ambulatory Care Reporting System and vital statistics databases to examine care and outcomes.

Results

Among 68,380 patients (76±12 years, 49.4% men), early mortality was stratified with death rates of 9.9%, 1.9%, 0.9%, and 0.5% at 1-day, and 17.2%, 5.9%, 3.8%, and 2.5% at 7-days, for CTAS 1, 2, 3, and 4–5, respectively. Compared to lower acuity (CTAS 4–5) patients, adjusted odds ratios (aOR) for 1-day death were 1.32 (95%CI; 0.93–1.88; p = 0.12) for CTAS 3, 2.41 (95%CI; 1.71–3.40; p<0.001) for CTAS 2, and highest for CTAS 1: 9.06 (95%CI; 6.28–13.06; p<0.001). Predictors of triage-critical (CTAS 1) status included oxygen saturation <90% (aOR 5.92, 95%CI; 3.09–11.81; p<0.001), respiratory rate >24 breaths/minute (aOR 1.96, 95%CI; 1.05–3.67; p = 0.034), and arrival by paramedic (aOR 3.52, 95%CI; 1.70–8.02; p = 0.001). While age/sex-adjusted CTAS score provided good discrimination for ED (c-statistic = 0.817) and 1-day (c-statistic = 0.724) death, mortality prediction was improved further after accounting for cardiac and non-cardiac co-morbidities (c-statistics 0.882 and 0.810, respectively; both p<0.001).

Conclusions

A semi-quantitative triage acuity scale assigned at ED presentation and based largely on respiratory factors predicted emergent death among HF patients.     

Age-Associated Changes In Oxidative Stress and NAD(+) Metabolism In Human Tissue

Nicotinamide adenine dinucleotide (NAD⁺) is an essential electron transporter in mitochondrial respiration and oxidative phosphorylation. In genomic DNA, NAD⁺ also represents the sole substrate for the nuclear repair enzyme, poly(ADP-ribose) polymerase (PARP) and the sirtuin family of NAD-dependent histone deacetylases. Age associated increases in oxidative nuclear damage have been associated with PARP-mediated NAD⁺ depletion and loss of SIRT1 activity in rodents. In this study, we further investigated whether these same associations were present in aging human tissue. Human pelvic skin samples were obtained from consenting patients aged between 15–77 and newborn babies (0–1 year old) (n = 49) previously scheduled for an unrelated surgical procedure. DNA damage correlated strongly with age in both males (p = 0.029; r = 0.490) and females (p = 0.003; r = 0.600) whereas lipid oxidation (MDA) levels increased with age in males (p = 0.004; r = 0.623) but not females (p = 0.3734; r = 0.200). PARP activity significantly increased with age in males (p<0.0001; r = 0.768) and inversely correlated with tissue NAD⁺ levels (p = 0.0003; r = −0.639). These associations were less evident in females. A strong negative correlation was observed between NAD⁺ levels and age in both males (p = 0.001; r = −0.706) and females (p = 0.01; r = −0.537). SIRT1 activity also negatively correlated with age in males (p = 0.007; r = −0.612) but not in females. Strong positive correlations were also observed between lipid peroxidation and DNA damage (p<0.0001; r = 0.4962), and PARP activity and NAD⁺ levels (p = 0.0213; r = 0.5241) in post pubescent males. This study provides quantitative evidence in support of the hypothesis that hyperactivation of PARP due to an accumulation of oxidative damage to DNA during aging may be responsible for increased NAD⁺ catabolism in human tissue. The resulting NAD⁺ depletion may play a major role in the aging process, by limiting energy production, DNA repair and genomic signalling.     

Evaluation of brain iron content based on magnetic resonance imaging (MRI): comparison among phase value, R2* and magnitude signal intensity

Background and Purpose

Several magnetic resonance imaging (MRI) techniques are being exploited to measure brain iron levels increasingly as iron deposition has been implicated in some neurodegenerative diseases. However, there remains no unified evaluation of these methods as postmortem measurement isn''t commonly available as the reference standard. The purpose of this study was to make a comparison among these methods and try to find a new index of brain iron.

Methods

We measured both phase values and R2* in twenty-four adults, and performed correlation analysis among the two methods and the previously published iron concentrations. We also proposed a new method using magnitude signal intensity and compared it with R2* and brain iron.

Results

We found phase value correlated with R2* in substantia nigra (r = −0.723, p<0.001) and putamen (r = −0.514, p = 0.010), while no correlations in red nucleus (r = −0.236, p = 0.268) and globus pallidus (r = −0.111, p = 0.605). And the new magnitude method had significant correlations in red nucleus (r = −0.593, p = 0.002), substantia nigra (r = −0.521, p = 0.009), globus pallidus (r = −0.750, p<0.001) and putamen (r = −0.547, p = 0.006) with R2*. A strong inverse correlation was also found between the new magnitude method and previously published iron concentrations in seven brain regions (r = −0.982, P<0.001).

Conclusions

Our study indicates that phase value may not be used for assessing the iron content in some brain regions especially globus pallidus. The new magnitude method is highly consistent with R2* especially in globus pallidus, and we assume that this approach may be acceptable as an index of iron content in iron-rich brain regions.     

Variability of NT-proBNP and its relationship with inflammatory status in patients with stable essential hypertension: a 2-year follow-up study

Background

The variability of NT-proBNP levels has been studied in heart failure, yet no data exist on these changes over time in hypertensive patients. Furthermore, studies on the relationship between natriuretic peptides and inflammatory status are limited.

Methodology/Principal Findings

220 clinically and functionally asymptomatic stable patients (age 59±13, 120 male) out of 252 patients with essential hypertension were followed up, and NT-proBNP was measured at baseline, 12 and 24 months. No differences in NT-proBNP were found with respect to the basal stage in the hypertrophic group, but significant changes were found in non-hypertrophic subjects. The reproducibility of NT-proBNP measurements was better in patients with hypertrophy than in the non-hypertrophic group for the three intervals (stage I-basal; stage II-stage I; stage II-basal) with a reference change value of 34%, 35% and 41%, respectively, in the hypertrophic group. A more elevated coefficient of correlation was obtained in the hypertrophic group than in patients without hypertrophy: basal versus stage I (r = 0.79, p<0.0001 and r = 0.59, p<0.0001) and stage I versus stage II (r = 0.86, p<0.0001 and r = 0.56, p<0.0001). Finally, levels of NT-proBNP significantly correlated with sTNF-R1 (p<0.0001) and IL-6 (p<0.01) during follow-up. A multivariate linear regression analysis showed that sTNF-R1 is an independent factor of NT-proBNP.

Conclusions/Significance

This work shows that there is good stability in NT-proBNP levels in a follow-up study of asymptomatic patients with stable hypertension and left ventricular hypertrophy. As a consequence, assessment of NT-proBNP concentrations may be a useful tool for monitoring the follow-up of hypertensive patients with hypertrophy. Measured variations in peptide levels, exceeding 35% in a 12-month follow-up and 41% in a 24-month follow-up, may indicate an increase in cardiovascular risk, and therefore implies adjustment in the medical treatment. In addition, this study shows a link between neurohormonal and inflammatory activation in these patients.     

Antiretroviral therapy outcomes in HIV-infected children after adjusting protease inhibitor dosing during tuberculosis treatment

Background

Modification of ritonavir-boosted lopinavir (LPV/r)-based antiretroviral therapy is required for HIV-infected children co-treated for tuberculosis (TB). We aimed to determine virologic and toxicity outcomes among TB/HIV co-treated children with the following modifications to their antiretroviral therapy (ART): (1) super-boosted LPV/r, (2) double-dose LPV/r or (3) ritonavir.

Methods and Findings

A medical record review was conducted at two clinical sites in Johannesburg, South Africa. The records of children 6–24 months of age initiating LPV/r-based therapy were reviewed. Children co-treated for TB were categorized based on the modifications made to their ART regimen and were compared to children of the same age at each site not treated for TB.Included are 526 children, 294 (56%) co-treated for TB. All co-treated children had more severe HIV disease, including lower CD4 percents and worse growth indicators, than comparisons.Children in the super-boosted group (n = 156) were as likely to be virally suppressed (<400 copies/ml) at 6 months as comparisons (69.2% vs. 74.8%, p = 0.36). Children in the double-dose (n = 47) and ritonavir groups (n = 91) were significantly less likely to be virally suppressed at 6 months (53.1% and 49.3%) than comparisons (74.8% and 82.1%; p = 0.02 and p<0.0001, respectively). At 12 months only children in the ritonavir group still had lower rates of virological suppression relative to comparisons (63.9% vs 83.3% p<0.05). Grade 1 or greater ALT elevations were more common in the super-boosted (75%) than double-dose (54.6%) or ritonavir (33.9%) groups (p = 0.09 and p<0.0001) but grade 3/4 elevations were observed in 3 (13.6%) of the super-boosted, 7 (15.9%) of the double-dose and 5 (8.9%) of the ritonavir group (p = 0.81 and p = 0.29).

Conclusion

Good short-term virologic outcomes were achieved in children co-treated for TB and HIV who received super-boosted LPV/r. Treatment limiting toxicity was rare. Strategies for increased dosing of LPV/r with TB treatment warrant further investigation.     

Kidney bean: a major sensitizer among legumes in asthma and rhinitis patients from India

Background

The prevalence of IgE mediated food allergies has increased over the last two decades. Food allergy has been reported to be fatal in highly sensitive individuals. Legumes are important food allergens but their prevalence may vary among different populations. The present study identifies sensitization to common legumes among Indian population, characterizes allergens of kidney bean and establishes its cross reactivity with other legumes.

Methodology

Patients (n = 355) with history of legume allergy were skin prick tested (SPT) with 10 legumes. Specific IgE (sIgE) and total IgE were estimated in sera by enzyme-linked immunosorbent assay. Characterization of kidney bean allergens and their cross reactivity was investigated by immunobiochemical methods. Identification of major allergens of kidney bean was carried out by mass spectrometry.

Principal Findings

Kidney bean exhibited sensitization in 78 (22.0%) patients followed by chickpea 65 (18.0%) and peanut 53 (15%). SPT positive patients depicted significantly elevated sIgE levels against different legumes (r = 0.85, p<0.0001). Sera from 30 kidney bean sensitive individuals exhibited basophil histamine release (16–54%) which significantly correlated with their SPT (r = 0.83, p<0.0001) and sIgE (r = 0.99, p<0.0001). Kidney bean showed eight major allergens of 58, 50, 45, 42, 40, 37, 34 and 18 kDa on immunoblot and required 67.3±2.51 ng of homologous protein for 50% IgE inhibition. Inhibition assays revealed extensive cross reactivity among kidney bean, peanut, black gram and pigeon pea. nLC-MS/MS analysis identified four allergens of kidney bean showing significant matches with known proteins namely lectin (phytohemagglutinin), phaseolin, alpha-amylase inhibitor precursor and group 3 late embryogenesis abundant protein.

Conclusion/Significance

Among legumes, kidney bean followed by chick pea and peanut are the major allergic triggers in asthma and rhinitis patients in India. Kidney bean showed eight major allergens and cross reacted with other legumes. A combination of SPT, sIgE and histamine release assay is helpful in allergy diagnosis.