Factor Xa generation by computational modeling: an additional discriminator to thrombin generation evaluation

Factor (f)Xa is a critical enzyme in blood coagulation that is responsible for the initiation and propagation of thrombin generation. Previously we have shown that analysis of computationally generated thrombin profiles is a tool to investigate hemostasis in various populations. In this study, we evaluate the potential of computationally derived time courses of fXa generation as another approach for investigating thrombotic risk. Utilizing the case (n = 473) and control (n = 426) population from the Leiden Thrombophilia Study and each individual''s plasma protein factor composition for fII, fV, fVII, fVIII, fIX, fX, antithrombin and tissue factor pathway inhibitor, tissue factor-initiated total active fXa generation was assessed using a mathematical model. FXa generation was evaluated by the area under the curve (AUC), the maximum rate (MaxR) and level (MaxL) and the time to reach these, TMaxR and TMaxL, respectively. FXa generation was analyzed in the entire populations and in defined subgroups (by sex, age, body mass index, oral contraceptive use). The maximum rates and levels of fXa generation occur over a 10- to 12- fold range in both cases and controls. This variation is larger than that observed with thrombin (3–6 fold) in the same population. The greatest risk association was obtained using either MaxR or MaxL of fXa generation; with an ∼2.2 fold increased risk for individuals exceeding the 90^th percentile. This risk was similar to that of thrombin generation(MaxR OR 2.6). Grouping defined by oral contraceptive (OC) use in the control population showed the biggest differences in fXa generation; a >60% increase in the MaxR upon OC use. FXa generation can distinguish between a subset of individuals characterized by overlapping thrombin generation profiles. Analysis of fXa generation is a phenotypic characteristic which may prove to be a more sensitive discriminator than thrombin generation among all individuals.     

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.     

MRI discriminates thrombus composition and ST resolution after percutaneous coronary intervention in patients with ST-elevation myocardial infarction

Histological composition of material obtained by thrombus aspiration during percutaneous coronary intervention (PCI) in patients with ST-segment elevation acute myocardial infarction (STEMI) is highly variable. We aimed to characterize this material using magnetic resonance imaging (MRI) and to correlate MRI findings with the success of PCI in terms of ST-segment resolution. Thrombus aspiration during primary or rescue PCI was attempted in 100 consecutive STEMI patients, of whom enough material for MRI was obtained in 59. MR images were obtained at 9.4T and T1 and T2 values were measured. Patients with (n = 31) and without (n = 28) adequate ST resolution 120 min after PCI (≥70% of pre-PCI value) had similar baseline characteristics except for a higher prevalence of diabetes mellitus in the latter (10 vs. 43%, p = 0.003). T1 values were similar in both groups (1248±112 vs. 1307±85 ms, respectively, p = 0.7). T2 values averaged 31.2±10.3 and 36.6±12.2 ms; in thrombus from patients with and without adequate ST resolution (p = 0.09). After adjusting for diabetes and other baseline characteristics, lower T2 values were significantly associated with inadequate ST resolution (odds ratio for 1 ms increase 1.08, CI 95% 1.01–1.16, p = 0.027). Histology classified thrombus in 3 groups: coagulated blood (n = 38), fibrin rich (n = 9) and lipid-rich (n = 3). Thrombi composed mostly of coagulated blood were characterized as being of short (n = 10), intermediate (n = 15) or long evolution (n = 13), T2 values being 34.0±13.2, 31.9±8.3 and 31.5±7.9 ms respectively (p = NS). In this subgroup, T2 was significantly higher in specimens from patients with inadequate perfusion (35.9±10.3 versus 28.6±6.7 ms, p = 0.02). This can be of clinical interest as it provides information on the probability of adequate ST resolution, a surrogate for effective myocardial reperfusion.     

Short fetal leukocyte telomere length and preterm prelabor rupture of the membranes

Background

Rupture of the fetal membranes is a common harbinger of imminent labor and delivery. Telomere shortening is a surrogate for oxidative stress (OS) and senescence. Fetal leukocyte and placental membrane DNA telomere lengths were evaluated to determine their association with preterm prelabor rupture of the membranes (pPROM) or spontaneous preterm births with intact membranes (PTB), compared to term birth.

Methods

Telomere lengths were quantified in cord blood leukocytes (n = 133) from three major groups: 1) pPROM (n = 28), 2) PTB (n = 69) and 3) uncomplicated full term births (controls, n = 35), using real-time quantitative PCR. Placental membrane specimens (n = 18) were used to correlate fetal leukocyte and placental telomere lengths. Telomere length differences among the groups were analyzed by ANOVA. Pearson correlation coefficients determined relationships between leukocyte and placental membrane telomere lengths.

Results

In pregnancies with intact membranes, fetal leukocyte telomere length was inversely proportional to gestational age. The mean telomere length decreased as gestation progressed, with the shortest at term. pPROM had telomere lengths (9962±3124 bp) that were significantly shorter than gestational age-matched PTB (11546±4348 bp, p = 0.04), but comparable to term births (9011±2497 bp, p = 0.31). Secondary analyses revealed no effects of race (African American vs. Caucasian) or intraamniotic infection on telomere length. A strong Pearson''s correlation was noted between fetal leukocyte and placental membrane telomere lengths (ρ = 0.77; p<0.01).

Conclusions

Fetal leukocyte telomere length is reduced in pPROM compared to PTB but is similar to term births. pPROM represents a placental membrane disease likely mediated by OS-induced senescence.     

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.     

A genetic risk score combining ten psoriasis risk loci improves disease prediction

Psoriasis is a chronic, immune-mediated skin disease affecting 2–3% of Caucasians. Recent genetic association studies have identified multiple psoriasis risk loci; however, most of these loci contribute only modestly to disease risk. In this study, we investigated whether a genetic risk score (GRS) combining multiple loci could improve psoriasis prediction. Two approaches were used: a simple risk alleles count (cGRS) and a weighted (wGRS) approach. Ten psoriasis risk SNPs were genotyped in 2815 case-control samples and 858 family samples. We found that the total number of risk alleles in the cases was significantly higher than in controls, mean 13.16 (SD 1.7) versus 12.09 (SD 1.8), p = 4.577×10⁻⁴⁰. The wGRS captured considerably more risk than any SNP considered alone, with a psoriasis OR for high-low wGRS quartiles of 10.55 (95% CI 7.63–14.57), p = 2.010×10⁻⁶⁵. To compare the discriminatory ability of the GRS models, receiver operating characteristic curves were used to calculate the area under the curve (AUC). The AUC for wGRS was significantly greater than for cGRS (72.0% versus 66.5%, p = 2.13×10⁻⁸). Additionally, the AUC for HLA-C alone (rs10484554) was equivalent to the AUC for all nine other risk loci combined (66.2% versus 63.8%, p = 0.18), highlighting the dominance of HLA-C as a risk locus. Logistic regression revealed that the wGRS was significantly associated with two subphenotypes of psoriasis, age of onset (p = 4.91×10⁻⁶) and family history (p = 0.020). Using a liability threshold model, we estimated that the 10 risk loci account for only11.6% of the genetic variance in psoriasis. In summary, we found that a GRS combining 10 psoriasis risk loci captured significantly more risk than any individual SNP and was associated with early onset of disease and a positive family history. Notably, only a small fraction of psoriasis heritability is captured by the common risk variants identified to date.     

Genome-wide association meta-analysis of cortical bone mineral density unravels allelic heterogeneity at the RANKL locus and potential pleiotropic effects on bone

Previous genome-wide association (GWA) studies have identified SNPs associated with areal bone mineral density (aBMD). However, this measure is influenced by several different skeletal parameters, such as periosteal expansion, cortical bone mineral density (BMD_C) cortical thickness, trabecular number, and trabecular thickness, which may be under distinct biological and genetic control. We have carried out a GWA and replication study of BMD_C, as measured by peripheral quantitative computed tomography (pQCT), a more homogenous and valid measure of actual volumetric bone density. After initial GWA meta-analysis of two cohorts (ALSPAC n = 999, aged ∼15 years and GOOD n = 935, aged ∼19 years), we attempted to replicate the BMD_C associations that had p<1×10⁻⁵ in an independent sample of ALSPAC children (n = 2803) and in a cohort of elderly men (MrOS Sweden, n = 1052). The rs1021188 SNP (near RANKL) was associated with BMD_C in all cohorts (overall p = 2×10⁻¹⁴, n = 5739). Each minor allele was associated with a decrease in BMD_C of ∼0.14SD. There was also evidence for an interaction between this variant and sex (p = 0.01), with a stronger effect in males than females (at age 15, males −6.77mg/cm³ per C allele, p = 2×10⁻⁶; females −2.79 mg/cm³ per C allele, p = 0.004). Furthermore, in a preliminary analysis, the rs1021188 minor C allele was associated with higher circulating levels of sRANKL (p<0.005). We show this variant to be independent from the previously aBMD associated SNP (rs9594738) and possibly from a third variant in the same RANKL region, which demonstrates important allelic heterogeneity at this locus. Associations with skeletal parameters reflecting bone dimensions were either not found or were much less pronounced. This finding implicates RANKL as a locus containing variation associated with volumetric bone density and provides further insight into the mechanism by which the RANK/RANKL/OPG pathway may be involved in skeletal development.     

Drug susceptibility in Leishmania isolates following miltefosine treatment in cases of visceral leishmaniasis and post kala-azar dermal leishmaniasis

Background

With widespread resistance to antimonials in Visceral Leishmaniasis (VL) in the Indian subcontinent, Miltefosine (MIL) has been introduced as the first line therapy. Surveillance of MIL susceptibility in natural populations of Leishmania donovani is vital to preserve it and support the VL elimination program.

Methodology and Principal Findings

We measured in vitro susceptibility towards MIL and paromomycin (PMM) in L. donovani isolated from VL and PKDL, pre- and post-treatment cases, using an amastigote-macrophage model. MIL susceptibility of post-treatment isolates from cured VL cases (n = 13, mean IC₅₀±SD = 2.43±1.44 µM), was comparable (p>0.05) whereas that from relapses (n = 3, mean IC₅₀ = 4.72±1.99 µM) was significantly higher (p = 0.04) to that of the pre-treatment group (n = 6, mean IC₅₀ = 1.86±0.75 µM). In PKDL, post-treatment isolates (n = 3, mean IC₅₀ = 16.13±2.64 µM) exhibited significantly lower susceptibility (p = 0.03) than pre-treatment isolates (n = 5, mean IC₅₀ = 8.63±0.94 µM). Overall, PKDL isolates (n = 8, mean IC₅₀ = 11.45±4.19 µM) exhibited significantly higher tolerance (p<0.0001) to MIL than VL isolates (n = 22, mean IC₅₀ = 2.58±1.58 µM). Point mutations in the miltefosine transporter (LdMT) and its beta subunit (LdRos3) genes previously reported in parasites with experimentally induced MIL resistance were not present in the clinical isolates. Further, the mRNA expression profile of these genes was comparable in the pre- and post-treatment isolates. Parasite isolates from VL and PKDL cases were uniformly susceptible to PMM with respective mean IC₅₀ = 7.05±2.24 µM and 6.18±1.51 µM.

Conclusion

The in vitro susceptibility of VL isolates remained unchanged at the end of MIL treatment; however, isolates from relapsed VL and PKDL cases had lower susceptibility than the pre-treatment isolates. PKDL isolates were more tolerant towards MIL in comparison with VL isolates. All parasite isolates were uniformly susceptible to PMM. Mutations in the LdMT and LdRos3 genes as well as changes in the expression of these genes previously correlated with experimental resistance to MIL could not be verified for the field isolates.     

Risk of all-cause mortality in HIV infected patients is associated with clinical, immunologic predictors and the CCR5 Δ32 deletion

Objective

Investigation of the interplay between the CCR5 Δ32/wt genotype and demographic, epidemiological, clinical and immunological factors associated with mortality in the cART era.

Design

Longitudinal data from 507 HIV-infected patients following the Δ32 allele detection were analyzed.

Methods

Cumulative 15 years mortality was calculated using Kaplan-Meyer methodology. Hazard ratios were estimated using univariate Cox models. Basing on Akakie information criteria and statistical significance multivariate Cox model was constructed and effect plots presenting adjusted hazard ratio time-dependency were drawn. Analysis of the association of all-cause mortality and CCR5 Δ32/wt genotype prior to the antiretroviral treatment (cART) initiation (n = 507) and on the therapy (n = 422) was also performed.

Results

A mortality rate of 2.66 (CI 2.57–3.19) per 100 person-years was observed. Univariate analysis factors modifying the risk of death included the CCR5 genotype, gender, history of cART, AIDS diagnosis and also CD4 lymphocyte nadir, zenith, the latest CD4 count and stable levels >500 cells/µl. For multivariate analysis the following predictors were selected: CCR5 genotype (HR for wt/wt 2.53, CI 1.16–5.53, p = 0.02), gender (HR for males 1.91, 95%CI 1.1–3.36, p = 0.023), introduction of combined antiretroviral treatment (HR 4.85, CI 3.0–7.89, if untreated or treated <1 month, p<0.0001) CD4 count of 500 cells/µl for six months or more (HR 4.16, CI 1.95–8.88 if not achieved, p = 0.028), the latest CD4 count (HR 5.44, CI 3.39–8.74 for <100 cells/µl, p<0.0001) and history of AIDS (HR 1.69, CI 1.03–2.79, p = 0.039). Among untreated individuals the Δ32/wt genotype was associated with notably better survival (p = 0.026), while among cART treated individuals the Δ32 mutation did not correlate significantly with higher survival rates (p = 0.23).

Conclusions

The Δ32 CCR5 allele is associated with a reduction of the risk of all-cause mortality in HIV (+) patients alongside clinical and immunologic predictors such as AIDS, history of cART, lymphocyte CD4 cell count and gender.     

Clinical outcome of high-risk patients with severe aortic stenosis and reduced left ventricular ejection fraction undergoing medical treatment or TAVI

Introduction

Reduced left ventricular function in patients with severe symptomatic valvular aortic stenosis is associated with impaired clinical outcome in patients undergoing surgical aortic valve replacement (SAVR). Transcatheter Aortic Valve Implantation (TAVI) has been shown non-inferior to SAVR in high-risk patients with respect to mortality and may result in faster left ventricular recovery.

Methods

We investigated clinical outcomes of high-risk patients with severe aortic stenosis undergoing medical treatment (n = 71) or TAVI (n = 256) stratified by left ventricular ejection fraction (LVEF) in a prospective single center registry.

Results

Twenty-five patients (35%) among the medical cohort were found to have an LVEF≤30% (mean 26.7±4.1%) and 37 patients (14%) among the TAVI patients (mean 25.2±4.4%). Estimated peri-interventional risk as assessed by logistic EuroSCORE was significantly higher in patients with severely impaired LVEF as compared to patients with LVEF>30% (medical/TAVI 38.5±13.8%/40.6±16.4% versus medical/TAVI 22.5±10.8%/22.1±12.8%, p <0.001). In patients undergoing TAVI, there was no significant difference in the combined endpoint of death, myocardial infarction, major stroke, life-threatening bleeding, major access-site complications, valvular re-intervention, or renal failure at 30 days between the two groups (21.0% versus 27.0%, p = 0.40). After TAVI, patients with LVEF≤30% experienced a rapid improvement in LVEF (from 25±4% to 34±10% at discharge, p = 0.002) associated with improved NYHA functional class at 30 days (decrease ≥1 NYHA class in 95%). During long-term follow-up no difference in survival was observed in patients undergoing TAVI irrespective of baseline LVEF (p = 0.29), whereas there was a significantly higher mortality in medically treated patients with severely reduced LVEF (log rank p = 0.001).

Conclusion

TAVI in patients with severely reduced left ventricular function may be performed safely and is associated with rapid recovery of systolic left ventricular function and heart failure symptoms.     

Chronic immune activation in HIV-1 infection contributes to reduced interferon alpha production via enhanced CD40:CD40 ligand interaction

Although a signature of increased interferon (IFN-)alpha production is observed in HIV-1 infection, the response of circulating plasmacytoid dendritic cells (PDC) to Toll-like receptor ligand stimulation is substantially impaired. This functional PDC deficit, which we specifically observed in HIV-1 infected individuals with less than 500 CD4+ T cells/µl, is not well understood. We provide evidence that the peripheral IFN-alpha production in HIV-1 infection is actively suppressed by the enhanced interaction of CD40 ligand (CD40L), a member of the tumor necrosis factor family, and its receptor CD40, which are both upregulated upon immune activation. Plasma levels of soluble CD40L were significantly higher in untreated HIV-1 infected individuals (n = 52) than in subjects on long-term antiretroviral therapy (n = 62, p<0.03) and in uninfected control donors (n = 16, p<0.001). Concomitantly, cell-associated CD40L and the expression of the receptor CD40 on the PDC were significantly upregulated in HIV-1 infection (p<0.05). Soluble and cell-associated CD40L inhibited the PDC-derived IFN-alpha production by CpG oligodeoxynucleotides dose-dependently. This suppressive effect was observed at much lower, physiological CD40L concentrations in peripheral blood mononuclear cells (PBMC) of HIV-1 infected individuals compared to controls (p<0.05). The CpG-induced IFN-alpha production in PBMC of HIV-1 infected donors was directly correlated with PDC and CD4+ T cell counts, and inversely correlated with the viral loads (p<0.001). In HIV-1 infected donors with less than 500 CD4+ T cells/µl, the CpG-induced IFN-alpha production was significantly correlated with the percentage of CD40-expressing PDC and the level of CD40 expression on these cells (p<0.05), whereas CD40L plasma levels played a minor role. In addition, low-dose CD40L contributed to the enhanced production of interleukin 6 and 8 in PBMC of HIV-1 infected donors compared to controls. Our data support the conclusion that the chronic immune activation in HIV-1 infection impairs peripheral PDC innate immune responses at least in part via enhanced CD40:CD40L interactions.     

Association of plasma Aß peptides with blood pressure in the elderly

Background

Aß peptides are often considered as catabolic by-products of the amyloid ß protein precursor (APP), with unknown physiological functions. However, several biological properties have been tentatively attributed to these peptides, including a role in vasomotion.We assess whether plasma Aß peptide levels might be associated with systolic and diastolic blood pressure values (SBP and DBP, respectively).

Methodology/Principal Findings

Plasma Aß_1-40 and Aß_1-42 levels were measured using an xMAP-based assay in 1,972 individuals (none of whom were taking antihypertensive drugs) from 3 independent studies: the French population-based 3C and MONA-LISA (Lille) studies (n = 627 and n = 769, respectively) and the Australian, longitudinal AIBL study (n = 576). In the combined sample, the Aß_1-42/ Aß_1-40 ratio was significantly and inversely associated with SBP (p = 0.03) and a similar trend was observed for DBP (p = 0.06). Using the median age (69) as a cut-off, the Aß_1-42/Aß_1-40 ratio was strongly associated with both SBP and DBP in elderly individuals (p = 0.002 and p = 0.03, respectively). Consistently, a high Aß_1-42/ Aß_1-40 ratio was associated with a lower risk of hypertension in both the combined whole sample (odds ratio [OR], 0.71; 95% confidence interval [CI], 0.56-0.90) and (to an even greater extent) in the elderly subjects (OR, 0.53; 95% CI, 0.37–0.75). Lastly, all these associations appeared to be primarily driven by the level of plasma Aß_1-40.

Conclusion

The plasma Aß_1-42/Aß_1-40 ratio is inversely associated with SBP, DBP and the risk of hypertension in elderly subjects, suggesting that Aß peptides affect blood pressure in vivo. These results may be particularly relevant in Alzheimer''s disease, in which a high Aß_1-42/Aß_1-40 plasma ratio is reportedly associated with a decreased risk of incident disease.     

Status of Systemic Oxidative Stresses in Patients with Primary Open-Angle Glaucoma and Pseudoexfoliation Syndrome

Background

The involvement of local and systemic oxidative stress in intraocular pressure (IOP) elevation and optic nerve damage has been hypothesized in the pathogenesis of glaucoma. To test this, we measured the systemic levels of prooxidants and antioxidants by analyzing the blood biochemistry in patients with glaucoma.

Methods

Peripheral blood samples were collected from Japanese patients with primary open-angle glaucoma (PG) (n = 206), exfoliation syndrome (EX) (n = 199), and controls (n = 126). Plasma levels of lipid peroxides, ferric-reducing activity, and thiol antioxidant activity were measured by diacron reactive oxygen metabolites (dROM), biological antioxidant potential (BAP), and sulfhydryl (SH) tests, respectively, using a free radical analyzer.

Results

In the PG, EX, and control groups, the mean ± standard deviation values were 355±63, 357±69, and 348±56 (U. Carr), respectively, for dROM; 1,951±282, 1,969±252, and 2,033±252 (µmol/L), respectively, for BAP (µmol/L); and 614±98, 584±91, and 617±99 (µmol/L), respectively, for SH. The differences in the BAP values were significant between the PG and control groups (p = 0.0062), for SH between the EX and control groups (p = 0.0017), and for SH between the PG and EX groups (p = 0.0026). After adjustment for differences in age and sex among groups using multiple regression analysis, lower BAP values were correlated significantly with PG (p = 0.0155) and EX (p = 0.0049). Higher dROM values with and without glaucoma were correlated with female gender, and lower SH values with older age. There were no significant differences between the higher (≥21 mmHg) and lower (<21 mmHg) baseline IOPs in the PG group or between the presence or absence of glaucoma in the EX group.

Conclusions

Lower systemic antioxidant capacity that measured by ferric-reducing activity is involved in the pathogenesis of PG and EX.     

Mitochondrial haplogroups and polymorphisms reveal no association with sporadic prostate cancer in a southern European population

Background

It is known that mitochondria play an important role in certain cancers (prostate, renal, breast, or colorectal) and coronary disease. These organelles play an essential role in apoptosis and the production of reactive oxygen species; in addition, mtDNA also reveals the history of populations and ancient human migration. All these events and variations in the mitochondrial genome are thought to cause some cancers, including prostate cancer, and also help us to group individuals into common origin groups. The aim of the present study is to analyze the different haplogroups and variations in the sequence in the mitochondrial genome of a southern European population consisting of subjects affected (n = 239) and non-affected (n = 150) by sporadic prostate cancer.

Methodology and Principal Findings

Using primer extension analysis and DNA sequencing, we identified the nine major European haplogroups and CR polymorphisms. The frequencies of the haplogroups did not differ between patients and control cohorts, whereas the CR polymorphism T16356C was significantly higher in patients with PC compared to the controls (p = 0.029). PSA, staging, and Gleason score were associated with none of the nine major European haplogroups. The CR polymorphisms G16129A (p = 0.007) and T16224C (p = 0.022) were significantly associated with Gleason score, whereas T16311C (p = 0.046) was linked with T-stage.

Conclusions and Significance

Our results do not suggest that mtDNA haplogroups could be involved in sporadic prostate cancer etiology and pathogenesis as previous studies performed in middle Europe population. Although some significant associations have been obtained in studying CR polymorphisms, further studies should be performed to validate these results.     

The prognostic value of non-linear analysis of heart rate variability in patients with congestive heart failure--a pilot study of multiscale entropy

Aims

The influences of nonstationarity and nonlinearity on heart rate time series can be mathematically qualified or quantified by multiscale entropy (MSE). The aim of this study is to investigate the prognostic value of parameters derived from MSE in the patients with systolic heart failure.

Methods and Results

Patients with systolic heart failure were enrolled in this study. One month after clinical condition being stable, 24-hour Holter electrocardiogram was recording. MSE as well as other standard parameters of heart rate variability (HRV) and detrended fluctuation analysis (DFA) were assessed. A total of 40 heart failure patients with a mea age of 56±16 years were enrolled and followed-up for 684±441 days. There were 25 patients receiving β-blockers treatment. During follow-up period, 6 patients died or received urgent heart transplantation. The short-term exponent of DFA and the slope of MSE between scale 1 to 5 were significantly different between patients with or without β-blockers (p = 0.014 and p = 0.028). Only the area under the MSE curve for scale 6 to 20 (Area_6–20) showed the strongest predictive power between survival (n = 34) and mortality (n = 6) groups among all the parameters. The value of Area_6–20 21.2 served as a significant predictor of mortality or heart transplant (p = 0.0014).

Conclusion

The area under the MSE curve for scale 6 to 20 is not relevant to β-blockers and could further warrant independent risk stratification for the prognosis of CHF patients.     

Prominent plasmacytosis following intravenous immunoglobulin correlates with clinical improvement in Guillain-Barré syndrome

Background

High doses of pooled polyclonal IgG are commonly used to treat numerous autoimmune diseases. Their mode of action nevertheless remains only partially explained. At the same time, until now, no early biological marker has been able to predict their efficacy.

Methodology/Principal Findings

In a first pilot retrospective analysis, we reviewed white blood cell counts and blood smears in consecutive patients with autoimmune disease (n = 202) and non-autoimmune disease (n = 104). Autoimmune patients received either intravenous immunoglobulin (IVIg, n = 103), plasma exchange (n = 78) or no specific treatment (n = 21). We then prospectively monitored consecutive autoimmune patients with IVIg injection (n = 67), or without any specific treatment (n = 10) using the same routine laboratory tests, as well as flow cytometry. Both retrospective and prospective analyses identified large plasma-cell mobilization exclusively in IVIg-treated autoimmune patients 7 days after initiation of treatment. The majority of IVIg-mobilized plasma cells were immature HLA-DR^high/CD138^low/CXCR4^low plasma cells expressing intracellular immunoglobulin G which were neither IVIg- nor human IgG-specific. Importantly, we found a strong negative correlation between the absolute number of IVIg-mobilized plasma cells and time to improve neurological function in both retrospective and prospective studies of Guillain-Barré syndrome (GBS), (r = −0.52, p = 0.0031, n = 30, r = −0.47, p = 0.0028, n = 40, respectively).

Conclusions/Significance

IVIg promotes immature plasma-cell mobilization in patients with GBS, chronic inflammatory demyelinating polyneuropathy, myasthenia gravis and inflammatory myopathy. Prominent day 7 plasma-cell mobilization is a favourable prognostic marker in patients with GBS receiving IVIg treatment.     

Genome-wide association studies of the PR interval in African Americans

The PR interval on the electrocardiogram reflects atrial and atrioventricular nodal conduction time. The PR interval is heritable, provides important information about arrhythmia risk, and has been suggested to differ among human races. Genome-wide association (GWA) studies have identified common genetic determinants of the PR interval in individuals of European and Asian ancestry, but there is a general paucity of GWA studies in individuals of African ancestry. We performed GWA studies in African American individuals from four cohorts (n = 6,247) to identify genetic variants associated with PR interval duration. Genotyping was performed using the Affymetrix 6.0 microarray. Imputation was performed for 2.8 million single nucleotide polymorphisms (SNPs) using combined YRI and CEU HapMap phase II panels. We observed a strong signal (rs3922844) within the gene encoding the cardiac sodium channel (SCN5A) with genome-wide significant association (p<2.5×10⁻⁸) in two of the four cohorts and in the meta-analysis. The signal explained 2% of PR interval variability in African Americans (beta  = 5.1 msec per minor allele, 95% CI  = 4.1–6.1, p = 3×10⁻²³). This SNP was also associated with PR interval (beta = 2.4 msec per minor allele, 95% CI = 1.8–3.0, p = 3×10⁻¹⁶) in individuals of European ancestry (n = 14,042), but with a smaller effect size (p for heterogeneity <0.001) and variability explained (0.5%). Further meta-analysis of the four cohorts identified genome-wide significant associations with SNPs in SCN10A (rs6798015), MEIS1 (rs10865355), and TBX5 (rs7312625) that were highly correlated with SNPs identified in European and Asian GWA studies. African ancestry was associated with increased PR duration (13.3 msec, p = 0.009) in one but not the other three cohorts. Our findings demonstrate the relevance of common variants to African Americans at four loci previously associated with PR interval in European and Asian samples and identify an association signal at one of these loci that is more strongly associated with PR interval in African Americans than in Europeans.     

Progression to AIDS in South Africa is associated with both reverting and compensatory viral mutations

We lack the understanding of why HIV-infected individuals in South Africa progress to AIDS. We hypothesised that in end-stage disease there is a shifting dynamic between T cell imposed immunity and viral immune escape, which, through both compensatory and reverting viral mutations, results in increased viral fitness, elevated plasma viral loads and disease progression. We explored how T cell responses, viral adaptation and viral fitness inter-relate in South African cohorts recruited from Bloemfontein, the Free State (n = 278) and Durban, KwaZulu-Natal (n = 775). Immune responses were measured by γ-interferon ELISPOT assays. HLA-associated viral polymorphisms were determined using phylogenetically corrected techniques, and viral replication capacity (VRC) was measured by comparing the growth rate of gag-protease recombinant viruses against recombinant NL4-3 viruses. We report that in advanced disease (CD4 counts <100 cells/µl), T cell responses narrow, with a relative decline in Gag-directed responses (p<0.0001). This is associated with preserved selection pressure at specific viral amino acids (e.g., the T242N polymorphism within the HLA-B*57/5801 restricted TW10 epitope), but with reversion at other sites (e.g., the T186S polymorphism within the HLA-B*8101 restricted TL9 epitope), most notably in Gag and suggestive of “immune relaxation”. The median VRC from patients with CD4 counts <100 cells/µl was higher than from patients with CD4 counts ≥500 cells/µl (91.15% versus 85.19%, p = 0.0004), potentially explaining the rise in viral load associated with disease progression. Mutations at HIV Gag T186S and T242N reduced VRC, however, in advanced disease only the T242N mutants demonstrated increasing VRC, and were associated with compensatory mutations (p = 0.013). These data provide novel insights into the mechanisms of HIV disease progression in South Africa. Restoration of fitness correlates with loss of viral control in late disease, with evidence for both preserved and relaxed selection pressure across the HIV genome. Interventions that maintain viral fitness costs could potentially slow progression.