Increased responsiveness to thrombin through protease-activated receptors (PAR)-1 and -4 in active Crohn's disease

Background and aimsPlatelets are essential in hemostasis and inflammation, thereby linking coagulation with inflammation. Abundant thrombin generation in association with inflammation is considered a major reason for the increased risk for thromboembolic events. We therefore investigated platelet responsiveness to thrombin.MethodsIn this case–control study 85 patients with Crohn's disease (active CD 42, remission 43) and 30 sex- and age-matched controls were enrolled. Clinical disease activity (Harvey–Bradshaw-Index) was assessed and CD-related data were determined by chart review. Platelets' response to protease activated receptor-1 and -4 (PAR-1, -4) was assessed by whole blood platelet aggregometry (MEA), levels of platelets adhering to monocytes (PMA), and platelet surface P-selectin.ResultsPlatelets' aggregation after activation with the specific PAR-1 agonist (SFLLRN) and PAR-4 agonist (AYPGKF) was higher in patients with active CD compared to patients in remission and controls (p = 0.0068 and p = 0.0023 for SFLLRN, p = 0.0019 and 0.0003 for AYPGKF). Likewise, levels of PMA after activation with PAR-1 and PAR-4 receptor agonists were higher in patients with active CD compared to patients in remission and controls (p = 0.0001 and p < 0.0001 for SFLLRN, p = 0.0329 and p = 0.0125 for AYPGKF). However, P-selectin expression on human platelets showed heterogeneous results. Only PAR-1 activation of platelets resulted in significant differences between CD patients and controls (p = 0.0001 and p = 0.0022 for active and inactive CD versus controls, respectively).ConclusionsOur data suggest a new mechanism of platelet activation which has the potential to increase risk for thromboembolism in patients with active CD which might be due to platelets poised for thrombin-inducible activation.     

Microparticle-associated P-selectin reflects platelet activation in preeclampsia

Platelet activation in preeclampsia is reflected by elevated levels of platelets exposing P-selectin. In plasma, a non-cell bound (soluble) form of P-selectin is present. Elevated levels of this soluble form have been reported in preeclampsia. Plasma P-selectin may consist of two fractions: microparticle (MP)--associated P-selectin and non-MP--associated P-selectin. In the present cross-sectional study, we investigated to which extent plasma P-selectin is MP--associated and whether such MP are elevated in preeclamptic patients. Preeclamptic patients (n?=?10) were matched with normotensive pregnant women (n?=?10) and non-pregnant controls (n?=?10). Plasma P-selectin was measured by ELISA. MP were isolated, double labelled with anti-CD61 (GPIIIa) and anti-CD62P (P-selectin) and subsequently analyzed with flowcytometry. Plasma P-selectin concentration was elevated in preeclamptic patients compared to non-pregnant controls (p?=?0.007), but not compared to normotensive pregnant women (p?=?0.210). Plasma P-selectin is partially MP--associated (3–5%). In pregnancy, the fraction of P-selectin exposing platelet-derived MP (PMP) (10.9%) was increased compared to non-pregnant controls (8%). This fraction further increased in preeclamptic patients (15.4%), and significantly differed from normotensive pregnant women (p?=?0.02). A minor fraction of plasma P-selectin is associated with PMP. The fraction of PMP exposing P-selectin is increased in preeclamptic patients and to a lesser extent in normotensive pregnancy. Because MP associated P-selectin exclusively originates from platelets, this fraction indicates platelet activation. Platelet activation is prominent in preeclampsia and this study proves that at least a part of the plasma P-selectin originates from platelets.     

Platelets and acute cerebral infarction

Stroke is worldwide a leading cause of death and disability. Its etiology is regarded as heterogeneous. Platelets are implicated in its pathophysiology, but our understanding of their specific role is incomplete. Only sparse and conflicting information exists about platelet reactivity and activity in acute stroke. Some scientists take the view that platelets activate in conjunction with acute cerebral infarctions. Others put forward evidence corroborating the contrary notion. Increased soluble P-selectin as a sign of platelet and/or endothelial activity seems to be a feature of the disease. The latter point of view is opposed by other researchers. Due to these conflicting opinions, this study is devoted to platelet characteristics in acute cerebral infarctions. We studied subjects (n?=?72; age 74?±?10(SD) years; 31 females) having acute stroke. As controls served atrial fibrillation (AF) patients (n?=?58; age 69?±?7(SD) years; 12 females) subject to electrical cardioversion, a flow cytometer was put to use for measuring platelet reactivity and activity. After agonist provocation, both platelet bound P-selectin and fibrinogen were employed as estimates of platelet reactivity. Dilutions of a thrombin-receptor-activating peptide (TRAP-6) (74 and 57?µmol/l) (P-selectin and fibrinogen) and ADP (8.5 and 1.7?µmol/l) (fibrinogen only) were put to use as platelet agonists. Membrane-bound P-selectin without agonist stimulation served as a measure of in vivo platelet activation. Soluble P-selectin, as determined from a commercial ELISA, was used to assess platelet and/or endothelial activity. In acute stroke neither platelet-bound P-selectin nor fibrinogen after stimulation, i.e. reactivity, differed from AF controls. In contrast, lower platelet activity as judged from surface attached and circulating P-selectin without agonist stimulation proved to be a feature of cerebral infarctions. The p-values were p?<?0.001 and p?<?0.01, respectively. It is concluded that acute stroke is not associated with platelet reactivity platelets circulate less activated during the disease. It is evident that the mechanisms reflecting platelet reactivity and activity being investigated in this study play minor roles in stroke pathophysiology. New powerful platelet inhibitory drugs are currently introduced. To avoid major bleeding studies on platelet, behavior in acute stroke are necessary before including these medications in stroke treatment protocols.     

ACUTE LEUKEMIAS XVI

Few studies have examined the prognostic impact of blood markers [other than the five factors in the enhanced International Prognostic Index (NCCN-IPI)] in elderly patients with diffuse large B cell lymphoma (DLBCL). We retrospectively analyzed 391 DLBCL patients receiving rituximab plus anthracycline-containing chemotherapy to examine the prognostic impact of simple blood markers. The NCCN-IPI was more accurate for discriminating prognoses than the original IPI. Multivariate analysis identified platelet count (<100,000/μl) and albumin (<3.5 g/dl) levels as significantly associated with lower overall survival (OS), independently of the NCCN-IPI. These parameters stratified patients into three risk groups: platelet–albumin (PA) score low (platelet count ≥100,000/μl, albumin ≥3.5 g/dl, n?=?243); intermediate (platelet count <100,000/μl, albumin ≥3.5 g/dl or platelet count ≥100,000/μl, albumin <3.5 g/dl, n?=?125); and high (platelet count <100,000/μl, albumin <3.5 g/dl, n?=?23). The 5-year OS rates were 81.5, 48.6, and 20.2 %, respectively (p?<?0.001). Notably, most patients with a low platelet count (n?=?30) were stratified into the high-risk subgroup, suggesting that platelet count was prognostic for high-risk patients with a dismal outcome. In elderly patients (n?=?291), the prognostic value of the NCCN-IPI might be diminished because the low-risk category was excluded; however, the PA score was predictive of survival: the 5-year OS rates for PA score low (n?=?171), intermediate (n?=?101), and high (n?=?19) groups were 77.6, 47.9, and 19.0 %, respectively (p?<?0.001). Platelet count and albumin levels are useful prognostic factors, and their combined use can predict survival, even in elderly patients.     

Downregulation of platelet activation markers during long-term immobilization

Immobilization and sedentary lifestyle are risk factors for venous thromboembolism and cardiovascular disease, yet little is known about platelet function during long-term physical inactivity. Our aim was to investigate platelet activation markers and their coupling to standardized immobilization: platelet-derived growth factor (PDGF-BB) and P-selectin. We studied 15 healthy females participating in the Women International Space simulation for Exploration study. Following a 20-day ambulatory control period, the subjects underwent 60 days of bed rest in head-down tilt position (?6°) 24 hours a day, finalized by 20 days of recovery. The subjects were randomized into two groups during bed rest: a control group (n?=?8) that remained physically inactive and an exercise group (n?=?7) that participated in both supine resistance and aerobic exercise training. Blood samples for the analysis of platelet activation markers were collected at baseline (5 days before bed rest), after 44 days of bed rest and 8 days into the recovery period. Compared to baseline, the levels of P-selectin and PDGF-BB decreased after bed rest (by 55%, p?=?0.01 and 73%, p?<?0.03, respectively) and remained decreased in the recovery period (by 76%, p?<?0.001 and 78%, p?<?0.02, respectively, compared to baseline). Platelet count (baseline value for the exercise group 260?000/µl?±?34?000 and baseline value for the control group 210?000/µl?±?30?000) did not change during the bed rest study (two-way repeated measurements ANOVA, p?=?ns). There were no statistical differences between the physically inactive and the exercise group. During long-term immobilization, a known risk factor for thrombosis, the levels of P-selectin and PDGF-BB decreased. Our findings indicate downregulation of platelet activation during immobilization.     

A platelet P-selectin test predicts adverse cardiovascular events in patients with acute coronary syndromes treated with aspirin and clopidogrel

Abstract

There is wide variation in response to antiplatelet therapy and high on-treatment platelet reactivity is associated with adverse cardiovascular events. The objective here was to determine whether the results of a novel strategy for assessing platelet reactivity (based on P-selectin measurement) are associated with clinical outcomes in patients with acute coronary syndromes (ACS). This was a prospective cohort study of 100 ACS patients taking aspirin and clopidogrel. P-selectin tests designed to assess response to P2Y₁₂ antagonists or aspirin were performed alongside light transmission aggregometry. For the P2Y₁₂ P-selectin test, an optimal cutoff for high platelet reactivity was determined by receiver operating characteristic (ROC) curve analysis. Patients were divided into two cohorts based on this value: patients with (n?=?42) or without (n?=?58) high platelet reactivity. The primary endpoint was defined as the composite of cardiovascular death, myocardial infarction and stent thrombosis. After 12 months, the primary endpoint occurred in 12 patients. ROC curve analysis determined that the P2Y₁₂ P-selectin test results were predictive of the primary endpoint (area under curve?=?0.69, p?=?0.046). The primary endpoint occurred more frequently in patients with high on-treatment platelet reactivity compared to those without (21.4% vs. 5.2%; hazard ratio (HR) 4.14; p?=?0.026). The P2Y₁₂ P-selectin test results correlated with light transmission aggregometry (Spearman p?<?0.0001). Using the Aspirin P-selectin test, only two patients demonstrated high on-treatment platelet reactivity. This study suggests that a P2Y₁₂ P-selectin test is capable of detecting high on-treatment platelet reactivity, which is associated with subsequent cardiovascular events.     

Platelet-monocyte cross talk and tissue factor expression in stable angina vs. unstable angina/non ST-elevation myocardial infarction

Tissue factor (TF), the major procoagulant in vivo, is usually absent from blood cells. However, since both monocyte TF (MoTF) expression and platelet activation are present in acute coronary syndrome we hypothesized that MoTF expression may in part depend on monocyte platelet aggregate (MPA) formation in coronary artery disease (CAD). Patients with unstable angina/non-ST-elevation myocardial infarction (UA/NSTEMI, n?=?20) had significantly higher levels of MoTF (17.4?±?3.1MFI) and MPAs (CD42b:273?±?183MFI; CD62P:256.3?±?48.5MFI) than patients with stable angina (SA, n?=?40; MoTF:13.2?±?2.2MFI, p?=?0.001; CD42b:160?±?113MFI, p?=?0.025; CD62P:118.7?±?24.5MFI, p?=?0.018) as measured by whole blood flow cytometry on CD14⁺-cells. TF-activity of isolated mononuclear cells (MNC) was elevated in UA/NSTEMI (75?±?27?pg/mL) in comparison to SA (47?±?17?pg/mL, p?=?0.001) as determined by chromogenic assay, and TF mRNA expression in isolated MNC was more frequent in UA/NSTEMI than in SA (50% vs. 18.2%; p?=?0.017). MoTF expression significantly correlated with the constitutive platelet marker CD42b (r?=?0.69, p?<?0.001) and the platelet activation marker CD62P (r?=?0.47, p?=?0.001) on CD14⁺-cells suggesting its association with MPAs in UA/NSTEMI. In addition, MoTF expression correlated with MoTF activity of isolated MNC (r?=?0.41, p?=?0.01) and plasma levels of the F1.2 prothrombin fragment (r?=?0.35, p?=?0.02). In conclusion, MoTF and MPAs are elevated in UA/NSTEMI compared with SA. MoTF expression correlates with platelet mass and activity attached to monocytes.     

Platelet hyperaggregability in patients with atrial fibrillation

Objective

Atrial fibrillation (AF) is a condition where platelet hyperaggregability is commonly present. We examined potential physiological bases for platelet hyperaggregability in a cohort of patients with acute and chronic AF. In particular, we sought to identify the impact of inflammation [myeloperoxidase (MPO) and C-reactive protein (CRP)] and impaired nitric oxide (NO) signaling.

Methods

Clinical and biochemical determinants of adenosine diphosphate (ADP)-induced platelet aggregation were sought in patients (n?=?106) hospitalized with AF via univariate and multivariate analysis.

Results

Hyper-responsiveness of platelets to ADP was directly (r?=?0.254, p?<?0.01) correlated with plasma concentrations of thrombospondin-1 (TSP-1), a matricellular protein that impairs NO responses and contributes to development of oxidative stress. In turn, plasma TSP-1 concentrations were directly correlated with MPO concentrations (r?=?0.221, p?<?0.05), while MPO concentrations correlated with those of asymmetric dimethylarginine (ADMA, r?=?0.220, p?<?0.05), and its structural isomer symmetric dimethylarginine (SDMA, r?=?0.192, p?=?0.05). Multivariate analysis identified TSP-1 (β?=?0.276, p?<?0.05) concentrations, as well as female sex (β?=?0.199, p?<?0.05), as direct correlates of platelet aggregability, and SDMA concentrations (β?=???0.292, p?<?0.05) as an inverse correlate.

Conclusion

We conclude that platelet hyperaggregability, where present in the context of AF, may be engendered by impaired availability of NO, as well as via MPO-related inflammatory activation.

     

New-onset systemic lupus erythematosus during pregnancy

Few studies have been published focusing on the clinical features of new-onset systemic lupus erythematosus (SLE) during pregnancy. This study examined the clinical characteristics of SLE during pregnancy or puerperium. The clinical characteristics and serological parameters of 48 patients with onset of SLE during pregnancy were retrospectively compared with those of age-matched new-onset SLE patients who were diagnosed in a period of more than 12 months without pregnancy (n?=?65) and age-matched preeclampsia patients (n?=?48). SLE tended to occur during the first and second trimesters (33 and 42 %, respectively). Lupus nephritis (LN) and severe thrombocytopenia were more commonly seen in new-onset SLE during pregnancy than in patients without pregnancy (68.8 vs 35.4 % and 25 vs 9.2 %, respectively, p?<?0.05). However, pregnant patients had lower frequency of fever, arthritis, arthralgia, alopecia, oral ulcer, and hypocomplementemia than the nonpregnant controls (p?<?0.05). Compared to LN patients without pregnancy (n?=?23), LN patients with pregnancy (n?=?33) had more prominent proteinuria and nephrotic syndrome (p?<?0.05). On the other hand, when compared to patients with preeclampsia, patients with new-onset SLE during pregnancy had early onset of symptoms during gestation and were characterized by presence of fever, malar lesion, autoantibodies, hypocomplementemia, hyperuricemia, active urinary sediment, and multi-organ involvement. In conclusion, patients with their first onset of lupus during pregnancy generally have more severe disease with higher prevalence of renal and platelet involvement.     

Increased mean platelet volume in primary Raynaud's phenomenon

We hypothesized that mean platelet volume (MPV), a reliable marker of platelet activation, might be elevated in primary Raynaud's phenomenon (PRP) even if there was no thrombotic complication in our subjects. In this retrospective-cohort study, we examined the clinical value of MPV in 200 patients with PRP and 116 clinical controls, and measured MPV and platelet P-selectin (CD62P) in all study participants. We also evaluated the effect of age, gender, and disease duration on these platelet activation markers in PRP. MPV and CD62 positivities were significantly (p?<?0.001) elevated in patients with PRP compared with controls. These differences retained when patients and controls were analyzed according to age, gender, and the disease duration. In logistic regression analysis, MPV (OR: 15.8, 95% CI: 8.14–30.64, p?<?0.001) and CD62P (OR: 11.3, 95% CI: 4.85–26.12, p?<?0.001) were found to be independently associated with PRP. In conclusion, increased MPV is independently related to PRP, and its level was not influenced by age, gender, and the duration of PRP.     

Incidence and predictors of hematological side effects in chronic HCV Egyptian patients treated with PEGylated interferon and ribavirin

Aim

The aim of this paper was to study the incidence and predictors of hematological abnormalities during treatment of chronic hepatitis C virus (HCV) patients with interferon and ribavirin.

Methods

One thousand and eighty-one chronic HCV patients who were treated with PEGylated interferon α-2a 180 μg (n?=?536) or α-2b 1.5 μg/kg (n?=?545) plus ribavirin for 48 weeks were included. Baseline demographic, laboratory, and histopathological data and, during treatment, hematological data were collected and analyzed using univariate and multivariate analyses to identify independent predictors of hematological side effects.

Results

During therapy, 168 of 1,018 (15.5 %) had moderate anemia (Hb?<10 and ≥8.5 g/dL) and 88 (8.1 %) had severe anemia (Hb?<8.5 g/dL). Two hundred and six patients (19.1 %) had moderate neutropenia (absolute neutrophil count (ANC)?<750 and ≥500/mm³); only 55 (5.1 %) had severe neutropenia (ANC?<500/mm³). Forty-three patients (4 %) had moderate (platelet <50,000 and ≥25,000/mm³) and 5 (1.4 %) had severe thrombocytopenia (platelet <25,000/mm³). Fibrosis stage, week 4 Hb level, and week 2 and 4 reduction level in Hb were independent predictors of moderate and severe anemia (p?<?0.001). Fibrosis stage and ANC at weeks 2 and 4 were predictors of neutropenia (p?<?0.001, 0.001, and 0.004, respectively). Fibrosis stage and platelet count at weeks 2 and 4 were predictors of thrombocytopenia (p?<?0.001, <0.001, and 0.005, respectively). There was no association between interferon type and anemia (p?=?0.57), neutropenia (p?=?0.6), or thrombocytopenia (p?=?0.79).

Conclusions

Fibrosis stage and week 2 and 4 hematological parameter reduction levels were independent predictors of hematological side effects, which are not related to interferon type.     

Metabolic syndrome in Sjögren’s syndrome patients: a relevant concern for clinical monitoring

Metabolic syndrome (MetS) has been described in autoimmune diseases. However, there are scarce data about MetS and adipocytokine profile in primary Sjögren’s syndrome (pSS). Seventy-one female pSS patients (American-European Consensus Group Criteria, 2002) aged 18–65 years and 71 age-, race-matched control women were enrolled in this case–control study. Clinical data were collected by a standardized protocol. Blood levels of glucose, cholesterol, low-density lipoprotein-cholesterol (LDL-C), high-density lipoprotein-cholesterol (HDL-C), triglycerides, interleukin-1beta (IL-1beta)/IL-6, B-cell activating factor (BAFF), insulin, and leptin/adiponectin/visfatin/resistin were determined. Patients and controls were comparable regarding body mass index (BMI), smoking, sedentariness, and menopause (p?>?0.05). MetS (39.4 vs. 16.9 %, p?=?0.005), hypertension (p?=?0.004), and dyslipidemia (p?=?0.002) were more frequent in patients than controls. IL-1beta, IL-6, BAFF, resistin, and adiponectin levels were higher in patients than controls (p?<?0.05). pSS patients with MetS (n?=?28) had higher BMI, waist circumference, cholesterol, LDL-C, triglycerides, insulin, leptin and HOMA-IR values, and greater hypertension and diabetes rates than pSS patients without MetS (n?=?43) (p?<?0.05). Current and/or previous prednisone use (75.0 vs. 62.8 %, p?=?0.313), current (3.0?±?4.5 vs. 1.6?±?3.2 mg/day, p?=?0.299), and cumulative prednisone doses (p?=?0.495) were similar in both groups. Otherwise, IL-1beta level was higher in MetS patients than in non-MetS patients (p?=?0.012), and this finding was confirmed (p?=?0.048) by multivariate analysis with adjustments for age, ethnicity, prednisone use, current and cumulative prednisone doses, and duration of use. We identified high MetS frequency and abnormal adipocytokine profile in pSS. The association of MetS with elevated IL-1beta level suggests that inflammation plays an important role in its pathogenesis.     

Relationship between serum leptin level and disease activity in patients with systemic sclerosis

To determine the relationship between serum leptin levels and disease activity in systemic sclerosis (SSc). A total of 60 subjects (30 controls and 30 patients) were included. The inflammatory markers and leptin levels were evaluated and body mass index (BMI) was measured for both groups. The assessment of the skin involvement was performed based on the modified Rodnan skin score (mRSS). Disease activity was evaluated according to the Valentini scleroderma disease activity index. There was a significant difference between the patient and control groups in terms of BMI (p?<?0.05); however there was no difference with regards to age and gender (p?>?0.05). Valentini scores and mRSS were determined to be significantly higher in active patients (n?=?14) than in inactive patients (n?=?16) (p?<?0.05). No significant difference was determined between groups in terms of leptin levels (p?>?0.05). However, leptin levels were significantly lower in active patients than in inactive patients (p?<?0.05). We found a significant positive correlation between serum leptin and BMI (p?<?0.05), and leptin and serum C3 levels (p?<?0.05); no relationship was detected between leptin and other parameters. Leptin can be used as an activity marker in SSc. Further studies, including larger series, should be carried out to clarify this relationship.     

Increased serum levels of adhesion molecules ICAM-1 and VCAM-1 in systemic sclerosis are not specific for pulmonary manifestations

Studies suggest elevated serum intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) levels may be markers of pulmonary arterial hypertension in systemic sclerosis (SSc-PAH). We sought to evaluate whether ICAM-1 and VCAM-1 levels are useful screening biomarkers for incident SSc-PAH. In this cross-sectional study, four groups were selected from the Australian Scleroderma Cohort Study: group 1 (n?=?15) had definite PAH; group 2 (n?=?19) had interstitial lung disease (ILD); group 3 (n?=?30) were SSc-controls; and group 4 (n?=?34) were healthy controls. Serum ICAM-1 and VCAM-1 levels were measured using the Millipore Milliplex MAP Human 2-Plex Panel. There were no differences in ICAM-1 levels in the PAH versus ILD group (263.0?±?85.4 vs 380.4?±?168.3 ng/mL, p?=?0.136), SSc-controls (263.0?±?85.4 vs 253.1?±?98.0 ng/mL, p?=?1.00), or healthy controls (263.0?±?85.4 vs 201.8?±?57.2 ng/mL, p?=?0.093). Similarly, there were no differences in VCAM-1 level in PAH versus ILD groups (1476.2?±?434.9 vs 1424.8?±?527.6 ng/mL, p?=?1.00) and SSc-controls (1476.2?±?434.9 vs 1409.5?±?341.1 ng/mL, p?=?1.00). SSc subjects had significantly higher levels of ICAM-1 (297.4?±?134.0 vs 201.8?±?57.2 ng/mL, p?<?0.0001) and VCAM-1 compared to healthy controls (1432.7?±?427.4 vs 1125.6?±?273.4 ng/mL, p?<?0.0001). Neither ICAM-1 nor VCAM-1 is a specific screening biomarker of SSc-PAH. Instead, increased levels of these adhesion molecules in SSc, irrespective of pulmonary complications, suggest that they may play a role in SSc pathogenesis.     

<Emphasis Type="Italic">Vegf-A</Emphasis> mRNA transfection as a novel approach to improve mouse and human islet graft revascularisation

Aims/hypothesis

The initial avascular period following islet transplantation seriously compromises graft function and survival. Enhancing graft revascularisation to improve engraftment has been attempted through virus-based delivery of angiogenic triggers, but risks associated with viral vectors have hampered clinical translation. In vitro transcribed mRNA transfection circumvents these risks and may be used for improving islet engraftment.

Methods

Mouse and human pancreatic islet cells were transfected with mRNA encoding the angiogenic growth factor vascular endothelial growth factor A (VEGF-A) before transplantation under the kidney capsule in mice.

Results

At day 7 post transplantation, revascularisation of grafts transfected with Vegf-A (also known as Vegfa) mRNA was significantly higher compared with non-transfected or Gfp mRNA-transfected controls in mouse islet grafts (2.11- and 1.87-fold, respectively) (vessel area/graft area, mean?±?SEM: 0.118?±?0.01 [n?=?3] in Vegf-A mRNA transfected group (VEGF) vs 0.056?±?0.01 [n?=?3] in no RNA [p?<?0.05] vs 0.063?±?0.02 [n?=?4] in Gfp mRNA transfected group (GFP) [p?<?0.05]); EndoC-bH3 grafts (2.85- and 2.48-fold. respectively) (0.085?±?0.02 [n?=?4] in VEGF vs 0.030?±?0.004 [n?=?4] in no RNA [p?<?0.05] vs 0.034?±?0.01 [n?=?5] in GFP [p?<?0.05]); and human islet grafts (3.17- and 3.80-fold, respectively) (0.048?±?0.013 [n?=?3] in VEGF vs 0.015?±?0.0051 [n?=?4] in no RNA [p?<?0.01] vs 0.013?±?0.0046 [n?=?4] in GFP [p?<?0.01]). At day 30 post transplantation, human islet grafts maintained a vascularisation benefit (1.70- and 1.82-fold, respectively) (0.049?±?0.0042 [n?=?8] in VEGF vs 0.029?±?0.0052 [n?=?5] in no RNA [p?<?0.05] vs 0.027?±?0.0056 [n?=?4] in GFP [p?<?0.05]) and a higher beta cell volume (1.64- and 2.26-fold, respectively) (0.0292?±?0.0032 μl [n?=?7] in VEGF vs 0.0178?±?0.0021 μl [n?=?5] in no RNA [p?<?0.01] vs 0.0129?±?0.0012 μl [n?=?4] in GFP [p?<?0.001]).

Conclusions/interpretation

Vegf-A mRNA transfection before transplantation provides a promising and safe strategy to improve engraftment of islets and other cell-based implants.

     

Aspirin Dose Increase from 75 to 150 mg Suppresses Red Blood Cell Contribution to Suboptimal Platelet Response to Aspirin in Patients with CAD

Purpose

To verify the hypothesis that erythrocytes play a role in suboptimal blood platelet response to acetylsalicylic acid (ASA, aspirin) in subjects with coronary artery disease (CAD).

Methods

In a cross-over randomized controlled intervention study we evaluated blood platelet response to 30-day treatment with 75 mg/d or 150 mg/d of ASA (enteric coated) in CAD patients (n?=?125). In vitro platelet response to collagen or arachidonic acid was monitored with impedance aggregometry and plasma thromboxane B₂ was assayed immunoenzymatically. Blood morphology and several plasma biochemical parameters were determined using routine diagnostic procedures.

Results

CAD patients demonstrated lower blood platelet responsiveness to 75 mg/d of ASA compared to healthy subjects. The improved platelet responsiveness to 150 mg/d of ASA was particularly evident in “poor” responding patients. Positive correlations between platelet “poor” response to lower (75 mg/d) ASA dose and red blood cell count (R_s?=?0.215; p?<?0.04), haemoglobin (R_s?=?0.232; p?<?0.02) and haematocrit (R_s?=?0.239; p?<?0.02) were found in CAD patients. Association between “poor” platelet response with lower ASA dose was confirmed by conditional maximum likelihood logistic regression, which showed the independency between erythrocyte-derived parameters, as the risk factors for suboptimal platelet response to ASA, and other risk factors, like CRP or LDL-cholesterol. In “poor” ASA responders taking the higher ASA dose (150 mg/d) the correlation between platelets’ response to ASA and erythrocyte-derived parameters was not significant.

Conclusions

Red blood cell parameters are associated with suboptimal blood platelet response to ASA in patients with CAD. Such a platelet refractoriness to ASA may be effectively overcome by increasing the ASA dose.     

Volatile Organic Compounds in Exhaled Breath of Idiopathic Pulmonary Fibrosis for Discrimination from Healthy Subjects

Purpose Human breath analysis is proposed with increasing frequency as a useful tool in clinical application. We performed this study to find the characteristic volatile organic compounds (VOCs) in the exhaled breath of patients with idiopathic pulmonary fibrosis (IPF) for discrimination from healthy subjects. Methods VOCs in the exhaled breath of 40 IPF patients and 55 healthy controls were measured using a multi-capillary column and ion mobility spectrometer. The patients were examined by pulmonary function tests, blood gas analysis, and serum biomarkers of interstitial pneumonia. Results We detected 85 VOC peaks in the exhaled breath of IPF patients and controls. IPF patients showed 5 significant VOC peaks; p-cymene, acetoin, isoprene, ethylbenzene, and an unknown compound. The VOC peak of p-cymene was significantly lower (p?<?0.001), while the VOC peaks of acetoin, isoprene, ethylbenzene, and the unknown compound were significantly higher (p?<?0.001 for all) compared with the peaks of controls. Comparing VOC peaks with clinical parameters, negative correlations with VC (r?=?0.393, p?=?0.013), %VC (r?=?0.569, p?<?0.001), FVC (r?=??0.440, p?=?0.004), %FVC (r?=?0.539, p?<?0.001), DLco (r?=?0.394, p?=?0.018), and %DLco (r?=?0.413, p?=?0.008) and a positive correlation with KL-6 (r?=?0.432, p?=?0.005) were found for p-cymene. Conclusion We found characteristic 5 VOCs in the exhaled breath of IPF patients. Among them, the VOC peaks of p-cymene were related to the clinical parameters of IPF. These VOCs may be useful biomarkers of IPF.     

Advanced glycation end products in myocardial reperfusion injury

Advanced glycation end products (AGEs) are associated with cardiovascular diseases. Whether the AGE levels change during myocardial reperfusion injury is currently unknown. The aim of our study was to investigate the dynamics of AGEs in myocardial reperfusion injury and to discuss potential reasons for these changes. The dynamics of AGEs, pentosidine and neopterin in the plasma of patients with acute myocardial infarction (AMI) treated using thrombolysis (n?=?40) were analyzed. In addition, AGEs were measured in patients with open heart surgery (n?=?12) and rabbits with induced AMI (n?=?9). In all three studies of myocardial reperfusion injury, a significant decrease of AGEs was observed (by 26?±?19% in patients with AMI, by 23?±?14% in patients with open heart surgery and by 39?±?10% in rabbits with AMI within 1?day of reperfusion; p?<?0.05 in all studies). In additional studies, an association between lower AGEs and an activated immune system (R ²?=?0.09; p?<?0.01) and fasting (decrease by 38%; p?<?0.01) was shown. AGEs decrease in reperfusion injury of the heart. Indices pointing towards the involvement of immune system activation and fasting are presented. Further studies focusing on the underlying mechanism and on the clinical value of the observed dynamics of AGEs are needed.     

Prevalence of irritable bowel syndrome and functional dyspepsia,overlapping symptoms,and associated factors in a general population of Bangladesh

Background

This community-based survey aimed to find out the prevalence of irritable bowel syndrome (IBS), functional dyspepsia (FD), overlapping symptoms, and associated factors for overlap.

Method

By cluster sampling method, 3,000 (1,523 male) randomly selected adult subjects in the Sylhet district of Bangladesh were interviewed by a questionnaire based on ROME III criteria. Multivariate logistic regression analyses were done to find out the factors for overlap with significance level set at ≤0.05.

Results

The mean age of the study population was 33.9?±?16.4 years. Prevalence of IBS and FD and IBS-FD were 12.9 % (n?=?387), 8.3 % (n?=?249), and 3.5 % (n?=?105), respectively. Approximately 27.1 % of IBS patients and 42.1 % of FD patients had overlapping IBS-FD. The odds ratio for IBS-FD overlap was 6.3 (95 % CI, 4.8–8.4). Mean age (p?=?0.011) and epigastric pain (p?=?0.002) were more in overlap patients than FD alone, whereas epigastric pain syndrome subtype (p?<?0.009) was more prevalent in lone FD subjects. In the multivariate logistic analysis, early satiety (OR, 3.0; 95 % CI, 1.2–7.5; p?=?0.018) and epigastric pain (OR, 14.5; 95 % CI, 5.0–42.1; p?=?0.000) in FD patients appeared as independent risk factors for overlap. Bloating (p?=?0.026), <3 stools per week (p?=?0.050), abdominal pain reduced by defecation (p?=?0.002), abdominal pain severity score (p?=?0.004), and overall symptom frequency score (p?=?0.000) were more in overlap patients than IBS-alone patients. In IBS patients, bloating (OR, 3.6; CI, 2.0–6.5; p?=?0.000) was found as potential symptom associated with IBS-FD overlap.

Conclusion

FD was a less prevalent disorder than IBS in our community, and significant overlap existed between the two disorders. Early satiety, epigastric pain, and bloating were important factors associated with overlap.     

Non-driver mutations in patients with <Emphasis Type="Italic">JAK2</Emphasis>V617F-mutated polycythemia vera or essential thrombocythemia with long-term molecular follow-up

JAK2V617F monitoring and NGS of non-driver genes was performed in 100 patients with polycythemia vera (PV) or essential thrombocythemia (ET) with long molecular follow-up. Patients who did not progress to myelofibrosis (MF) or acute myeloid leukemia (AML) after more than 10 years (n?=?50) showed a low frequency of mutations at first sample (18%) and an incidence rate of 1.7 new mutations?×?100 person-years. Mutations were detected at first sample in 83% of PV/ET patients who later progressed to AML (n?=?12) with these patients having a rate of 25.6 mutations?×?100 person-years. Presence of mutations at diagnosis was the unique risk factor for acquiring a new genetic event (HR 2.7, 95% CI 1.1–6.8, p?=?0.03) after correction for age, PV diagnosis, and total duration of hydroxyurea (HU) exposure. Patients with additional mutation at first sample showed a higher probability of developing cytopenia under HU therapy and a higher risk of AML (HR 12.2, 95% CI 2.6–57.1, p?=?0.001) with mutations in ASXL1 (p?<?0.0001), TP53 (p?=?0.01), SRSF2 (p?<?0.0001), IDH1/2 (p?<?0.0001), and RUNX1 (p?<?0.0001) being associated with a higher probability of AML. Myelofibrotic transformation was more frequent in patients with additional mutations, especially in SF3B1 (p?=?0.02) and IDH1/2 (p?<?0.0001) although a persistently high or a progressive increase of the JAK2V617F allele burden while receiving cytoreduction was the strongest predictor of MF transformation (HR 10.8, 95% CI 2.4–49.1, p?=?0.002). In conclusion, NGS may be useful to identify a minority of PV and ET patients with high genetic instability and increased risk of AML transformation.