Revised response criteria for polycythemia vera and essential thrombocythemia:an ELN and IWG-MRT consensus project

Standardized response criteria to interpret and compare clinical trials are needed for approval of new therapeutic agents by regulatory agencies. The European LeukemiaNet (ELN) response criteria for essential thrombocythemia (ET) and polycythemia vera (PV) issued in 2009 have been widely adopted as end points in a number of recent clinical trials. However, evidence exists that they do not predict response or provide clinically relevant measures of benefit for the patients. This article presents revised recommendations for assessing response in ET and PV provided by a working group established by ELN and International Working Group-Myeloproliferative Neoplasms Research and Treatment. New definitions of complete and partial remission incorporate clinical, hematological, and histological response assessments that include a standardized symptom assessment form and consider absence of disease progression and vascular events. We anticipate that these criteria will be adopted widely to facilitate the development of new and more effective therapies for ET and PV.

The Role of K+ and Cl- Channels in the Regulation of Retinal Arteriolar Tone and Blood Flow

Purpose: This study tested the role of K(+)- and Cl(-)-channels in retinal arteriolar smooth muscle in the regulation of retinal blood flow.

Methods: Studies were carried out in adult Male Hooded Lister rats. Selectivity of ion channel blockers was established using electrophysiological recordings from smooth muscle in isolated arterioles under voltage clamp conditions. Leukocyte velocity and retinal arteriolar diameters were measured in anesthetised animals using leukocyte fluorography and fluorescein angiography imaging with a confocal scanning laser ophthalmoscope. These values were used to estimate volumetric flow, which was compared between control conditions and following intravitreal injections of ion channel blockers, either alone or in combination with the vasoconstrictor potent Endothelin 1 (Et1).

Results: Voltage activated K(+)-current (IKv) was inhibited by correolide, large conductance (BK) Ca(2+)-activated K(+)-current (IKCa) by Penitrem A, and Ca(2+)-activated Cl(-)-current (IClCa) by disodium 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS). Intravitreal injections (10µl) of DIDS (estimated intraocular concentration 10mM) increased flow by 22%, whereas the BK-blockers Penitrem A (1µM) and iberiotoxin (4µM), and the IKv-inhibitor correolide (40µM) all decreased resting flow by approximately 10%. Et1 (104nM) reduced flow by almost 65%. This effect was completely reversed by DIDS but was unaffected by Penitrem A, iberiotoxin or correolide.

Conclusions: These results suggest that Cl(-)-channels in retinal arteriolar smooth muscle limit resting blood flow and play an obligatory role in Et1 responses. K(+)-channel activity promotes basal flow but exerts little modifying effect on the Et1 response. Cl(-)-channels may be appropriate molecular targets in retinal pathologies characterised by increased Et1 activity and reduced blood flow.

Children's views on microneedle use as an alternative to blood sampling for patient monitoring

Objectives: To explore children's views on microneedle use for this population, particularly as an alternative approach to blood sampling, in monitoring applications, and so, examine the acceptability of this approach to children.

Methods: Focus groups were conducted with children (aged 10-14 years) in a range of schools across Northern Ireland. Convenience sampling was employed, i.e. children involved in a university-directed community-outreach project (Pharmacists in Schools) were recruited.

Key findings: A total of 86 children participated in 13 focus groups across seven schools in Northern Ireland. A widespread disapproval for blood sampling was evident, with pain, blood and traditional needle visualisation particularly unpopular aspects. In general, microneedles had greater visual acceptability and caused less fear. A patch-based design enabled minimal patient awareness of the monitoring procedure, with personalised designs, e.g. cartoon themes, favoured. Children's concerns included possible allergy and potential inaccuracies with this novel approach; however, many had confidence in the judgement of healthcare professionals if deeming this technique appropriate. They considered paediatric patient education critical for acceptance of this new approach and called for an alternative name, without any reference to 'needles'.

Conclusions: The findings presented here support the development of blood-free, minimally invasive techniques and provide an initial indication of microneedle acceptability in children, particularly for monitoring purposes. A proactive response to these unique insights should enable microneedle array design to better meet the needs of this end-user group. Further work in this area is recommended to ascertain the perspectives of a purposive sample of children with chronic conditions who require regular monitoring. © 2013 Royal Pharmaceutical Society.

Contractile properties of human renal cell carcinoma recruited arteries and their response to nicotinamide

Background and purpose: The manipulation of tumour blood supply and thus oxygenation is a potentially important strategy for improving the treatment of solid tumours by radiation. Increased knowledge about the characteristics that distinguish the tumour vasculature from its normal counterparts may enable tumour blood flow to be more selectively modified, Nicotinamide (NA) causes relaxation of preconstricted normal and tumour-supply arteries in rats. It has also been shown to affect microregional blood flow in human tumours. Direct effects of NA on human tumour supply arteries have not previously been reported. This paper describes our evaluation of the effects of NA on two parameters: 'spontaneous', oscillatory contractile activity and agonist (phenylephrine)-induced constriction in the arteries supplying human renal cell carcinomas.

Materials and methods: Isolated renal cell carcinoma feeder vessels were perfused in an organ bath with the alpha(1)-adrenoceptor agonist phenylephrine (PE). When the arteries had reached a plateau of constriction, nicotinamide (8.2 mM) was added to the perfusate and changes in perfusion pressure were measured.

Results: PE (10 mu M) induced a sustained constriction in the majority of the renal cell carcinoma feeder vessels examined, demonstrating that they retain contractile characteristics, at least in response to this alpha(1)-adrenoceptor agonist. In combination with NA (8.2 mM) the constriction was significantly attenuated in half of the preparations. In addition, seven arteries exhibited spontaneous contractile activity which was significantly attenuated by NA in six of them.

Conclusions: NA can significantly attenuate both 'spontaneous' and agonist-induced constrictions in tumour-recruited human arteries, though not all arteries are sensitive. Published by Elsevier Science Ireland Ltd.

On the antioxidant properties of erythropoietin and its association with the oxidative-nitrosative stress response to hypoxia in humans

Aim: The aim of this study was to examine if erythropoietin (EPO) has the potential to act as a biological antioxidant and determine the underlying mechanisms.

Methods: The rate at which its recombinant form (rHuEPO) reacts with hydroxyl (HO center dot), 2,2-diphenyl-1-picrylhydrazyl (DPPH center dot) and peroxyl (ROO center dot) radicals was evaluated in-vitro. The relationship between the erythopoietic and oxidative-nitrosative stress response to poikilocapneic hypoxia was determined separately in-vivo by sampling arterial blood from eleven males in normoxia and following 12 h exposure to 13% oxygen. Electron paramagnetic resonance spectroscopy, ELISA and ozone-based chemiluminescence were employed for direct detection of ascorbate (A(center dot-)) and N-tert-butyl-a-phenylnitrone spin-trapped alkoxyl (PBN-OR) radicals, 3-nitrotyrosine (3-NT) and nitrite (NO2-).

Results: We found rHuEPO to be a potent scavenger of HO center dot (k(r) = 1.03-1.66 x 10(11) M-1 s(-1)) with the capacity to inhibit Fenton chemistry through catalytic iron chelation. Its ability to scavenge DPPH. and ROO center dot was also superior compared to other more conventional antioxidants. Hypoxia was associated with a rise in arterial EPO and free radical-mediated reduction in nitric oxide, indicative of oxidative-nitrosative stress. The latter was confirmed by an increased systemic formation of A(center dot-), PBN-OR, 3-NT and corresponding loss of NO2- (P <0.05 vs. normoxia). The erythropoietic and oxidative-nitrosative stress responses were consistently related (r =-0.52 to 0.68, P <0.05).

Conclusion: These findings demonstrate that EPO has the capacity to act as a biological antioxidant and provide a mechanistic basis for its reported cytoprotective benefits within the clinical setting.

Increasing Fruit and Vegetable Intake Has No Dose-Response Effect on Conventional Cardiovascular Risk Factors in Overweight Adults at High Risk of Developing Cardiovascular Disease

BACKGROUND: Improving diet and lifestyle is important for prevention of cardiovascular disease (CVD). Observational evidence suggests that increasing fruit and vegetable (FV) consumption may lower CVD risk, largely through modulation of established risk factors, but intervention data are required to fully elucidate the mechanisms by which FVs exert benefits on vascular health.

OBJECTIVE: The aim of this study was to examine the dose-response effect of FV intake on cardiovascular risk factors in adults at high CVD risk.

METHODS: This was a randomized controlled parallel group study involving overweight adults (BMI: >27 and ≤35 kg/m(2)) with a habitually low FV intake (≤160 g/d) and a high total risk of developing CVD (estimated ≥20% over 10 y). After a 4-wk run-in period where FV intake was limited to <2 portions/d (<160 g/d), 92 eligible participants were randomly assigned to 1 of 3 groups: to consume either 2, 4, or 7 portions (equivalent to 160 g, 320 g, or 560 g, respectively) of FVs daily for 12 consecutive weeks. Fasting venous blood samples were collected at baseline (week 4) and post-intervention (week 16) for analysis of lipid fractions and high-sensitivity C-reactive protein (hsCRP) concentrations. Compliance with the FV intervention was determined with use of self-reported FV intake and biomarkers of micronutrient status. Ambulatory blood pressure and body composition were also measured pre- and post-intervention.

RESULTS: A total of 89 participants completed the study and body composition remained stable throughout the intervention period. Despite good compliance with the intervention, no significant difference was found between the FV groups for change in measures of ambulatory blood pressure, plasma lipids, or hsCRP concentrations.

CONCLUSIONS: There was no evidence of a dose-response effect of FV intake on conventional CVD risk factors measured in overweight adults at high CVD risk. This trial was registered at clinicaltrials.gov as NCT00874341.

Monitoring of lineage-specific chimaerism allows early prediction of response following donor lymphocyte infusions for relapsed chronic myeloid leukaemia

Donor lymphocyte infusions (DLI) have been shown to enhance the graft-versus-leukaemia (GVL) effect and induce haematological and molecular remission in patients with relapsed CML following allogeneic bone marrow transplantation (BMT). The potent donor cell-mediated cytolysis following DLI may lead to a short period of aplasia before the re-establishment of donor haematopoiesis. The absence of detectable donor cells in patients prior to DLI infusion may result in permanent aplasia in certain patients. We report on four patients who relapsed 1, 3, 6.5 and 7 years post-BMT for chronic phase CML and were treated with DLI from their original BMT donor. Polymorphic short tandem repeats (STRs) were used to assess haematological chimaerism both prior to and following DLI. At the time of relapse, STR-PCR indicated the presence of donor cells in all four patients, at levels ranging from 1-40%. A clinical and molecular response was seen in 4/4 patients following a short period of cytopenia and all patients remain in clinical remission with a follow-up of 2 months-3 years post-DLI. STR-PCR indicated that a response was occurring during the period of pancytopenia when metaphase analysis was unsuccessful. Lineage-specific analysis of the cellular response to DLI was monitored using STR-PCR of peripheral blood (PB) and bone marrow (BM) lymphocyte-enriched fractions and CD2-positive and -negative T cell fractions. In one patient BM and PB CD34-positive and -negative fractions were also assessed. A change in the ratio of donor:recipient cells in the PB lymphocyte fraction was the earliest molecular indication of an anti-leukaemic response. Subsequent conversion to donor chimaerism occurred in the other lineages and the granulocyte fraction was the last lineage to convert. In conclusion, lineage-specific STR-PCR permits detailed monitoring of subtle changes in donor/recipient cell dynamics in specific lineages following DLI during the crucial pancytopenic phase and may be a useful predictor of haematological response to DLI therapy.

Starting slow: the effects of response-switching frequency on patterns of cardiovascular reactivity

Research findings suggest that switching between competing response sets can be resource demanding. The current study focused on concurrent health-relevant physiological effects of task switching by assessing cardiovascular response at varying levels of switch frequency. The participants performed a response-switching task at three different levels of response set switching frequency (low, medium and high) while measurements of blood pressure and heart rate were taken. One group was exposed to response-switching frequency conditions in the order low → medium → high, while the other group was exposed to the same task conditions in the reverse order (i.e. high → medium → low). The results showed that the participants in the low → medium → high switch frequency group recovered faster from initially heightened systolic blood pressure when compared with participants in the high → medium → low group. It is concluded that the results point to a physiological "carry over" effect associated with beginning a task at rapid response switching frequency levels, and suggest the importance of habituation to task demands as a means of offsetting potentially unhealthy levels of reactivity. Implications for modern work environments are discussed.

Wavelet entropy of Doppler ultrasound blood velocity flow waveforms distinguishes nitric oxide-modulated states

Wavelet entropy assesses the degree of order or disorder in signals and presents this complex information in a simple metric. Relative wavelet entropy assesses the similarity between the spectral distributions of two signals, again in a simple metric. Wavelet entropy is therefore potentially a very attractive tool for waveform analysis. The ability of this method to track the effects of pharmacologic modulation of vascular function on Doppler blood velocity waveforms was assessed. Waveforms were captured from ophthalmic arteries of 10 healthy subjects at baseline, after the administration of glyceryl trinitrate (GTN) and after two doses of N(G)-nitro-L-arginine-methyl ester (L-NAME) to produce vasodilation and vasoconstriction, respectively. Wavelet entropy had a tendency to decrease from baseline in response to GTN, but significantly increased after the administration of L-NAME (mean: 1.60 ± 0.07 after 0.25 mg/kg and 1.72 ± 0.13 after 0.5 mg/kg vs. 1.50 ± 0.10 at baseline, p < 0.05). Relative wavelet entropy had a spectral distribution from increasing doses of L-NAME comparable to baseline, 0.07 ± 0.04 and 0.08 ± 0.03, respectively, whereas GTN had the most dissimilar spectral distribution compared with baseline (0.17 ± 0.08, p = 0.002). Wavelet entropy can detect subtle changes in Doppler blood velocity waveform structure in response to nitric-oxide-mediated changes in arteriolar smooth muscle tone.

Oxidation and glycoxidation of phosphatidylserine and their effect in immune response : a lipidomic approach

Phosphatidylserine (PS) is a member of the class of phospholipids, and is distributed among all cells of mammalians, playing important roles in diverse biological processes, including blood clotting and apoptosis. When externalized, PS is a ligand that is recognized on apoptotic cells. It has been considered that before externalization PS is oxidized and oxPS enhance the recognition by macrophages receptors, however the knowledge about oxidation of PS is still limited. PS, like others phospholipids, has two fatty acyl chains and one polar head group, in this case is the amino acid serine. The modifications in PS structure can occur by oxidation of the unsaturated fatty acyl chains and by glycation of the polar head group, due to free amine group, thus increasing the susceptibility to oxidative events. The main goal of this work was to characterize and identify oxidized and glycoxidized PS, contributing to the knowledge of the biological role of oxidation products of PS, as well as of glycated PS, in immune and inflammatory processes. To achieve this goal, PS standards (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho- L-serine (POPS), 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS), 1- palmitoyl-2-linoleoyl-sn-glycero-3-phospho-L-serine (PLPS) and 1-palmitoyl-2- arachidonoyl-sn-glycero-3-phospho-L-serine (PAPS)) and glycated PS (PAPS and POPS) were induced to oxidize in model systems, using different oxidant reagents: HO• and 2,2'-azobis-2-methyl-propanimidamide dihydrochloride (AAPH) . The detailed structural characterization of the oxidative products was performed by ESI-MS and MS/MS coupled to separation techniques such as off line TLC-MS and on line LC-MS, in order to obtained better characterization of the larger number of PS and glycated PS oxidation products. The results obtained in this work allowed to identify several oxidation products of PS and glycated PS with modifications in unsaturated fatty acyl chain. Also, oxidation products formed due to structural changes in the serine polar head with formation of terminal acetamide, terminal hydroperoxyacetaldehyde.and terminal acetic acid (glycerophosphacetic acid, GPAA) were identified. The mass spectrometric specific fragmentation pathway of each type of oxidation product was determined using different mass spectrometry approaches. Based on the identified fragmentation pathways, targeted lipidomic analysis was performed to detect oxidation products modified in serine polar head in HaCaT cell line treated with AAPH. The GPAA was detected in HaCaT cells treated with AAPH to induce oxidative stress, thus confirming that modifications in PS polar head is possible to occur in biological systems. Furthermore, it was found that glycated PS species are more prone to oxidative modifications when compared with non glycated PS. During oxidation of glycated PS, besides the oxidation in acyl chains, new oxidation products due to oxidation of the glucose moiety were identified, including PS advanced glycation end products (PSAGES). To investigate if UVA oxidative stress exerted changes in the lipidome of melanoma cell lines, particularly in PS profile, a lipidomic analysis was performed. The lipid profile was obtained using HILIC-LC-MS and GC-MS analysis of the total lipid extracts obtained from human melanoma cell line (SKMEL- 28) after UVA irradiation at 0, 2 and 24 hours. The results did not showed significant differences in PS content. At molecular level, only PS (18:0:18:1) decreased at the moment of irradiation. The most significant changes in phospholipids content occurred in phosphatidylcholines (PC) and phosphatidylinositol (PI) classes, with an increase of mono-unsaturated fatty acid (MUFA), similarly as observed for the fatty acid analysis. Overall, these data indicate that the observed membrane lipid changes associated with lipogenesis after UVA exposure may be correlated with malignant transformations associated with cancer development and progression. Despite of UVA radiation is associated with oxidative damage, in this work was not possible observe oxidation phospholipids. The anti/pro-inflammatory properties of the oxidized PLPS (oxPLPS) versus non-oxidized PLPS were tested on LPS stimulated RAW 264.7 macrophages. The modulation of intracellular signaling pathways such as NF-kB and MAPK cascades by oxPLPS and PS was also examined in this study. The results obtained from evaluation of anti/pro-inflammatory properties showed that neither PLPS or oxPLPS species activated the macrophages. Moreover only oxidized PLS were found to significantly inhibit NO production and iNOS and il1β gene transcription induced by LPS. The analysis at molecular level showed that this was the result of the attenuation of LPS-induced c-Jun-N-terminal kinase (JNK) and p65 NF-kB nuclear translocation. Overall these data suggest that oxPLPS, but not native PLPS, mitigates pro-inflammatory signaling in macrophages, contributing to containment of inflammation during apoptotic cell engulfment. The results obtained in this work provides new information on the modifications of PS, facilitating the identification of oxidized species in complex samples, namely under physiopathologic conditions and also contributes to a better understanding of the role of oxPS and PS in the inflammatory response, in the apoptotic process and other biological functions.

The effect of a selective CXCR2 antagonist (AZD5069) on human blood neutrophil count and innate immune functions.

Abstract AIMS: The aim of the present study was to investigate whether selective antagonism of the cysteine-X-cysteine chemokine receptor-2 (CXCR2) receptor has any adverse effects on the key innate effector functions of human neutrophils for defence against microbial pathogens. METHODS: In a double-blind, crossover study, 30 healthy volunteers were randomized to treatment with the CXCR2 antagonist AZD5069 (100 mg) or placebo, twice daily orally for 6 days. The peripheral blood neutrophil count was assessed at baseline, daily during treatment and in response to exercise challenge and subcutaneous injection of granulocyte-colony stimulating factor (G-CSF). Neutrophil function was evaluated by phagocytosis of Escherichia coli and by the oxidative burst response to E. coli. RESULTS: AZD5069 treatment reversibly reduced circulating neutrophil count from baseline by a mean [standard deviation (SD)] of -1.67 (0.67) ×10(9) l(-1) vs. 0.19 (0.78) ×10(9) l(-1) for placebo on day 2, returning to baseline by day 7 after the last dose. Despite low counts on day 4, a 10-min exercise challenge increased absolute blood neutrophil count, but the effect with AZD5069 was smaller and not sustained, compared with placebo treatment. Subcutaneous G-CSF on day 5 caused a substantial increase in blood neutrophil count in both placebo- and AZD5069-treated subjects. Superoxide anion production in E. coli-stimulated neutrophils and phagocytosis of E. coli were unaffected by AZD5069 (P = 0.375, P = 0.721, respectively vs. baseline, Day 4). AZD5069 was well tolerated. CONCLUSIONS: CXCR2 antagonism did not appear adversely to affect the mobilization of neutrophils from bone marrow into the peripheral circulation, phagocytosis or the oxidative burst response to bacterial pathogens. This supports the potential of CXCR2 antagonists as a treatment option for diseases in which neutrophils play a pathological role.

Immune cell changes in response to a swimming training session during a 24-h recovery period

Understanding the impact of training sessions on the immune response is crucial for the adequate periodization of training, to prevent both a negative influence on health and a performance impairment of the athlete. This study evaluated acute systemic immune cell changes in response to an actual swimming session, during a 24-h recovery period, controlling for sex, menstrual cycle phases, maturity, and age group. Competitive swimmers (30 females, 15 ± 1.3 years old; and 35 males, 16.5 ± 2.1 years old) performed a high-intensity training session. Blood samples were collected before, immediately after, 2 h after, and 24 h after exercise. Standard procedures for the assessment of leukogram by automated counting (Coulter LH 750, Beckman) and lymphocytes subsets by flow cytometry (FACS Calibur BD, Biosciences) were used. Subjects were grouped according to competitive age groups and pubertal Tanner stages. Menstrual cycle phase was monitored. The training session induced neutrophilia, lymphopenia, and a low eosinophil count, lasting for at least 2 h, independent of sex and maturity. At 24 h postexercise, the acquired immunity of juniors (15-17 years old), expressed by total lymphocytes and total T lymphocytes (CD3+), was not fully recovered. This should be accounted for when planning a weekly training program. The observed lymphopenia suggests a lower immune surveillance at the end of the session that may depress the immunity of athletes, highlighting the need for extra care when athletes are exposed to aggressive environmental agents such as swimming pools

New insights into host-parasite ubiquitin proteome dynamics in P. falciparum infected red blood cells using a TUBEs-MS approach

Malaria, caused by Plasmodium falciparum (P. falciparum), ranks as one of the most baleful infectious diseases worldwide. New antimalarial treatments are needed to face existing or emerging drug resistant strains. Protein degradation appears to play a significant role during the asexual intraerythrocytic developmental cycle (IDC) of P. falciparum. Inhibition of the ubiquitin proteasome system (UPS), a major intracellular proteolytic pathway, effectively reduces infection and parasite replication. P. falciparum and erythrocyte UPS coexist during IDC but the nature of their relationship is largely unknown. We used an approach based on Tandem Ubiquitin-Binding Entities (TUBEs) and 1D gel electrophoresis followed by mass spectrometry to identify major components of the TUBEs-associated ubiquitin proteome of both host and parasite during ring, trophozoite and schizont stages. Ring-exported protein (REX1), a P. falciparum protein located in Maurer's clefts and important for parasite nutrient import, was found to reach a maximum level of ubiquitylation in trophozoites stage. The Homo sapiens (H. sapiens) TUBEs associated ubiquitin proteome decreased during the infection, whereas the equivalent P. falciparum TUBEs-associated ubiquitin proteome counterpart increased. Major cellular processes such as DNA repair, replication, stress response, vesicular transport and catabolic events appear to be regulated by ubiquitylation along the IDC P. falciparum infection.