Functional molecules to test the free radical theory of ageing

Oxidative stress at a cellular level resulting from reactive oxygen species (ROS) has been implicated as a key factor in the ageing process and the development of age-related diseases. Mitochondria are the primary source of endogenously generated ROS through the leakage of electrons from the respiratory chain to molecular oxygen. Therefore there is a lot of interest in investigating ROS production and their biological impacts on both a cellular level and whole organism level. A mitochondria-targeted hydrogen peroxide (H2O2) probe (MitoB) i and a mitochondria-targeted nitric oxide (•NO) probe (MitoDA) iii have been prepared to allow the detection and quantification of mitochondrial concentrations of these species in vitro and in vivo. These utilise the selective reactivity of boronic acids with H2O2 and 1,2-dianilines with the combination of •NO and oxygen respectively to afford specific reaction products. These conversions are readily detectable by ESI-MS and quantified using deuterated internal standards. The mitochondrial membrane potential (ΔΨm) is directly linked to levels of ROS production, with an elevated ΔΨm resulting in increased ROS production. Mitochondrial membrane potential moderators that release uncoupling molecules from caged precursors selectively in the presence of H2O2 were developed. Two untargeted analogues, DNP-SUM v and FCCP-SUM viii were prepared to release 2,4-dinitrophenoxyl anions (DNP–, vi) or carbonyl cyanide-p-trifluoromethoxyphenylhydrazonyl anions (FCCP–, ix) respectively and their pseudo-first order kinetics assessed. Two mitochondria-targeted analogues, MitoDNP-SUM(1) x and MitoDNP-SUM(2) xi designed to release DNP– vi inside the mitochondrial matrix were prepared. Their abilities to lower the ΔΨm and therefore the ROS producing capabilities in isolated rat skeletal muscle mitochondria in response to endogenously generated H2O2 were assessed. Work was also conducted towards the development of mitochondria-targeted photo-activatable probes that could potentially release small functional bioactive compounds from caged precursors inside individual mitochondria upon selective irradiation

Polymorphism in TGFB1 is associated with worse non-relapse mortality and overall survival after stem cell transplantation with unrelated donors.

Transforming growth factor beta-1, encoded by the TGFB1 gene, is a cytokine that plays a central role in many physiological and pathogenic processes. We have sequenced TGFB1 regulatory region and assigned allelic genotypes in a large cohort of hematopoietic stem cell transplantation patients and donors. In this study, we analyzed 522 unrelated donor-patient pairs and examined the combined effect of all the common polymorphisms in this genomic region. In univariate analysis, we found that patients carrying a specific allele, 'p001', showed significantly reduced overall survival (5-year overall survival 30.7% for p001/ p001 patients vs. 41.6% others; P=0.032) and increased non-relapse mortality (1-year nonrelapse mortality: 39.0% vs. 25.4%; P=0.039) after transplantation. In multivariate analysis, the presence of a p001/ p001 genotype in patients was confirmed as an independent factor for reduced overall survival [hazard ratio=1.53 (1.04-2.24); P=0.031], and increased non-relapse mortality [hazard ratio=1.73 (1.06-2.83); P=0.030]. In functional experiments we found a trend towards a higher percentage of surface transforming growth factor beta-1-positive regulatory T cells after activation when the cells had a p001 allele (P=0.07). Higher or lower production of transforming growth factor beta-1 in the inflammatory context of hematopoietic stem cell transplantation may influence the development of complications in these patients. Findings indicate that TGFB1 genotype could potentially be of use as a prognostic factor in hematopoietic stem cell transplantation risk assessment algorithms

Carmustine, Etoposide, Cytarabine, and Melphalan (BEAM)–Campath Allogeneic Stem Cell Transplantation for Aggressive Non-Hodgkin Lymphoma: An Analysis of Outcomes from the British Society of Blood and Marrow Transplantation

AbstractThe role of allogeneic stem cell transplantation (SCT) in the management of aggressive non-Hodgkin lymphoma (NHL) remains to be defined, but the number of procedures performed continues to increase. We report here the outcomes of allogeneic SCT using carmustine, etoposide, cytarabine, and melphalan (BEAM)-Campath (Genzyme Corporation, Cambridge, MA) conditioning for aggressive NHL as reported to the British Society of Blood and Marrow Transplantation (BSBMT). This retrospective study identified 46 patients who reported to the BSBMT registry as having undergone BEAM-Campath conditioned allogeneic SCT for aggressive NHL between 1999 and 2010. Disease histology was diffuse large B cell lymphoma (DLBCL, n = 25), DLBCL/Burkitt lymphoma (n = 5), and T cell lymphoma (n = 16). At diagnosis, the median age was 42.5 (range, 17 to 59), 37 had advanced stage disease (Ann Arbor III/IV), 28 had 2 or more extra-nodal sites of disease, and 23 had elevated lactate dehydrogenase. International prognostic index was high or high/intermediate in 58%. The median number of prior therapies was 3 (range, 1 to 5) and 5 patients had previously undergone transplantation (4 autologous, 1 allogeneic). The median age at transplantation was 44.8 (range, 18 to 59), with 34 patients demonstrating chemo-sensitive disease and 22 undergoing transplantation in first response. Performance score was good in 40 patients and all engrafted with a median of 14 days (range, 11 to 27) to neutrophil recovery. At latest follow-up, 20 patients were alive with 17 in complete remission. Acute graft-versus-host disease (GVHD) developed in 7 patients and chronic GVHD developed in 13 (7 limited, 6 extensive). Five patients died from nonrelapse causes, with a cumulative incidence of nonrelapse mortality of 7% at 100 days and 11% at 3 and 5 years. Twenty-one patients died after lymphoma relapse, with a cumulative incidence of relapse/progression of 51% at 1 year and 53% at 5 years. Disease status at transplantation had no impact on relapse rate. Progression-free survival was 41% at 1 year and 36% at 5 years. Overall survival was 54% at 1 year and 42% at 5 years. Overall, BEAM-Campath–conditioned allogeneic SCT is well tolerated and able to deliver durable disease-free survival to a subset of patients with aggressive NHL. However, the high relapse rates indicate further investigation is needed to identify those patients most likely to benefit

Using exomarkers to assess mitochondrial reactive species in vivo

Background: The ability to measure the concentrations of small damaging and signalling molecules such as reactive oxygen species (ROS) in vivo is essential to understanding their biological roles. While a range of methods can be applied to in vitro systems, measuring the levels and relative changes in reactive species in vivo is challenging. Scope of review: One approach towards achieving this goal is the use of exomarkers. In this, exogenous probe compounds are administered to the intact organism and are then transformed by the reactive molecules in vivo to produce a diagnostic exomarker. The exomarker and the precursor probe can be analysed ex vivo to infer the identity and amounts of the reactive species present in vivo. This is akin to the measurement of biomarkers produced by the interaction of reactive species with endogenous biomolecules. Major conclusions and general significance: Our laboratories have developed mitochondria-targeted probes that generate exomarkers that can be analysed ex vivo by mass spectrometry to assess levels of reactive species within mitochondria in vivo. We have used one of these compounds, MitoB, to infer the levels of mitochondrial hydrogen peroxide within flies and mice. Here we describe the development of MitoB and expand on this example to discuss how better probes and exomarkers can be developed. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn. Abbreviations: EPR, electron paramagnetic resonance; GFP, green fluorescent protein; 4-HNE, 4-hydroxynonenal; MitoB, 3-(dihydroxyboronyl)benzyltriphenylphosphonium bromide; MitoP, (3-hydroxybenzyl)triphenylphosphonium bromide; ROS, reactive oxygen species; SOD, superoxide dismutase; TPMP, methyltriphenylphosphonium; TPP, triphenylphosphonium catio

Measurement of H₂O₂ within living drosophila during aging using a ratiometric mass spectrometry probe targeted to the mitochondrial matrix

Hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>) is central to mitochondrial oxidative damage and redox signaling, but its roles are poorly understood due to the difficulty of measuring mitochondrial H<sub>2</sub>O<sub>2</sub> in vivo. Here we report a ratiometric mass spectrometry probe approach to assess mitochondrial matrix H<sub>2</sub>O<sub>2</sub> levels in vivo. The probe, MitoB, comprises a triphenylphosphonium (TPP) cation driving its accumulation within mitochondria, conjugated to an arylboronic acid that reacts with H<sub>2</sub>O<sub>2</sub> to form a phenol, MitoP. Quantifying the MitoP/MitoB ratio by liquid chromatography-tandem mass spectrometry enabled measurement of a weighted average of mitochondrial H<sub>2</sub>O<sub>2</sub> that predominantly reports on thoracic muscle mitochondria within living flies. There was an increase in mitochondrial H<sub>2</sub>O<sub>2</sub> with age in flies, which was not coordinately altered by interventions that modulated life span. Our findings provide approaches to investigate mitochondrial ROS in vivo and suggest that while an increase in overall mitochondrial H<sub>2</sub>O<sub>2</sub> correlates with aging, it may not be causative

High-dose therapy and autologous stem cell transplantation in patients with POEMS syndrome: a retrospective study of the Plasma Cell Disorder sub-committee of the Chronic Malignancy Working Party of the European Society for Blood & Marrow Transplantation

POEMS syndrome is a rare para-neoplastic syndrome secondary to a plasma cell dyscrasia. Effective treatment can control the diseaserelated symptom complex. We describe the clinical outcome of autologous stem cell transplantation for patients with POEMS syndrome, determining the impact of patient- and disease-specific factors on prognosis. One hundred and twenty-seven patients underwent an autologous stem cell transplantation at 1997-2010 with a median age of 50 years (range 26-69 years). Median time from diagnosis to autologous stem cell transplantation was 7.5 months with 32% of patients receiving an autologous stem cell transplantation more than 12 months from diagnosis. Engraftment was seen in 97% patients and engraftment syndrome was documented in 23% of autologous stem cell transplantation recipients. Hematologic response was characterized as complete response in 48.5%, partial response in 20.8%, less than partial response in 30.7%. With a median follow up of 48 months (95%CI: 38.3, 58.6), 90% of patients are alive and 16.5% of patients have progressed. The 1-year non-relapse mortality was 3.3%. The 3-year probabilities of progression-free survival and overall survival are 84% and 94%, respectively, with 5-year probabilities of progression-free survival and overall survival of 74% and 89%. In a cohort of graft recipients, detailed organ-specific symptom response demonstrated clear symptom benefit after autologous stem cell transplantation especially in relation to neurological symptom control. The data analysed in this study demonstrate the clinical utility of autologous stem cell transplantation for patients with POEMS syndrome

Post-transplantation Cyclophosphamide-based Haploidentical Transplantation As Alternative To Matched Sibling Or Unrelated Donor Transplantation For Hodgkin Lymphoma: A Registry Study Of The Lymphoma Working Party Of The European Society For Blood And Marrow Transplantation

Purpose: To compare the outcome of patients with Hodgkin lymphoma who received post-transplantation cyclophosphamide-based haploidentical (HAPLO) allogeneic hematopoietic cell transplantation with the outcome of patients who received conventional HLA-matched sibling donor (SIB) and HLA-matched unrelated donor (MUD). Patients and Methods: We retrospectively evaluated 709 adult patients with Hodgkin lymphoma who were registered in the European Society for Blood and Marrow Transplantation database who received HAPLO (n = 98), SIB (n = 338), or MUD (n = 273) transplantation. Results: Median follow-up of survivors was 29 months. No differences were observed between groups in the incidence of acute graft-versus-host disease (GVHD). HAPLO was associated with a lower risk of chronic GVHD (26%) compared with MUD (41%; P =.04). Cumulative incidence of nonrelapse mortality at 1 year was 17%, 13%, and 21% in HAPLO, SIB, and MUD, respectively, and corresponding 2-year cumulative incidence of relapse or progression was 39%, 49%, and 32%, respectively. On multivariable analysis, relative to SIB, nonrelapse mortality was similar in HAPLO (P =.26) and higher in MUD (P =.003), and risk of relapse was lower in both HAPLO (P =.047) and MUD (P,.001). Two-year overall survival and progression-free survival were 67% and 43% for HAPLO, 71% and 38% for SIB, and 62% and 45% for MUD, respectively. There were no significant differences in overall survival or progression-free survival between HAPLO and SIB or MUD. The rate of the composite end point of extensive chronic GVHD and relapse-free survival was significantly better for HAPLO (40%) compared with SIB (28%; P =.049) and similar to MUD (38%; P =.59). Conclusion: Post-transplantation cyclophosphamide-based HAPLO transplantation results in similar survival outcomes compared with SIB and MUD, which confirms its suitability when no conventional donor is available. Our results also suggest that HAPLO results in a lower risk of chronic GVHD than MUD transplantation

ESHAP and G-CSF is a superior blood stem cell mobilizing regimen compared to cyclophosphamide 1.5 g m−2 and G-CSF for pre-treated lymphoma patients: a matched pairs analysis of 78 patients

Cyclophosphamide 1.5 g m−2followed by granulocyte colony-stimulating factor (G-CSF) is an effective peripheral blood stem cell (PBSC) mobilizing regimen, but has limited anti-lymphoma activity. We therefore assessed the mobilizing potential of ESHAP (etoposide, ara-C, methylprednisolone and cisplatin), a potent second-line lymphoma regimen followed by G-CSF. The results were compared in 78 patients with relapsed or resistant lymphomas with the use of cyclophosphamide 1.5 g m−2followed by G-CSF in a matched pairs analysis, matching the ESHAP recipients (for predetermined prognostic factors) from a cohort of 178 lymphoma patients mobilized with cyclophosphamide and G-CSF. The total numbers of mononuclear cells collected at apheresis was similar with both regimens but ESHAP plus G-CSF resulted in a significantly higher percentage of CD34+ cells, absolute number of CD34+ cells and GM-CFC (all with P -values < 0.001). The number of patients requiring only one apheresis harvest to achieve a CD34+ cell yield of > 2.0 × 106kg−1was greatly increased in the ESHAP recipients (56/78 vs 17/78, P< 0.001). The total number of progenitor cells collected was not significantly different with the two mobilization regimens because of this higher number of apheresis in the cyclophosphamide group. The proportion of patients who failed to achieve a minimum CD34+ cell target of 1 × 106kg−1with the pooled harvests was less in the ESHAP arm (four patients vs nine patients) despite an increased number of aphereses in the cyclophosphamide recipients. ESHAP plus G-CSF is well tolerated and is an excellent mobilization regimen in patients with pre treated lymphoma. © 2000 Cancer Research Campaig

Mechanisms of relapse in acute leukaemia: involvement of p53 mutated subclones in disease progression in acute lymphoblastic leukaemia

Mutations of the p53 tumour suppressor gene are infrequent at presentation of both acute myeloblastic leukaemia (AML) and acute lymphoblastic leukaemia (ALL), being found in between 5–10% of AML and 2–3% of ALL. Here we have studied the frequency of detection of p53 mutations at relapse of both AML and B-precursor ALL. In those patients with detectable mutations at relapse we investigated whether the mutation was detectable at presentation and was thus an early initiating event or whether it had arisen as a late event associated with relapse. Bone marrow samples from 55 adults and children with relapsed AML (n = 41) or ALL (n = 14) were analysed for p53 gene alterations by direct sequencing of exons 5–9. For samples where a p53 mutation was found at relapse, analysis of presentation samples was carried out by direct sequencing of the exon involved, or by allele-specific polymerase chain reaction (PCR) if the mutation could not be detected using direct sequencing. A p53 mutated gene was found at relapse in seven out of 55 cases. The frequency was higher in relapsed ALL (four out of 14 cases; 28.6%) compared to AML (three out of 41 cases; 7.3%). In five out of the seven cases presentation samples were available to study for the presence of the mutation. In two out of two AML patients the p53 mutation was detectable in the presentation sample by direct sequencing. In three ALL patients analysis of presentation material by direct sequencing showed a small mutant peak in one case, the other two being negative despite the sample analysed containing > 90% blast cells. However in both of these patients, the presence of p53 mutation was confirmed in the presentation sample using allele-specific PCR. In one of these patients the emergence of a subclone at relapse was confirmed by clonality analysis using IgH fingerprinting. Our results confirm that in ALL p53 mutations are present in a proportion of patients at relapse. Furthermore cells carrying the mutation are detectable at presentation in a minor clone suggesting that p53 mutations in ALL may be a mechanism contributing to disease relapse. © 1999 Cancer Research Campaig