Inhibition of Mitochondrial Complex I Impairs Release of α-Galactosidase by Jurkat Cells.

Fabry disease (FD) is caused by mutations in the GLA gene that encodes lysosomal α-galactosidase-A (α-gal-A). A number of pathogenic mechanisms have been proposed and these include loss of mitochondrial respiratory chain activity. For FD, gene therapy is beginning to be applied as a treatment. In view of the loss of mitochondrial function reported in FD, we have considered here the impact of loss of mitochondrial respiratory chain activity on the ability of a GLA lentiviral vector to increase cellular α-gal-A activity and participate in cross correction. Jurkat cells were used in this study and were exposed to increasing viral copies. Intracellular and extracellular enzyme activities were then determined; this in the presence or absence of the mitochondrial complex I inhibitor, rotenone. The ability of cells to take up released enzyme was also evaluated. Increasing transgene copies was associated with increasing intracellular α-gal-A activity but this was associated with an increase in Km. Release of enzyme and cellular uptake was also demonstrated. However, in the presence of rotenone, enzyme release was inhibited by 37%. Excessive enzyme generation may result in a protein with inferior kinetic properties and a background of compromised mitochondrial function may impair the cross correction process.

X chromosome inactivation analysis reveals a difference in the biology of ET patients with JAK2 and CALR mutations.

Calreticulin mutations (CALR(MUT)) are found in a significant proportion of patients with essential thrombocythemia (ET) lacking JAK2(V617F) or MPL mutations. They are associated with substantially different hematological and clinical features and define a distinct subtype of ET. We show here that their presence is significantly correlated with a clonal X chromosome inactivation pattern (XCIP). Of 105 female ET patients investigated, 61 had an interpretable XCIP, and a clonal pattern was observed in 88% of CALR(MUT) patients compared with 26% of JAK2(V617F) (P = .0002) and 9% of JAK2(V617F)/MPL/CALR wild-type patients (P < .0001). Neutrophil CALR(MUT) level was significantly higher than JAK2(V617F) level (median, 50% vs 18%; P < .0001), and wild-type myelopoiesis was suppressed in CALR(MUT) but not JAK2(V617F) patients. These data are suggestive of truly monoclonal hematopoiesis in CALR(MUT) patients and provide further evidence that the biology associated with CALR mutations is markedly different from that of JAK2(V617F) mutations.

The clinical utility of Multiplate analyser measurement in platelet function testing following stroke and transient ischaemic attack.

BACKGROUND: Platelet responsiveness to aspirin in people with cerebrovascular disease is poorly understood. OBJECTIVES: To determine: (i) normal reference range, imprecision and reproducibility of the Multiplate instrument in healthy volunteers naive to aspirin; (ii) imprecision and reproducibility of the Multiplate instrument in acute stroke and transient ischaemic attack (TIA); (iii) the relationship between aspirin responsiveness and clinical outcome. MATERIALS AND METHODS: We evaluated platelet function response to three agonists [Adenosine Diphosphate (ADP), Arachidonic Acid (AA), Collagen (Col)] using the Mulitplate platelet function analyser in a two-phase pilot study. In phase 1, we recruited healthy volunteers to determine the normal reference range and imprecision of the Multiplate instrument. In phase 2, we assessed platelet function in acute stroke or TIA patients presenting to hospital. These patients were bled within 24 h of presentation and between 12 and 24 h after ≥75 mg dose of Aspirin. Patients were followed up to 1 yr to assess mortality and recurrent cardiovascular event. RESULTS: Overall, 29 healthy volunteers and 81 stroke/TIA patients were recruited. On assessing components of variance, Multiplate testing is reproducible and precise in volunteers and stroke/TIA patients. In stroke patients receiving aspirin, Bland-Altman plots show initial day 1 measurement provided a reliable measure of continuing response to aspirin at day 3. We defined one-third of patients as aspirin resistant [31.8% (95% CI: 22.1%-42.8%)] using cut off mean aggregation of ≥23.08% for AA and mean aggregation of ≥80.76% for ADP. CONCLUSION: The Multiplate device gives reproducible, precise results in volunteers and stroke/TIA patients.

Prognostic role of PET scanning before and after reduced-intensity allogeneic stem cell transplantation for lymphoma.

Allogeneic stem cell transplantation (SCT) is an established therapy for patients with relapsed lymphoma, but the role of positron emission tomography (PET) scanning preallogeneic and postallogeneic SCT is uncertain. We investigated whether pretransplantation PET status predicted outcome after allogeneic SCT and whether PET surveillance after transplantation provided additional information compared with computed tomography (CT) scanning. Eighty consecutive patients with lymphoma who received a reduced-intensity allogeneic SCT were entered onto a prospective trial. PET and CT scans were performed before transplantation and up to 36 months after transplantation. Forty-two patients were PET-positive before transplantation. Pretransplantation PET status had no significant impact on either relapse rate or overall survival. Thirty-four relapses were observed, of which 17 were PET-positive with a normal CT scan at relapse. Donor lymphocyte infusion (DLI) was administered in 26 episodes of relapse and was guided by PET alone in 14 patients. These findings suggest that, in contrast to autologous SCT, pretransplantation PET status is not predictive of relapse and survival after allogeneic SCT for lymphoma. Posttransplantation surveillance by PET detected relapse before CT in half of episodes, often allowing earlier administration of DLI in patients with recurrent lymphoma, and permitted withholding of potentially harmful DLI in those with PET-negative masses on CT scans.

In essential thrombocythemia, multiple JAK2-V617F clones are present in most mutant-positive patients: a new disease paradigm.

In essential thrombocythemia (ET), the JAK2-V617F mutation is usually restricted to a subpopulation of neutrophils and platelets, and production of JAK2 wild-type (WT) platelets is not suppressed. Nonmutated precursor cells may, therefore, be susceptible to the acquisition of further JAK2 mutations. We used a common single nucleotide polymorphism (SNP) in the JAK2 coding sequence to genotype V617F alleles obtained either by allele-specific restriction enzyme digestion (RED) or by cloning. Both SNP alleles were detected in JAK2 mutant-positive alleles from neutrophils of 10 of 11 ET patients studied using RED compared with 0 of 5 with polycythemia vera. These results were confirmed in cloned products from 5 ET patients and indicate the occurrence of at least 2 separate JAK2 mutation events in the majority of ET patients investigated. In a further ET patient, JAK2 mutant-positive erythroid colonies with either X-allele inactivated were detected, demonstrating they could not have arisen from a common clonal precursor. These results indicate that at least 2 independent JAK2-V617F events occur commonly in ET patients, and they may arise on a polyclonal background. The presence of a JAK2 mutation in ET patients should not, therefore, be equated with a malignant disease.

The production of JAK2 wild-type platelets is not downregulated in patients with JAK2 V617F mutant-positive essential thrombocythaemia.

To clarify the relationship between the levels of JAK2 wild-type (WT) and V617F mutant-positive platelets in patients with essential thrombocythaemia (ET), we quantified mutant levels in purified cells from 10 V617F-positive patients prior to receiving cytoreductive therapy. Mutant levels were significantly higher in platelet than neutrophil RNA (P = 0.002), but the mutation was still only present in a sub-population of platelets (median 54%). When the absolute number of WT platelets was calculated, it was always within or above the normal platelet range, indicating that there is an aberration in the negative feedback to JAK2 WT platelets in ET.

Challenges in glucoCEST MR body imaging at 3 Tesla.

BACKGROUND: The aim of this study was to translate dynamic glucose enhancement (DGE) body magnetic resonance imaging (MRI) based on the glucose chemical exchange saturation transfer (glucoCEST) signal to a 3 T clinical field strength. METHODS: An infusion protocol for intravenous (i.v.) glucose was optimised using a hyperglycaemic clamp to maximise the chances of detecting exchange-sensitive MRI signal. Numerical simulations were performed to define the optimum parameters for glucoCEST measurements with consideration to physiological conditions. DGE images were acquired for patients with lymphomas and prostate cancer injected i.v. with 20% glucose. RESULTS: The optimised hyperglycaemic clamp infusion based on the DeFronzo method demonstrated higher efficiency and stability of glucose delivery as compared to manual determination of glucose infusion rates. DGE signal sensitivity was found to be dependent on T2, B1 saturation power and integration range. Our results show that motion correction and B0 field inhomogeneity correction are crucial to avoid mistaking signal changes for a glucose response while field drift is a substantial contributor. However, after B0 field drift correction, no significant glucoCEST signal enhancement was observed in tumour regions of all patients in vivo. CONCLUSIONS: Based on our simulated and experimental results, we conclude that glucose-related signal remains elusive at 3 T in body regions, where physiological movements and strong effects of B1 + and B0 render the originally small glucoCEST signal difficult to detect.