The imbalance between reactive oxygen species (ROS) production and their elimination by antioxidants leads to oxidative stress. Depending on their concentration, ROS can trigger apoptosis or stimulate cell proliferation. We hypothesized that oxidative stress and mitochondrial dysfunction may participate not only in apoptosis detected in some myelodysplastic syndrome (MDS) patients, but also in increasing proliferation in other patients. We investigated the involvement of oxidative stress and mitochondrial dysfunction in MDS pathogenesis, as well as assessed their diagnostic and prognostic values. Intracellular peroxides, superoxide, superoxide/peroxides ratio, reduced glutathione (GSH), and mitochondrial membrane potential (Δψ(mit)) levels were analyzed in bone marrow cells from 27 MDS patients and 12 controls, by flow cytometry. We observed that all bone marrow cell types from MDS patients had increased intracellular peroxide levels and decreased GSH content, compared with control cells. Moreover, oxidative stress levels were MDS subtype- and risk group-dependent. Low-risk patients had the highest ROS levels, which can be related with their high apoptosis; and intermediate-2-risk patients had high Δψ(mit) that may be associated with their proliferative potential. GSH levels were negatively correlated with transfusion dependency, and peroxide levels were positively correlated with serum ferritin level. GSH content proved to be an accurate parameter to discriminate patients from controls. Finally, patients with high ROS or low GSH levels, as well as high superoxide/peroxides ratio had lower overall survival. Our results suggest that oxidative stress and mitochondrial dysfunction are involved in MDS development, and that oxidative stress parameters may constitute novel diagnosis and/or prognosis biomarkers for MDS.

Oxidative stress and abnormal DNA methylation have been implicated in some types of cancer, namely in myelodysplastic syndromes (MDS). Since both mechanisms are observed in MDS patients, we analyzed the correlation of intracellular levels of peroxides, superoxide anion, and glutathione (GSH), as well as ratios of peroxides/GSH and superoxide/GSH, with the methylation status of P15 and P16 gene promoters in bone marrow leukocytes from MDS patients. Compared to controls, these patients had lower GSH content, higher peroxide levels, peroxides/GSH and superoxide/GSH ratios, as well as higher methylation frequency of P15 and P16 gene promoters. Moreover, patients with methylated P15 gene had higher oxidative stress levels than patients without methylation (peroxides: 460 ± 42 MIF vs 229 ± 25 MIF, p = 0.001; superoxide: 383 ± 48 MIF vs 243 ± 17 MIF, p = 0.022; peroxides/GSH: 2.50 ± 0.08 vs 1.04 ± 0.34, p < 0.001; superoxide/GSH: 1.76 ± 0.21 vs 1.31 ± 0.10, p = 0.007). Patients with methylated P16 and at least one methylated gene had higher peroxide levels as well as peroxides/GSH ratio than patients without methylation. Interestingly, oxidative stress levels allow the discrimination of patients without methylation from ones with methylated P15, methylated P16, or at least one methylated (P15 or P16) promoter. Taken together, these findings support the hypothesis that oxidative stress is correlated with P15 and P16 hypermethylation.

A 69-year-old Caucasian woman with a 15-year history of refractory chronic lymphocytic B-cell leukaemia (CLL), treated with alemtuzumab in the past 10 months presented with a subacute right foot drop. Initial evaluation with a brain CT scan, lumbosacral MRI, nerve conduction studies and LP was negative. In the following months, progressive right hemibody weakness and dysarthria developed. Brain MRI showed a bilateral parasagittal frontal lesion. Alemtuzumab treatment was withdrawn. Progressive multifocal leukoencephalopathy (PML) was confirmed by PCR. Attempted antiviral therapies proved fruitless. Inexorable clinical deterioration ensued and the patient passed away 10 months after the presentation. This case report intends to call attention for PML as a potential fatal complication of severe immunosuppression, including the possible role of new monoclonal antibodies (such as alemtuzumab) in its pathogenesis.

One of the most severe side effects of the immunosuppressive agent, cyclosporin A (CsA), is increased risk of thromboembolic complications and drug-related hypertension. Because platelets might be involved in these processes, we tested the possibility of CsA affecting platelet activation, which might contribute to these adverse drug reactions. The experiments were done using Wistar rats, treated or not (control) with CsA (Sandimmun Neoral), 5 and 30 mg/kg/day, for 7 weeks. Systolic, diastolic, and mean blood pressures, intracellular free calcium concentration ([Ca2+]i), platelet serotonin (5-HT) contents, and aggregation were determined, at weeks 0, 2, and 7 of treatment. Inositol phosphates (InsP) production, platelet thromboxane A2 (TXA2) generation, and morphology of platelets, through electron microscopy studies, also were compared. It was demonstrated that blood pressures increased in the CsA-treated groups, when compared with the control group, after 2 and 7 weeks of administration. CsA at both "attack" and "maintenance" doses increased basal, 5-HT, and thrombin-evoked [Ca2+]i after 2 and 7 weeks versus the control group. However, basal and evoked InsP production was stimulated by 5 mg/kg of CsA, but inhibited by 30 mg/kg, when compared with the control. Platelet 5-HT contents decreased significantly after 2 and 7 weeks in the CsA-treated groups, when compared with the control group. Collagen-induced whole blood platelet aggregation increased drastically in the "attack" CsA-treated group, whereas adenosine diphosphate (ADP)-induced platelet aggregation did not reach statistical significance. Finally, in vitro basal, collagen-, and ADP-evoked platelet TXA2 generation increased in both CsA concentrations, versus the control. In conclusion, our study demonstrates that both CsA doses alter platelet calcium homeostasis (even affecting the calcium fluxes differently), 5-HT and TXA2 contents and aggregation, which might contribute to the development and/or maintenance of high blood pressures and increased risk of thromboembolic complications.

Onze doentes evoluíram para leucemia aguda; estes têm, em média, eritropoietina sérica superior (p < 0,05). Adicionalmente, a eritropoietina sérica acima do limite superior da normalidade associa-se a menor sobrevivência (p = 0,0336). Após ajuste do modelo de regressão de Cox, o valor preditivo da eritropoietina para a sobrevivência global manteve-se (p < 0,001). Em análise multivariada, a eritropoietina sérica demonstrou ser um factor de prognóstico independente (p < 0,001). Discussão: A eritropoietina sérica é um factor preditivo de resposta à terapêutica com eritropoietina subcutânea, sendo que os doentes com síndrome mielodisplásica com valores mais elevados de eritropoietina apresentam uma pior resposta à administração de eritropoietina, mesmo com doses mais elevadas. A nossa amostra demonstra que a eritropoietina sérica apresenta também valor prognóstico, e em todos os subtipos de síndrome mielodisplásica. Além disso, isoladamente ou em associação com outros factores ou índices de prognóstico, poderá melhorar o valor prognóstico de índices como o International Prognostic Scoring System, uma vez que valores elevados de eritropoietina estão associados a progressão para leucemia aguda e, consequentemente, a menor sobrevivência. Conclusão: Os resultados sugerem que o aumento dos níveis séricos de eritropoietina ao diagnóstico pode constituir um factor de mau prognóstico em doentes com síndrome mielodisplásica, associando-se a maior risco de evolução para leucemia aguda e menor sobrevivência global. Palavras-chave: Eritropoietina; Prognóstico; Síndrome Mielodisplásica. ABSTRACT Introduction:This myelodysplastic syndromes are a heterogeneous entity characterized by dysplasia, hypercellular bone marrow, cytopenias and risk of transformation to acute leukaemia. Prognostic factors, such as bone marrow fibrosis, lactate dehydrogenase and β2-microglobulin elevation have been described, but treatment is mainly based in the International Prognostic Scoring System. Material and Methods:Our aim was to analyze serum´s erythropoietin at diagnosis in de novo myelodysplastic syndromes patients, through its impact in overall survival and possible implementation as prognostic marker. Clinical and laboratorial data from 102 patients with de novo myelodysplastic syndromes diagnosed between October/2009 and March/2014 were collected. Survival analysis was performed according to serum erythropoietin level stratification, using Kaplan-Meier methodology. Results: Our 102 patients had a median age of 74 years, with a male:female ratio of 0.8. Mean erythropoietin was significantly lower in refractory cytopenia with unilineage dysplasia patients in contrast with the higher values observed in 5q-syndrome (p < 0.05). Eleven patients progressed to acute leukaemia; these have higher mean erythropoietin values (p < 0.05). In addition, elevated serum erythropoietin was associated with lower survival rates (p = 0.0336). Predictive value of serum erythropoietin was maintained after Cox regression adjustment. In multivariate analysis, serum ery...

commencing treatment. Three patients (21.4%) relapsed with median time to relapse of 3.6 years. 15 patients (34.9%) achieved complete response after 30 days of commencing therapy and 5 patients (33.3%) relapsed with median time of 2 years. Five patients achieved very good partial response (VGPR), six patients with limited or no response. Summary/Conclusion: Renal disease is the most common presentation of AL amyloidosis followed by cardiac involvement. 2/3 of patients have elevated serum free lambda light chain and most of them have either MGUS or smouldering myeloma on bone marrow biopsy. Diagnoses are mostly made on renal biopsy but rectal biopsy seems to be sensitive for diagnosis of amyloidosis infiltration as well. Bortezomib based therapy appeared to be effective treatment as first line therapy with 79% achieving good haematological response with sustainable remission status. Ongoing follow up and larger cohort will be required for more conclusive results.

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B-cell lymphocytic leukaemia (B-CLL) is an indolent non-Hodgkin’s lymphoma and the most frequent leukaemia. Even though the capacity of B-CLL leukemic cells to proliferate has been underestimated until recently, the accumulation of tumor cells mostly results from a defect in the apoptotic program. The ubiquitin/proteasome system is important in the turnover of regulatory proteins and as regulator of cell proliferation and apoptosis. This critical link between the apoptotic machinery and the ubiquitin/proteasome system led to an increased interest in designing inhibitory agents that target these pathways. A promising new drug, bortezomib (BZ), a dipeptidyl boronic acid proteasome inhibitor, was approved for treatment of relapsed multiple myeloma. Extensive preclinical data are being developed to study the potential application of this new drug in other cancers. Our preliminary results show that BZ induces a marked decrease in LLC-B cell viability by increasing the percentage of apoptosis. The aim of this study is to evaluate the potential therapeutic value of BZ in B-CLL patients studying the cytotoxicity mechanisms induced by this proteasome inhibitor. For this purpose, mononuclear cells isolated from 22 patients with B-CLL (14 without and 8 with conventional therapy) were cultured in absence and presence of bortezomib (BZ) (ranging concentration from 0.01 μM to 10 μM), as single agent, or plus 75 or 150 μM fludarabine (FDN), during 24 hours. Directly conjugated monoclonal antibodies to CD5 and CD19 was used to identify LLC-B cells. Cell death was evaluated by annexin V incorporation and detected by flow cytometry. The expression of the proteins involved in apoptosis regulation, namely the proapoptotic proteins, Bax and p53, and the antiapoptotic protein Bcl-2 was determined by flow cytometry using monoclonal antibodies. Our results show that BZ induces a marked decrease in B-CLL cell viability by increasing the percentage of apoptosis in a dose dependent manner (20 to 80% apoptosis). However, in concentrations higher than 1 mM a saturable effect is observed. On the other hand, a lesser sensitivity to BZ was observed in normal mononuclear cells (≤ 30% apoptosis). BZ apoptotic effect seems to be independent of previous therapy. We observed higher levels of apoptosis in B-CLL cells treated with BZ compared with cells not treated or in response to treatment with FDN, which may relate with the increase in Bax expression (average difference of 37.3 ± 18%). However, we observe identical apoptosis levels in B-CLL cells, as opposed to others (Duechler, M. et al., 2005), and an increase in apoptotic T cells levels in the presence of treatment with BZ plus FDN compared with those observed in cells treated with BZ alone. On the other hand, when compared with the results obtained with FDN alone (in 150 μM), an increase in apoptosis levels was detected (average difference of 69.5 ± 28% increase in apoptosis, for B-CLL cells and 33.6 ± 11.6%, for T cells). Our results support that bortezomib induces apoptosis as a single agent in a Bax-dependent way. The apoptotic effect show some selectivity for the transformed cells (B-CLL). These results suggests that this proteasome inhibitor may be useful as a therapeutic approach in B-CLL patients.