CXCL10 treatment promotes reduction of IL-10+ regulatory T (Foxp3+ and Tr1) cells in the spleen of BALB/c mice infected by Leishmania infantum.

American visceral leishmaniasis is caused by the protozoan Leishmania infantum. The control of the disease depends on the magnitude of the Th1 cell response and IL-10 producing regulatory T cells. Administration of chemokine, such as CXCL10, has shown promising results in the leishmaniasis treatment. Previous studies from our group have shown that CXCL10 induces a reduction in parasite burden in the spleen and a decrease in IL-10 and TGF-ß production in L. infantum-infected BALB/c mice. This work investigated whether CXCL10-treatment reduces IL-10 + Treg cell populations (CD4+CD25+Foxp3+ and Tr1) and induces morphological changes in the spleen. BALB/c mice were infected and treated or not with CXCL10 on the 1st, 3rd and 7th days of infection. CXCL10-treatment was able to reduce the parasite load in the spleen in L. infantum-infected BALB/c mice and this decrease in the number of parasites correlated with the decrease in size of this organ in treated animals compared to untreated animals. 7, 23, and 45 days post-treatment (p.t.), the phenotype and frequency of IL-10 + Treg cells were evaluated by flow cytometry, and the morphological changes of the spleen were analyzed by optical microscopy. After 7 and 23 days p.t., CXCL10-treated animals showed a significant reduction of CD25-Foxp3-IL-10+ (Tr1) cells in the spleen when compared to untreated animals, whereas CD4+CD25+Foxp3+IL-10+ Treg cells reduced later at 23rd and 45th days p.t. Furthermore, while untreated animals showed a significant positive correlation between IL-10 production and Tr1 cells, in CXCL10-treated group this correlation was negative. Thus, these findings show that treatment with CXCL10 chemokine in L. infantum-infected BALB/c mice results in suppression of IL10+ Treg (Foxp3+ and Tr1) cells in the spleen, associated with a reduction in parasite load and splenomegaly.

HFE gene mutation and oxidative damage biomarkers in patients with myelodysplastic syndromes and its relation to transfusional iron overload: an observational cross-sectional study.

OBJECTIVE: A relation between transfusional IOL (iron overload), HFE status and oxidative damage was evaluated. DESIGN, SETTING AND PARTICIPANTS: An observational cross-sectional study involving 87 healthy individuals and 78 patients with myelodysplastic syndromes (MDS) with and without IOL, seen at University Hospital of the Federal University of Ceará, Brazil, between May 2010 and September 2011. METHODS: IOL was defined using repeated measures of serum ferritin ≥1000 ng/mL. Variations in the HFE gene were investigated using PCR/restriction fragment length polymorphism (RFLP). The biomarkers of oxidative stress (plasmatic malonaldehyde (MDA), glutathione peroxidase (GPx) and superoxide dismutase (SOD)) were determined by spectrophotometry. RESULTS: The HFE gene variations were identified in 24 patients (30.77%) and 5 volunteers (5.74%). The H63D variant was observed in 35% and the C282Y variant as heterozygous in 5% of patients with MDS with IOL. One patient showed double heterozygous variant (C282Y/H63D) and serum ferritin of 11,649 ng/mL. In patients without IOL, the H63D variant was detected in 29.34%. Serum MDA levels were highest in patients with MDS with IOL, with a significant difference when compared with patients without IOL and healthy volunteers, pointing to the relationship between IOL and oxidative stress. The GPx and SOD were also significantly higher in these patients, indicating that lipid peroxidation increase was followed by an increase in antioxidant capacity. Higher ferritin levels were observed in patients with HFE gene variation. 95.7% of patients with MDS with the presence of HFE gene variations had received more of 20 transfusions. CONCLUSIONS: We observed a significant increase in MDA levels in patients with MDS and IOL, suggesting an increased lipid peroxidation in these patients. The accumulation of MDA alters the organisation of membrane phospholipids, contributing to the process of cellular degeneration. Results show that excess iron intensifies the process of cell damage through oxidative stress. TRIAL REGISTRATION NUMBER: Local Ethics Committee (licence 150/2009).

Proteins of the mitotic checkpoint and spindle are related to chromosomal instability and unfavourable prognosis in patients with myelodysplastic syndrome.

AIMS: To study the immunoexpression of proteins related to the mitotic checkpoint (cell division cycle 20 (CDC20), mitotic arrest deficient 2 (MAD2)) and the mitotic spindle (Aurora-B) in patients with myelodysplastic syndrome (MDS). METHODS: Protein expression was analysed in bone marrow tissue samples from 40 patients with MDS using immunohistochemistry. Prognostic markers (transfusion dependency, depth of cytopenias, chromosomal abnormalities and survival) were also studied. RESULTS: Higher MAD2 expression was observed among patients with platelets <50×10(9)/L than among patients with platelets ≥50×10(9)/L (42.6±22.8% vs 22.7±19.1%, respectively). Higher CDC20 expression was identified among patients with three dysplasias compared with patients who presented with one or two dysplasias (33.9±24.1% vs 10.5±5.7% vs 12.8±7.8%, respectively), among patients who exhibited a complex versus non-complex karyotype (50.0±30.2% vs 18.4±14%, respectively) and among patients with platelets <50×10(9)/L vs platelets ≥50×10(9)/L (38.2±26.2% vs 16.1±12.4%, respectively). Higher Aurora-B expression was found in patients with an abnormal versus normal karyotype (21.2±13.2% vs 7.5±5.0%, respectively). High expression of MAD2 and CDC20 (≥50%) was associated with severe thrombocytopenia. We also found statistically significant differences in the overall survival rate when comparing different degrees of CDC20, MAD2 and Aurora-B protein expression. CONCLUSIONS: To the best of our knowledge, this is the first report to demonstrate that these proteins are associated with chromosomal abnormalities and poor prognosis in patients with MDS.

Polymorphisms of DNA repair genes are related to the pathogenesis of myelodysplastic syndrome.

Some studies show that alterations in DNA repair genes polymorphisms are associated with the pathogenesis and susceptibility of Myelodysplastic Syndrome (MDS). We genotyped 60 MDS patients for six DNA repair gene polymorphisms: BRCA1 rs4793191, BRCA2 rs9567623, RAD51 rs1801320, XRCC5 rs3835, XRCC6 rs2267437 and LIG4 rs1805388. The G/C heterozygote genotype of rs1801320 polymorphism was associated with a decreased chance of developing MDS (p = 0.05). Additionally, the G/G homozygous genotype was associated with the presence of one cytopenia in whole blood. The genotype C/G and CG + GG of the rs2267437 polymorphism was associated with normal karyotype (p = 0.010) and bone marrow cellularity normocellular + hypercellular (p = 0.023). We found that the A/G heterozygous genotype of the rs3835 polymorphism is associated with decreased chance of developing MDS (p < 0.001). These results support the importance of RAD51, XRCC5 and XRCC6 genes polymorphisms in the maintenance of genomic stability promoting a better understanding of the genesis and etiology of MDS.

Proteins related to the spindle and checkpoint mitotic emphasize the different pathogenesis of hypoplastic MDS.

Some studies show that alterations in expression of proteins related to mitotic spindle (AURORAS KINASE A and B) and mitotic checkpoint (CDC20 and MAD2L1) are involved in chromosomal instability and tumor progression in various solid and hematologic malignancies. This study aimed to evaluate these genes in MDS patients. The cytogenetics analysis was carried out by G-banding, AURKA and AURKB amplification was performed using FISH, and AURKA, AURKB, CDC20 and MAD2L1 gene expression was performed by qRT-PCR in 61 samples of bone marrow from MDS patients. AURKA gene amplification was observed in 10% of the cases, which also showed higher expression levels than the control group (p=0.038). Patients with normo/hypercellular BM presented significantly higher expression levels than hypocellular BM patients, but normo and hypercellular BM groups did not differ. After logistic regression analysis, our results showed that HIGH expression levels were associated with increased risk of developing normo/hypercellular MDS. It also indicated that age is associated with AURKA, CDC20 and MAD2L1 HIGH expression levels. The distinct expression of hypocellular patients emphasizes the prognostic importance of cellularity to MDS. The amplification/high expression of AURKA suggests that the increased expression of this gene may be related to the pathogenesis of disease.

Estudo de proteínas relacionadas ao fuso mitótico (AURORA A e AURORA B) e ponto de checagem mitótico (CDC20 e MAD2L1) em pacientes portadores de Síndrome mielodisplásica / Mitotic spindle (AURORA A e AURORA B) and mitotic checkpoint (CDC20 and MAD2L1) related protein study in patients with myelodysplastic syndrome

Síndrome mielodisplásica (SMD) representa um grupo heterogêneo de doenças hematopoéticas clonais caracterizadas por alterações displásicas e citopenias no sangue periférico. A patogênese da SMD envolve múltiplas etapas e ainda não está claramente definido como a doença progride para o estágio avançado e para leucemia mielóide aguda. A presença de alterações citogenéticas desempenha importante papel no prognóstico e seguimento desses pacientes. Alguns estudos demonstram que alterações na expressão de proteínas relacionadas ao fuso mitótico (AURORA A e AURORA B) e ao ponto de checagem mitótico (CDC20 e MAD2L1) estão envolvidas na instabilidade cromossômica, aneuploidia e progressão tumoral em diferentes tumores sólidos e neoplasias hematológicas. No entanto, pesquisas que investiguem a expressão destes genes em pacientes com SMD são escassas, e estudos que avaliem essas proteínas podem contribuir para o endentimento da fisiopatologia da doença. Nesse sentido, este trabalho teve como objetivo avaliar o perfil de expressão destes genes em pacientes com SMD, e investigar sua relação com as variáveis clínicas e laboratoriais da doença. O estudo das alterações cromossômicas foi realizado por bandamento G, a análise de amplificação para os genes AURKA e AURKB foi realizada através da técnica de hidridização in situ por fluorescência (FISH), e a expressão dos genes AURKA, AURKB, CDC20 e MAD2L1 foi realizada, através de PCR Quantitativo em Tempo Real, em 61 amostras de medula óssea de pacientes portadores de SMD. Assim como em outras neoplasias, a maioria dos pacientes estudados (55,8%) apresentaram cariótipo alterado, refletindo a instabilidade cromossômica decorrentes do processo de patogênese da doença...