Effects of Chronic Exercise on Endothelial Progenitor Cells and Microparticles in Professional Runners / Efeitos de Exercício Crônico Sobre Células Progenitoras Endoteliais e Micropartículas em Corredores Profissionais

Abstract Background: The effects of chronic exposure to exercise training on vascular biomarkers have been poorly explored. Objective: Our study aimed to compare the amounts of endothelial progenitor cells (EPCs), and endothelial (EMP) and platelet (PMP) microparticles between professional runners and healthy controls. Methods: Twenty-five half-marathon runners and 24 age- and gender-matched healthy controls were included in the study. EPCs (CD34+/KDR+, CD133+/KDR+, and CD34+/CD133+), EMP (CD51+) and PMP (CD42+/CD31+) were quantified by flow-cytometry. All blood samples were obtained after 12 h of fasting and the athletes were encouraged to perform their routine exercises on the day before. Results: As compared with controls, the CD34+/KDR+ EPCs (p=0.038) and CD133+/KDR+ EPCs (p=0.018) were increased, whereas CD34+/CD133+ EPCs were not different (p=0.51) in athletes. In addition, there was no difference in MPs levels between the groups. Conclusion: Chronic exposure to exercise in professional runners was associated with higher percentage of EPCs. Taking into account the similar number of MPs in athletes and controls, the study suggests a favorable effect of exercise on these vascular biomarkers.

Resumo Fundamento: Os efeitos da exposição crônica ao exercício sobre biomarcadores vasculares foram pouco estudados. Objetivo: Nosso estudo teve como objetivo comparar as quantidades de células progenitoras endoteliais (CPEs), e de micropartículas endoteliais (MPEs) e plequetárias (MPPs) de corredores profissionais com controles sadios. Métodos: Vinte e cinco corredores de meia maratona e 24 controles pareados quanto à idade e ao sexo foram incluídos no estudo. CPEs (CD34+/KDR+, CD133+/KDR+ e CD34+/CD133+), MPE (CD51+) e MPPs (CD42+/CD31+) foram quantificadas por citometria de fluxo. Todas as amostras de sangue foram obtidas após 12 horas de jejum, e os atletas foram incentivados a realizar seus exercícios de rotina no dia anterior à coleta. Resultados: Em comparação aos controles, CPEs CD34+/KDR+ (p=0,038) e CD133+/KDR+ (p=0,018) estavam aumentados, e CPEs CD34+/CD133+ não foram diferentes (p=0,51) nos atletas. As concentrações de MP não diferiram entre os grupos. Conclusão: A exposição crônica ao exercício em corredores profissionais associou-se a uma maior porcentagem de CPEs. Considerando o número similar de MPs entre atletas e controles, o estudo sugere um efeito favorável do exercício sobre esses biomarcadores vasculares.

Mobilization of hematopoietic progenitor cells for autologous transportation: consensus recommendations / Mobilização de células progenitoras hematopoéticas para transplante autólogo: recomendações de consenso

SUMMARY Selected patients with certain hematological malignancies and solid tumors have the potential to achieve long-term survival with autologous hematopoietic progenitor cell transplant. The collection of these cells in peripheral blood avoids multiple bone marrow aspirations, results in faster engraftment and allows treatment of patients with infection, fibrosis, or bone marrow hypocellularity. However, for the procedure to be successful, it is essential to mobilize a sufficient number of progenitor cells from the bone marrow into the blood circulation. Therefore, a group of Brazilian experts met in order to develop recommendations for mobilization strategies adapted to the reality of the Brazilian national health system, which could help minimize the risk of failure, reduce toxicity and improve the allocation of financial resources.

RESUMO Pacientes selecionados com certas neoplasias hematológicas e tumores sólidos têm o potencial de alcançar sobrevida de longo prazo com o transplante autólogo de células progenitoras hematopoéticas. A coleta dessas células no sangue periférico evita múltiplas aspirações de medula óssea, resulta em enxertia mais rápida, e permite o tratamento de pacientes com infiltração, fibrose ou hipocelularidade medular. Contudo, para o sucesso desse procedimento, é essencial mobilizar um número suficiente de células progenitoras da medula óssea para a circulação sanguínea. Por isso, um painel de especialistas brasileiros se reuniu com o objetivo de desenvolver recomendações para estratégias de mobilização adaptadas à realidade do sistema de saúde nacional, que pudessem contribuir para minimizar os riscos de falha, reduzir a toxicidade e melhorar a alocação de recursos financeiros.

Markers of neural degeneration and regeneration in Down syndrome patients

On the trisomy Down syndrome Critical Region [DSCR1] is located the APP gene, which accelerates amyloid peptide protein [APP] expression leading to cerebral accumulation of APP-derived amyloid-beta peptides [Abeta] and age-dependent cognitive sequelae. Also DSCR1 attenuates endothelial cell proliferation and angiogenesis required for tissue repair. The aim of the present work is to determine markers of neural degeneration and regeneration in the blood of young and adolescent Down syndrome [DS] patients as well as controls. Markers of regeneration were measured in terms of circulating mononuclear cells expressing Nestin and CD34, while markers of degeneration were measured in terms of plasma Abeta[42] and advanced glycation end products receptors [RAGES]. Results showed a significant increase in plasma Ap[42] [20 +/- 5.1 vs. 11.9 +/- 3.4] and RAGES leucocytes mRNA relative expression [1.9 +/- 0.2 vs. 1.1 +/- 0.6] in adolescent DS patients compared to young DS. Both parameters were also significantly increased in DS compared to controls: Abeta[42] [15.4 +/- 5.9 vs. 12. 3 +/- 4.5]; RAGES [1.4 +/- 0.5 vs. 0.7 +/- 0.2]. Nestin [5.2 +/- 1.4 vs. 6.3 +/- 0.6] and CD34 [52 +/- 2.5 vs. 53 +/- 4.7] were non-significantly lower in adolescent DS patients compared to young DS, but significantly lower in DS patients compared to controls: Nestin [6.3 +/- 1.5 vs. 9 +/- 4.4]; CD34 [54 +/- 3.4 vs. 60 +/- 4.8]. The significant decrease in the number of mononuclear cells bearing Nestin and CD34 markers accompanied by a significant increase in Abeta[42] and RAGES indicate that degeneration in DS is an ongoing process, which is not counterbalanced by the regenerative mechanism

Assessment of cord blood vascular endothelial growth factor levels and circulating CD34[+] cells in preterm infants with respiratory distress syndrome

Respiratory distress syndrome [RDS] secondary to surfactant deficiency is a common cause of mobility and mortality in premature infants. Vascular endothelial growth factor [VEGF] is a major angiogenic factor and prime regulator of endothelial cells proliferation. So, VEGF may contribute to surfactant secretion and pulmonary maturation. Additionally, circulating CD34[+] stem - progenitor cells are elevated along with its mobilizing cytokines in neonatal RDS. This study aimed to elucidate the role of cord blood VEGF and the circulating CD34[+] cells in preterm infants with and without RDS. This study was conducted on 55 preterm neonates divided into 25 preterm [15 males/ 10 females] without RDS with mean age of 31.60 +/- 1.56 weeks and 30 preterm neonates with RDS [18 males/ 12 females] with mean age of 29.95 +/- 1.09 weeks. Twenty healthy neonates [14 males/ 6 females] served as controls with mean age of 38.20 +/- 3.57 weeks. All neonates were subjected to full history taking; thorough clinical examination and laboratory investigations including determination of VEGF levels in cord blood samples using ELISA and circulating CD34[+] cells in peripheral blood by flowcytometery. The results of this study revealed that cord blood VEGF levels were significantly decreased in preterms with RDS versus preterms without RDS and controls with p values of both < 0.0001. Furthermore, the circulating CD34[+] cells were significantly increased in preterm infants with RDS versus preterm infants without RDS and controls [p < 0.05 and < 0.0001 respectively]. Premature rupture of the membrane, gender of the newborn, birth weight and antenatal steroid administration had neither significant effect on the cord blood VEGF nor on the number of CD34[+] cells. There was inverse significant correlation between GA and the number of CD34[+] cells. It was concluded that low cord blood VEGF is associated with RDS and its level negatively correlated with the severity of the disease. Thus, it may play a role in recovery from acute lung injury in preterm infants. Moreover, the marked high level of circulating CD34[+] cells in preterms with RDS may give clear evidence of its promise therapeutic role in the future

Serine protease [TPS]: a diagnostic and prognostic marker in pediatric patients with acute non-lymphoblastic leukemia

The Serine protease, TPS [tryptase], is a specific marker for mast cells and mast cell-associated disorders. However, substantial amounts of TPS are also expressed in neoplastic myeloid, non-mast cell lineage. The aim of this study is determination and quantitation of TPS expression in patients with acute non-lymphoblastic leukemia [ANLL]; to evaluate its prognostic value and its relevance as a genetic marker for detection of minimal residual blast cells. Reverse transcriptase-polymerase chain reaction [RT-PCR] was used to detect levels of TPS from 30 newly diagnosed ANLL children and 10 normal children served as controls. TPS levels for positive cases were reevaluated after induction chemotherapy; after onset of relapse or by the end of the study. Our results showed that the gene transcripts were detected in 56.7% of patients but were not expressed by normal controls. The highest frequency of TPS was recorded in patients with M4 showing significantly higher levels compared to other FAB [French-American-British Classification] subtypes. TPS levels were directly correlated to TLC, absolute blast counts in peripheral blood, levels of CD34 and CD117. After induction chemotherapy, levels of TPS decreased significantly in those who achieved complete remission while it increased significantly in relapsed patients, with a risk estimate of relapse six times higher in positive cases than TPS negative patients. 84.6% of the patients negative for TPS achieved complete remission with better disease free survival. In conclusion, TPS is expressed in a group of patients with ANLL thus serves as a disease related marker; it could be used as a prognostic indicator in evaluating remission status and early relapse as its elevated levels are associated with high risk of relapse; again, it is useful in predicting disease outcome

Growth advantage of CD34+cells in trisomy 8 high risk mids cases despite enhanced apoptotic signals

The essence of myelodysplastic syndrome [MDS] pathogenesis is damage of colony forming unit [CFU]with numerous reports implicating apoptosis. While low risk MDS showed enhanced intramedullary apoptosis, high risk MDS was associated with cellular proliferation giving the abnormal clone a growth advantage. Ninteen high risk MDS patients were studied for cellular apoptosis of bone marrow progenitors using tn-color flow cytometric quantification of CD34/Annexin/PU cells. Presence of cytogenetic abnormalities was detected using conventional analysis and FISH while bone marrow mononuclear cells' [BMMNC] survivin expression was evaluated using immunocytochemical examination of bone marrow cytospin preparations. The capacity for hematopoietic colony formation was performed using long term stem cell cultures. High risk MDS cases showed significantly higher percent of both apoptotic CD34/Annex1n*/PI cells and anti-apoptotic survivin cells compared to controls with significantly higher percent among trisomy 8 cases. Trisomy 8 cells showed a significant positive correlation with percent of apoptotic CD34/Annexin/PI cells and capacity for colony formation. The latter was significantly lower in MDS patients negative for trisomy 8 as compared to normal controls, while that of trisomy 8 cases was comparable to controls. Although trisomy 8 cells are in a pro-apoptotic state, they are checked by the enhanced expression of antiapoptotic signals which provide them with their proliferative advantage

Preliminary study of CD34 stem cells in rheumatoid arthritis patients

To study CD34+ stem cells count in the peripheral blood [PB] of patients with rheumatoid arthritis [RA] and to correlate it with the activity and severity of the disease as a preliminary study for their role in the disease pathogenesis. This study was conducted on 20 RA patients in addition to 10 healthy subjects as a control group. All patients were subjected to full history taking and thorough clinical examination. Assessment was done using modified DAS far disease activity, Speed severity index [SSI] for disease severity, and Larsen score for radiological assessment of the plain x-ray findings of both hands. Assessment of CD34+ stem cells count in the PB was done by using fluorescence-activated cell sorting [FACS]. In this study there was a significantly higher count of CD34+ stem cells in the PB of RA patients compared to the controls. Stem cells absolute count and percentage were significantly negatively correlated with modified DAS, SSI and Larsen score. Bone marrow stem cells [CD34 cells] could play a crucial role in RA. Their level is elevated in the PB of RA patients in comparison with controls. It is suggested that the reduced number of CD34[+] cells in the PB of patients with more severely destructed joints is due to their recruitment to sites of inflammation. Studies are required to further investigate the role of the bone marrow and stem cells in the disease pathogenesis of RA. It could be a future target of treatment in these patients

Quantitative and qualitative characterization of hematopoietic stem cells from umbilical cord blood according to aldehyde dehydrogenase activity

Aldehyde dehydrogenase [ALDH] is a cytosolic enzyme that is responsible for the oxidation of intracellalar aldehydes. Elevated levels of ALDH have been demonstrated in murine and human progenitor cells compared with other hematopoietic cells, and this is thought to be important in chemoresistance and purification techniques and an indication of the proper function of the cell. A Flowcytometric method for the assessment of ALDH activity in viable cells recently has been developed. Forty six cord blood samples from mothers underwent normal delivery of full term infants were obtained, after informed consent. Mononuclear cells were obtained by Ficoll-Paque density centrifugation and ammonium chloride red cell lysis. Percentage of viable cells was determined by trypan blue exclusion dye. Cells were labeled with Aldefluor reagent [Stem cell technology, Vancouver., Canada] as described by the manufacturer. Cells were then stained with phycoerythrin [PE]-conjugated anti-CD34 [Miltenyi Biotec, Cologne, Germany] antibodies for 30 minutes at 4°C. Cells were washed and resuspended in phosphate-buffered saline [PBS] with 2% fetal calf serum. Cells were then analyzed on coulter epics flow cytometer. The mean percentage of ALDH enzyme expression among the, CD34[+] cells in the cord blood samples was 61.3% with a minimum of 6 and a maximum of 94.6%. Significant correlations were found between the wbcs count in the cord blood samples and both the CD34[+] cell count and the count of ALDH expressing cells, while, No correlation was found between the CD34[+] cells count or the ALDH expressing cells count in the cord blood samples and either the sex or the weight of the newborn. Identification and isolation of cells on the basis of ALDH activity provides a tool for their isolation and further analysis. In summary, a high ALDH-1 activity identifies CD34[+] cells in cord blood

Associations among white blood cells, CD34+ cells and GM-CFU in predicting the optimal timing of peripheral blood stem cell collections by apheresis.

AIMS: To analyze preapheresis blood CD34+ cells and corresponding apheresis products in order to investigate whether peripheral blood CD34+ cell counts correlate with peripheral blood progenitor cell (PBPC) yields and to determine the optimal timing for starting PBPC collections in our clinical setting. MATERIAL AND METHODS: Thirty-eight patients with hematological malignancies and undergoing varied mobilization regimens were enrolled. White blood cell counts (WBC), CD34+ cells and granulocyte-macrophage colony-forming units (GM-CFU) enumeration were performed on blood samples taken immediately prior to each apheresis procedure and in the corresponding PBPC collection. Results: A total of 61 apheresis procedures were performed, with a median of two collections per patient (range 1-3). The number of CD34+ cell/ml in the preapheresis blood correlated closely with CD34+ cells/kg and, to a lesser degree, with GM-CFU/kg in the apheresis products (r = 0.81 and r = 0.67, respectively, P < 0.0001). WBC showed significant but poor correlation with CD34+ cells/kg and GM-CFU/kg (r = 0.43 and r = 0.45, respectively, P = 0.004). A significant correlation was also found between CD34+ cells/kg and GM-CFU/kg in PBPC collections (r = 0.62, P < 0.0001). Linear regression analysis indicated that the minimum threshold of 2 x 10(6) CD34+ cells/kg might be attained with a single apheresis if the CD34+ cells/ml in the peripheral blood measured prior to apheresis on the day of collection is > or =26 x 10(3) CD34+ cells/ml. CONCLUSION: Collectively, these data demonstrate that circulating CD34+ cells is more useful than GM-CFU or WBC for predicting the optimal timing of PBPC harvests.

Role of interleukin-3 and signaling pathways on beta-thalassemia/HbE erythroid progenitor cell in culture.

In order to study the role of the cytokine interleukin-3 (IL-3) and its signaling pathways in erythropoiesis of beta-thalassemia/HbE erythroid progenitor cells, CD34 positive cells were isolated from peripheral blood of patients and healthy subjects. After culturing the cells in the presence or absence of IL-3, cell viability was measured by trypan blue staining and apoptotic cells were analyzed by flow cytometry. After 7 days of culture the highest percent erythroid progenitor cell viability was obtained with cells from healthy subjects, while the lowest percentage was found in those from splenectomized beta-thalassemia/HbE. Viability of beta-thalassemia/HbE erythroid progenitor cells in the presence of IL-3 was higher than that of nonsupplemented cells. In addition, specific inhibitors of protein kinase C (Ro-318220), phospholipase C (U-73122) and Janus kinase 2 (AG-490) were used to investigate the involvement of signaling pathways in erythropoiesis. Percent apoptosis of erythroid progenitor cells from splenectomized beta-thalassemia/HbE subjects treated with RO-318220 was higher than those of nonsplenectomized beta-thalassemia/HbE and healthy subjects. Treatment with U-73122 resulted in enhanced percent apoptotic cells from normal and beta-thalassemia/HbE subjects. All these effects were independent of IL-3 treatment.

Diagnostic value of CD34, epidermal growth factor receptor [EGFR] expression and proliferation in vesicular mole and persistent gestational trophoblastic disease

Identifying patients with increased risk for developing persistent trophoblastic disease [PTD] following evacuation of hydatiform mole, whether partial or complete, is essential to prevent unnecessary prophylactic chemotherapy. It is challenging to find an adequate diagnostic modality that identifies patients at increased risk for developing PTD. This study was investigating villous angiogenesis by identification of CD34 antigen, trophopastic epidermal growth factor receptor [EGFr] as well as trophoblastic cell proliferation to predict the risky patients with molar pregnancy. Thirty aborted patients were conducted for clinical examination, ultrasonography, and estimation of serum human chorionic gonadotropin [HCG] before abortion and frequent measurement after evacuation for six months. Contents of evacuation were fixed in buffered formalin [10%], processed, paraffin embedded and 4 sets of sections were subjected for hematoxylin and eosin stain, immunohistochemistry using monoclonal CD34 and EGFr antibodies and silver stain to asses proliferative potentiality by counting the Argerophilic nucleolar organizer regions [AgNor]. Clinical, ultrasonographic examination revealed molar pregnancy in 25 cases. Histopathologic examination of post evacuation tissue revealed normal pattern of chorionic villi in 5 cases and they were considered as control. Patterns consistent with partial vesicular mole [PVM] were detected in 12 case and features of complete vesicular mole [CVM] were seen in 13 cases. Persistent elevation of HCG-alpha titer after evacuation was detected in 4 molar cases [16%] [one out of 12 partial mole cases [8.33%] and 3 of the 13 complete partial moles [23.08%]. Immunohistochemistry study comprised frequent expression of CD34 with increased microvessel density per villous [MVD/villous] in control cases [mean 7.5 +/- 0.56]. The microvessels located at the periphery beneath vasculosyncytial membrane to perform margination. However, the mean MVD/Villous was significantly reduced in PVM and CVM [3 +/- 0.75 and 1.75 +/- 0.68 respectively]. In 5 out of 12 PVM [41.7%] the micro vessels were located centrally while rest cases perform margination. Contrary, in all CVM specimens the micro vessels if present were located centrally. In control group, EGFr The trophoblasts were negative for EGFr expression, while in PVM group, trophoblasts displayed mild to moderate expression in 5/12 [41.67%] with a mean of 1.8 +/- 0.58. Contrary, all CVM showed moderate to strong expression of EGFr [mean=2.56 +/- 0.75]. High expression of EGFr was in parallel with decreased MVD/villous. There was increased EGFr expression in 3 cases that were associated with persistent elevation of serum HCG following evacuation [1/12 [8.3%] was PVM and 2/13 [15.4%] were CVM]. Proliferative potential was noticed to be increased with increased AgNor count in the trophblasts of CVM versus PVM and control groups [4.29 +/- 1.25, 1.98 +/- 0.32 and 1.1 +/- 0.07 respectively]. Significant reduction of MVD/Villous and increased expression of EGFr confirm diagnosis h of CVM. In addition, absence of CD34 positive micro vessels and high expression of EGFr could be used as markers to predict the possibility for persistent trophoblastic disease, providing better chance for early medical intervention

Clinical Significance of Platelet Count at day +60 After Allogeneic Peripheral Blood Stem Cell Transplantation

Thrombocytopenia (TP) is a frequent complication after allogeneic stem cell transplantation (SCT) and regarded as a poor prognostic factor, especially in patients with chronic graft-versus-host disease (GVHD), although various factors were related to the development of TP after allogeneic SCT. Sixty-three patients receiving allogeneic peripheral blood stem cell transplantation (PBSCT) were stratified according to platelet count (PC) at day +60 and analyzed in terms of overall survival (OS) and the incidence of non-relapse mortality (NRM). Ten patients (15.9%) were stratified in group 1 (PC or =80 x 10(9)/L). Group 3 was associated with lower incidence of extensive chronic GVHD (p=0.013), better 3-yr OS (p=0.0030), and lower NRM rate (p<0.0001). In multivariate analyses, the PC at day +60 was identified as an independent prognostic factor (p=0.003) together with CD34+ cell dose (p<0.001), disease risk (p=0.004), and acute GVHD (p=0.033) in terms of NRM, and the PC (p=0.047) and CD34+ cell dose (p=0.026) in terms of incidence of infectious events. Measuring the platelet count at day +60 is a simple method for predicting the risk of chronic GVHD development and prognosis after allogeneic PBSCT.

Effect of G-CSF on peripheral blood progenitor cell mobilization and collection from healthy donors.

We studied granulocyte colony-stimulating factor (G-CSF) mediated peripheral blood progenitor cells (PBPC), which were mobilized and collected from healthy donors for allogeneic transplantation. A total of 26 donors, age ranged from 21-41 years were mobilized with G-CSF at a dose of 7.5 microg/kg/day subcutaneously for 5 days and the collection was started on day 5. The CD34 cell counts reached a maximum on day 5 and subsequently declined despite continually given G-CSF. White blood cells (WBC), absolute neutrophil counts (ANC), absolute lymphocytes (AL) and their subsets, absolute mononuclear cells (AMNC), colony-forming unit-granulocyte, macrophage (CFU-GM) and CD34+ cells were increased about 6, 9, 2, 3, 34 and 40-fold, respectively, but red blood cells (RBC), hematocrit (Hct) and platelets (Pit) decreased on day 5 when compared to day 0. All parameters decreased after stem cell collection. For stem cell collection by Cobe Spectra, we used a blood volume of 19.27 +/- 4.65 liters, flow rate of 60.53 +/- 10.03 ml/minute, acid citrate dextrose solution (ACD)/blood ratio of 1:13.31, the final product volume was 314.14 +/- 72.24 ml, collection time was 325.40 +/- 73.36 minutes and one or two procedures were sufficient. The correlation between the number of CD34+ cells/kg, CFU-GM/kg and MNC/kg found in the harvested product and CD34 cells can be used for determining the necessary amount of progenitor cells for transplantation.