[Show abstract][Hide abstract] ABSTRACT: As the history of the cord blood banking system has lengthened, the number of cord blood units (CBUs) cryopreserved for years has increased. The global expansion of cord blood banking resulted in active international exchange of CBUs. To determine whether long-term cryopreservation and international shipment of CBUs affect the quality of the units and outcome after transplantation, we retrospectively analyzed the quality of 95 CBUs and the hematologic recovery of 127 patients with hematological malignancy following single-unit cord blood transplantation. Of the 127 CBUs used to transplant, 42 units were cryopreserved for long periods (5-11.8 years), and 44 units were shipped from distant countries. We found that length of cryopreservation and origin of CBUs did not affect the ratio of viable total-nucleated cells after thawing. Also, neutrophil engraftment was not affected by long-term cryopreservation (> 5 years) or origin (from distant countries), (hazard ratio, 0.91 and 1.2; P=0.65 and 0.41; respectively). The number of CD34(+) cells before freezing (> 1.4 cells/kg recipient) was the only factor that enhanced neutrophil engraftment (hazard ratio, 1.8; P<0.01). This suggests that length of cryopreservation and origin need not be prioritized over the CD34(+) cell dose when selecting CBUs.Bone Marrow Transplantation advance online publication, 23 March 2015; doi:10.1038/bmt.2015.56.
Bone marrow transplantation 03/2015; 50(6). DOI:10.1038/bmt.2015.56 · 3.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Aim: Despite its enormous relevance, homing of hematopoietic stem cells (SCs) remains relatively uncertain due to the limitations of measuring small number of systemically administered cells in the different organs. Despite its high sensitivity, radionuclide detection has been relatively underutilized to this purpose since it cannot differentiate hematopietic SCs recruited by target tissues from those circulating in the blood pool. Our study aims to verify the potential of tracer kinetic approaches in estimating the recruitment of labeled SCs after their systemic administration. Methods: Twenty-four Lewis rats underwent administration of 2 millions cells labeled with 37 MBq of 99mTc-exametazime. Animals were divided into 2 groups according to administered cells: hematopoietic SCs or cells obtained from a line of rat hepatoma. Cell injection was performed during a planar dynamic acquisition. Regions of interest were positioned to plot time activity curves on heart, lungs, liver and spleen. Blood cell clearance was evaluated according to common stochastic analysis approach. Either fraction of dose in each organ at the end of the experiment or computing the slope of regression line provided by Patlak or Logan graphical approach estimated cell recruitment. At the end of the study, animals were sacrificed and the number of cells retained in the same organs was estimated by in vitro counting. Results: Cell number, documented by the dose fraction retained in each organ at imaging was consistently higher with respect to the "gold standard" in vitro counting in all experiments. An inverse correlation was observed between degree of overestimation and blood clearance of labeled cells (r=-0.56, P<0.05). Logan plot analysis consistently provided identifiable lines, whose slope values closely agreed with the "in vitro" estimation of hepatic and splenic cell recruitment. Conclusion: The simple evaluation of organ radioactivity concentration does not provide reliable estimates of local recruitment of systemically administered cells. Yet, the combined analysis of temporal trends of tracer (cell) tissue accumulation and blood clearance can provide quantitative estimations of cell homing in the different organs.
The quarterly journal of nuclear medicine and molecular imaging: official publication of the Italian Association of Nuclear Medicine (AIMN) [and] the International Association of Radiopharmacology (IAR), [and] Section of the Society of.. 06/2013; 57(2):207-215. · 2.03 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background:
Unrelated cord blood transplantation (UCBT) is associated with delayed hematopoietic recovery. Intrabone injection of cord blood cells (IB-UCBT) and double-UCBT (dUCBT) are designed to circumvent this problem.
In a retrospective registry-based analysis, we compared outcomes of 87 IB-UCBT with 149 dUCBT recipients, after myeloablative conditioning regimen adjusting for the differences between the two groups. Median-infused total nucleated cells were 2.5×10/kg for IB-UCBT and 3.9×10/kg for dUCBT (P<0.001).
At day +30, cumulative incidence (CI) of neutrophil recovery was 76% and 62% (P=0.014) with a median time to engraftment of 23 and 28 days (P=0.001), after IB-UCBT and dUCBT, respectively. At day +180, CI of platelets recovery was 74% after IB-UCBT, and 64%, after dUCBT (P=0.003). In multivariate analysis, IB-UCBT was associated with neutrophil and platelets recovery and lower acute graft versus host disease (II-IV) (P<0.01). At 2 years, CI of nonrelapse mortality and relapse incidence were 30% and 25% after IB-UCBT and 34% and 29% after dUCBT, and disease-free survival was 45% and 37%, respectively. However, after landmark analysis at 4.7 months from transplantation, in multivariate analysis, relapse incidence was reduced (P=0.03), and there was a trend for better disease-free survival after IB-UCBT (P=0.09).
Both approaches expand the possibility of offering UCBT to patients with hematopoietic malignancies; IB-UCBT is associated with faster myeloid and platelet recovery and lower acute graft versus host disease and may reduce the total cost. However, studies on cost effectiveness are needed to compare both strategies.
[Show abstract][Hide abstract] ABSTRACT: Background. Several reports established an association between iron chelation therapy with deferasirox and hematopoietic improvement in patients with myelodysplastic syndromes. Data in β-thalassemia major patients is absent. Design and Methods. In a cross-sectional study, we evaluated the absolute number of several hematopoietic peripheral progenitors (Colony Forming Unit-Granulocyte/Macrophage, Erythroid Burst-Forming Unit, Colony Forming Unit-Granulocyte/Erythrocyte/Macrophage/Megakaryocyte, and Long Term Culture-Initiating Cells) in 30 β-thalassemia major patients (median age 29.5 years, 40% males) and 12 age-matched controls. In β-thalassemia major patients, data on splenectomy status, the type of iron chelator used, and serum ferritin levels reflecting changes in iron status on the chelator were also retrieved. All patients had to be using the same iron chelator for at least 6 months with >80% compliance. Results. The absolute number of all hematopoietic peripheral progenitors were higher in β-thalassemia major patients than controls, and varied between splenectomized and nonsplenectomized patients (lower Erythroid Burst-Forming Unit and higher Colony Forming Unit-Granulocyte/Macrophage, Colony Forming Unit-Granulocyte/Erythrocyte/Macrophage/Megakaryocyte, and Long Term Culture-Initiating Cells). Patients using deferasirox (n=10) showed significantly higher levels of BFU-E compared with both deferoxamine (n=10) and deferiprone (n=10) treated patients (p<0.05). After adjusting for age, sex, splenectomy status, and serum ferritin changes, the association between higher Erythroid Burst-Forming Unit absolute number and deferasirox compared with deferoxamine or deferiprone therapy remained statistically significant (p=0.011). Conclusions. Deferasirox therapy in β-thalassemia major patients is associated with higher levels of circulating Erythroid Burst-Forming Unit than other iron chelators. Such variation is independent of iron status changes, and is more likely attributed to the type of chelator.
[Show abstract][Hide abstract] ABSTRACT: Background. Despite advancements in comprehension of molecular mechanisms governing bone marrow (BM) homing of hematopoietic stem cells, cord blood transplant (CBT) suffers from a slow rate of hematopoietic recovery. Intrabone (IB) injection has been proposed as a method able to improve speed of BM engraftment with respect to conventional IV protocols. However, the mechanisms underlying this benefit are largely unknown.
Aim. To verify whether IB-CBT determines a local engraftment able to predict the reconstitution of recipient hematopoiesis.
Design and Methods. Twenty-one patients with hematologic malignancies received IB injection into both iliac crests of 3.2 ± 0.68 ∗ 107/kg cord blood cells. One month following IB-CBT, PET-CT imaging was performed. Maximal standardized uptake values (SUVs) were assessed in BM of both iliac crests and in all lumbar vertebrae.
Results. Maximal SUV within iliac crests was higher than in lumbar vertebrae (4.1 ± 1.7 versus 3.2 ± 0.7, resp., P = 0.01). However, metabolic activity in these two different BM districts was significantly correlated (r = 0.7, P < 0.001). Moreover, FDG uptake values within the injection site closely predicted platelet recovery 100 days after IB-CBT (r = 0.72, P < 0.01).
Conclusions. The metabolic activity of injected BM predicts the subsequent rate of hematopoietic recovery after IB-CBT, suggesting a pivotal role of the local engraftment in the reconstitution of recipient hematopoiesis.
BioMed Research International 10/2012; 2012:767369. DOI:10.1155/2012/767369 · 2.71 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Natural killer (NK) cells play a crucial role in early immunity after hematopoietic stem cell transplantation because they are the first lymphocyte subset recovering after the allograft. In this study, we analyzed the development of NK cells after intrabone umbilical cord blood (CB) transplantation in 18 adult patients with hematologic malignancies. Our data indicate that, also in this transplantation setting, NK cells are the first lymphoid population detectable in peripheral blood. However, different patterns of NK-cell development could be identified. Indeed, in a group of patients, a relevant fraction of NK cells expressed a mature phenotype characterized by the KIR(+)NKG2A(-) signature 3-6 months after transplantation. In other patients, most NK cells maintained an immature phenotype even after 12 months. A possible role for cytomegalovirus in the promotion of NK-cell development was suggested by the observation that a more rapid NK-cell maturation together with expansion of NKG2C(+) NK cells was confined to patients experiencing cytomegalovirus reactivation. In a fraction of these patients, an aberrant and hyporesponsive CD56(-)CD16(+)p75/AIRM1(-) NK-cell subset (mostly KIR(+)NKG2A(-)) reminiscent of that described in patients with viremic HIV was detected. Our data support the concept that cytomegalovirus infection may drive NK-cell development after umbilical CB transplantation.
[Show abstract][Hide abstract] ABSTRACT: Patients treated with low-dose anthracyclines often show late onset cardiotoxicity. Recent studies suggest that this form of cardiotoxicity is the result of a progenitor cell disease. In this study we demonstrate that Cord Blood Endothelial Progenitor Cells (EPCs) exposed to low, sub-apoptotic doses of doxorubicin show a senescence phenotype characterized by increased SA-b-gal activity, decreased TRF2 and chromosomal abnormalities, enlarged cell shape, and disarrangement of F-actin stress fibers accompanied by impaired migratory ability. P16( INK4A) localizes in the cytoplasm of doxorubicin-induced senescent EPCs and not in the nucleus as is the case in EPCs rendered senescent by different stimuli. This localization together with the presence of an arrest in G2, and not at the G1 phase boundary, which is what usually occurs in response to the cell cycle regulatory activity of p16(INK4A), suggests that doxorubicin-induced p16( INK4A) does not regulate the cell cycle, even though its increase is closely associated with senescence. The effects of doxorubicin are the result of the activation of MAPKs p38 and JNK which act antagonistically. JNK attenuates the senescence, p16( INK4A) expression and cytoskeleton remodeling that are induced by activated p38. We also found that conditioned medium from doxorubicin-induced senescent cardiomyocytes does not attract untreated EPCs, unlike conditioned medium from apoptotic cardiomyocytes which has a strong chemoattractant capacity. In conclusion, this study provides a better understanding of the senescence of doxorubicin-treated EPCs, which may be helpful in preventing and treating late onset cardiotoxicity.
PLoS ONE 12/2010; 5(12):e15583. DOI:10.1371/journal.pone.0015583 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Despite enormous advancements in our comprehension of molecular mechanisms governing hematopoietic stem cells (HSCs) engraftment in the bone marrow, current clinical protocols of intravenous (IV) transplantation suffer from a relatively low seeding efficiency. To solve this problem, intrabone (IB) injection of HSCs has been proposed. However, the mechanisms underlying the benefit provided by this procedure remain unknown. This study aims to evaluate the effect of IB on HSCs trafficking and homing features in the living rat.
A total of 35 Lewis rats underwent IB or IV administration of HSCs harvested from syngeneic animals and purified according to CD90 expression. These cells were labeled with 37 MBq 99mTc-exametazime and injected either IV or IB. Cell trafficking and distribution in heart, lung, spleen, liver, and forelimb was evaluated by dynamic radionuclide imaging. Logan graphical approach was used to estimate tissue recruitment of HSCs.
More than 90% of cells escaped from the injected bone to the bloodstream in <15 seconds. However, this short contact profoundly modified HSCs kinetics, reducing their lung sequestration and shortening their blood persistence with respect to IV. More importantly, IB passage resulted in reduced lung uptake and in a fourfold increase in homing of remote bone marrow sites. CD90(+) cells transplantation restored hematopoiesis in eight further rats previously exposed to lethal irradiation.
The first-entry contact with the hematopoietic microenvironment immediately readdresses the fate of transplanted HSCs, providing them with "the final destination stamp" to define their bone marrow homing.
[Show abstract][Hide abstract] ABSTRACT: Cord blood transplant (CBT) in adult patients is scarcely utilized because of the risk of graft failure or very delayed platelet recovery. To improve the capacity and the speed to engraft, we have developed an intra-bone (IB) cord blood transplant technique. 75 patients with hematological malignancies, categorized by disease phase as early (18%), intermediate (20%) and advanced (62%), were transplanted. The median cell dose (TNC) infused was: 2.6 (1.35-5.4)×10(7)/kg; the HLA disparity was: 12 cases=5/6, 62 cases=4/6 and 1 case=3/6 matched antigens. 72/75 patients engrafted (96%); median day of recovery of neutrophils (PMN) >500×10(9)/L and platelets (PLT) >20 000×10(9)/L was: 23 (14-44) and 35 (16-70) days respectively. The outcomes at 2 years according to Kaplan-Meier are: OS=46%±5; RI=18%±2; NRM=39%±5. Acute GVHD incidence/severity was: grade 0-I=64%, II=14%, III-IV=0%. The incidence of Chronic GVHD was globally low but in 3 cases was very severe. Intra-bone CBT is associated with high rate of engraftment, early and robust platelet recovery, low incidence of acute GVHD. A very promising aspect is that the relapse rate is low considering the advanced phase of the disease in two/thirds of patients. A suitable CBU was found for nearly every patient searching for a CBU. Therefore, IB CBT extends the possibility to transplant any patient for whom this approach represents the sole possibility of long-term survival.
Best practice & research. Clinical haematology 06/2010; 23(2):237-44. DOI:10.1016/j.beha.2010.06.003 · 2.12 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Critical size segmental bone defects are still a major challenge in reconstructive orthopedic surgery. Transplantation of human mesenchymal stem cells (hMSC) has been proposed as an alternative to autogenous bone graft, as MSC can be expanded in vitro and induced to differentiate into bone-regenerating osteoblasts by several bone morphogenetic proteins (BMP). The aim of this study was to investigate whether the association of hMSC and BMP-7, with providing the necessary scaffold to fill the bone loss, improved bone regeneration in a rat model of critical size segmental bone defect, compared to treatment with either hMSC or BMP-7 and the matrix. In addition, we tested whether pre-treatment of hMSC with cyclic ADP-ribose (cADPR), an intracellular Ca2+ mobilizer previously shown to accelerate the in vitro expansion of hMSC (Scarfì S et al, Stem Cells, 2008), affected the osteoinductive capacity of the cells in vivo. X-ray analysis, performed 2, 10 and 16 weeks after transplantation, revealed a significantly higher score in the rats treated with hMSC and BMP-7 compared to controls, receiving either hMSC or BMP-7. Microtomography and histological analysis, performed 16weeks after transplantation, confirmed the improved bone regeneration in the animals treated with the association of hMSC and BMP-7 compared to controls. Pre-treatment with cADPR to stimulate hMSC proliferation in vitro did not affect the bone regenerating capacity of the cells in vivo. These results indicate that the association of in vitro expanded hMSC with BMP-7 provide a better osteoinductive graft compared to either hMSC or BMP-7 alone. Moreover, cADPR may be used to stimulate hMSC proliferation in vitro in order to reduce the time required to obtain a transplantable number of cells, with no adverse effect on the bone regenerating capacity of hMSC.
Bone 03/2010; 47(1):117-26. DOI:10.1016/j.bone.2010.03.023 · 3.97 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Abscisic acid (ABA) is a hormone involved in pivotal physiological functions in higher plants, such as response to abiotic stress and control of seed dormancy and germination. Recently, ABA was demonstrated to be autocrinally produced by human granulocytes, beta pancreatic cells, and mesenchymal stem cells (MSC) and to stimulate cell-specific functions through a signaling pathway involving the second messenger cyclic ADP-ribose (cADPR). Here we show that ABA expands human uncommitted hemopoietic progenitors (HP) in vitro, through a cADPR-mediated increase of the intracellular calcium concentration ([Ca(2+)](i)). Incubation of CD34(+) cells with micromolar ABA also induces transcriptional effects, which include NF-kappaB nuclear translocation and transcription of genes encoding for several cytokines. Human MSC stimulated with a lymphocyte-conditioned medium produce and release ABA at concentrations sufficient to exert growth-stimulatory effects on co-cultured CD34(+) cells, as demonstrated by the inhibition of colony growth in the presence of an anti-ABA monoclonal antibody. These results provide a remarkable example of conservation of a stress hormone and of its second messenger from plants to humans and identify ABA as a new hemopoietic growth factor involved in the cross-talk between HP and MSC.
[Show abstract][Hide abstract] ABSTRACT: Various lines of evidence suggest that substantial numbers of very primitive normal hematopoietic cells persist in the marrow of most patients with CML, despite the presence of an expanded Philadelphia-Chromosome (Ph) positive population, and that normal clones might, in certain circumstances, have a proliferative advantage over leukemic populations. We have recently demonstrated in 5/8 CML patients with blastic phase (BP) that the blood progenitor cells/(BPC) harvested during early recovery from marrow aplasia were Ph-negative on cytogenetic analysis, suggesting that leukapheresis may provide a useful source of 'normal' progenitors for subsequent reinfusions. We report here an update on 40 patients with Ph + CML and 9 patients with ALL in first or subsequent relapses with associated cytogenetic translocations including t(8;14) t(4;8) t(4;11) and t(9;22). All these patients received intensive conventional chemotherapy and during early recovery from marrow aplasia, when the WBC reached 0.5–2.0 × 109/L, BPC were collected by 4–8 leukapheresis and tested for the persistence of the marker translocations and, when possible, for the presence of the hybrid bcr/abl transcripts by polymerase chain reaction (PCR). In seven out of 10 patients with chronic phase CML, BPC were Ph-negative and in 5 PCR negative. In both accelerated phase patients, BPC were Ph-negative but PCR-positive and in eight out of 28 blastic CML patients, BPC were Ph-negative and in two cases also PCR-negative. Six out of 9 ALL patients, lost the cytogenetic translocations. After complete recovery, 16 patients were subsequently given high-dose therapy followed by reinfusion of 'normal' BPC. Two patients in CP-CML and 2 out of six patients with ALL maintain clinical and cytogenetic remission at 3 and 10 months and 16 months respectively. All the patients transplanted in BP-CML relapsed 5–18 months post-transplant. These data suggest that intensive conventional chemotherapy can lead to a precocious overshoot of cytogenetically normal BPC.
Leukemia and Lymphoma 06/2009; 9(6):477-483. DOI:10.3109/10428199309145754 · 2.89 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cord-blood transplants are associated with delayed or failed engraftment in about 20% of adult patients. The aim of this phase I/II study was to establish the safety and efficacy of a new administration route (intrabone) for cord-blood cells, measured by the donor-derived neutrophil and platelet engraftment.
Adult patients with acute leukaemia, for whom an unrelated stem-cell transplantation was indicated and no suitable unrelated human leucocyte antigen (HLA)-matched donor had been identified, were included in the study and underwent a cord-blood transplant in San Martino Hospital, Genoa, Italy. Eight patients were in first complete remission, ten in second complete remission, and 14 had advanced-stage, refractory disease. HLA matching was 5/6, 4/6, and 3/6 for 9, 22, and one patient, respectively. Cord-blood cells were concentrated in four 5-mL syringes, and were infused in the superior-posterior iliac crest under rapid general anaesthesia. Median transplanted cell dose was 2.6 x 10(7)/kg (range 1.4-4.2). The primary endpoint was the probability of neutrophil and platelet recovery after intrabone cord-blood transplantantion. Secondary endpoints included the incidence of acute graft-versus-host disease, relapse, and overall survival. This trial is registered on the ClinicalTrials.gov website, number NCT 00696046.
Between March 31, 2006, and Jan 25, 2008, 32 consecutive patients with acute myeloid leukaemia (n=20) or acute lymphoblastic leukaemia (n=12) underwent a cord-blood transplant (median age 36 years [range 18-66]). No complications occurred during or after the intrabone infusion of cells. Four patients with advanced-stage disease died within 12 days of the procedure. Median time to recovery of neutrophils in 28 patients (>/=0.5 x 10(9)/L) was 23 days (range 14-44) and median time to recovery of platelets in 27 patients (>/=20 x 10(9)/L) was 36 days (range 16-64). All patients were fully chimeric from 30 days after transplantation to the last follow-up visit, suggesting an early complete donor engraftment. No patient developed grade III-IV acute graft-versus-host disease. Causes of death were transplant related (n=5), infection (n=7), and relapse (n=4). 16 patients were alive and in haematological remission at a median follow-up of 13 months (range 3-23).
Our preliminary data suggest that direct intrabone cord-blood transplantation overcomes the problem of graft failure even when low numbers of HLA-mismatched cord-blood cells are transplanted, thus leading to the possibility of use of this technique in a large number of adult patients.
The Lancet Oncology 09/2008; 9(9):831-9. DOI:10.1016/S1470-2045(08)70180-3 · 24.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mesenchymal stromal cells are multipotent cells considered to be of great promise for use in regenerative medicine. However, the cell dose may be a critical factor in many clinical conditions and the yield resulting from the ex vivo expansion of mesenchymal stromal cells derived from bone marrow may be insufficient. Thus, alternative sources of mesenchymal stromal cells need to be explored. In this study, mesenchymal stromal cells were successfully isolated from second trimester amniotic fluid and analyzed for chromosomal stability to validate their safety for potential utilization as a cell therapy product.
Mesenchymal stromal cells were expanded up to the sixth passage starting from amniotic fluid using different culture conditions to optimize large-scale production.
The highest number of mesenchymal stromal cells derived from amniotic fluid was reached at a low plating density; in these conditions the expansion of mesenchymal stromal cells from amniotic fluid was significantly greater than that of adult bone marrow-derived mesenchymal stromal cells. Mesenchymal stromal cells from amniotic fluid represent a relatively homogeneous population of immature cells with immunosuppressive properties and extensive proliferative potential. Despite their high proliferative capacity in culture, we did not observe any karyotypic abnormalities or transformation potential in vitro nor any tumorigenic effect in vivo.
Fetal mesenchymal stromal cells can be extensively expanded from amniotic fluid, showing no karyotypic abnormalities or transformation potential in vitro and no tumorigenic effect in vivo. They represent a relatively homogeneous population of immature mesenchymal stromal cells with long telomeres, immunosuppressive properties and extensive proliferative potential. Our results indicate that amniotic fluid represents a rich source of mesenchymal stromal cells suitable for banking to be used when large amounts of cells are required.
[Show abstract][Hide abstract] ABSTRACT: Bone marrow (BM) cells from 10 patients with Ph1-positive chronic granulocytic leukaemia (CGL) were placed in long-term cultures in the presence of fetal calf serum (FCS) and horse serum (HoS), or in the presence of human AB serum. The long-term cultures were started with three different cell combinations: (1) CGL BM cells (four cases), (2) CGL BM cells + normal BM cells (1:1 ratio) from an HLA identical sex-matched sibling (five cases), (3) CGL BM cells + normal BM cells (1:1 ratio) from an HLA identical sex mismatched sibling (five cases). Cytogenetic studies were performed at weeks 0, 3, 4 and 5 of culture. The results of this study can be summarized as follows: (a) Ph1-positive cells could be detected at any time of culture in all three of the described cell combinations; (b) a population of Ph1-negative cells of patient origin could be detected after 3–5 weeks of culture; (c) there was a trend for a better survival of Ph1-negative cells in cultures supplemented with FCS + HoS and, conversely, of Ph1-positive cells in cultures containing human serum. These results warrant further studies on the possibility of manipulating survival and proliferation of normal and leukaemic cells by varying the culture conditions.
British Journal of Haematology 03/2008; 63(1):135 - 141. DOI:10.1111/j.1365-2141.1986.tb07503.x · 4.71 Impact Factor