[Show abstract][Hide abstract] ABSTRACT: Previously, we had reported the role of tacrolimus (TAC) versus sirolimus (SRL) on the generation of regulatory T cells (Tregs) in primary MLR assays with SRL, demonstrating a uniquely supportive effect. However, the mechanisms associated with their actions on alloreactive human are not fully understood. Therefore, we tested whether TAC and SRL differentially affect already alloactivated human CD4 T-cell subsets.
Alloreactive CD4CD45RA/CD45RO T cells generated in 9-day MLR were cocultured with anti-CD3 and autologous antigen presenting cells plus interleukin (IL)-2 in presence of TAC, SRL, or both, and the Tregs generated after another 5 to 6 days were phenotypically, molecularly, and functionally characterized.
Tacrolimus significantly and SRL modestly inhibited interferon (IFN)-γ (Th1) and IL-17 (Th17)-producing cells. At clinical therapeutic concentrations, SRL, however, significantly increased forkhead/winged helix transcription factor P3 (FOXP3) Tregs, whereas TAC inhibited this T-cell population dose dependently and significantly. When used in combination, TAC and SRL had additive effects on inhibition of IFN-γ- and IL-17-producing cells. This was in contrast to the ability of SRL to reverse TAC-mediated inhibition of FOXP3-expressing cells. Proinflammatory cytokines (IL-1β, IL-6, and tumor necrosis factor-α) added to cultures caused significant decrease in FOXP3 Tregs that was again reversed by SRL. Sirolimus-derived Tregs were phenotypically normal, anergic to allostimulation, and suppressed proliferation of allogeneic effector T-cells.
Thus, although TAC inhibits all alloreactive T cells, SRL promotes the differentiation and expansion of donor-specific Tregs without secondary reprogramming to IFN-γFOXP3 and IL-17FOXP3 Treg subsets. These results, although performed in an artificial in vitro model, add clinically applicable information on how these agents affect T-cell subpopulations.
[Show abstract][Hide abstract] ABSTRACT: Nineteen subjects have more than 18 months follow-up in a phase IIb tolerance protocol in HLA-mismatched recipients of living donor kidney plus facilitating cell enriched hematopoietic stem cell allografts (FCRx). Reduced intensity conditioning preceded a kidney allograft, followed the next day by FCRx. Twelve have achieved stable donor chimerism and have been successfully taken off immunosuppression (IS). We prospectively evaluated immune reconstitution and immunocompetence. Return of CD4 and CD8 T central and effector memory cell populations was rapid. T-cell receptor (TCR) Excision Circle analysis showed a significant proportion of chimeric cells produced were being produced de novo. The TCR repertoires posttransplant in chimeric subjects were nearly as diverse as pretransplant donors and recipients, and were comparable to subjects with transient chimerism who underwent autologous reconstitution. Subjects with persistent chimerism developed few serious infections when off IS. The majority of infectious complications occurred while subjects were still on conventional IS. BK viruria and viremia resolved after cessation of IS and no tissue-invasive cytomegalovirus infections occurred. Notably, although 2 of 4 transiently or nonchimeric subjects experienced recurrence of their underlying autoimmune disorders, none of the chimeric subjects have, suggesting that self-tolerance is induced in addition to tolerance to alloantigen. No persistently chimeric subject has developed donor-specific antibody, and renal function has remained within normal limits. Patients were successfully vaccinated per The American Society for Blood and Marrow Transplantation guidelines without loss of chimerism or rejection. Memory for hepatitis vaccination persisted after transplantation. Chimeric subjects generated immune responses to pneumococcal vaccine. These data suggest that immune reconstitution and immunocompetence are maintained in persistently chimeric subjects.
[Show abstract][Hide abstract] ABSTRACT: To describe the clinical outcomes and science behind a CD8/TCR facilitating cell-based hematopoietic stem cell transplant approach (termed FCRx) to induce tolerance to renal allografts without graft-versus-host disease (GVHD) and avoidance of long-term immunosuppressant drugs in living donor kidney transplant recipients.
Successful solid organ transplantation currently requires the life-long use of medications to suppress the immune system to prevent transplant rejection. Drug-based immunosuppression significantly increases the risk of infection and cancer, as well as being very costly. Development of new therapies to minimize or eliminate entirely the need for antirejection drugs is of great interest to the transplant community. Therapeutic cell transfer for the control of the human immune system represents a compelling approach to reduce or eliminate the need for antirejection drugs.
Establishment of durable hematopoietic macrochimerism under nonmyeloablative conditioning is achievable in mismatched recipients using facilitating cells and stem cells obtained from donor mobilized peripheral blood mononuclear cells. Persistently chimeric recipients developed donor-specific tolerance and were weaned off of immunosuppressive drugs over 12 months. They maintained stable renal function without development of acute or chronic GVHD.
Current Opinion in Organ Transplantation 12/2014; 20(1). DOI:10.1097/MOT.0000000000000156 · 2.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The cellular immune response is the most important mediator of allograft rejection and is a major barrier to transplant tolerance. Delineation of the depth and breadth of the alloreactive T cell repertoire and subsequent application of the technology to the clinic may improve patient outcomes. As a first step toward this, we have used MLR and high-throughput sequencing to characterize the alloreactive T cell repertoire in healthy adults at baseline and 3 months later. Our results demonstrate that thousands of T cell clones proliferate in MLR, and that the alloreactive repertoire is dominated by relatively high-abundance T cell clones. This clonal make up is consistently reproducible across replicates and across a span of three months. These results indicate that our technology is sensitive and that the alloreactive TCR repertoire is broad and stable over time. We anticipate that application of this approach to track donor-reactive clones may positively impact clinical management of transplant patients.
PLoS ONE 11/2014; 9(11):e111943. DOI:10.1371/journal.pone.0111943 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Tacrolimus and sirolimus are commonly used maintenance immunosuppressants in kidney transplantation. As their effects on immune cells and allograft molecular profiles have not been elucidated, we characterized the effects of tacrolimus to sirolimus conversion on the frequency and function of T cells, and on graft molecular profiles. Samples from renal transplant patients in a randomized trial of 18 patients with late sirolimus conversion and 12 on tacrolimus maintenance were utilized. Peripheral blood was collected at 0, 6, 12, and 24 months post randomization, with T-cell subpopulations analyzed by flow cytometry and T-cell alloreactivity tested by IFN-γ ELISPOT. Graft biopsy samples obtained 24 months post randomization were used for gene expression analysis. Sirolimus conversion led to an increase in CD4(+)25(+++)Foxp3(+) regulatory T cells. While tacrolimus-maintained patients showed a decrease in indirect alloreactivity over time post transplant, sirolimus conversion increased indirect alloreactive T-cell frequencies compared with tacrolimus-maintained patients. No histological differences were found in graft biopsies, but molecular profiles showed activation of the antigen presentation, IL-12 signaling, oxidative stress, macrophage-derived production pathways, and increased inflammatory and immune response in sirolimus-converted patients. Thus, chronic immune alterations are induced after sirolimus conversion. Despite the molecular profile being favorable to calcineurin inhibitor-based regimen, there was no impact in renal function over 30 months of follow-up.Kidney International advance online publication, 29 October 2014; doi:10.1038/ki.2014.350.
Kidney International 10/2014; 87(4). DOI:10.1038/ki.2014.350 · 8.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND: Human leukocyte antigen (HLA)-DQ has emerged as the alloantibody most frequently associated with the generation of de novo donor-specific antibody (DSA), antibody-mediated-rejection, and unfavorable transplantation outcome. METHODS: The generation of HLA-DQ de novo DSA was interrogated in 40 transplant recipients who were immunologically naive before their failed transplantation. Eplet and epitope analyses were performed using HLAMatchmaker and Cn3D software. RESULTS: Ten DQA and thirteen DQB eplets or eplet combinations were identified. All but one revealed an epitope footprint that includes both the DQα and DQβ chains. Four examples are illustrated in detail, representing a range of different epitope landscapes. A disparity between antigen density and mean fluorescence intensity values for some alleles within an eplet group was noted, with mean fluorescence intensity values of the lowest fluorescence bead being one tenth of the highest fluorescence bead, despite the fact that the amount of antigen on these beads were not significantly different. CONCLUSION: Our data support the need for changing the manner in which HLA-DQ antigens and antibodies are evaluated for organ transplantation. The current nomenclature system does not reflect the true nature of HLA-DQ polymorphism. Moreover, epitope immunogenicity likely involves more than the mere presence of a specific eplet. Because our field contemplates the use of epitope matching as an approach to improve organ allocation and overall outcomes, it is imperative to have accurate characterization of the immunogenicity of each epitope. This will pave the way to identifying acceptable mismatches and will allow risk stratification for generating de novo HLA-DSA after transplantation.
[Show abstract][Hide abstract] ABSTRACT: Recently, The Transplantation Society convened a workshop to address the question, "What do we need to have in place to make tolerance induction protocols a 'standard of care' for organ transplant recipients over the next decade?" In a productive 2-day meeting, there was wide-ranging discussion on a broad series of topics, resulting in five consensus recommendations as follows: (1) establish a registry of results for patients enrolled in tolerance trials; (2) establish standardized protocols for sample collection and storage; (3) establish standardized biomarkers and assays; (4) include children 12 years and older in protocols that have been validated in adults; and (5) establish a task force to engage third-party payers in discussions of how to fund tolerance trials. Future planned workshops will focus on progress in implementing these recommendations and identifying other steps that the community needs to take.
[Show abstract][Hide abstract] ABSTRACT: Traditionally, chronic calcineurin inhibitor (CNI) nephrotoxicity has been considered to be one of the main nonimmune mechanisms causing chronic renal allograft dysfunction. CNI minimization and withdrawal strategies have yielded inconsistent results. Few studies address the feasibility of CNI elimination in a prednisone-free regimen. We report a prospective, randomized trial in 200 patients evaluating the impact on renal function and incidence of acute rejection after conversion from tacrolimus (Tac) to sirolimus (SRL). Patients with recent (<3 months) acute rejection episodes or with >0.5 g/day of proteinuria were excluded. All were induced with alemtuzumab, underwent rapid steroid elimination and were maintained on mycophenolate mofetil and Tac. At 12 months posttransplant, patients were randomized 2:1 to SRL (n = 123) or maintained on Tac (n = 64). Mean follow-up was 41.1 ± 15.8 months in the SRL group and 40.7 ± 14.4 months in the Tac group. Biopsy-proven acute rejection at 24 months postrandomization was similar between the groups. Patient survival, graft survival and estimated GFR were also not statistically different. Our study demonstrates that in a prednisone-free immunosuppressive regimen, conversion from Tac to SRL at 12 months posttransplantation is not associated with increased rates of acute rejection and graft loss. However, despite CNI elimination, renal allograft function is equally maintained in both groups.
American Journal of Transplantation 09/2013; 13(11). DOI:10.1111/ajt.12437 · 6.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Panel-reactive antibody (PRA) testing provides assessment of the breadth of sensitization a patient might have against human leukocyte antigen (HLA) antigens. The evolution of calculated PRA (cPRA) reflects the commitment of the transplant community to increase accessibility and promote equity to all patients awaiting kidney transplantation. Recent data from our center and others, however, suggested that a significant diversity of HLA-DQ antigens is not captured, which may lead to inequity in allocating cPRA points.
HLA-DRB1-DQA1-DQB1 typing of 2182 individuals was evaluated for this study using Luminex-based sequence-specific oligonucleotide typing. A total of 3182 haplotypes were confirmed to have the level of resolution required for this study.
The diversity of HLA-DQαβ alleles is greater than what is apparent using the serologic equivalents. The distribution of these alleles within a serologic group varies, with some alleles being more frequent than others; therefore, their representation within the current cPRA system is inaccurate. Three informative examples are given. Haplotypes of DR antigens with DQαβ alleles did not always follow the common published linkage disequilibrium, especially in populations where there is greater genetic diversity.
The current cPRA system does not take into account the distribution of molecular equivalents within DQ serologic specificities. This can result is inequitable allocation of sensitization points and disadvantaging the more sensitized patients. To ameliorate this situation, the United Network for Organ Sharing system should allow inputting HLA-DQαβ alleles both for donor typing and as antibody specificities, which will lead to better representation of unacceptable DQ alleles and improve organ allocation equity.
[Show abstract][Hide abstract] ABSTRACT: The ability to achieve immunologic tolerance after transplantation is a therapeutic goal. Here, we report interim results from an ongoing trial of tolerance in HLA-identical sibling renal transplantation. The immunosuppressive regimen included alemtuzumab induction, donor hematopoietic stem cells, tacrolimus/mycophenolate immunosuppression converted to sirolimus, and complete drug withdrawal by 24 months post-transplantation. Recipients were considered tolerant if they had normal biopsies and renal function after an additional 12 months without immunosuppression. Of the 20 recipients enrolled, 10 had at least 36 months of follow-up after transplantation. Five of these 10 recipients had immunosuppression successfully withdrawn for 16-36 months (tolerant), 2 had disease recurrence, and 3 had subclinical rejection in protocol biopsies (nontolerant). Microchimerism disappeared after 1 year, and CD4(+)CD25(high)CD127(-)FOXP3(+) regulatory T cells and CD19(+)IgD/M(+)CD27(-) B cells were increased through 5 years post-transplantation in both tolerant and nontolerant recipients. Immune/inflammatory gene expression pathways in the peripheral blood and urine, however, were differentially downregulated between tolerant and nontolerant recipients. In summary, interim results from this trial of tolerance in HLA-identical renal transplantation suggest that predictive genomic biomarkers, but not immunoregulatory phenotyping, may be able to discriminate tolerant from nontolerant patients.
Journal of the American Society of Nephrology 06/2013; DOI:10.1681/ASN.2013010068 · 9.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND: The United Network for Organ Sharing algorithm for deceased-donor kidney allocation considers only the human leukocyte antigen (HLA)-A, HLA-B, and HLA-DR loci. Although HLA-DQ serologic specificities can be entered as unacceptable antigens, they are assigned only by the identity of the DQβ chain, disregarding the role of the similarly polymorphic α chain. DQα/β combinations result in unique antigenic epitopes, which serve as targets to different antibodies. Therefore, the presence of HLA antibodies to one DQα/β combination should not preclude negative crossmatch (XM) against another combination. In this retrospective analysis, patients were allowed XM against a particular donor if they had antibodies to some, but not all, DQα/β allele combinations with the donor serologic HLA-DQ antigens. METHODS: HLA antibody signature was obtained using solid-phase Luminex-based antibody analysis. Results were captured at the high-resolution level (as provided by the positive beads). Potential donors were typed to include information on both HLA-DQA and HLA-DQB alleles. RESULTS: Of the 1130 flow XM assays performed, 147 patients had antibodies to donor serologic HLA-DQ antigens. Thirty-five of those patients had antibodies to an allelic DQα/β combination within the donor serologic DQ specificity that were different from the donor's DQα/β, leading to negative flow XM results (24%). Virtual XM, accounting for donor DQα/β combinations, successfully predicts more than 98% of XM outcomes. CONCLUSIONS: In patients with allelic DQα/β antibodies, denying the opportunity for XM based on serologically defined unacceptable antigens can disadvantage the patient. Larger cohort studies are required to substantiate our observation. Introducing DQα/β combination information may increase virtual XM accuracy and organ allocation equity.
[Show abstract][Hide abstract] ABSTRACT: Immunosuppression (IS) withdrawal from calcineurin inhibitors is only possible in ∼20% of liver transplant recipients. However, mammalian target of rapamycin inhibitors (e.g., sirolimus; SRL) appear to be more immunoregulatory and might promote a tolerant state for withdrawal. Our aim was to determine whether systemic (i.e., blood, marrow, and allograft) signatures of immunoregulation are promoted by conversion from tacrolimus (TAC) to SRL. We therefore performed the following serial assays before and after SRL conversion in liver transplant recipients to test for enhanced markers of immunoregulation: (1) flow-cytometry immunophenotyping of peripheral blood mononuclear cells (PBMCs) and bone marrow aspirates for regulatory T cells (Tregs) (e.g., CD4(+) CD25(+++) FOXP3(+) ) and regulatory dendritic cells (DCregs) (immunoglobulin-like transcript 3(+) /4(+) ); (2) liver biopsy immunohistochemical staining (e.g., FOXP3:CD3 and CD4:CD8 ratios) and immunophenotyping of biopsy-derived Tregs after growth in culture; (3) effects of pre- versus postconversion sera on Treg generation in mixed lymphocyte reactions; (4) peripheral blood nonspecific CD4 responses; and (5) peripheral blood gene transcripts and proteomic profiles. We successfully converted 20 nonimmune, nonviremic recipients (age, 57.2 ± 8.0; 3.5 ± 2.1 years post-liver transplantation) from TAC to SRL for renal dysfunction. Our results demonstrated significant increases in Tregs in PBMCs and marrow and DCregs in PBMCs (P < 0.01) after conversion. In biopsy staining, FOXP3:CD3 and CD4:CD8 ratios were significantly higher after conversion and a number of biopsy cultures developed new or higher FOXP3(+) cell growth. Nonspecific CD4 responses did not change. Both pre- and postconversion sera inhibited mixed lymphocyte reactions, although only TAC sera suppressed Treg generation. Finally, 289 novel genes and 22 proteins, several important in immunoregulatory pathways, were expressed after conversion. Conclusions: TAC to SRL conversion increases systemic Tregs, DCregs, and immunoregulatory proteogenomic signatures in liver transplant recipients and may therefore facilitate IS minimization or withdrawal. (HEPATOLOGY 2012).
[Show abstract][Hide abstract] ABSTRACT: In this chapter, we describe studies on non-chimeric human leukocyte antigen (HLA) identical tolerance and chimeric HLA disparate tolerance brought about by infusions of hematopoietic stem cells from the renal donor (DHSC). In our HLA identical series, 4 DHSC infusions were administered during the first 9 months posttransplant in a highly immunoregulatory environment using alemtuzumab induction and rapid conversion from early tacrolimus to mycophenolate and sirolimus. This resulted in the generation of recipient T regulatory cells accompanied by genomic indicators, but only transient chimerism. Seven of the first 12 recipients have been immunosuppression-free between 1 1/2 - 4 years with transplant biopsies free of rejection one year after immunosuppression withdrawal. The HLAdisparate group was treated by non-myeloablative conditioning consisting of: 200cGy whole body irradiation; fludarabine; cyclophosphamide; and, perioperative infusion of a product termed FCRx that contained DHSC, T cells, and a unique fraction of bone marrow derived CD8+TCR-alphabeta-negative cells. Five of the first 8 subjects became 100% chimeric in the peripheral blood and have been immunosuppression-free for 2 to 4 years without graft-versus-host-disease and with normal function and transplant biopsies. An additional 12 recipients with shorter follow-up have had similar courses. Those with non-durable chimerism have not been able to have immunosuppression withdrawn but maintain normal renal transplant function. We conclude that non-HLA disparities in renal transplants between HLA identical pairs may not need durable chimerism to induce tolerance provided by DHSC and temporary immunosuppression supporting the development of regulatory T cells. However, more intense conditioning and infusion of FCRx leading to durable chimerism in the absence of graft versus host disease is necessary to induce tolerance in HLA disparate pairs.
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND: We recently reported that durable chimerism can be safely established in mismatched kidney recipients through nonmyeloablative conditioning followed by infusion of a facilitating cell (FC)-based hematopoietic stem cell transplantation termed FCRx. Here we provide intermediate-term follow-up on this phase II trial. METHODS: Fifteen human leukocyte antigen-mismatched living donor renal transplant recipients underwent low-intensity conditioning (fludarabine, cyclophosphamide, 200 cGy TBI), received a living donor kidney transplant on day 0, then infusion of cryopreserved FCRx on day +1. Maintenance immunosuppression, consisting of tacrolimus and mycophenolate, was weaned over 1 year. RESULTS: All but one patient demonstrated peripheral blood macrochimerism after transplantation. Engraftment failure occurred in a highly sensitized (panel reactive antibody [PRA] of 52%) recipient. Chimerism was lost in three patients at 2, 3, and 6 months after transplantation. Two of these subjects had received either a reduced cell dose or incomplete conditioning; the other two had PRA greater than 20%. All demonstrated donor-specific hyporesponsiveness and were weaned from full-dose immunosuppression. Complete immunosuppression withdrawal at 1 year after transplantation was successful in all patients with durable chimerism. There has been no graft-versus-host disease or engraftment syndrome. Renal transplantation loss occurred in one patient who developed sepsis following an atypical viral infection. Two subjects with only transient chimerism demonstrated subclinical rejection on protocol biopsy despite donor-specific hyporesponsiveness. CONCLUSIONS: Low-intensity conditioning plus FCRx safely achieved durable chimerism in mismatched allograft recipients. Sensitization represents an obstacle to successful induction of chimerism. Sustained T-cell chimerism is a more robust biomarker of tolerance than donor-specific hyporeactivity.
[Show abstract][Hide abstract] ABSTRACT: BK virus nephropathy (BKVN) is a recognized cause of graft failure in kidney transplant recipients. There are limited data on the epidemiology of BK virus (BKV) infection after alemtuzumab induction. By clinical protocol, the kidney transplant recipients at our center were screened with BKV plasma PCR monthly for the first 4 months posttransplant then every 2-3 months for 2 years. A single center retrospective cohort study of all kidney transplant recipients from January 2008 to August 2010 was conducted to determine incidence and outcomes of BKV infection. Descriptive statistics and Kaplan-Meier analysis was performed. Of 666 recipients, 250 (37.5%) developed viruria, 80 (12%) developed viremia and 31 (4.7%) developed BKVN at a median of 17, 21 and 30 weeks, respectively. Induction with alemtuzumab did not significantly affect incidence of BKVN. Increased recipient age, African American race, acute graft rejection and CMV infection were significantly associated with the development of BKVN in multivariate analysis. The incidence of BK viruria, viremia and nephropathy was not significantly different among kidney transplant recipients who received alemtuzumab induction compared to patients receiving less potent induction.
American Journal of Transplantation 11/2012; 13(1). DOI:10.1111/j.1600-6143.2012.04314.x · 6.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The immunoregulatory properties of hematopoietic stem cells (HSCs) have been recognized for more than 60 years, beginning in 1945, when Owen reported that genetically disparate freemartin cattle sharing a common placenta were red blood cell chimeras. In 1953, Billingham, Brent, and Medawar demonstrated that murine neonatal chimeras prepared by infusion of donor-derived hematopoietic cells exhibited donor-specific tolerance to skin allografts. Various approaches using HSCs in organ transplantation have gradually brought closer to reality the dream of inducing donor-specific tolerance in organ transplant recipients. Several hurdles needed to be overcome, especially the risk of graft-versus-host disease (GVHD), the toxicity of ablative conditioning, and the need for close donor-recipient matching. For wide acceptance, HSC therapy must be safe and reproducible in mismatched donor-recipient combinations. Discoveries in other disciplines have often unexpectedly and synergistically contributed to progress in this area. This review presents a historic perspective of the quest for tolerance in organ transplantation, highlighting current clinical approaches.Clinical Pharmacology & Therapeutics (2012); advance online publication 5 December 2012. doi:10.1038/clpt.2012.201.
[Show abstract][Hide abstract] ABSTRACT: Graft-versus-host disease is one of the major transplant-related complications in allogeneic hematopoietic stem cell transplantation. Continued efforts have been made to prevent the occurrence of severe graft-versus-host disease by eliminating or suppressing donor-derived effector T cells. Conventional immunosuppression does not adequately prevent graft-versus-host disease, especially in mismatched transplants. Unfortunately, elimination of donor-derived T cells impairs stem cell engraftment, and delays immunologic reconstitution, rendering the recipient susceptible to post-transplant infections and disease relapse, with potentially lethal consequences. In this review, we discuss the role of dynamic immune regulation in controlling graft-versus-host disease, and how cell-based therapies are being developed using regulatory T cells and other tolerogenic cells for the prevention and treatment of graft-versus-host disease. In addition, advances in the design of cytoreductive conditioning regimens to selectively target graft-versus-host disease-inducing donor-derived T cells that have improved the safety of allogeneic stem cell transplantation are reviewed. Finally, we discuss advances in our understanding of the tolerogenic facilitating cell population, a phenotypically and functionally distinct population of bone marrow-derived cells which promote hematopoietic stem cell engraftment while reducing the risk of graft-versus-host disease.
BMC Medicine 05/2012; 10(1):48. DOI:10.1186/1741-7015-10-48 · 7.28 Impact Factor