bluuebunny/crossref_metadata_2025_split huggingface dataset:
Prevention and Reversal of Lupus in NZB/NZW Mice by Transplantation of Purified Allogeneic Hematopoietic Stem Cells (HSC) with Non-Myeloablative Conditioning (NMT).
Authors: Smith-Berdan Stephanie , Gille Daphne , Christensen Julie L. 
Published: October 2019

Abstract
Systemic lupus erythematosus (SLE) is a multifactorial autoimmune disease. Patients’ refractory to therapy may be considered for hematopoietic stem cell transplantation. Using lupus prone female NZB x NZW (NZBW) mice, we tested the ability of highly enriched, allogeneic HSC to prevent and reverse autoimmune symptoms with FACS purified haplo-identical allogeneic HSC. Ablative conditioning: 92 animals were given lethal TBI (14.5 Gy) and divided among 4 groups (Table 1). Urine and serology tested monthly, final time point before death tabulated. Transplantation with either syngeneic HSC or WBM accelerated disease in these mice, resulting in a rate of death exceeding age matched controls. Allogeneic transplanted mice had significantly greater survival above all groups (p= 0.0243). Proteinuria, elevated levels of circulating immune complexes (CIC), and auto-antibodies to dsDNA, nuclear antigens (ANA) and histones were lower in allo-HSC animals compared to the other ablative conditioning groups (p≤ 0.0001). Overall survival (OS) in allo-HSC animals was still unexceptional, possibly due to regimen related toxicity (TRM).
Table 1: Ablative age@TX=75 days Age Matched Control Syngeneic WBM Syngeneic HSC Allogeneic HSC N 15 21 28 28 OS@420 days of age 20% 0% 0% 53% Proteinuria 100% 74% 75% 15% CIC 93% 81% 100% 25% Anti-dsDNA 100% 91% 100% 39% Anti-ANA 100% 86% 93% 39% Anti-Histone 93% 91% 100% 46%
NMT conditioning: To determine if we could attenuate disease in NZBW mice already progressing into lupus-like disease with transplantation of allogeneic, purified HSC and reduce TRM, we developed a non-myeloablative conditioning protocol (2x5 Gy TBI + ATG + a-ASIALO-GM1) achieving an average mixed chimerism of 50%. Animals were treated at ~241 days with established symptoms of lupus (Table 2). While the group receiving conditioning alone, had a slight survival advantage over age matched control mice, the transplanted mice had greatly increased OS with 70% living well beyond 500 days of age (>250 days from transplant). Allo-HSC mice showed reversal or stabilization of their lupus symptoms including proteinuria, CIC, dsDNA and histone.
Table 2: NMT age@Tx=241 days Age Matched Control Allogeneic HSC Conditioned Only N 10 33 30 OS@500 days of age 0% 70% 0% Proteinuria@Tx 20% 49% 47% Final Proteinuria 100% 39% 67% CIC 67% 50% 74% Anti-dsDNA 85% 15% 44% Anti-Histone 100% 37% 66%
Conclusions:
Ablative and NMT transplant can treat lupus; OS after NMT exceeds ablative conditioning; Induction of mixed chimerism with purified allogeneic HSC using NMT conditioning treats established lupus.
The ability of pure HSC transplant and establishment of durable mixed chimerism to reverse established lupus makes it a reasonable strategy to test in man.
Repertoire Analysis of Ex Vivo Polyclonally Expanded Regulatory T Cells.
Authors: Zilberberg Jenny , Goldgirsh Kira , Korngold Robert , Friedman Thea M. 
Published: October 2019

Abstract
CD4+CD25+ regulatory T cells (Treg) are essential for the maintenance of self-tolerance and have also been implicated in the control of alloreactive immune responses. Several studies using murine models of graft-vs.-host disease (GVHD) have shown that addition of equivalent numbers of Treg to the donor T cell inoculum at time of hematopoietic stem cell transplantation can significantly reduce the incidence of GVHD. In addition, in an MHC-matched, minor histocompatibility disparate model, the infusion of Treg ten days post-transplantation was shown to ameliorate the progression of GVHD while permitting a graft-versus-leukemia effect. However, because Treg constitute <5% of peripheral CD4+ T cells in humans, the use of freshly isolated Treg to treat and/or prevent GVHD, as well as other diseases in the clinical situation, is limited. Therefore, much effort is now under way to expand Treg in order to have sufficient numbers for therapeutic use. There is little available information regarding the repertoire complexity of ex vivo, polyclonally expanded regulatory T cells. We hypothesize that like their CD4+CD25− T cell counterparts, the diversity of the Treg T cell receptor (TCR) repertoire will also be complex. To this end, CD4+CD25− and CD4+CD25+ T cells from B10.BR mice were purified using fluorescence activated cell sorting; both populations were polyclonally expanded using CD3/CD28 paramagnetic microbeads in combination with high levels (100 IU/ml) of hrIL-2. After achieving a greater than 50 fold expansion, RNA from 1–1.5×107 cells was isolated for RT-PCR. The complexity of the T cell repertoire of expanded CD4+CD25− and CD4+CD25+ was determined using TCR Vb CDR3-size spectratype analysis. The PCR products were run on a sequencing gel and analyzed by the GeneMapper Software from Applied Biosystems. This comparison revealed that the number of resolvable Vb families is more heterogeneous in the CD25− populations. Whether this reflected a lack of complexity in the regulatory repertoire warrants further investigation. However, for the resolvable Vb families there were no significant differences in the complexity indexes between these two groups.
Imaging the Differential Engraftment of GFP-Expressing Spleen and Bone Marrow Cells in Various Organs after Reduced-Intensity Stem Cell Transplantation in Mice Model.
Authors: Heike Yuji , Yang Zhijian , Ma Huaiyu , Hoffmann Robert , Morita Yuriko , Soeda Atsuko , Makiyama Hiroaki , Tobinai Kensei , Takaue Yoichi 
Published: October 2019

Abstract
[Background] Reduced-intensity allogeneic stem cell transplantation is of critical importance in the treatment of hematological malignancies. The object of this experiment is to establish fludrabine-based reduced-intensity allogeneic stem cell transplantation model using donor cells labeled with GFP and image the engraftment process of donor cells in various organs.
[Materials and Methods] GFP transgenic C57/BL6 mice (GFP-Tgm) were used as donors and C57/BL6 × DBA F1 mice (BDF1) were used as recipients. Recipient mice were pretreated with fludarabine (Flu) 150 mg/kg/day × 6 days i.p. and cyclophosphamide (CPA) 150 mg/kg/day × 2 days i.p. On day 0, 107 GFP splenocytes (Group 1) and GFP bone marrow cells (Group 2) were injected in the tail vein. Whole body and intravital imaging were used to visualize the migration of GFP-Tgm cells into various organs including the brain, femur, intestine, liver, lung, ovary, pelvic bone, ribs, skin, skull, spine, spleen and uterus on days-7 and -14. The macro images were obtained with the Olympus OV100 Small Animal Imaging System. GFP-Tgm cell migration, particularly CD34+ cells in the various organs was also analyzed by flow cytometry (FACS) using APC-labeled anti-CD34 monoclonal antibodies.
[Results and Discussion] On day-7, the migration of donor GFP-Tgm cells in peripheral lymph nodes, intestine, lung, ovary, skin and uterus were detected in both groups. Migration of GFP-Tgm cells in the femur, pelvic bone, ribs and skull were clearly detected in Group 2, but not in Group 1. On day-14 GFP donor cells were imaged in the lung, ovary, skin and uterus both groups. However, GFP-Tgm cells were no longer imaged in the intestine in Group 2 except in Payer patches. In both groups the GFP-Tgm cells were strongly detected in the femur, pelvic bone, skull and spine on day-14. We also analyzed the migration of CD34+ GFP-Tgm cells by FACS analysis. On day-14, the percentage of GFP-Tgm cells in the bone marrow and spleen was, respectively, 14% and 53% in Group 1, and 10% and 13% in Group 2. The percentage of CD34+ GFP-Tgm cells among total CD34+ cells in the bone marrow was 10% in Group 1 and 14% in Group 2, and in the spleen was 24% and 19%, respectively. Those results suggested that in this model, donor bone marrow and spleen cells, but not purified CD34+ cells, have different engraftment kinetics in various organs including the intestine, which is a target organ for graft-versus host disease. These results clearly suggest that caution should be paid to evaluate engraftment kinetics of infused cells, at least in mice model.
Adoptive Infusion of Alloreactive Donor NK Cells Reduces GVHD, Mediates Anti-Tumor Effects and Prolongs Survival in Recipients of MHC-Matched Hematopoietic Cell Transplantation.
Authors: Lundqvist Andreas , McCoy Philip , Samsel Leigh , Smith Aleah , Srivastava Shivani , Harashima Nanae , Yokoyama Hisayuki , Berg Maria , Childs Richard 
Published: October 2019

Abstract
Alloreactive natural killer (NK) cells with killer immunoglobulin-like receptor (KIR) ligand incompatibility have been implicated to reduce graft-vs-host disease (GVHD) and leukemia relapse in humans undergoing MHC-mismatched T-cell depleted allogeneic hematopoietic cell transplantation (HCT). Analogous to human NK KIR, murine NK cells express subgroups of MHC-specific receptors of the C-type lectin superfamily, termed Ly49, that regulate their function. In MHC-mismatched HCT, the infusion of alloreactive Ly49-mismatched NK cells decreases the incidence of GVHD. We investigated if an infusion of alloreactive NK cells would reduce GVHD and further mediate anti-tumor effects in mice undergoing MHC-matched allogeneic HCT. Balb/c mice were injected intravenously with 105 RENCA tumor cells. Ten days following tumor injection, mice were irradiated (950cGy) and transplanted with 15×106 and 8×106 splenocytes and bone-marrow cells respectively from MHC-matched B10.d2 donors. Five days following transplantation, recipients were given a single infusion of either 0.5–1×106 Ly49 ligand-matched (Ly49G2; non-alloreactive to Balb/c) or Ly49 ligand-mismatched (Ly49C; alloreactive to Balb/c) donor NK cells. Recipients of Ly49 ligand-mismatched NK cells had a significant reduction in the incidence of GVHD (p<0.01-figure), and prolonged survival (median 84 vs 39 days; p<0.01-figure) compared to HCT recipients not receiving NK cells. Recipients of Ly49 ligand-matched NK cells had the same incidence of GVHD and similar survival compared to controls not receiving NK cells. Pulmonary tumor burden was significantly (p<0.01) lower in recipients of Ly49-mismatched or Ly49-matched NK cells compared to mice not receiving NK cells. These data provide in vivo evidence that a single infusion of alloreactive donor NK cells can prolong survival by both reducing GVHD and mediating anti-tumor effects following MHC-matched allogeneic HCT.
Figure: Significant reduction in GVHD and improved survival in RENCA tumor bearing mice undergoing an allogeneic HCT that received an infusion of alloreactive NK cells (Ly49C:thick line) compared to transplant recipients receiving non-alloreactive NK cells (Ly49G2:thin line) or no NK cells (dotted line) Figure:. Significant reduction in GVHD and improved survival in RENCA tumor bearing mice undergoing an allogeneic HCT that received an infusion of alloreactive NK cells (Ly49C:thick line) compared to transplant recipients receiving non-alloreactive NK cells (Ly49G2:thin line) or no NK cells (dotted line)
Allogeneic Stem Cell Transplantation for MDS and sAML Following Reduced Intensity Conditioning and Preemptive Donor Lymphocyte Transfusion.
Authors: Kolb Hans-Jochem , Schmid Christoph , Tischer Johanna , Haussmann Andreas , Rank Andreas , Schleuning Michael , Ledderose Georg 
Published: October 2019

Abstract
Myelodysplastic syndrome (MDS) and secondary acute myeloid leukemia (sAML) are diseases of the hematopoietic stem cell that are potentially curable by allogeneic stem cells transplantation (ASCT). MDS and sAML are most frequent in elderly patients with significant co-morbidity. Here we report our results with reduced intensity conditioning regimen and preemptive donor lymphocyte transfusions that have been successful in patients with advanced and refractory AML. Sixty seven patients were treated for MDS and 90 patients for sAML. The FLAMSA regimen consisted of a 4 day course of chemotherapy (Ara-C 2g/m2, Fludarabin 30 mg/m2, and amsacrin 100 mg/m2) followed by 3 days rest and reduced intensity conditioning with 4 Gy total body irradiation (TBI), cyclophosphamide (CY) +/− antithymocyte globulin (ATG). Overall survival at 10 years was 50% for MDS and 26% for sAML (p=0.014, log rank). In the MDS group most patients had preleukemic forms (IPSS 3 and 4). Survival at 5 years was 58% with FLAMSA, 44% with myeloablative conditioning with TBI and 67% with Busulfan (BUS). Non-relapse mortality at 5 years was 41% with FLAMSA, 20% for BUS and 50% for TBI. The relapse or progression rate was 0% for FLAMSA, 24 % for BUS and TBI respectively. In sAML the 5 year survival was 17% for FLAMSA, 57 % for BUS and 30% for TBI. Non-relapse mortality was 60% for FLAMSA, 36% for BUS and 66% for TBI, whereas the relapse rate was 62% for FLAMSA, 12.5 % for BUS and 37% for TBI (p=0.04). In multivariate analysis adjusted for disease, conditioning treatment and source of stem cells – marrow vs. mobilized blood – differences were not significant and the only significant prognostic factor was age less than 30 years (p=0.003). Therefore patients of the age of 60 years and older with AML were treated with FLAMSA containing BUS (8mg/kg in two days) instead of 4 Gy TBI. The overall survival at 2 years was 64%, 61% with FLAMSA-TBI and 70% with FLAMSA-BUS. The non-relapse mortality was 9% for FLAMSA-BUS and 37% for FLAMSA-TBI; the relapse rate was 25% at 2 years. At day 120 patients received donor lymphocytes (DLT) in 3 escalating doses from 1 × 106/kg, 5 × 106/kg on day 150 and 1 × 107/kg on day 180, if they had no GVHD, infection or relapse. Preemptive DLT appear to prevent relapse when compared to a historical control and contemporary patients not given DLT. We conclude from these data that MDS/sAML may have a different biology in young patients, and that in the elderly we can optimize the treatment with FLAMSA-BUS as a dose reduced regimen and preemptive immunotherapy for maintenance of remission.
SNP12 and SNP13 Variants of NOD2/CARD15 Gene of Donor and Recipient as Independent Risk Factors for Severe Intestinal and Pulmonary GVHD after Allogeneic Hematopoietic Stem Cell Transplantation.
Authors: Holowiecka-Goral Alexandra , Wojnar Jerzy , Giebel Sebastian , Grudziecka Elzbieta , Oczko Malgorzata , Jarzab Barbara , Sankowska Monika 
Published: October 2019

Abstract
Background: Presence of any of single nucleotide polymorphism (SNPs) of the NOD2/CARD15 gene have been recently proven to correlate with higher rate of GvHD after allogeneic hematopoietic stem cell transplantation - alloHSCT (Holler E et al; Blood2006,107,4189). Mutated variants lead to impaired antibacterial response. As NOD2/CARD15 is expressed not only in monocytets/macrophages but also in both intestinal and bronchial epithelial cells these organs seem to be a major target for developing advanced GvHD.
Patients and methods: The impact of each single donor (D) and recipient (R) SNPs (8,12,13) of NOD2/CARD15 gene on the incidence of acute GvHD (grade II-IV, grade III-IV, intestinal), chronic GvHD (overall, extensive, pulmonary), survival, and non-relapse mortality was evaluated in a setting of 72 patients treated with alloHSCT in single BMT center in Katowice between Jan 2000 and Jun 2005. Median patient age was 33 (11–52) years. In 70/72 cases hematological malignancies were indication for alloHSCT. Conditioning regimen was myeloablative in all cases, bone marrow was used as the only source of stem cells in 74% of patients. GVHD prophylaxis consisted of CsA, Mtx and, in case of URD-HSCT - antithymocyte globulin.
Results: In univariate and multivariate analysis, including other potential risk factors, the presence of SNP12 in the recipient was associated with increased incidence of intestinal acute GVHD (75% vs. 29%; RR 4,37, p=0,03), overall chronic GVHD (100% vs. 50%, RR 4,72; p=0,003), extensive chronic GVHD (80% vs. 24%; RR 5,12; p=0,02), and pulmonary chronic GVHD (47% vs. 9%; RR 5,97; p=0,02). SNP13 in the recipient resulted in increased incidence of grade III–IV acute GVHD (45% vs. 12%; RR 4,66; p=0,01), intestinal acute GVHD (64% vs. 24%; RR 4,21, p=0,005), and extensive chronic GVHD (55% vs. 25%; RR 3,59; p=0,03). SNP13D in the donor contributed to increased risk of pulmonary chronic GVHD (36% vs. 8%; RR 6,58; p=0,01). In this single centre analysis we were not able to demonstrate the impact of NOD2/CARD15 SNPs on survival.
Conclusion: The presence of particular mutations of NOD2/CARD15 in the recipient and/or the donor is associated with increased risk of severe acute and chronic GVHD. In particular, intestines and lungs appear to be target organs of these complications. Our findings may contribute to optimization of immunosuppressive and gastrointestinal decontamination regimens based on individual risk assessment for GVHD.
Non-Random Integration and Clone Selection by Lentiviral SIN-LTR Vectors.
Authors: Bartholomae Cynthia C. , Deichmann Annette , Schmidt Manfred , Yanez-Munoz Rafael J. , Howe Steven J. , Schmidt Sonja , Prinz Claudia , Thrasher Adrian J. , von Kalle Christof 
Published: October 2019

Abstract
The need for safer gene therapy vectors was highlighted by the occurrence of three cases of retroviral vector-induced leukemia in children after the cure of severe combined immunodeficiency by gene therapy. These severe adverse events enhanced the development of new gene therapy vector systems, which aim to reduce influence of insertions on integrity and expression of genomic host DNA. Lentiviral vectors with self-inactivating (SIN) long terminal repeats (LTRs) have no LTR enhancer activity and are not expected to produce insertional gene activation. In our study we analyzed the integrations sites of three different lentiviral SIN-HIV-based- vectors. These vectors differed in their internal elements (Promotor, Transgene, WPRE) which allowed us to investigate whether internal elements are influencing target site selection and clone survival. A total of 1422 integration sites were analyzed at three different time points (1 day, 30 days, 60 days) after transduction of identical HeLa cells by LAM- PCR. Our results showed similar gene involvement and chromosomal distribution of integration sites for all vector types analyzed, independent of vector composition. 62–63% of the integrations were detected in Refseq genes, 82–86% were found within Refseq genes and their surrounding 10kb. Surprisingly, 271 of 1422 integration sites were clustered as common integration sites (CIS). Computer simulations allowed us to show that this high number of CIS was significantly different from a modeled random distribution of integration sites. Gene ontology analysis showed no difference in significantly overrepresented gene categories between the distinct vector types. Interestingly, we detected a time dependent increase or decrease in significance. Specific gene categories like phosphorylation, protein kinase or ATP binding activity increased in significance from freshly transduced cells (1 day) to 30 days. This observation was even more pronounced at the latest time point analyzed (60 days). Other gene categories showed the exactly opposite effect. Gene ontology analysis of freshly transduced cells showed a significant overrepresentation of genes involved in cell cycle regulation, whereas the analysis of the later time points did not show overrepresentation of this gene category. These results suggest that lentiviral SIN-HIV-based vectors may induce clonal selection in vitro independently of internal vector elements. The character of such effects as well as any putative relevance of such genotoxicity for the in vivo situation will have to be investigated in detail for the role of individual vector/cell type configurations.
Long-Term Polyclonal and Stable Gene Transfer into Rhesus Repopulating Hematopoietic Stem Cells Using a Simian Immunodeficiency Virus-Based Lentiviral Vector System.
Authors: Kim Yoo-Jin , Hussein Nadia L. , Hematti Peiman , Hong Bum-Kee , Calmels Boris , Donahue Robert E. , Hanawa Hideki , Nienhuis Arthur W. , Dunbar Cynthia E. 
Published: October 2019

Abstract
Murine leukemia virus (MLV) vectors have been studied extensively in animal models and utilized for over a decade in clinical trials of gene therapy directed at hematopoietic stem and progenitor cells MLV have a number of limitations, including inefficient transduction of quiescent cells and difficulty in maintaining stable high-level expression. More recently concerns have arisen regarding their safety regarding activation of adjacent proto-oncogenes and resultant leukemogenesis. We have previously reported that lentiviral vectors derived from the simian immunodeficiency virus (SIV) were efficient at transducing rhesus CD34+ cells, resulting in high-level in vivo marking with transduced progeny cells up to one year post-transplantation.(Hanawa et al, 2004) A comparison of vector integration sites in these animals compared to animals receiving MLV-transduced cells revealed different patterns, showing that SIV integrants strongly favored entire transcription units and gene-dense regions of the genome, compared to MLV that favored regions surrounding transcription start sites.(Hematti et al, 2004). Animals receiving MLV-transduced cells had highly non-random engraftment with integrants in or near the the MDS1/EVI1 gene complex. To evaluate long-term safety implications of the SIV vector-mediated CD34+ cell gene transfer, we analyzed the insertional sites in granulocytes, T cell, and B cells from 3 rhesus macaques which were transplanted three years ago with transduced, autologous cytokine-mobilized peripheral blood CD34+ cells. All three animals continued to show significant marking and expression levels in T cells, B cells and granulocytes, with mean GFP + levels of 6.7% (range, 3.3–13.0%), 7.4% (4.2–13.4%) and 5.6% (3.1–10.5%), respectively. Vector insertion site analysis by linear amplification-mediated PCR method at three years continued to show highly polyclonal reconstitution. Subsequent cloning and sequencing data confirmed long-term polyclonality with vector-containing cells and there was no evidence for any worrisome common integration sites, with no integrants detected in the MDS1/EVI1 region, in contrast to results with the MLV vector. These results indicate that the SIV vector system can result in stable and efficient long-term expression in progeny of transduced CD34+ cells, without the worrisome integration profile previously reported in our model with MLV vectors.