bluuebunny/crossref_metadata_2025_split huggingface dataset:
Epitope-Specific Typing (EST) for HLA-DPB1 Matching: Proof of Principle for an Innovative Approach to Unrelated Hematopoietic Stem Cell Donor Selection.
Authors: Zino Elisabetta , Vago Luca , Di Terlizzi Simona , Mazzi Benedetta , Amato Luigi D’ , Rossini Silvano , Ciceri Fabio , Roncarolo Maria G. , Bordignon Claudio , Fleischhauer Katharina 
Published: October 2019

Abstract
Background. Conventional matching of patients and their unrelated donor (UD) hematopoietic stem cells (HSC) by 4-digit molecular HLA typing is associated with lengthy donor searches and elevated social costs. 80% of UD transplants are performed across DPB1 mismatches which, if involving disparity in host versus graft (HvG) direction for an immunogenic T cell epitope, have been shown to be associated with poor clinical outcome of transplantation for hematopoietic malignancies and beta-thalassemia. In this study we have developed an innovative approach of DPB1 epitope- rather than allele-specific matching, by only two PCR reactions (epitope-specific typing; EST). Moreover, we have determined allelic DPB1 frequencies in Italy, confronted them with the ones previously reported for other ethnic groups, and calculated the probability of finding non-permissive DPB1 mismatches in unrelated HSC donor searches.
Methods. High resolution genomic DPB1 typing and EST were performed in parallel on blood samples taken from 112 healthy unrelated Italian blood donors.
Results. EST of DPB1 alleles encoding the immunogenic T cell epitope yielded 100% concordant results with high resolution DPB1 typing in all 112 samples studied, and is therefore suitable to univocally determine the presence or absence of non-permissive DPB1 disparities. The overall frequency of DPB1 alleles encoding the shared T cell epitope in the Italian population was 23.15%. Importantly, we show that based on DPB1 allelic polymorphism in the four ethnic groups representative of the world-wide UD registries, over 75% of UD matched for the other HLA loci will not present a DPB1 epitope disparity in HvG direction, demonstrating that prospective UD-recipient DPB1 matching by EST does not significantly limit the number of suitable donors, and has a negligible impact on the time and cost of the search.
Conclusions. EST is a challenging alternative to conventional tissue typing which, if applied more broadly to HLA loci other than DPB1, could fundamentally change current approaches to UD searches.
Transplant-Related Mortality in Patients ≥50 Years of Age Is No Lower after Transplantation from Young Fully Matched Unrelated Donors (MUD) Than from Matched Siblings When All Patients Are Given the FLUBUP Regimen with Thymoglobulin.
Authors: Russell James A. , Chaudhry M. Ahsan , Quinlan Diana M. , Turner A. Robert , Larratt Loree M. , Geddes Michelle , Storek Jan , Savoie Mary Lynn , Brown Christopher , Bahlis Nizar J. , Stewart Douglas A. 
Published: October 2019

Abstract
There are reports indicating that older patients (pts) may have better outcomes after stem cell transplantation (SCT) from young fully matched unrelated donors (MUD) than from matched siblings (MRD) perhaps because of donor age. In some of these studies MUD and MRD recipients may have been treated differently, for example with more intense GVHD prophyaxis for MUD SCT such as antithymocyte globulin (ATG). We have compared outcomes of SCT pts receiving myeloablative fludarabine & busulfan based conditioning (FLUBUP) between 05/99 and 05/05. Only 10/10 (HLA–A, −B, C, DR & DQ) matched SCT were considered. Recipients of MRD SCT were ≥50 years old, MUD SCT recipients were unselected for age but their donors were ≤30 years old. All pts received fludarabine 50mg/m2 on days −6 to −2 and IV busulfan (Busulfex, PDL Pharma) at a myeloablative dose of 3.2 mg/kg once daily days −5 to −2 inclusive (FLUBUP) +/− TBI 200cGy x 2 on day −1 or 0. Prophylaxis for GVHD was cyclosporine A, methotrexate with folinic acid and Thymoglobulin (Genzyme) 4.5 mg/kg in divided doses over 3 consecutive days pretransplant finishing D0.[table 1]These data demonstrate that TRM for older SCT pts given the FLUBUP regimen with ATG is similar to that for adults in general transplanted from young MUD. When using these protocols in older pts there is usually no justification for the expense and inconvenience of using a young MUD in preference to a matched sibling.
Patient, donor & SCT characteristics and outcomes Unrelated Related p * at 5 years Number 41 63 Patient age median (range) 44 (16–61) 55 (50–65) <0.0001 Donor age median (range) 24 (19–30) 52 (37–71) <0.0001 Low risk (Acute leukemia CR1/2, CML CP1) 22 (54%) 14 (22%) 0.0015 CMV+ve recipient or donor 31 (76%) 53 (84%) ns Female to male SCT 8 (20%) 18 (29%) ns Blood cells 20 (49%) 56 (89%) <0.0001 CD 34+ cell dose x 10e6 median (range) 5.35 (0.91–15.47) 4.23 (0.83–13.52) 0.08 TBI (not TRM risk factor) 19 (46%) 10 (16%) 0.0014 Acute GVHD II–IV 21±7% 16±5% ns Acute GVHD III–IV 10±5% 5±3% ns Chronic GVHD 64±7% 61±9% ns Pts≥50 TRM * 26±15% (n = 12) 16±5% ns Low risk TRM 5±5% 0% ns High risk TRM 31±12% 23±7% ns BCT TRM 10±7% 19±6% ns All pts TRM 16±6% 16±5% ns
TNFR25 Expression on CD4+CD25+ T Cells: Down Modulation of Regulatory Activity.
Authors: Deyev Vadim , Roskos Melinda , Levy Robert B. , Podack Eckhard R. 
Published: October 2019

Abstract
TNFR25 (“DR3”) is a member of the TNF receptor family that is expressed by activated CD4+ and CD8+ T cells. To determine if activated CD4+CD25+ T cells also expressed this TNFR family molecule, B6 CD4+CD25+ T cells were stimulated with anti-CD3/CD28 coated beads (kind gift of Dr. B. Blazar, U. Minn.) and expanded for 3–4 days. TNFR25 expression was readily detected on CD4+CD25+ FoxP3+ T cells. Since other members of the TNF receptor family (GITR, OX40, 4–1BB) are known to influence T regulatory cell function, we investigated whether TNFR25 signaling can regulate CD4+CD25+ T cell activity. TNFR25 triggering in B6-wt T regulatory CD4+CD25+ cells with the recombinant TNFR25 ligand TL1A or agonistic anti-TNFR25 antibody (4C12) resulted in reduction of their ability to suppress anti-CD3 induced ex-vivo proliferation of CD4+CD25− cells. 4C12 mediated TNFR25 signaling also reduced B6-wt Treg mediated inhibition of peptide induced proliferation of OVA-specific B6 CD8+ (OT-I) cells. To further investigate a role for TNFR25 in Treg cell regulation, TNFR25 (full length) transgenic mice were generated and bred onto the BL/6 background. CD4+CD25+ cells from these TNFR25 tg mice were found to possess diminished T regulatory activity in vitro as determined by their diminished inability to regulate proliferation by B6-wt CD4+ and OT-I CD8+ T cells. To assess their in vivo regulatory activity, B6-wt and B6 TNFR25 tg Treg cells were examined for their ability to inhibit graft vs. host disease (GVHD) following allogeneic MHC class I/II mismatched BMT. In contrast to B6-wt Treg cells, TNFR25 tg Treg cells exhibited significantly diminished ability to regulate the onset of GVHD in vivo as assessed by weight loss and clinical symptoms. Using agonistic antibody, stimulation of TNFR25 on transgenic Treg cells was also found to effectively remove the ex-vivo regulatory activity expressed by this population. To exclude any possible direct co-stimulatory effects of 4C12 antibody on the responding proliferating cells, CD4+CD25−T cells from TNFR25 dominant negative transgenic mice were employed. 4C12 mab again abolished Treg cell inhibitory activity. The effect of TNFR25 agonists on T reg cell activity in vivo is being further investigated in both mouse models of GVHD and IBD diseases. Initial observations administering 4C12 post-allogeneic BMT together with B6-wt Treg cells indicate a reduction in their ability to regulate GVHD. In total, these studies identify TNFR25 as a new potential target for augmenting CD4+ and CD8+ responses by concomitant direct co-stimulation of effecter cells and inhibition of T regulatory cell function.
Busulfan Selectively Induces Cellular Senescence Via a p53-Independent but Erk-p38 MAPK-Dependent Mechanism.
Authors: Probin Virginia , Bai Aiping , Zhou Daohong , Wang Yong 
Published: October 2019

Abstract
Busulfan (BU), an alkylating agent, has been used extensively for the depletion of leukemia cells and normal hematopoietic stem cells (HSCs) prior to bone marrow transplantation. However, its mechanism(s) of action is unknown. Our laboratory has previously shown that BU primarily depletes HSCs by induction of senescence, but not apoptosis. In the present study, we investigated the molecular mechanisms whereby BU induces cellular senescence utilizing WI38 human diploid fibroblasts as a model system. We found that WI38 fibroblasts incubated with BU (from 7.5 to 120μM) for 24 h underwent senescence but not apoptosis in a dose-independent manner, whereas cells incubated with 80μM and 20μM etoposide (Etop) committed to apoptosis and senescence, respectively. The induction of WI38 cell senescence by Etop was associated with p53 activation and could be attenuated by down-regulation of p53 using a-PFT or p53 siRNA. In contrast, WI38 cell senescence induced by BU was associated with prolonged activation of Erk, p38 and JNK, and could be suppressed by the inhibition of Erk and p38 MAPKs with PD98059 and SB203580, respectively. Upon release from Erk and p38 inhibition, BU-treated cells proceeded to DNA synthesis and cell division. However, inhibition of p53 with a-PFT or p53 siRNA, or JNK with SP600125, failed to protect WI38 cells from BU-induced senescence. These findings suggest that BU is a distinctive chemotherapeutic agent that can selectively induce cellular senescence through the Erk and p38 MAPK pathways.
Generation and Functional Characterization of T Cells Against Candida albicans as Potential Immunotherapy after Allogeneic Stem Cell Transplantation.
Authors: Lehrnbecher Thomas , Beck Olaf , Koehl Ulrike , Roeger Frauke , Hunfeld Klaus-Peter , Latge Jean Paul , Sarfati Jaqueline , Klingebiel Thomas , Tramsen Lars 
Published: October 2019

Abstract
Invasive fungal infections (IFI), in particular infections due to Aspergillus spp and Candida spp, still pose considerable problems in patients undergoing allogeneic stem cell transplantation (SCT). Despite the availability of new antifungal agents, morbidity and mortality of IFI are still unacceptable high. Although neutropenia is known as the single most important risk factor for IFI, there is a growing body of evidence that T cells play a major role in the defense against fungi. Therefore, adoptive immunotherapy with T cells against Candida spp. might be an interesting therapeutic option in patients undergoing allogeneic SCT. After overnight incubation of 1×108 peripheral blood mononuclear cells from 4 healthy individuals with cellular extracts of C.albicans, activated T cells were selected using the IFN-γ secretion-assay (Miltenyi Biotec, Bergisch Gladbach, Germany). After 14 days of culture, T cell clones were generated by limiting dilution and incubated for another 14 days. The median number of cells obtained was 2.6×107 (range, 0.85–5.75×107). Flow cytometry revealed a highly homogenous population of CD3+CD4+ cells (97.2% ± 2.6; n=6), of which an average of 8.6% (range, 4.8–58.2%) produced IFN-gamma on re-stimulation with C.albicans antigens, as assessed by intracellular cytokine staining assay. 20.5% (range, 5.8–72.4%) of the generated cells produced TNF-alpha, whereas no significant number of cells produced TH2 cytokines such as IL-4 and IL-10, indicating that the generated T cell clones were TH1 cells. The percentage of IFN-gamma producing T cells was significant upon stimulation with C.albicans and C.tropicalis, whereas less than 1% of cells produced IFN-gamma upon stimulation with antigens of other yeasts such as C.glabrata, Debaryomyces hansenii and Kluyveromyces lactis and molds such as A.fumigatus, Penicillium chrysogenum and Alternaria alternata. Compared to CD4+ T cells of the original fraction, the isolated and expanded anti-Candida T cells showed reduced alloreactivity, as assessed by means of CSFE. In addition, a strong proliferation of the generated anti-Candida T cells was seen after re-stimulation with C.albicans antigens. The potency of the generated T-cells to damage C.albicans was evaluated using the XTT assay. Compared to polymorphonucelar cells (PMNs), APCs and T-cells alone or to the combination of PMNs with T cells or APCs, respectively, the combination of PMNs, APCs and T-cells showed highest fungal damage (n=4).
In conclusion, our data suggest that the isolation and expansion of anti-Candida T cells is possible and feasible. The generated T cells show low alloreactivity in vitro and increase the antimycotic potential of phagocytes. Thus, antimycotic T cells might become an important tool in the prophylaxis and therapy of IFI in patients after allogeneic SCT.
Association of TLR-4 Mutations in Host and Donor Cells and the Risk for Acute GVHD after Allogeneic Hematopoietic Stem Cell Transplantation.
Authors: Imado Takehito , Iwasaki Tsuyoshi , Kuroiwa Takanori , Kitano Sachie , Sano Hajime , Ogawa Hiroyasu 
Published: October 2019

Abstract
Lipopolysaccharide (LPS) has been implicated in the pathogenesis of acute graft-versus-host disease (GVHD). The toll-like receptor (TLR)-4 has been recently identified as a major receptor for LPS. It is possible that mutations of TLR-4 have been associated with LPS hyporesponsiveness, thereby reducing the risk of acute GVHD in allogeneic hematopoietic stem cell transplantation (HSCT). However, stimulation of TLRs may also initiates a signaling cascade for induction of various pro-inflammatory cytokines, leading to microbial eradication. Therefore, mutations of TLR-4 may increase risk of microbial infection, thereby accelerating acute GVHD in allogeneic HSCT recipients. Retrospective studies to determine the frequency of TLR-4 mutations and their possible association with acute GVHD contradict each other. In the present study, we examined the association of TLR-4 mutations in host and donor cells and the risk for acute GVHD after allogeneic HSCT. To examine the role of TLR-4 in host cells, we analyzed TLR-4 expression in the intestine by immunohistochemistry. Lethally irradiated C3H/HeN mice received allogeneic HSCT from C57BL/6 donors. TLR-4 expression in the intestinal epithelial cells was significantly increased after GVHD induction. Next, we used a mouse strain (C3H/HeJ) that has a single amino acid substitution in its TLR-4 as recipients in an allogeneic HSCT. Lethally irradiated either C3H/HeN or C3H/HeJ hosts received HSCT from C57BL/6 donors. C3H/HeN recipients receiving C57BL/6 donors developed significantly less GVHD as measured by mortality and intestinal histopathology compared with C3H/HeJ recipients receiving C57BL/6 donors. TNF-a mRNA expression in C3H/HeJ recipients was stronger in the intestine but weaker in the spleen and the liver comparing to that in C3H/HeN recipients. These results suggest that TLR-4 mutation in host cells reduces the expression of pro-inflammatory cytokines in the spleen and the liver, while it also reduces microbial eradication in the intestine, increasing the risk of LPS stimulation of macrophages in the intestine, thereby augmenting TNF-a expression and damages of the intestine. Next, we examined the role of TLR-4 in donor cells. Lethally irradiated C57BL/6 hosts received allogeneic HSCT from either C3H/HeN or C3H/HeJ donors. Mice receiving C3H/HeJ donors developed significantly less GVHD as measured by mortality and intestinal histopathology compared with mice receiving C3H/HeN donors. Taken together, these results suggest that TLR-4 in host cells is crucial in the microbial eradication in the intestine and the protection of intestinal GVHD, while responsiveness of donor cells to LPS may be an important risk factor for acute GVHD.
Effect of Aging on Host Dendritic Cell (DC) Numbers and Function, and Response to Regulatory DC Treatment in a Murine Allogeneic Transplant Model.
Authors: Schlueter Annette J. , Park Joon Seong , Wilke Werner W. , Fan Ji , Gingrich Roger D. 
Published: October 2019

Abstract
Graft versus host disease (GVH) incidence and severity increases with older patient age. Host dendritic cells (DC) are responsible for initiating GVH, presumably as a result of activation at the time of transplant conditioning (as they are short lived after transplantation). Age-related alterations in host DC function have not been studied in a transplant setting. In this study, young (8–14 wk) and older (12–16 mo) mice were treated with the conditioning regimen of lethal irradiation (1100 cGy) and assessed at 6 h later for changes in DC costimulatory molecule expression and cytokine secretion. Older mice had slightly higher numbers of splenic classical DC (cDC; CD11chi Cl II+ B220−) and plasmacytoid DC (pDC; CD11clo Cl IIlo B220+) both before and after irradiation. No differences were observed in CD40, CD80, or CD86 expression between young and older mice, either before or after irradiation. IL-12, IFNγ, and TNFα producing cDC were more numerous in young untreated mice than in older mice. However, these cells decreased in young mice and increased in older mice after irradiation, resulting in generally higher numbers of cells producing these cytokines in older mice. IL-12, IFNγ, and TNFα producing pDC were more frequent in older mice than in young mice prior to irradiation, and both older and young mice showed increased frequency of cells producing these cytokines after irradiation. Overall, the highest numbers of cDC and pDC producing IL-12, IFNγ, and TNFα were present in older mice after irradiation. In both cDC and pDC populations, older mice had a higher frequency of IL-10+ cells prior to irradiation. After irradiation, young mice had a higher frequency of IL-10+ cells. These findings indicate that the balance of cytokine production in older mice after conditioning with irradiation is shifted to immunostimulatory cytokines rather than immunosuppressive cytokines. Murine GVH can be prevented by treatment with regulatory DC (rDC) in young animals (Sato et al., Immunity 2003). To determine whether this treatment is feasible in older animals, older BALB/c mice were transplanted with young C57Bl/6 BM. A subset of each group received age-matched host derived rDC. As expected, among mice that did not receive rDC, old recipients got more rapid and severe GVH than young recipients. This correlated with an increased frequency of residual host DC at the time of GVH in the older group. rDC were used to successfully treat GVH in older (as well as young) transplant recipients. These results provide the foundation for a cell based therapeutic approach to the treatment of GVH in older individuals.
Induction of Anti-Cancer Effects by Targeting Alloreactive Intentionally Mismatched T Cells and NK Cells to Tumor Cells Using Bi-Specific Trifunctional Antibodies.
Authors: Morecki Shoshana , Lindhofer Horst , Yacovlev Elena , Gelfand Yael , Slavin Shimon 
Published: October 2019

Abstract
Engraftment of donor stem cells induces host vs graft unresponsiveness thus allowing durable engraftment of donor lymphocytes which can induce graft vs malignancy (GVM) effects following transplantation of MHC compatible donor stem cells. Unfortunately, graft vs host disease (GVHD) frequently results in major toxicity and mortality and limits the clinical efficacy of cell therapy. Although the intensity of GVM effects can be improved by using mismatched donors GVM effects cannot be induced in recipients of haploidentically mismatched allografts because of anticipated lethal GVHD. We have attempted to induce GVM effects by intentionally mismatched donor lymphocytes by targeting donor derived T cells and NK cells activated by rIL-2 to cancer cells, in order to improve the efficacy of the GVL effects on the one hand and prevent or minimize GVHD by targeting alloreactive cells to the tumor cells that need to be eliminated using bispecific tri-functional antibodies, on the other. A trifunctional bispecific antibody (BiLu) directed against murine CD3 and human epithelial-cell adhesion molecule (EpCAM), was administered concomitantly with naïve or IL-2 activated donor splenocytes to mice inoculated with a murine model of melanoma cells transfected with human EpCAM (B16-EpCAM). A total of 10/20 and 32/38 mice treated with BiLu and naïve or IL-2 activated donor splenocytes, respectively, were tumor-free survivors without GVHD for >250 days following tumor inoculation. Out of 28 of the disease free survivors (DFS) without GVHD, treated with IL-2 activated splenocytes and BiLu, 24 mice were resistant to a second tumor inoculum (104) given >250 days following the first tumor inoculation. In contrast, only 8 of the 18 DFS mice treated with naïve splenocytes and BiLu, and 5 of the 10 DFS controls treated with BiLu only, resisted the second tumor challenge. Induction of anti-tumor immunity by IL-2 activated cells and BiLu was more efficient and long-lasting in mice previously injected with a lethal first tumor cell dose (5×104) than in mice inoculated with a sub-lethal tumor cell dose (5×103). Interestingly, a similar infusion of donor cells labeled with bi-specific anti-EpCAM and anti-CD3 following allogeneic stem cell transplantation for metastatic breast cancer did not result in augmentation of GVHD. In conclusion, concomitant inoculation of alloreactive donor lymphocytes and trifunctional bispecific antibody can be effective for targeting of killer T and NK cells to tumor cells while avoiding GVHD, as well as for induction of long-lasting anti-cancer immunity, most likely due to presentation of tumor antigen to T cells by antigen presenting cells (dendritic cells) expressing Fc receptor binding to the Fc portion of the bi-specific antibody.
Six-Year Follow-Up of Inhibitor Prone Hemophilia B Dogs Treated with Muscle and Liver-Directed AAV2 Mediated Factor IX Gene Therapy.
Authors: Niemeyer Glenn P. , Herzog Roland W. , Mount Jane D. , Arruda Valder , Tillson Michael , Hathcock John , High Katherine A. , Lothrop Clinton D. 
Published: October 2019

Abstract
Hemophilia B, caused by factor IX (FIX) deficiency, is intensively studied as a model for gene therapy. Preclinical studies in mice, dogs and initial clinical trials have documented the feasibility of adeno-associated virus (AAV) mediated gene therapy for hemophilia B. The purpose of this study is to report 6 year follow-up of inhibitor prone hemophilia B dogs treated with muscle or liver directed AAV2 FIX gene therapy. Short term, one year follow-up of these dogs (n=6) has been previously reported. The dogs treated with liver directed AAV2 FIX have not had a single bleed requiring FIX replacement in the 6 years since vector administration. The dogs undergoing muscle directed gene therapy have continued to have a bleed frequency similar to untreated FIX deficient dogs. The whole blood clotting time (WBCT), activated clotting time (ACT), and activated partial thromboplastin time (APTT) have remained at the upper limits of the normal ranges for the six year follow-up in the two liver treated dogs. The FIX activity has remained stable between 4–10% in both liver treated dogs but undetectable in the dogs undergoing muscle directed therapy. The vector/FIX sequences have persisted in liver biopsies but were undetectable in WBC and sperm DNA. Administration of purified canine FIX protein resulted in an immune response to FIX in the muscle treated dogs but not the dogs treated with liver directed gene therapy, suggesting FIX tolerance had been established in the hemophilia B dogs with liver directed gene therapy. A complete clinical evaluation of the dogs undergoing liver directed gene therapy including CBC, serum chemistries, bile acid profile, hepatic MRI and CT scans and liver biopsy was normal with no evidence for tumor formation. These results demonstrate that AAV mediated liver directed gene therapy completely corrects the hemophilia phenotype without toxicity or inhibitor development in the inhibitor prone null mutation dogs for more than six years.