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Qualificação das unidades de SCUP criopreservadas no banco de sangue de cordão umbilical e placentário da Fundação HEMOPE no período de dezembro de 2014 a junho de 2017 / Qualification of cryopreserved SCUP units in the umbilical cord and placental blood bank of the HEMOPE Foundation from December 2014 to June 2017Costa, Ana Maria do Nascimento 07 November 2018 (has links)
Qualificar as primeiras unidades de Sangue de Cordão Umbilical e Placentá- rio (SCUP) criopreservadas no Banco de Sangue Umbilical e placentário da Fundação de Hematologia e Hemoterapia de Pernambuco (BSCUP/HEMOPE), no período de dezembro de 2014 a junho de 2017. Justificativa: O sangue do cordão é rico em células progenitoras hematopoéticas (CPH), utilizado para transplante e tratamento de patologias benignas e malignas. Avaliar as primeiras unidades coletadas no BSCUP/ HEMOPE será de grande valia para sua caracterização, planejamento de ações de melhoria do processo, contribuindo para o aumento da eficácia dos transplantes. Métodos: Foram triadas gestantes sem histórico de diagnóstico para doenças transmissíveis pelo sangue, com gestação terminada em recém-nascido (RN) vivo; caracterizadas através das variáveis sociodemográficas: idade, idade gestacional, antecedentes obstétricos e cor da pele dos RN foram caracterizados pelas variáveis explicativas: Gênero e Peso; as amostras das mães foram coletadas no dia do parto ou em até 48h após. O tempo entre o término da coleta e o início da criopreservação da unidade das CPH não excedeu 48 horas. As unidades de SCUP foram avaliadas quanto ao volume, total de células nucleadas (TCN), quantificação de células CD 34+, contagem de eritroblastos viabilidade celular e contaminação microbiológica. Neste estudo foi avaliada a variável \"unidades adequadas ao uso em relação ao peso do receptor, tomando com o base o TCN e o peso de um provável receptor, assim categorizados: unidades <12,5 x10e8 para peso 12,5 para peso >50kg; Resultados: Volume Inicial (ml):(min.50,0; max.166,80); Volume final(mL): (min.19,07;max.21,75); TCN pré (x108): (min.6,0;max.27,80);TCN pós(x108): (min.5,0; max.22,6); Recuperação Celular (%): (min.0,67; max.39,50); CD34+(x106):(min.0,67;max.39,50);Viabilidade(%): (min.71,35max.100); Conclusão: Das 113 unidades armazenadas, 89 (78,76%) atendem a receptores < 50kg (crianças) e 24 (21,24%) atendem a receptores >50k (adultos). O inventário apresentou resultados em conformidades com o especificado na legislação vigente, portanto, qualificadas para atender à demanda transfusional de transplantes de CPH. / To qualify the first units of cryopreserved Umbilical Cord and Placental Blood (SCUP) in the Umbilical and Placental Blood Bank of the Hematology and Hemotherapy Foundation of the State of Pernambuco (BSCUP / HEMOPE), from December 2014 to June 2017 Rationale: Cord blood is rich in hematopoietic progenitor cells (HPC), used for transplantation and treatment of benign and malignant pathologies. Evaluating the first units collected in the BSCUP / HEMOPE will be of great value for its characterization, planning of actions to improve the process, contributing to increase the efficiency of transplants. Methods: Pregnant women with no history of diagnosis for blood-borne diseases were screened, with gestation terminated in live newborn (NB); characterized by the sociodemographic variables: age, gestational age, obstetric history and skin color, NB were characterized by the explanatory variables: Gender and Weight; the samples of the mothers were collected on the day of delivery or within 48 hours after delivery. The time between the end of the collection and the beginning of the cryopreservation of the MHC unit did not exceed 48 hours. SCUP units were evaluated for volume, total nucleated cells (TNC), CD34 + cell count, cell viability erythroblast counts, and microbiological contamination. In this study, the variable \"units suitable for use in relation to the weight of the receptor\" was evaluated, taking as base the TNC and the weight of a probable receiver, as follows: units <12.5 x10e8 for weight 12 , 5 for weight> 50 kg; Results: Initial volume (ml): (min.50.0, max.166.80); Final volume (mL): (min.19.07, max.21.75); TCN pre (x108): (min.6,0; max.27,80); TCN post (x108): (min.5,0; max.22,6); Cellular Recovery (%): (min.0.67, max.39,50); CD34 + (x106): (min.0.67, max.39.50); Viability (%): (min.71,35max.100); : Of the 113 stored units, 89 (78.76%) attend to receptors <50kg (children) and 24 (21.24%) attend receptors> 50k (adults). Conclusion: The inventory presented results in compliance with that specified in current legislation, therefore, qualified to meet the transfusional demand for HPC transplants.
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The role of Notch and GATA3 in postnatal and adult haematopoiesisDuarte, Sara January 2011 (has links)
The role of Notch in cell fate determination and lineage restriction in the bone marrow (BM) is controversial in the field. Recent studies have convincingly shown that Notch is dispensable for haematopoietic stem cell (HSC) regulation in adult haematopoiesis (Maillard et al., 2008). In contrast, Notch signaling has been proposed to be of importance in the regulation of BM megakaryocyte progenitor differentiation, based on dominant negative genetic approaches, identifying a potentially distinct role for Notch in adult BM haematopoiesis (Mercher et al., 2008). Here, I found that by selectively ablating the gene coding the transcription factor recombination signal-binding protein J kappa (RBP-Jk), to which all canonical Notch signaling converges, canonical Notch signaling does not mediate HSC maintenance, neither in steady state nor in conditions of stress. Furthermore, I propose, in contrast with previous studies (Mercher et al., 2008), that canonical Notch signaling plays no role in myeloerythropoiesis cell lineage commitment in the BM. My data also show that key Notch target genes are suppressed by RBP-Jk, as their expression is unaffected in Notch1-deficient BM progenitors, while target genes are upregulated in Rbp-Jk-deleted megakaryocyte and erythroid progenitors. This establishes for the first time in mammalian cells in vivo, that Notch target genes are kept in a suppressed state by RBP-Jk, potentially restricting T cell commitment to the thymus and not to the BM, at the expense of myeloerythropoiesis. Notch signaling and GATA3 are two master regulators in T cell commitment (Han et al., 2002; Ho et al., 2009; Pui et al., 1999; Radtke et al., 1999; Zhu et al., 2004). However, although very well established as being involved in the thymic stages of T cell restriction, there is little evidence of Notch and GATA3 being involved in the migration of a thymus settling progenitor (TSP) from the BM to the thymus or in the establishment of the earliest thymic progenitor (ETP) in the thymus. From this thesis work, I conclude that Notch signaling is essential for the emergence of ETPs in the thymus in a NOTCH1-independent manner. Moreover, I demonstrate, as supported by a very recent published study (Hosoya et al., 2009), that GATA3 is important for the development of the earliest T cell progenitor. GATA1 and GATA2 mediate haematopoietic stem cell maintenance in the BM. GATA1 is required for erythropoiesis, megakaryocytes and eosinophils while GATA2 is important for the proliferation and survival of HSCs. In contrast, a role for GATA3 in the BM has never been established. By using a Gata3-conditional knockout mouse model, I demonstrate that GATA3 is dispensable for HSC maintenance in steady state and following active haematopoietic regeneration as well as for HSC self-renewal in the BM.
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A study of regulatory T cells in allogeneic haematopoietic stem cell transplantationDanby, Robert David January 2012 (has links)
Allogeneic haematopoietic stem cell transplantation (alloHSCT) is an established therapy for many haematological disorders. Unfortunately, the new donor-derived immune system may damage host cells (graft-versus-host disease (GvHD)), causing significant morbidity and mortality. Since regulatory T cells (Tregs) can modulate immune responses, it was hypothesised that Treg numbers in the haematopoietic stem cell grafts and/or peripheral blood may influence the development of GvHD and other transplant-related complications. In this project, a prospective observational clinical study of putative Tregs in human alloHSCT was performed in Oxford. Flow cytometry and methylation-specific qPCR assays were developed to quantify putative Tregs and lymphocyte populations within the grafts and post-transplant blood samples. Although low CD4(+)CD25(+)FOXP3(+)CD127(-/dim) T-cell numbers were not associated with increased incidence of GvHD, low proportions of CD25(+)FOXP3(+)CD127(-/dim) cells in the graft (as a percentage of total CD4(+) T cells) were independently associated with poor engraftment, increased non-relapse mortality and inferior overall survival. Similarly, falling CD4(+)CD25(+)FOXP3(+)CD127(-/dim) T-cell counts over the first three months post-transplant were associated with higher non-relapse mortality and inferior overall survival. In view of these novel findings, strategies that increase CD4(+)CD25(+)FOXP3(+)CD127(-/dim) T cells in alloHSCT may improve clinical outcomes. One possible route for increasing Tregs is through cellular therapy. This project therefore tested the hypothesis that CD4(+)CD25(+)FOXP3(+) Tregs can be produced in vitro from conventional CD4(+) T cells. In the presence of TGFβ and Azacitidine, FOXP3 was expressed in the majority of activated CD4(+) T cells. These cells also had a demethylated FOXP3 TSDR enhancer which is specific to natural Tregs. However, most of these cells produced pro-inflammatory cytokines, for example, TNFα. Therefore, under these conditions, FOXP3 expression was not sufficient to produce a Treg phenotype. It is proposed that current focus for generating Tregs for human clinical trials should be directed towards improving isolation and expansion of ex vivo isolated Tregs.
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The mechanism of Nov (CCN3) function in haematopoiesisGuo, Yanping January 2012 (has links)
Haematopoietic stem cells (HSC) are strictly regulated by intrinsic regulators and extrinsic signals from the microenvironment. Nov (CCN3), a matricellular protein of the CCN family, has been reported as a suppressor gene in solid tumours and chronic myeloid leukaemia (CML). Recent study identified Nov as a positive regulator in human cord blood CD34+ stem cells. However, the functions of Nov in haematopoiesis and adult HSC remain largely unknown.
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Genotypová analýza lidského cytomegaloviru u pacientů po allogenní transplantaci kmenových buněk krvetvorby. / Genotypic analysis of human cytomegalovirus in the patients after allogeneic haematopoietic stem cell transplatation.Javornická, Tereza January 2014 (has links)
In patients after allogeneic haematopoietic stem cell transplantation (HSCT) is a human cytomegalovirus (CMV) one of the most important viral pathogens. Its detailed characteristic could provide information about the impact of each CMV genotype on overall survival of the patient, and some serious complications, such as graft versus host disease (GvHD). This thesis deals with retrospective genetic analysis of samples from 1877 patients transplanted at the Clinic of Pediatric Hematology and Oncology, University Hospital Motol and the Institute of Hematology and Blood Transfusion since 2002. DNA from biological samples (especially whole blood) was isolated kit Qiagen DNA Blood Mini or Qiagen DNA Mini and samples were prospectively detected presence of CMV DNA. Samples were subsequently stored at -20 řC. Genotyping was performed using real-time PCR technologies to the genes of 2 structural proteins glycoprotein B, glycoprotein H and using sequence specific primers and probes. In 1343 samples (71.6%) from 390 patients there was only one strain of CMV; in 256 (13.6%) samples from 113 patients have detected mixed infection caused by two or more strains of CMV. The most common genotype demonstrated in "single" infection was in pediatric and adult patients gB1/gH2 detected in 118 (28.4%) patients. Most...
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Effects of IL-2,IL-6,IL-7 and IFN on the proliferation,survival,induction and reduction of spontaneous in-vitro apoptosis of B CLL cellsSeahloli, Michael Sello 14 February 2007 (has links)
Student Number : 9708297R -
MSc (Med) dissertation -
School of Medicine -
Faculty of Health Sciences / B chronic lymphocytic leukaemia (B-CLL) is a monoclonal haematopoietic disorder with expansion of small lymphocytes of B-cells. B-CLL cells accumulate in blood, bone marrow, lymph nodes and spleen, resulting in enlargement of these organs and decreased bone marrow function. B-CLL is the most common leukaemia, with an annual incidence of 1.8 to 3.0 per 100 000 population in the United States. It is characterised by the accumulation of long-lived monoclonal CD5+ B lymphocytes. In vivo normal B-lymphocytes derive growth factors through interactions with T-cells and monocytes. In culture however, survival and growth of activated B-cells depends on the availability of external factors such as interleukins.
B-CLL cells populations are unable to survive in culture long enough to respond to the addition of growth factors. Such factors are important for the proliferation and survival of many cell types and in the absence of cytokines, these cells die as a result of apoptosis. Chronic lymphocytic leukaemia cells are influenced in vitro by a number of exogenously added cytokines that include IFN- α, IFN-γ, IL-2, IL-4, IL-10, IL-13, IL-15, TGF- β and TNF- α.
The aim of this study was to investigate the effect of cytokines e.g., IFN, IL-2, IL-6, IL7 and IL-10 on the proliferation and survival of B-CLL cells and furthermore to compare the induction and reduction of spontaneous and induced apoptosis in vitro. Patients with B-CLL were recruited from three centres. Thirty blood samples were collected, separated using Ficoll Hypaque Gradient and purified by rosetting with AET treated SRBC. The proliferation and survival of B-CLL cells were studied in vitro in response to GM-CSF, IFN, IL-2, IL-6, IL7 and IL-10,. The survival and apoptosis of B-CLL cells in cultures with or without interleukins and other growth factors were studied under microscopic examinations and DNA agarose gel electrophoresis.
It was observed in B-CLL cells cultures that IFN and IL-2 enhanced proliferation significantly. IL6, IL-7 and GM-CSF also enhanced proliferation of B-CLL cells but not to the greater extent than IL2 and IFN. IL-10 inhibited proliferation of B-CLL cells when compared to controls. In a long-term (5-day) culture, survival of B-CLL cells was greatly enhanced by IFN and followed by IL-2. Therefore it appeared that IFN and IL-2 are the two most potent growth factors tested in this study to promote B-CLL cells proliferation and survival. The combination of these mitogens did not further enhanced proliferation. IL-6 and GM-CSF enhanced proliferation and survival of B-CLL cells. IL-7 promoted proliferation but had no effect on survival of B-CLL cells in-vitro. IL-10 enhanced apoptosis and did not promote survival of B-CLL cells in-vitro.
IFN and IL2 are survival and promoting growth factors for B-CLL cells in culture. In contrast, IL-10 has demonstrated to induce apoptotic cell death of B-CLL cells. In conclusion B-CLL cells proliferated equally well with IFN and IL-2. IL-6, IL-7 and GM-CSF had a much lower proliferation and survival effect with noticeable antiapototic activity when compared to IFN and IL-2. IL-7 was found not to promote survival of B-CLL cells and IL-10 enhanced cell death by apoptosis.
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Molecular regulation and function of Gata2 in the programming of haemogenic endotheliumDobrzycki, Tomasz January 2017 (has links)
Haematopoietic stem cells (HSCs) maintain the vertebrate blood system throughout life. Exploiting their clinical potential requires a thorough understanding of the natural origins of the HSCs. They first arise from the haemogenic endothelium (HE), located in the main embryonic artery, the dorsal aorta. Our understanding of the genetic mechanisms underlying HE specification remains incomplete, but one of the crucial transcription factors is Gata2. We found that a conserved enhancer of zebrafish gata2a gene (i4 enhancer) is active in vivo specifically in endothelial cells, including the HE. To unravel the function of gata2a in specifying the HSCs, we have targeted the i4 enhancer with CRISPR/Cas9, generating the first reported genomic deletion of an endogenous cis-regulatory region in zebrafish. Deletion of the i4 enhancer leads to a decrease in endothelial gata2a expression and a concomitant transient decrease in the number of HSCs. This is marked by an early decrease in the expression of gata2b, a gata2a paralogue previously shown to be required for the initiation of the haematopoietic programme. Our results suggest non-redundant roles of both zebrafish gata2 paralogues in programming of HSCs, providing insights into different roles of GATA2 throughout the programming of HSCs. We also confirmed the previously reported loss of HSCs upon MO-mediated knockdown of lmo4a, associated with increased gata2a expression in HE. We validated the increase in gata2a levels in TALEN-generated lmo4a mutants. To identify the links between lmo4a, gata2a and the HE programming, we have profiled the transcriptome of lmo4a-deficient endothelial cells, including the HE. Our results suggest that Lmo4a may be a global regulator of the transcriptional programming of the HE. Moreover, Wnt signalling pathway may regulate gata2a downstream of lmo4a. This provides novel insights into the gene regulatory network orchestrating the generation of HSCs in the embryo.
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Prerequisites for establishing a public human UCB SCB; assessment of public acceptance and resistance of UCB to HIVMeissner-Roloff, Madelein 26 April 2013 (has links)
South Africa is in dire need of a public umbilical cord blood stem cell bank (UCB SCB). A severe shortage of genetically compatible samples for BM transplantation precludes the majority of South Africans from receiving the relevant medical care. UCB is a viable alternative to BM but is currently disposed of post-delivery. UCB could furthermore serve as a resource of genetically compatible haematopoietic progenitor cells (HPCs) that could be used in gene therapy approaches directed towards a cure for HIV-1. Knowing whether HIV-1 affects or infects primitive HPCs is vital to determine the course of action for transplantation of UCB-derived genetically resistant HPCs. Collecting and storing UCB in a public UCB bank could thus serve as a vital resource of genetically compatible samples for BM transplantation. It was thought that the high incidence of HIV-1 in South African patients and the persistent stigma surrounding HIV-1 would be problematic for collecting sustainable numbers of UCB units and subjecting units to compulsory screening for infectious diseases. This was however, not the case. In the South African context, we are faced with unique and rich challenges relating to cultural and religious differences that are further augmented by linguistic constraints and educational insufficiencies. Nevertheless, the majority of patients within the interviewed patient cohort were supportive of the idea of establishing a public UCB SCB in SA and were willing to undergo additional HIV-1 screening. The Ultrio-Plus® assay was verified in this study for screening UCB units for HIV-1 and could be used in routine analyses of UCB units prior to banking. Conflicting results in the literature exist with regard to HIV-1’s ability to infect or affect haematopoietic progenitor cells. Results from this study revealed that HIV-1 was not only able to affect HPCs’ ability to form colonies in vitro, but was also capable of infecting CD34+ HPCs in some individuals. These results substantiate the theory that some CD34+ HPCs serve as viral reservoirs which could account for residual viraemia in patients on antiretroviral therapy. Results suggest that allogeneic transplantation of HIV-1 infected individuals with UCB-derived, genetically modified HPCs, should be pursued. / Thesis (PhD)--University of Pretoria, 2012. / Immunology / unrestricted
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Decellularised extracellular matrices as instructive microenvironments for bone marrow derived stem cellsPrewitz, Marina 19 December 2011 (has links)
The regenerative potential of adult stem cell populations within the human body bears great promises for their use in regenerative medicine. The bone marrow (BM) harbours two different types of adult stem cells, haematopoietic stem and progneitor cells (HSPCs) and multipotent mesenchymal stromal cells (MSCs), which are tightly regulated in their distinct anatomically defined niches by multiple cues such as cytokines, cell-cell contacts, the extracellular matrix (ECM) and the physical microenvironment. The ex vivo expansion of these cells for applications in regenerative therapies is of great interest and several biomaterial approaches attempt to mimic the natural BM niche and its components to control stem cell maintenance and differentiation. However, as of now the complexity of such stem cell niches is hard to recapitulate. Towards this goal, this work was focussing on the ECM environment of BM stem cells and was set out to engineer improved in vitro culture systems. MSC themselves are one of the most important cell types within the BM that secrete and construct ECM-networks and thereby shape the microenvironment of the residing cells. The potential of primary human BM-MSC to secrete ECM in vitro has been exploited to generate niche-like ECM surrogates in a robust and versatile format. Application of decellularisation regimes allowed the fabrication of complex matrices which demonstrated suprastructural, compositional and physicochemical properties compareable to those of the native BM-ECM environment. Reliable stability and reproduciblity was achieved by a dedicated procedure of maleic anhydride co-polymer-mediated covalent binding of fibronectin and subsequent anchorage of cell-secreted ECM molecules. As a result of the high reproducibility, a complete proteomic register of ECM molecules was obtained in combination with determining the complex fibrillar and soft gel-like characteristics of MSC-derived matrices. Based on the established BM niche-like substrate, the impact of extracellular matrices on MSC and HSPC ex vivo behavior has been explored. Both cell types demonstrated strong adhesion to ECM substrates and depicted a changed cellular morphology upon contact with native ECM structures compared to standard culture substrates or simple ECM protein coatings, indicating an intense interplay between the cell and the microenvironment. MSC that re-grew into their own matrices have shown advantageous proliferation and cytokine secretion levels as well as enhanced differentiation intensity (upon differentiation induction) compared to MSC that were cultured on less complex substrates. Similarly, HSPC were also instructed for enhanced expansion on MSC-derived matrices without exhaustion of stem cell-marker expressing progenitor cells. The efficiency of these matrices was related to their ability to mimic the native composite suprastructure, ligand nano-topography, molecular composition and physical properties of natural BM ECM environments. The data obtained within this thesis set the ground for a more rational design of artificial stem cell niches with defined and distinct properties, offering exciting options for the in-depth analysis and understanding of stem cell regulation by exogenous cues.
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L'interleukine-22 dans la maladie du greffon contre l'hôte après allogreffe de cellules souches hématopoïétiques / Interleukine-22 in graft-versus-host disease after allogeneic stem cell transplantationLamarthee, Baptiste 28 October 2014 (has links)
La maladie du greffon contre l’hôte (GVHD) reste la complication majeure de l’allogreffe de cellules soucheshématopoïétiques (allo-CSH). La GVHD résulte de l’activation de la réponse immunitaire et de la reconnaissanced’alloantigènes par les lymphocytes T (LT) du donneur, entrainant ainsi des lésions tissulaires principalement auniveau de la peau, des intestins et du foie. L’interleukine-22 (IL-22) est une cytokine sécrétée par les LT Th1,Th17 et les cellules de l’immunité innée (ILC). Compte tenu des propriétés de l’IL-22 dans les tissus cibles de laGVHD, nous avons évalué sa contribution dans la physiopathologie de la maladie à l’aide de modèlesexpérimentaux murins. Il apparaît que les souris qui reçoivent des lymphocytes T invalidés pour l’IL-22développent une maladie moins sévère, et leur mortalité est diminuée. L’IL-22 issue du greffon participe donc à lasévérité de la GVHD en favorisant l’inflammation systémique, mais aussi locale au niveau des organes cibles. Deplus, dans les intestins, l’IL-22 agit en synergie avec les interférons de type I pour amplifier l’inflammation de typeTh1 au cours de la GVHD. Chez l’homme, la GVHD est associée à une modification du microbiote intestinal.Nous avons montré que l’absence d’IL-22 semble favoriser la colonisation de lactobacilles au détriment declostridiums, ce qui pourrait également participer à la diminution de la GVHD intestinale. Enfin, nous avonsmontré que l’effet anti-tumoral est préservé malgré l’absence d’IL-22. Ces résultats permettent donc d’envisagerde nouvelles perspectives thérapeutiques dans le traitement de la GVHD. / Graft-versus-host disease (GVHD) is still the major complication after allogeneic stem cell transplantation. GVHDresults from the activation of the immune response and the recognition by donor T cells of alloantigens leading totissue injury, especially in skin, gut and liver. Interleukin-22 (IL-22) is a cytokine secreted by CD4+ T cells Th1 andTh17 but also by innate lymphoid cells (ILC). Given that IL-22 functions in the GVHD target tissues, weinvestigated its contribution in GVHD physiopathology using mouse experimental models. We showed that IL-22deficiency in donor cells reduced the severity of GVHD by limiting systemic and local inflammation. Moreover, inthe large intestine, IL-22 acts in synergy with type I interferon to increase Th1-like inflammation. In humans,GVHD severity is associated with microbiotal modification in the intestine. We demonstrated that IL-22 deficiencyin donor cells seems to favor lactobacillus colonization instead of clostridium. These changes of microbiotacomposition may reduce the severity of intestinal GVHD. Finally, we showed that the antitumor effect is preservedeven in absence of IL-22 donor cells. Overall, our data support the design of new clinical approaches aiming totarget IL-22 pathways in GVHD patients.
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