scholarly journals TNFα Controls the Delicate Balance between Erythropoiesis and Stem Cell Exhaustion during Inflammatory Stress

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2184-2184
Author(s):  
Amaliris Guerra ◽  
Vania Lo Presti ◽  
Ana Catarina Martins ◽  
Carlo Castruccio Castracani ◽  
Ritama Gupta ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Stem Cell ◽  
T Lymphocytes ◽  
Iron Homeostasis ◽  
Bone Marrow Failure ◽  
White Pulp ◽  
Severe Anemia ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract Anemia of Inflammation (AI) is prevalent in patients with chronic inflammatory states, such as infection, autoimmunity, or cancer. Induced expression of hepcidin by pro-inflammatory cytokines results in iron-restricted anemia. In particular, abnormally elevated levels of the cytokine Tumor Necrosis Factor-α (TNFα) is a hallmark of AI, however its contribution to the pathophysiology of AI is not well understood. In this study, we investigated the role of TNFα in the development of anemia in a TNFα knockout (TNFαKO) mouse model of AI, which is induced with a single intraperitoneal (i.p.) injection of heat-killed Brucella Abortus (BA) (Kim et al. Blood). We hypothesized that TNFαKO mice would show a less severe form of AI compared to WT animals when challenged with BA. Our results showed that WT-BA mice developed severe anemia within 2 weeks, which was resolved by 8 weeks, whereas the TNFαKO mice developed leukocytosis and an irreversible macrocytic, hyperchromic anemia. Serum analysis at 8 weeks showed that erythropoietin (EPO) and iron parameters were elevated in TNFαKO compared to WT mice, which ruled out iron-restriction as the cause for the persistent anemia. However, serum cytokine measurements of TNFαKO mice at 4 weeks showed continual elevation of interleukin (IL)-12p40 and Interferon-γ (IFNγ) compared to WT-BA controls. We hypothesized that TNFα served an anti-inflammatory role that restrained prolonged inflammation after BA, and in its absence, pro-inflammatory macrophages continuously secreted IL-12p40 levels and induced the proliferation of IFNγ secreting cells. To test if concurrent loss of IFNγ would correct the inflammatory phenotype, we crossed TNFαKO with IFNγKO mice (DKO). Indeed, IFNγKO and DKO mice challenged with BA showed complete reversal of the anemic phenotype present in WT-BA and TNFαKO-BA at 2 weeks. Additionally, serum levels of IL-12p40 were normalized by 4 weeks in IFNγKO-BA and DKO-BA compared to TNFαKO-BA mice. Flow cytometry analysis of the bone marrow (BM) and spleen (SPL) at 8 weeks showed T-lymphocytes and macrophages were markedly expanded, whereas erythrocytes and B-lymphocytes were reduced in TNFαKO-BA mice. However, only modest differences in erythrocytes, macrophage, T- and B-lymphocyte in WT-BA, IFNγKO-BA and DKO-BA in the BM and SPL were detected. Additionally, we performed immunohistochemistry using an anti-CD3 antibody in SPL and livers of BA treated mice sacrificed at 8 weeks. We found complete disorganization of the white pulp in the SPL and infiltration of T-Lymphocytes in livers of TNFαKO-BA but not in WT-BA, IFNγKO-BA or DKO-BA animals. These results led us to question if lack of TNFα skewed the BM towards T-Lymphocytes. We investigated the hematopoietic stem cell (HSC) LSK, multipotent myeloid progenitors (MPP) LK, and common lymphoid progenitor (CLP) compartments by flow cytometry. Shockingly, the TNFαKO had over a 30% increase of Lin-cKit+Sca1+ LSKs, compared to TNFαKO controls, while the Lin-cKit+Sca1- LK population was drastically reduced. By contrast, the Lin-CD217-cKit+Sca1+ CLP population was expanded by more than 40% in the TNFαKO-BA compared to TNFαKO controls. Considering this data, it is difficult to disentangle the effects of TNFα in AI in vivo from its role in regulating upstream stem and progenitor cell differentiation using a germline KO strategy. However, our observations reinforce the link between iron homeostasis and HSC self-renewal and provide a new model to study inflammation associated bone marrow failure. The TNFα-BA mice displayed severe anemia, which seems to result from persistent IFNγ elevation. A recent study identified TNFα as a major pro-survival and pro-regeneration factor for HSCs (Yamashita & Passegue, Cell Stem Cell). Other studies have shown that IFNγ restricts HSCs self-renewal (Chen et al. Blood). Our results in the TNFαKO-BA treated mice suggest that TNFα preserves balanced progenitor output by countering the action of IFNγ at the HSC level. Ongoing work aims to understand the relationship between TNFα and IFNγ in regulating HSC quiescence, self-renewal, and overall pool size. Disclosures Paulson: Forma Therapeutics: Consultancy. Rivella: Meira GTx: Consultancy; Ionis Pharmaceuticals: Consultancy.

Crispr-Cas9 Mediated Disruption of Dnmt3a in JakV617F Hematopoietic Stem Cells Accelerates Disease Phenotype and Induces Lethal Myelofibrosis

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 794-794
Author(s):  
Sebastien Jacquelin ◽  
Emma Dishington ◽  
Therese Vu ◽  
Axia Song ◽  
Matthew Heidecker ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Polycythemia Vera ◽  
Essential Thrombocythemia ◽  
Early Stage ◽  
Bone Marrow Failure ◽  
Self Renewal ◽  
Early Stage Disease ◽  
Hematopoietic Stem ◽  
Stage Disease

Abstract Myeloid malignancies arise following the sequential acquisition of somatic mutations within hematopoietic stem and progenitor cells (HSPC). JAK2V617F is commonly found in myeloproliferative neoplasms (MPN) such as polycythemia vera, essential thrombocythemia and myelofibrosis. While other mutations (e.g. TET2, DNMT3A) have been found to co-occur in MPN HSPC, it remains unclear how they impact disease biology or progression from early stage disease (i.e. polycythemia or essential thrombocythemia) to advanced stage disease such as myelofibrosis or acute myeloid leukemia. DNMT3A methylates cytosine rich DNA residues (known as CpG islands, and often found in promoters of genes) leading to transcriptional repression. DNMT3A is also recurrently mutated at relatively low frequency in polycythemia vera (5-7%) but mutations are more common in advanced MPN (approximately 15% of MF and 17% of AML, Stegelmann et al. Leukemia 2011; Abdel-Wahab et al. Leukaemia 2011). These mutations are found in the methyltransferase domain and cluster around arginine 882 (e.g. R882H), resulting in loss of DNA binding and reduced catalytic activity. We used CRISPR-Cas9 gene editing technology to disrupt Dnmt3a function in mouse HSPC and assessed for cooperativity together with a conditional, knockin Jak2V617F allele. Jak2V617F/∆Dnmt3a-Cas9 but not Jak2V617F/Cas9 controls demonstrated increased HSPC self-renewal and proliferation properties in vitro as evidenced by serial replating in methylcellulose (>5 weeks) and increased colony forming unit capacity. Flow-cytometry analysis of Jak2V617F/∆Dnmt3a-Cas9 revealed enrichment in LKS+ (Lin-Sca-1highKithigh) cells 5 weeks after CRISPR-Cas9disruption of Dnmt3a, and this was associated with increased expression of stemness markers Kit and Cd34 in Jak2V617F/∆Dnmt3a-Cas9 cells. RNAseq was performed on early (week 1, P1) and late culture HSPC (week 5, P5) from Jak2V617F-Cas9 (P1 only) and Jak2V617F/∆Dnmt3a-Cas9 (P1, P5). This confirmed deletion of Dnmt3a in Jak2V617F/∆Dnmt3a-Cas9 but not in Jak2V617F/Cas9 controls. Transcriptional upregulation of Kit and Cd34 were confirmed, as well as other key stem cell genes such as Erg and Angpt1 in Jak2V617F/∆Dnmt3a-Cas9 P5. We observed denovo expression of imprinted genes Igf2 and H19 in Jak2V617F/∆Dnmt3a P5, suggesting impaired DNA methylation in this group. Jak2V617F/∆Dnmt3a-Cas9 P5 were significantly enriched for transcriptional pathways controlling cell cycle progression, oncogenic signatures, and DNA damage. Conversely, Jak2V617F/Cas9 controls were enriched for myeloid differentiation and normal progenitor cell signatures. To assess the effect of Dnmt3a loss on Jak2V617F driven MPN, we transplanted Jak2V617F/∆Dnmt3a-Cas9 or Jak2V617F/Cas9 LKS+ into irradiated B6 recipients. Recipients of Jak2V617F/Cas9 LKS+ developed early stage MPN reminiscent of polycythemia vera with high hemoglobin, white cell count and platelets and was sustained >32 weeks. In contrast, Jak2V617F/∆Dnmt3-Cas9 recipients exhibited a biphasic disease, reminiscent of human myelofibrosis. At 8 weeks, Jak2V617F/∆Dnmt3-Cas9 showed panmyelosis with thrombocytosis (1.38x106/µl vs. 1.14x106/µl controls, p=0.057). However, by 32 weeks, this mice became severely pancytopenic with progressive bone marrow failure (Hemoglobin 121g/L vs. 210g/L controls, p =0.0011; platelets 0.338x106/µl vs. 1.343x106/µl controls, p <0.0001). Jak2V617F/∆Dnmt3-Cas9 mice exhibited extreme splenomegaly associated with reticulin fibrosis and the accumulation of myeloid cells. Bone marrow histology of Jak2V617F/∆Dnmt3-Cas9 revealed osteosclerosis and disorganized architecture and a dense fibrocellular infiltrate and reticulin fibrosis. Flow cytometry revealed impaired erythropoiesis and blocked differentiation. AML was not seen. These data demonstrate new evidence linking loss of Dnmt3a with acquisition of self-renewal in combination with constitutively active Jak2V617F. Importantly, in vivo loss of Dnmt3a accelerates or induces myelofibrotic transformation of the underlying MPN. This work provides new understanding to the factors that promote advanced disease in MPN. Ultimately, such knowledge has the potential to inform the development of novel targeted therapeutic approaches for the treatment of transformed MPN, a highly chemorefractory disease associated with extremely poor prognosis in patients. Disclosures Lane: Janssen: Other: i have done consulting (once) for janssen..

Dual Role Of MERIT40 In Hematopoietic Stem and Progenitor Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 797-797
Author(s):  
Krasimira Rozenova ◽  
Jing Jiang ◽  
Chao Wu ◽  
Junmin Wu ◽  
Bernadette Aressy ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Dna Damage ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Bone Marrow Failure ◽  
Adaptor Protein ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract The balance between self-renewal and differentiation of hematopoietic stem cells (HSCs) is maintained by cell intrinsic and extrinsic mechanisms, including tight regulation of signaling pathways such as Tpo-Mpl and SCF-ckit. Posttranslational modifications, such as phosphorylation and ubiquitination, regulate these pathways. While the role of protein phosphorylation is well established, the importance of ubiquitination in HSC self-renewal has not been well addressed. It is known that of the seven different lysines on ubiquitin, Lys48 polyubiquitination is a marker for protein degradation, and Lys63 polyubiquitination is associated with regulation of kinase activity, protein trafficking, and localization. In this study, we provide evidence that the adaptor protein MERIT40 has multiple roles in hematopoietic stem/progenitor cells (HSPCs). MERIT40 is a scaffolding protein shared by two distinct complexes with Lys63 deubiquitinase (DUB) activities: the nuclear RAP80 complex with a known role in DNA damage repair in breast/ovarian cancer cells, whereas the functions of the cytoplasmic BRISC remains less characterized. MERIT40 is important for integrity of both complexes, and its deficiency leads to their destabilization and a >90% reduction in deubiquitinase activity. By using MERIT40 knockout (M40-/-) mice, we found that lack of MERIT40 leads to a two-fold increase in phenotypic and functional HSCs determined by FACS and limiting dilution bone marrow transplantation (BMT), respectively. More importantly, M40-/- HSCs have increased regenerative capability demonstrated by increased chimerism in the peripheral blood after BMT of purified HSCs. The higher self-renewal potential of these HSCs provides a survival advantage to M40-/- mice and HSCs after repetitive administration of the cytotoxic agent 5-flurouracil (5FU). MERIT40 deficiency also preserves HSC stemness in culture as judged by an increase in peripheral blood chimerism in recipient mice transplanted with M40-/- Lin-Sca1+Kit+ (LSK) cells cultured in cytokines for nine days compared to recipient mice receiving cultured wildtype (WT) LSK cells. In contrast to the increased HSC homeostasis and superior stem cell activity due to MERIT40 deficiency, M40-/- mice are hypersensitive to DNA damaging agents caused by inter-cross linking (ICL), such as Mitomycin C (MMC) and acetaldehydes that are generated as side products of intracellular metabolism. MMC injection caused increased mortality in M40-/- mice compared to WT controls attributable to DNA damage-induced bone marrow failure. MMC-treated M40-/- mice showed marked reduction in LSK progenitor numbers accompanied by increased DNA damage, in comparison to WT mice. Consistent with the in vivo studies, M40-/- progenitor cells are hypersensitive to MMC and acetaldehyde treatment in a cell-autonomous manner in colony forming assays. ICL repair is known to require Fanconi Anemia (FA) proteins, an ICL repair network of which mutations in at least 15 different genes in humans cause bone marrow failure and cancer predisposition. Thus, M40-/- mice represent a novel mouse model to study ICL repair in HSPCs with potential relevance to bone marrow failure syndromes. Taken together, our data establishes a complex role of MERIT40 in HSPCs, warranting future investigation to decipher functional events downstream of two distinct deubiquitinating complexes associated with MERIT40 that may regulate distinct aspects of HSPC function. Furthermore, our findings reveal novel regulatory pathways involving a previously unappreciated role of K63-DUB in stem cell biology, DNA repair regulation and possibly bone marrow failure. DUBs are specialized proteases and have emerged as potential “druggable” targets for a variety of diseases. Hence, our work may provide insights into novel therapies for the treatment of bone marrow failure and associated malignancies that occur in dysregulated HSCs. Disclosures: No relevant conflicts of interest to declare.

Erg is required for self-renewal of hematopoietic stem cells during stress hematopoiesis in mice

Blood ◽  
2011 ◽  
Vol 118 (9) ◽  
pp. 2454-2461 ◽  
Author(s):  
Ashley P. Ng ◽  
Stephen J. Loughran ◽  
Donald Metcalf ◽  
Craig D. Hyland ◽  
Carolyn A. de Graaf ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Steady State ◽  
Hematopoietic Stem Cells ◽  
Blood Cells ◽  
Bone Marrow Failure ◽  
Mutant Mice ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract Hematopoietic stem cells (HSCs) are rare residents of the bone marrow responsible for the lifelong production of blood cells. Regulation of the balance between HSC self-renewal and differentiation is central to hematopoiesis, allowing precisely regulated generation of mature blood cells at steady state and expanded production at times of rapid need, as well as maintaining ongoing stem cell capacity. Erg, a member of the Ets family of transcription factors, is deregulated in cancers; and although Erg is known to be required for regulation of adult HSCs, its precise role has not been defined. We show here that, although heterozygosity for functional Erg is sufficient for adequate steady-state HSC maintenance, Erg+/Mld2 mutant mice exhibit impaired HSC self-renewal after bone marrow transplantation or during recovery from myelotoxic stress. Moreover, although mice functionally compromised for either Erg or Mpl, the receptor for thrombopoietin, a key regulator of HSC quiescence, maintained sufficient HSC activity to sustain hematopoiesis, Mpl−/−Erg+/Mld2 compound mutant mice displayed exacerbated stem cell deficiencies and bone marrow failure. Thus, Erg is a critical regulator of adult HSCs, essential for maintaining self-renewal at times of high HSC cycling.

The p110α Catalytic Isoform of PI3 Kinase Is Important for Erythropoiesis, but Has a Minimal Role in Hematopoietic Stem Cell Self-Renewal.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3620-3620
Author(s):  
Kira Gritsman ◽  
Tulasi Khandan ◽  
Rachel Okabe ◽  
Maricel Gozo ◽  
Mahnaz Paktinat ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Hematopoietic Cells ◽  
Pi3 Kinase ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cell Counts

Abstract Abstract 3620 Poster Board III-556 PIK3CA, which encodes the p110α catalytic isoform of PI3 kinase (PI3K), is mutated in many human cancers, and is an attractive therapeutic target. However, PI3K may also be important during hematopoiesis, as it is activated by hematopoietic growth factor receptors which control hematopoietic stem cell (HSC) and progenitor proliferation, differentiation, and self-renewal, such as erythropoietin receptor (epoR), c-kit receptor, and fms-like tyrosine kinase 3 (FLT3). In hematopoietic cells, receptor tyrosine kinases signal through the catalytic p110 subunit of PI3K, which has 3 isoforms (α, β, δ). However, the roles of PI3K and its specific catalytic isoforms in normal HSC function are poorly understood. We hypothesized that signaling through the p110α isoform is important for hematopoiesis and HSC self-renewal. We have used the Cre-loxP system to delete p110α in the HSCs of adult mice by breeding p110αF/F mice to Mx1-Cre transgenic mice. p110αF/F;Mx1-Cre+ (Cre+) mice and their p110αF/F (Cre-) littermates were injected with PolyI-PolyC (pIpC) at 4-6 weeks of age to induce Cre-mediated excision at the PIK3CA locus specifically in hematopoietic cells. Deletion of p110α in the bone marrow (BM) was verified by PCR and by immunoblot. We observed that, by four weeks after pIpC treatment, Cre+ mice developed microcytic anemia compared with Cre- littermates, characterized by a decreased mean hemoglobin (p<0.0001) and decreased mean corpuscular volume, while white blood cell counts and platelet counts were unaffected. Cre+ mice also had significantly decreased spleen, liver, and thymus weights. Flow cytometry analyses of bone marrow and spleen cells revealed a relative block in erythropoiesis in the spleens of Cre+ mice, with expansion of the basophilic erythroblast population, and a decrease in the most mature nucleated erythroblast population. Colony assays of splenocytes in erythropoietin-containing methylcellulose medium revealed a 52% decrease in BFU-E colony formation by p110α-deleted cells in response to erythropoietin, suggesting that loss of p110α may lead to blunted epoR signaling (p=0.009). Multiparameter flow cytometry revealed that the overall number of Lin-Sca1+ckit+ (LSK) cells, which contains the HSC population, was increased two-fold in the BM of Cre+ mice compared with Cre- littermates (p=0.01). To determine whether loss of p110α affects long-term HSC self-renewal in vivo, we performed competitive repopulation assays, in which CD45.2+ BM cells from PIPC-treated Cre+ mice or Cre- controls were transplanted together with CD45.1+CD45.2+ competitor BM cells into lethally irradiated CD45.1+ recipient mice. Donor BM chimerism (%CD45.2+ cells) at 16 weeks was mildly reduced in the absence of p110α, but Cre+ cells were still capable of long-term reconstitution. In summary, we have found that the p110α catalytic isoform is specifically required for erythropoiesis, but has only a small role in HSC homeostasis and in differentiation of the other hematopoietic lineages. This suggests that pharmacologic targeting of p110α in cancer therapy may result in mild anemia, but should otherwise have minimal hematologic toxicity. Disclosures: Gilliland: Merck Research Laboratories: Employment. Roberts:Novartis Pharmaceuticals, Inc.: Consultancy. Zhao:Novartis Pharmaceuticals, Inc.: Consultancy.

scholarly journals ALLOGENEIC HEMATOPOIETIC STEM CELL TRANSPLANTATION FOR ADULT PATIENTS WITH FANCONI ANEMIA.

2016 ◽  
Vol 8 ◽  
pp. 2016054 ◽  
Author(s):  
Hosein Kamranzadeh fumani ◽  
Mohammad Zokaasadi ◽  
Amir Kasaeian ◽  
Kamran Alimoghaddam ◽  
Asadollah Mousavi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cell Transplantation ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Hematopoietic Stem Cell ◽  
Fanconi Anemia ◽  
Bone Marrow Failure ◽  
Term Survival ◽  
Hematopoietic Stem

Background & objectives: Fanconi anemia (FA) is a rare genetic disorder caused by an impaired DNA repair mechanism which leads to an increased tendency toward malignancies and progressive bone marrow failure. The only curative management available for hematologic abnormalities in FA patients is hematopoietic stem cell transplantation (HSCT). This study aimed to evaluate the role of HSCT in FA patients.Methods: Twenty FA patients with ages of 16 or more who underwent HSCT between 2002 and 2015 enrolled in this study. All transplants were allogeneic and the stem cell source was peripheral blood and all patients had a full HLA-matched donor.Results: Eleven patients were female and 9 male (55% and 45%). Mean age was 24.05 years. Mortality rate was 50% (n=10) and the main cause of death was GVHD. Survival analysis showed an overall 5-year survival of 53.63% and 13 year survival of 45.96 % among patients.Conclusion: HSCT is the only curative management for bone marrow failure in FA patients and despite high rate of mortality and morbidity it seems to be an appropriate treatment with an acceptable long term survival rate for adolescent and adult group.

scholarly journals Detection of Mutations in Inherited Bone Marrow Failure and Myelodysplastic Syndrome Genes Using Genomic Capture and Massively Parallel Sequencing in Clinical Diagnostics

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1507-1507
Author(s):  
Siobán B. Keel ◽  
Tom Walsh ◽  
Colin Pritchard ◽  
Akiko Shimamura ◽  
Mary-Claire King ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Genetic Testing ◽  
Hematopoietic Stem Cell ◽  
Copy Number ◽  
Bone Marrow Failure ◽  
Copy Number Variants ◽  
Donor Selection ◽  
Hematopoietic Stem ◽  
Pathogenic Mutations

Abstract Accurate and timely diagnosis of inherited bone marrow failure (BMF) and myelodysplastic syndromes (MDS) ensures appropriate clinical management. The correct diagnosis allows appropriate monitoring for both hematopoietic (i.e. clonal evolution and progressive marrow failure) and extra-hematopoietic complications, informs the timing of hematopoietic stem cell transplant, donor selection and transplant regimen planning, and ensures appropriate genetic counseling of family members. Substantial phenotypic overlap among these disorders and the variable expressivity within syndromes complicate their diagnosis based purely on physical exam and standard laboratory testing and provide the rationale for comprehensive genetic diagnostic testing. We report here our initial one-year experience utilizing a targeted capture assay of known inherited BMF/MDS genes for clinical diagnostic purposes at the University of Washington. The assay sequences all exons and 20 base pairs of intronic sequence flanking each exon, as well as several regulatory and intronic regions of specific genes containing known pathogenic variants of 85 known inherited BMF/MDS genes (Zhang M. et al. Haematologica 2016). Between June 2015 and July 2016, 81 individual patients were referred for clinical testing (median age: 15 years-old, range: 0.6-76 years-old). For all samples evaluated, median coverage across the 383kb targeted region was 1887X. This depth of coverage enabled identification of all classes of mutations, including point mutations, small indels, copy number variants, and genomic rearrangements. Pathologic mutations in known inherited BMF/MDS genes were identified in 12 of 82 (14.6%) individuals (median age 13 years-old, range: 1.25-43 years-old) across a broad number of genes and of multiple classes including copy number variants (Table). Among the twelve patients with pathogenic mutations in inherited BMF/MDS genes, genetic testing was consistent with the prior clinical diagnoses of eight patients, including two Fanconi anemia patients subtyped as complementation group A, one of whom demonstrated reversion to wild-type resulting in mosaicism in the peripheral blood. Importantly, four patients carried no specific inherited BMF/MDS diagnosis prior to testing and were found to have pathogenic mutations in RPS10, RTEL1 and RUNX1 (ID 005, 008, 009, 010), suggesting additional diagnostic value to a multiplexed genetic approach in the clinical setting. Detailed clinical information was available for nine of the patients diagnosed with pathogenic mutations, two of whom have or will undergo a sibling or haploidentical hematopoietic stem cell transplantation (009 and 012, respectively) and thus genetic testing informed donor selection. To improve diagnostic accuracy, we are now updating the capture design to include newly discovered inherited BMF/MDS genes and intronic regions to optimize copy number variant detection. We are additionally pursuing CLIA-certified RNA analyses to characterize whether several variants bioinformatically predicted to affect splicing are functionally deleterious. Next-generation sequencing for mutations involved in hereditary marrow failure and MDS may also become increasingly important in the context of precision-medicine in which germline mutations are unexpectedly identified in somatic testing. Disclosures No relevant conflicts of interest to declare.

scholarly journals T Cell Subsets and Associated Cytokines in Chronic Graft-Versus-Host-Disease Using G-CSF Primed Bone Marrow in Allogeneic Hematopoietic Stem Cell Transplantation

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4580-4580
Author(s):  
Monica M Rivera Franco ◽  
Eucario Leon Rodriguez ◽  
Diana Gomez Martin ◽  
Javier Merayo Chalico ◽  
Jorge Alcocer Varela
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Stem Cell ◽  
Th17 Cells ◽  
Mononuclear Cells ◽  
Chronic Gvhd ◽  
The Other ◽  
Hematopoietic Stem ◽  
Graft Versus Host ◽  
Ifn Γ

Abstract Background Graft versus host disease (GVHD) is the major complication of allogeneic hematopoietic stem cell transplantation. It is characterized by an imbalance between the effector and regulatory arms of the immune system which results in the over production of inflammatory cytokines. Regulatory T (T regs) cells and T helper 17 (Th17) cells are two recently described lymphocyte subsets with opposing actions. Both can develop from naïve CD4+ T cell precursors under the influence of TGFβ1. Th17 lymphocytes, are key effector cells in rodent models of human diseases including GVHD. The other subset, T regs, is essential for dominant immunologic tolerance. At our institution, patients transplanted using G-CSF primed bone marrow (G-BM), have a lower incidence of acute and chronic GVHD when compared to those transplanted with peripheral blood and not primed bone marrow. Some microenvironment characteristics of this hematopoietic stem cells (HSC) source remain unknown, as well as the difference between Tregs, Th17 and cytokine levels in patients who develop GVHD and those who do not. Objective To analyze the characteristics of thirty-eight G-BM donor samples, identifying lymphocytes subsets and associated cytokines, and comparing patients who developed chronic GVHD (cGVHD) and those who did not. Materials and Methods A prospective analysis was performed in 38 G-BM samples from donors from 1999 to 2016. Mononuclear cells were defrosted, counted, and viability was evaluated. A 24 hour resting with RPMI, and posterior activation with PMA (50 ng/ml) for 48 hours was performed. Cells were harvested and cytokines were evaluated by flow cytometry (CBA assay). From each sample, one million mononuclear cells were permeabilized, fixed, and stained with CD4-FITC, IL17A-PE, IFN-γ APC, and IL-4 PECy7, for their posterior phenotipication by flow cytometry. The samples were obtained in a BD LSR Fortessa cytometry, and analyzed with the Flow-Jo software. Patients (recipients) information was analyzed using SPSS v.21. Results GVHD incidence was reported as following: Three (8%) patients developed acute GVHD (2 grade II, and 1 grade IV), 11 patients (29%) developed chronic GVHD (9% extensive, and 91% limited), and 24 patients did not present either. Mononuclear cells from G-BM from donors of patients who developed cGVHD showed a pro inflammatory response, characterized by an increased concentration of IL-17A (15.5 vs 0.71 pg/mL, p=0.013), TNF-α (80.27 vs 0.13 pg/mL, p=0.001), and IL-6 (4953.6 vs 11.75 pg/mL, p=0.025), after a mitogenic stimulation, compared to cells from donors of patients who did not developed GVHD. On the other hand, a decreased IL-10 production (2.62 vs 52.81 pg/mL, p=0.001) was documented in mononuclear cells from donors of patients who developed chronic GVHD, compared to donor cells of patients who did not. No significant difference in the production of IL-2, IL-4, and IFN-γ was observed. There was no difference in Th1 and Th2 between both groups, but mononuclear cells from donors of patients who developed chronic GVHD had a higher percentage of Th17 (1.02% vs 0.46%, p<0.001), and less Tregs (0.88% vs 1.95%, p<0.001), compared to those who did not developed GVHD. Conclusions Patients who develop cGVHD (29%) are characterized by a pro inflammatory response with an increased production of IL-17A, IL-6, and IFN-γ, and also a major percentage of Th17 cells. Also, a decreased suppressive response was documented with reduced IL-10 and Tregs levels. The low incidence of cGVHD show that G-CSF primed bone marrow is an excellent source for allogeneic HSC transplantations, and would be useful to compare these results with other HSC sources. Disclosures No relevant conflicts of interest to declare.

Late Donor Bone Marrow Failure After Allogeneic Hematopoietic Stem Cell Transplantation

2014 ◽  
Vol 97 (12) ◽  
pp. e75-e77 ◽  
Author(s):  
Mathieu Meunier ◽  
Anne-Claire Manez ◽  
Aliénor Xhaard ◽  
Régis Peffault de Latour ◽  
Flore Sicre de Fontbrune ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cell Transplantation ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Hematopoietic Stem Cell ◽  
Bone Marrow Failure ◽  
Hematopoietic Stem ◽  
Donor Bone Marrow
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