Pathogenesis Of Aplastic Anemia Research Articles

NOTCH signaling in immune-mediated bone marrow failure of aplastic anemia.

Severe aplastic anemia is a rare bone marrow failure disease with the majority of cases caused by aberrant immune destruction of blood progenitors. Although the Th1-mediated pathology of aplastic anemia is well-described, the molecular mechanisms that drive disease progression remain ill-defined. The NOTCH signaling pathway mediates Th1 differentiation in the presence of polarizing cytokines, an action requiring enzymatic processing of NOTCH receptors by γ- secretase. We used a mouse model of aplastic anemia to demonstrate that expression both of intracellular NOTCH1 (NOTCH1IC) and T-BET, a key transcription factor regulating Th1 differentiation, were increased in T cells in the spleen and bone marrow during active disease. Conditionally deleting NOTCH1 or administering γ-secretase inhibitors (GSI) in vivo, attenuated disease and rescued mice from lethal bone marrow failure. In peripheral T cells from patients with untreated aplastic anemia, NOTCH1IC was significantly elevated and was detected at the TBX21 promoter, showing NOTCH1 directly regulates the gene encoding T-BET. Treating patients’ cells ex vivo with GSI lowered NOTCH1IC levels, decreased the level of NOTCH1 detectable at the TBX21 promoter, and also decreased T-BET expression, indicating NOTCH1 signaling is responsive to GSI during active disease. Collectively, these results identify NOTCH1 signaling as a primary driver of Th1-mediated pathogenesis in aplastic anemia and may represent a novel target for therapeutic intervention.

Correlation of transforming growth factor beta-1 gene polymorphisms C-509T and aplastic anemia

Autoreactive cytotoxic T cells play a key role in the pathogenesis of aplastic anemia (AA) by myelosuppressive cytokines including interferon gamma, tumor necrosis factor alpha, and transforming growth factor beta. As an important cytokine that modulates the cell cycle, the involvement of transforming growth factor beta-1 (TGF-β1) in AA has been studied. The purpose of our study was to investigate the role of (TGF-β1/2509) gene polymorphisms in patients with acquired AA and to assess whether genotype was associated with higher or lower production and compare it with established control populations. Thirty-one patients with acquired aplastic anemia and 30 age- and sex-matched healthy controls were analyzed consecutively using PCR–restriction fragment length polymorphism (PCR-RFLP). TGF-β1 polymorphism C-509T was identified using PCR-RFLP. Significantly different distributions of the gene were demonstrated between the case and the control. The frequencies of −509CT and −509TT genotypes (74.2 versus 30 % and 16.2 versus 3.3 %, respectively) were significantly higher in cases than in controls (P < 0.001), while the frequency of the −509 CC genotype is higher in the control than in cases (66.7 versus 9.6 %, P < 0.001). Our data showed a genotypic profile associated with high TGF-β production in patients with bone marrow failure and suggest that the genetic regulation of inflammatory and T cell-mediated immunological pathways could be involved in the pathogenesis of bone marrow failure, reinforcing the view that AA are organ-specific autoimmune disorders.

Functional characterization of CD4+ T cells in aplastic anemia

AbstractThe role of CD4+ T cells in the pathogenesis of aplastic anemia (AA) is not well characterized. We investigate CD4+ T-cell subsets in AA. Sixty-three patients with acquired AA were studied. Th1 and Th2 cells were significantly higher in AA patients than in healthy donors (HDs; P = .03 and P = .006). Tregs were significantly lower in patients with severe AA than in HDs (P < .001) and patients with non-severe AA (P = .01). Th17 cells were increased in severe AA (P = .02) but normal in non-severe AA. Activated and resting Tregs were reduced in AA (P = .004; P = .01), whereas cytokine-secreting non-Tregs were increased (P = .003). Tregs from AA patients were unable to suppress normal effector T cells. In contrast, AA effector T cells were suppressible by Tregs from HDs. Th1 clonality in AA, investigated by high-throughput sequencing, was greater than in HDs (P = .03). Our results confirm that Th1 and Th2 cells are expanded and Tregs are functionally abnormal in AA. The clonally restricted expansion of Th1 cells is most likely to be antigen-driven, and induces an inflammatory environment, that exacerbate the functional impairment of Tregs, which are reduced in number.

Hydrogen therapy may be an effective and specific novel treatment for aplastic anemia

SummaryAplastic anemia (AA) is a rare bone marrow failure disorder with high mortality rate, which is characterized by pancytopenia and an associated increase in the risk of hemorrhage, infection, organ dysfunction and death. The oxidation phenomenon and/or the formation of free radicals have been suggested to be causally related to various hematological disorders, including aplastic anemia. TNF-α, IL-6, and IL-2 also play important roles in the pathogenesis of AA. Recent studies have provided evidence that hydrogen inhalation can selectively reduce cytotoxic oxygen radicals and exert antioxidant effects. It was also reported that hydrogen could suppress the levels of TNF-α and IL-6. Based on these findings, we hypothesize that hydrogen therapy may be an effective, simple, economic and novel strategy in the treatment of aplastic anemia.

Pathogenesis of aplastic anaemia and paroxysmal nocturnal haemoglobinuria: the concept of clonality in stem cell disease

Molecular technologies have been applied not only to the investigations of haematological malignancies but also bone marrow failure syndromes, including aplastic anaemia (AA), paroxysmal nocturnal haemoglobinuria (PNH) and single lineage cytopenias, often associated with T cell large granular lymphocyte leukaemia (T-LGL). One of the main diagnostic problems is the distinction of hypoplastic clonal processes consistent with the diagnosis of myelodysplasdtic syndrome (MDS) from true idiopathic AA. Due to the paucity of the marrow, pathomorpho-logical diagnosis may be difficult and metaphase cytogenetics may not yield informative results, precluding proper diagnostic case assignment. Single nucleotide polymorphisms arrays (SNP-A) do not require cell division and may help to detect clonal chromosomal abnormalities. Systematic application of this technology has shown that chromosomal defects consistent with the diagnosis of MDS can be detected in a proportion of otherwise typical AA patients, initially or throughout the clinical course. At presentation, clonal abnormalities, in particular non-recurrent/random defects may be transient and reflect oligoclonality of the stem cell compartment rather than true malignant clonal process. Using SNP-A, submicroscopic chromosomal microdeletions can be detected; we have found recurrent microdeletions involving HLA and PIG-A loci on chromosomes 6 and X, respectively. These lesions likely evolve as a result of immune selection pressure and facilitate escape of the immune-resistant haematopoietic clones. Manifest evolution of MDS in the course of AA can be expected in up to 20% of patients with AA. The most common abnormality is monosomy-7, present in most cases of MDS derived from antedescent AA. In this context, monosomy-7 is associated with a distinct mutational pattern that includes presence of CBL mutations and RUNX1, while common mutations such as TET2, U2AF1, SRSF2 and ASXL1 are frequent in primary MDS with monosomy-7 or deletion7q. In contrast, childhood cases of juvenile myelomonocytic leukemia with monosomy-7 have a similar mutational pattern as seen in AA. Whole genome sequencing technologies including exome sequencing have led to identification of new mutations, not only in MDS but also in T-LGL which is characterised by frequent somatic activating mutation in STAT3. By ultra-deep sequencing of T cell receptor (TCR) VB chains, one can also characterise TCR repertoire and identify oligoclonal T cell mediated processes. The presence of STAT3 mutations distinguish clonal processes from semi-reactive oligoclonal T cell expansions.

Frequent loss of HLA alleles associated with copy number-neutral 6pLOH in acquired aplastic anemia

AbstractIdiopathic aplastic anemia (AA) is a common cause of acquired BM failure. Although autoimmunity to hematopoietic progenitors is thought to be responsible for its pathogenesis, little is known about the molecular basis of this autoimmunity. Here we show that a substantial proportion of AA patients harbor clonal hematopoiesis characterized by the presence of acquired copy number-neutral loss of heterozygosity (CNN-LOH) of the 6p arms (6pLOH). The 6pLOH commonly involved the HLA locus, leading to loss of one HLA haplotype. Loss of HLA-A expression from multiple lineages of leukocytes was confirmed by flow cytometry in all 6pLOH(+) cases. Surprisingly, the missing HLA-alleles in 6pLOH(+) clones were conspicuously biased to particular alleles, including HLA-A*02:01, A*02:06, A*31:01, and B*40:02. A large-scale epidemiologic study on the HLA alleles of patients with various hematologic diseases revealed that the 4 HLA alleles were over-represented in the germline of AA patients. These findings indicate that the 6pLOH(+) hematopoiesis found in AA represents “escapes” hematopoiesis from the autoimmunity, which is mediated by cytotoxic T cells that target the relevant auto-antigens presented on hematopoietic progenitors through these class I HLAs. Our results provide a novel insight into the genetic basis of the pathogenesis of AA.

Functional Characterization of CD4+ T-Cells in Aplastic Anemia (AA)

Abstract 1340We have examined the role of CD4+ T-cells in the pathogenesis of AA in 63 patients, 48 of whom were analyzed at diagnosis and 15 following immunosuppressive therapy (IST). Absolute numbers of CD4+ regulatory T cells (Tregs, defined as CD3+CD4+CD25highCD27+Foxp3+) were lower in pre-treatment AA patients compared to 10 healthy donors (HDs) (5.5 × 106 v 1.4 × 107)(p=0.01). In patients with severe (SAA) and very severe AA (VSAA), the absolute number and frequency of Tregs were lower than non-severe AA (NSAA) (4.4 × 106/L v 1 × 107/L)(p=0.01) and HDs (4.4 × 106/L v 3 × 107/L) (p<0.001). Absolute numbers of Th1 and Th2 cells in all pre-treatment patients were higher compared to HDs (6.4 × 107/L v 1.8 × 107/L)(p=0.03) for Th1 and (2.6 × 107/L v 2.4 × 106/L)(p=0.006) Th2 cells. Although mean percentages of AA Th17 cells were higher than in HDs (1.5% v 0.15%)(p=0.04), differences in absolute numbers were not significant. Absolute numbers of Th2 and Th17 cells were increased in SAA (1.3 × 107/L v 7.4 × 106/L for Th2)(p=0.01) compared to NSAA (5.7 × 106/L v 2.15 × 106/L for Th17)(p=0.02). Ratios of Th1/Tregs (p=0.003), Th2/Tregs (p=0.02), and Th17/Tregs (p=0.001) were higher in SAA and VSAA compared to NSAA. Percentage of both activated (CD4+CD45RA−CD25highFoxp3high) and resting (CD4+CD45RA+ CD25highFoxp3low) Tregs was decreased in AA patients, compared to HDs (p=0.004 and p=0.01), whereas cytokine secreting Tregs (CD4+CD45RA−CD25high Foxp3low) were increased in AA (p<0.003). Sorted Tregs from AA patients did not suppress cytokine secretion by autologous or HD T effectors (Te) cells in 1:1 co-cultures, whereas IL-2 and IFN-γ secretion by AA Te (CD4+CD25lowCD127high) was suppressible by allogeneic Tregs from HDs, confirming Tregs dysfunction. AA Tregs did not inhibit either CD154 or CD69 expression on Te cells. Tregs from AA patients secreted significantly more IFN-γ, TNF-α and IL-17 (p=0.02, p=0.02 and p=0.01, respectively) after 4 hours stimulation with PMA/Ionomycine compared to HDs. Expression levels of FoxP3, ROR□c and T-bet in AA Tregs was normal. IFN-γ secreting cells (Th1) were enriched using enrichment kit then further enriched by FACS sorting. CDR3 region products of TCR Vβ-chain were amplified using Vβ specific forward and Cβ reverse primers. CDR3 PCR products from AA patients and HDs were subjected 454 sequencing (Roche GS FLX titanium). Sequencing was performed to yield an average ‘depth' in excess of 1000 clonally reads (1000x) for each sample specific CDR3 PCR amp icon. Reads were processed using Roche Amp icon Variant Analyzer software (AVA). Diversity of TCR receptors (measured by spectratyping and confirmed by high throughput deep sequencing) in AA Th1 cells was lower than HDs (p=0.037), as shown by the percentage and number of consensus clusters in total sequence reads. Interestingly, percentages of the most dominant CDR3 clones, revealed by high throughput sequencing, were higher in AA compared to HDs, regardless of spectratyping pattern. Global gene expression of Tregs was compared in 3 pre-IST AA patients and 5 HDs. A unique gene signature consisting of 86 genes that were significant was identified. There were 8 down regulated genes (fold change) in the pre-treatment group; PIN4 (−4.1), OR2T12 (−3.3), AMAC1 (−2.73), PERP (−2.69), UTS2 (−2.27), RNF139 (−2.13), COMMD9 (−2.09) and LOC100128356 (−2.01). The top 10 of 78 up-regulated genes in the pre-treatment group were HBB (19.5), PSME2 (13.8), CSDA (13.07), FAM127A (7.78), EXOSC1 (7.73), BPGM (7.43), CYSLTR1 (7.17), CHPT1 (6.96) and PLAC8 (6.71). qPCR analysis for CSDA, HBB, PSMiE2, PERP, PIN4, and UTS2 confirmed a similar trend to the microarray results. Interestingly absolute number of Tregs, and Th2/Treg ratio were higher in 10 IST responsive patients compared to 5 non-responsive patients (p=0.005 and 0.02, respectively).We show that expansion of Th1, Th2, Th17, and decreased/skewed Tregs immunophenotype and function are a consistent and defining feature of SAA and VSAA. Clonal expansion of Th1 cells is likely to be antigen driven and the presence of dysfunctional Tregs aggravates this autoimmune response.Increases of Tregs, and Th2/Treg ratios following IST predicts a favourable response to this treatment. Disclosures:No relevant conflicts of interest to declare.

Decreased Expression of HOXB4 Gene in Aplastic Anemia; Regulatory Interaction with GATA-2,

Abstract 3436 Background:Aplastic anemia (AA) is characterized by a reduced number of hematopoietic stem cells (HSCs). It has been proposed that immunological injury in HSCs leads to reduced numbers of stem cells in the bone marrow. In addition, expression of the critical regulator of hematopoiesis GATA-2 is decreased in CD34-positive cells in AA (Fujimaki et al. Br J Haematol 2001). Despite the compelling results described above, only limited information has emerged regarding intrinsic abnormalities of hematopoietic stem cells in AA. It has been demonstrated that HOXB4 induces HSC expansion ex vivo (Antonchuk et al. Cell 2002), and restoring HOXB4 protein in HSCs from bone marrow failure patients promotes HSC expansion (Tang et al. Br J Haematol 2009). In conjunction with the evidence that recent genome-wide analysis of GATA factor chromatin occupancy identified GATA-2 peak at HOXB4 promoter (Fujiwara et al. Mol Cell 2009), we hypothesized that GATA-2 directly regulates HOXB4 expression in HSCs, which might contribute to the pathogenesis of AA. Here, we investigated possible link between GATA-2 and HOXB4, and also tested if HOXB4 is deregulated in CD34-positive cells from patients with AA. Method:For GATA-2 overexpression, human GATA-2 coding sequence was cloned into pcDNA3.1 expression vector as well as MSCV retroviral expression vector (Clontech). For GATA-2 knockdown, siRNA specific for human GATA-2 was transfected into CD34-positive cells or K562 cells by Amaxa Nucleofector kit (Amaxa Inc.). For promoter assay, DNA fragment of the HOXB4 gene promoter region (up to −262 from 1st ATG) was cloned into pGL3-Basic (Promega), and the GATA deletion construct was subsequently created with QuickChange™Site-Directed Mutagenesis Kit (Stratagene). Quantitative chromatin immunoprecipitation (ChIP) analysis was performed using antibodies for GATA-2 (H-116, Santa Cruz). For analyzing clinical samples, informed consent was obtained in all cases and ethical considerations according to the declaration of Helsinki were followed. Results:To examine if GATA-2 and HOXB4 are functionally linked, we transfected a GATA-2 expression vector into K562 cells, and demonstrated that GATA-2 significantly upregulated endogeneous HOXB4 expression. Furthermore, siRNA-mediated GATA-2 knockdown in K562 cells significantly reduced HOXB4 expression, indicating that HOXB4 is a GATA-2 target gene. We overexpressed/reduced GATA-2 in cord blood-derived CD34+ cells, which also provided evidence for GATA-2 regulation of HOXB4 expression. Promoter analyses revealed that GATA sequence located at −160/-157 of the HOXB4 gene promoter region was required to confer luciferase activity in K562 cells. In vitro DNA binding studies and quantitative ChIP analysis revealed specific GATA-2 occupancy at a chromatin region containing this element. Finally, we demonstrated that HOXB4 gene expression was significantly decreased in CD34+ cells from patients with AA (n=10) compared to those with ITP (n=13). The expression levels of HOXB4 and GATA-2 also correlated in these populations (r=0.6573, p<0.01). Conclusion:Based on these findings, we propose that decreased expression of GATA-2 in hematopoietic stem cells of AA leads to reduced HOXB4 transcription, which may have an important role in the development and/or progression of the disease. Disclosures:No relevant conflicts of interest to declare.

Impact of Cytokine Gene Polymorphisms on Risk and Treatment Outcomes of Aplastic Anemia

Abstract 4369Immunosuppressive therapy (IST) is one of the main treatment modalities for acquired aplastic anemia (AA). Approximately 70% of AA patients have been known to achieve clinical improvements with IST consisting of antithymocyte globulin (ATG) and cyclosporine. This remarkably high response rate supports us to tell the immunogenic pathophysiology of AA. Autoreactive cytotoxic T cells play a key role in this immunogenic pathogenesis of AA by working with myelosuppressive cytokines like interferon-gamma (IFN-γ), tumor necrosis factor alpha (TNFα), and transforming growth factor beta (TGFβ), which induce apoptosis in hematopoietic stem cells, partially through the Fas-dependent pathway. The purpose of this study is to find out which single nucleotide polymorphisms (SNPs) in cytokine genes were relevant to the risk of AA and whether the relevant SNPs were associated with response to IST in AA patients.Between January 2000 and December 2008 84 patients were screened and 80 patients confirmed as having acquired AA by bone marrow biopsy, and 84 age- and sex-matched healthy controls were analyzed consecutively. We genotyped the polymorphisms in three cytokine genes (IFNG, TNF, and TGFB1) and FAS gene, which are known to be involved in T cell-mediated marrow destruction. We assessed the association between polymorphisms in those selected genes and risk for AA, and the association between those polymorphisms and response to IST in three genetic models (dominant, recessive, and additive).The IFNG -2353 T allele (dominant model, OR=0.43, p=.012) and TCA haplotype (dominant model, OR=0.50, p=.038) were significantly associated with the development of AA. In addition, this relevant IFNG -2353 T allele and TCA haplotype were related to the response of IST (dominant model, OR=0.076, p=.034). The presence of the minor T allele was protective and related to a 2.3-fold reduction in the risk for AA (p=.012), and the presence of the IFNG TCA haplotype was related to a 2-fold reduced risk for AA (p=.038). Concerning TGFB1, although its polymorphisms are not related to AA susceptibility, P10L T allele (recessive model, OR=0.18, p=.038) and CT haplotype (dominant model, OR=5.68, p=.038) were associated with response to IST. The T allele was related to a 4.3-fold reduced response to IST at 3 months (p=.038) and the presence of the CT haplotype was favorable to IST and was related to a 5.7-fold higher response at 3 months compared with the response in patients without the CT haplotype (p=.038).This exploratory study concurred with prior studies indicating that polymorphisms in IFNG are related to AA susceptibility. In addition, it was found that polymorphisms in IFNG and TGFB1 are associated with response to IST. AA patients with intracellular IFN-γ expression in peripheral lymphocytes showed almost 3-fold higher response to IST than patients without IFN-γ-expressing lymphocytes (p<0.0001) and this finding puts weight on the immunogenic association of responsiveness. Regarding TGFβ, the functionality of polymorphisms and in TGFB1 has rarely been reported. However, the significant relationship between TGFB1 polymorphisms and therapeutic response to IST in our study suggests their late effects on marrow suppression. Our results may help explain the variability of response to IST in AA and suggests that patients with a high probability of response might be treated first with IST rather than conventional marrow transplantation.Table 1Factors relevant to response to ISTFactorCorrected for Patient CharacteristicsUncorrected DataGenotypeOR (95% CI)POR (95% CI)PResponse at 3-month after IST (n=43)Age1.00 (0.96–1.05).841.02 (0.98–1.06).46Sex (male vs female)1.13 (0.26–4.84).871.26 (0.37–4.23).71Severity (severe vs non-severe)1.03 (0.23–4.53).0971.63 (0.48–5.47).43TGFB P10L C/TTT vs CT + CC0.18 (0.03–0.90).0380.23 (0.06–0.95).043TGFB haplotypeCT-CT + CT-other vs Other-other5.68 (1.11–29.15).0384.28 (1.05–17.42).043Response at 6-month after IST (n=43)Age1.00 (0.95–1.05).911.02 (0.97–1.06).45Sex (male vs female)0.38 (0.072–2.01).250.60 (0.16–2.21).44Severity (severe vs non-severe)2.53 (0.47–13.62).282.22 (0.59–8.26).24IFNG -2353 A/TTT+AT vs AA0.076 (0.007–0.82).0340.40 (0.10–1.56).19IFNG haplotypeTCA-TCA + TCA-other vs Other-other0.076 (0.007–0.82).0340.40 (0.10–1.56).19TGFB haplotypeTC-TC vs TC-other + Other-other0.22 (0.05–0.90).0360.19 (0.03–1.09).063 Disclosures:No relevant conflicts of interest to declare.

Expression and clinical significance of TRAIL and Caspase-3 in the BMMNC of aplastic anemia

Objective To study the expression and clinical significance of TNF related apoptosis-inducing ligand(TRAIL) and Caspase-3 in the bone marrow mononuclear cell(BMMNC) of aplastic anemia(AA) ,and to approach their effect on the apoptosis of haemopoietic stem cell in AA patients.Methods The TRAIL and Caspase-3 expression in 15 cases with newly diagnosed AA patients and remission patients,and 15 cases with normal bone marrow patients were detected by flow cytometry and microplate reader. Results The TRAIL expression of newly diagnosed AA patient, emission AA patients and control group was ( 5.94 ± 2. 57 ) ％, ( 2. 87 ± 1. 72 ) ％ and ( 3.01 ±2. 06)％ respectively,the Caspase-3 expression of newly diagnosed AA patient, emission AA patients and control group was ( 1. 3840 ± 0. 0236), (0. 8018 ± 0. 0126) and ( 0. 7441 ± 0. 0112 ) respectively. The TRAIL and Caspase-3 expression of newly diagnosed AA patient were significantly higher than the remission AA patients and control group,the difference was significant( all P ＜0. 05) ;The TRAIL and Caspase-3 expression of control group were higher than the remission AA patients, but the difference was not significant ( all P ＞ 0. 05 ). Conclusion The TRAIL and Caspase-3 had high expression in the BMMNC of AA patients, and the apoptosis induced by TRAIL and Caspase-3 should play an important role in the pathogenesis of AA. Key words: Anemia aplastic ; TNF related apoptosis-inducing ligand; Caspase-3

Tcf-1 gene silence suppresses downstream gene expression in CD4+ T cells from bone marrow of aplastic anemia patients

CD4(+) T cells play a crucial role in the pathogenesis of acquired aplastic anemia (AA). Tcf-1 gene regulates T cell development and function, and it is significantly upregulated in the bone marrow CD4(+) T cells from patients with acquired AA. To explore the role of Tcf-1 in the pathogenesis of AA, we knocked down Tcf-1 gene in CD4(+) T cells of AA patients and studied the effects of Tcf-1 silencing on its downstream gene expression. Upon transfection of psiRNA into marrow CD4(+) T cells from bone marrow of aplastic anemia patients, the expression of Tcf-1 was significantly knocked down; consequently, expressions of c-Myc and CD44 were also significantly reduced. Our results suggest that Tcf-1 may contribute to pathogenesis of AA by regulating downstream gene expression such as c-myc and CD44.

Interleukin‐27 enhances the production of tumour necrosis factor‐α and interferon‐γ by bone marrow T lymphocytes in aplastic anaemia

Aplastic anaemia (AA) is considered as an immune-mediated bone marrow failure syndrome. The mechanism is involved with a variety of immune molecules including interferon-γ (IFN-γ), tumour necrosis factor-α (TNF-α) and interleukins (ILs). IL-27 is a novel member of the IL-12 family, which mediates T cell response and enhances the production of IFN-γ. However, little is known about the role of IL-27 in the development of AA. This study investigated the role of IL-27 and its receptor IL-27R in the pathogenesis of AA. Both the mRNA expression of IL-27/IL-27R subunits in the bone marrow mononuclear cells (BMMNCs) and the levels of IL-27 in the marrow plasma in AA were found to be higher than in controls. Increased IL-27 correlated with the disease severity of AA. Subsequently, we stimulated marrow T lymphocytes with recombinant human (rh)IL-27 and found that rhIL-27 enhanced the production of TNF-α and IFN-γ in both CD4(+) and CD8(+) T lymphocytes from AA patients. We also detected increased TNF-α and IFN-γ in the supernatants of BMMNCs from AA patients after IL-27 stimulation. In conclusion, our data suggest that elevated IL-27 and IL-27-induced TNF-α and IFN-γ overproduction might be involved in the pathogenesis of AA.

Efficacy and Safety of the Treatment with Bone Marrow Mesenchymal Stem Cells of the Patients with Aplastic Anemia

AbstractAbstract 5144 ObjectiveSummarizing the features of bone marrow mesenchymal stem cells, the pathogenesis of aplastic anemia, and the characteristics of bone marrow mesenchymal stem cells in patient affected by aplastic anemia;investigating the feasibility of implanting bone marrow mesenchymal stem cells for the treatment of aplastic anemia, providing a new mentality for the aplastic anemia's clinical treatment. MethodsSearch the documents utilized by the CNKI, CBM, PubMED and Highwire Press database. According to the retrieved result, a summary about the bone marrow mesenchymal stem cells characteristic and the feasibility of implanting bone marrow mesenchymal stem cells for the treatment of aplastic anemia was made. Results1. The bone marrow mesenchymal stem cells(bone marrow mesenchymal stem cells, BMSC)is another important ingredient in the bone marrow, besides hematopoietic stem cells. It is easy to separate, raise, multiply and purify mesenchymal stem cells. It still carrys the qualities of stem cells after many generations of rising. When raised in vitro, BMSC may secrete many kinds of cell factors and the growth factors,and has the long-term hematopoietic supporting function, which does not only participate in the production hemopoiesis micro environment support hemopoiesis directly, but also able to grow and differentiate into various hematopoiesis supporting bone marrow matrix cells in the suitable condition. 2. BMSC has the great potential in self-renewal and multi-direction differentiation. Not only can it take function as substitute cells in the tissue framework, it also present good application prospect with regard to hematopoietic reconstruction, transplant immunity, and genetic treatment. 3. BMSC can repair the damage of hematopoietic micro environment(HME), and promote the reconstruction of hematopoiesis and immunity after transplant. 4. The low immunogenic and antigen present ability of BMSC provide the molecular foundation for the wide application of BMSC in stem cells transplant. 5. The pathogenesis of aplastic anemia(aplastic anemia, AA)is very complicated. The current belief is that aplastic anemia is related to the intrinsic multiplication flaw of hematopietic stem cells, the inadequate function of the hematopoietic micro environment, and the disorder of organism immune function. Studies have proved that there's the remarkable decrease of ectogenous hematopoietic supporting function and immuno-suppression capability of BMSC in aplastic anemia. Nowadays, methods have been established in separating, raising, and multiplying BMSC outside the human body. Besides, studies have revealed that transplanting BMSC can restore the hematopoietic micro environment, and the BMSC plays an important part in modulating transplant immunity. 6. Observe the efficacy and safety of the treatment on 11 patients of aplastic anemia with BMSC and the outcome indicate that the treatment is effective and safe. Accordingly, there's a good prospect for applying BMSC transplant as the treatment for aplastic anemia. ConclusionsIt has a good effect and prospect to apply bone mesenchymal stem cells transplant to the treatment of aplastic anemia. It provides a new approach to the clinical treatment of aplastic anemia. Disclosures:No relevant conflicts of interest to declare.

Impact of Cytokine Gene Polymorphisms on Risk and Treatment Outcomes of Aplastic Anemia In Korea

AbstractAbstract 4432 IntroductionThe clinical evidence of responsiveness to immunosuppressive therapy (IST) supports immune pathophysiology for acquired aplastic anemia (AA). Recent studies suggested that auto-reactive cytotoxic T-cells against hematopoietic cells play a key role in the pathogenesis of AA with various cytokines. The purpose of this study is to investigate whether single nucleotide polymorphisms (SNP) of cytokine genes are related to the risk of AA and, furthermore, the response to IST. MethodsWe analyzed 80 adult patients diagnosed as acquired AA. The 84 unrelated, age and sex matched healthy subjects served as a control group. In 3 cytokine genes (IFN-γ, TNF-α, TGF-β) and one FAS gene, we selected 10 polymorphisms on the basis of allelic frequency and the assumption of clinical relevance from previously reported associations. To assess the association between polymorphisms and risk of AA, we calculated statistical differences in allele, genotype, and haplotype distributions between patients and controls using chi-square test in 3 genetic models (dominant, recessive, and additive). For the association between polymorphisms and response to initial course of IST, we analyzed 44 patients who were treated with IST using a multivariate logistic regression model. ResultsAmong 10 SNPs in 4 genes, one SNP and one haplotype in IFN-γ gene were significantly associated with the development of AA: IFN-γ -2353A/T; the presence of the T allele in dominant model was protective and was related to a 2.3-fold reduction in the risk for AA (P =.012); the presence of the IFN-γ TCA haplotype was related to a two-fold reduced risk for AA (P =.038).The IFN-γ -2353 T allele was shown to be resistant to IST; the presence of T allele and TCA haplotype in IFN-γ gene (dominant model) was related to a 13.2-fold reduced hematologic response at 6 months following initial IST (P =.034). However, 4 SNPs and 2 haplotypes in TNF-α gene did not show any significant associations with the response at 3 and 6 months. In terms of 2 SNPs in TGF-β gene, TGF-β P10L T/C was independently related; the T allele (recessive model) was related to a 4.3-fold reduced response to IST at 3 months (P =.038). Accordingly, the response was related to the TGF-β haplotype; the TC haplotype homozygote (recessive model) was related to a 4.6-fold reduced response to IST at 6 months (P =.036); the presence of CT haplotype (dominant model) was favorable to IST and was related to a 5.7-fold higher response at 3 months than the absence of the CT haplotype (P =.038). ConclusionThis exploratory study found that the genetic polymorphism of IFN-γ was susceptible to the development of AA and was related to the hematologic response following initial IST. In case of TGF-β, the polymorphisms were related to the response to IST, though they were not be related to the disease susceptibility. Large studies with a prospective design are needed to better understand the determinants of risk of AA and responsiveness to IST. Disclosures:No relevant conflicts of interest to declare.

Impact of cytokine gene polymorphisms on risk and treatment outcomes of aplastic anemia

Autoreactive cytotoxic T cells play a key role in the pathogenesis of aplastic anemia (AA) by myelosuppressive cytokines including interferon-gamma, tumor necrosis factor alpha, and transforming growth factor beta. The purpose of this study is to determine which single nucleotide polymorphisms (SNPs) in cytokine genes were relevant to AA risk and whether the relevant SNPs were associated with response to immunosuppressive therapy (IST). Among 84 screened patients, 80 patients confirmed as having acquired AA, and 84 age- and sex-matched healthy controls were analyzed consecutively. We genotyped ten polymorphisms in three cytokine genes (IFNG, TNF, and TGFB1) and FAS gene. We assessed the association between polymorphisms and AA risk, and the association between polymorphisms and response to IST in three genetic models (dominant, recessive, and additive). The IFNG -2,353 T allele (dominant model, OR = 0.43, p = .012) and TCA haplotype (dominant model, OR = 0.50, p = .038) were significantly associated with the development of AA. In addition, this relevant IFNG -2,353 T allele and TCA haplotype were related to the response of IST (dominant model, OR = 0.076, p = .034). Concerning TGFB1, although its polymorphisms are not related to AA susceptibility, P10L T allele (recessive model, OR = 0.18, p = .038) and CT haplotype (dominant model, OR = 5.68, p = .038) were associated with response to IST. This exploratory study concurred with prior studies indicating that polymorphisms in IFNG are related to AA susceptibility. In addition, it was found that polymorphisms in IFNG and TGFB1 are associated with response to IST.

Progress of pathogenesis of aplastic anemia

Aplastic anemia is an acquired bone marrow failure syndrome,characterized by an empty bone marrow,pancytopenia,as well as anemia,bleeding,infection syndrome.The pathogenesis of aplastic anemia has not yet been completely clear.Some of the proposed causes include hematopoietic stem/ progenitor cell deficiency,immune disorders,and abnormalities in the hematopoietic microenvironment.In recent years,further clinical and experimental studies have accumulated to recognize the pathogenesis of aplastic anemia. Key words: Aplastic anemia; Pancytopenia; Hematopoietic stem cells; Immune disorders

Alteration in marrow stromal microenvironment and apoptosis mechanisms involved in aplastic anemia: an animal model to study the possible disease pathology.

Aplastic anemia (AA) is a heterogeneous disorder of bone marrow failure syndrome. Suggested mechanisms include a primary stem cell deficiency or defect, a secondary stem cell defect due to abnormal regulation between cell death and differentiation, or a deficient microenvironment. In this study, we have tried to investigate the alterations in hematopoietic microenvironment and underlying mechanisms involved in such alterations in an animal model of drug induced AA. We presented the results of studying long term marrow culture, marrow ultra-structure, marrow adherent and hematopoietic progenitor cell colony formation, flowcytometric analysis of marrow stem and stromal progenitor populations and apoptosis mechanism involved in aplastic anemia. The AA marrow showed impairment in cellular proliferation and maturation and failed to generate a functional stromal microenvironment even after 19 days of culture. Ultra-structural analysis showed a degenerated and deformed marrow cellular association in AA. Colony forming units (CFUs) were also severely reduced in AA. Significantly decreased marrow stem and stromal progenitor population with subsequently increased expression levels of both the extracellular and intracellular apoptosis inducer markers in the AA marrow cells essentially pointed towards the defective hematopoiesis; moreover, a deficient and apoptotic microenvironment and the microenvironmental components might have played the important role in the possible pathogenesis of AA.

Contrasting Roles of Th1 and Th17 Cells in Aplastic Anaemia (AA) and Myelodysplastic Syndrome (MDS).

Abstract 1090Poster Board I-112 IntroductionAutoimmunity is believed to play an important role in the pathogenesis of both idiopathic aplastic anaemia (AA) as well as low-risk MDS (L-MDS). While there is a significant overlap in some of the clinical features between AA and L-MDS, there is a striking difference in response to immunosuppressive therapy (IST) (70-80% in AA vs 20% in L-MDS).We have previously shown that Tregulatory cells are significantly reduced in L-MDS (Kordasti Blood 2008), while other groups have suggested that Tregs are decreased in AA. We hypothesised that differences in the immune regulatory profile as well as the cytokine environment, may differentially influence the pathogenesis of AA and MDS. In a prospective study we examined the immunological and cytokine profiles within a cohort of newly diagnosed AA and L-MDS patients. Patients and Methods33 Acquired aplastic anaemia(22 at diagnosis and pre-treatment and 11 post IST), 18 low risk MDS (IPSS=0) and 5 healthy controls were recruited.The percentage and absolute number of different CD4+ T cell subsets (Th1, Th2, Th17, TNF-αa producing CD4+ T cells and Foxp3+ Tregs) in peripheral blood, were investigated by flow cytometry. T cells were stimulated first and then stained intracellularly for IFN-γ, IL-4, IL-17 & TNF-αa. The serum level of 30 different cytokines was also measured by 30 Plex bead analysis (Luminex). NK cells were defined as CD3– CD56+. B cells were defined as CD3-CD19+. CD3+ CD4+ T-cell subsets were defined as CD45RO–CD27+ naïve, CD45RO+ CD27+ CD62L+ central memory,CD45RO+ CD27+ CD62L– effector memory, CD45RO+CD27– effectors and CD45RO– CD27– terminal effectors. CD4 regulatory T cells were defined as CD4+ CD25highFoxp3+. ResultsThe absolute number of Th1 cells and TNF-αa producing CD4+ T cells were significantly higher in AA patients compared to healthy controls(42 × 107/L v 29 × 107/L) (p=0.001, p=0.005). Although the number of Th17 cells was the same as healthy controls, this number was significantly lower than low risk MDS patients (0.86 × 107/L v 2.7 × 107/L) (p=0.009). Amongst AA patients, the number of Th2 cells and Foxp3+ Tregs were not significantly different from healthy controls. Following IST, the number of Tregs was significantly higher in responders than non responders (0.02 × 107/L v 0.001 × 107/L) (p=0.009). Interestingly the ratio of Th17/ Tregs was higher in non-responsive patients (15.2 v 2.8)(p=0.01). The number of T cell subsets, NK and B cells were not significantly different from healthy controls in our cohort of AA patients.The serum levels of proliferative cytokines, EGF (p=0.01), HGF(p=0.01), VEGF (p=0.01) and pro-inflammatory cytokines IL-13 (p=0.02), IL-8 (p<0.001) were significantly higher in AA patients than healthy controls and this was different from low risk MDS patients in whom the levels of IL-12 (P < 0.01), IL-7 (P < 0.005), IFN-γ (P < 0.01) and RANTES (P < 0.005) were elevated. ConclusionsOur data suggests that despite the presence of autoimmunity in both low risk MDS and AA, the involved mechanisms are significantly different. In AA the main mechanism is a Th1 derived (IFN-γ and TNF-αa production) response whereas in low risk MDS, Th17 cells may play an important role in creating a more indolent inflammatory environment. The serum cytokine profiles in these diseases are different too. These data also suggests a significant role for Tregs (absolute number as well as ratio of Th1 and Th17 to Tregs) in the prediction and evaluation of response to IST. DisclosuresNo relevant conflicts of interest to declare.

Hematogones are markedly reduced in pediatric acquired aplastic anemia: multiparametric flow cytometric analysis

Acquired aplastic anemia (AA) and myelodysplastic syndromes (MDS) are bone marrow (BM) failure syndromes with overlapping clinical features, and at least a subset appears to share common pathophysiologic mechanisms. Recent studies of MDS have shown down-regulation of genes involved in B-cell development and decreased B-cell precursors (hematogones). We explored the possibility that AA, similar to MDS, might also be associated with defects in development of lymphoid cells, especially B-cells, by using flow cytometry to assess the presence of hematogones and mature lymphocytes in BM samples from 25 children with AA and 41 age-matched controls. We observed that the percentage of total and early (stage I) hematogones were significantly decreased in AA compared to controls, and they returned to normal numbers after hematopoietic stem-cell transplant. This demonstrates early B-cell lineage involvement in AA, similar to recent findings in MDS. Our findings suggest dysfunction of an early multilineage progenitor in the pathogenesis of AA.

Aplastic Anemia: Pathophysiology and Treatment

An immune basis for most patients with aplastic anemia (AA) provides a rationale for immunosuppressive therapy (IST), using antithmyocyte globulin and cyclosporine as one therapeutic modality; hematologic response is observed in up to 75% of patients. Recent advances in understanding the pathogenesis of AA have identified defective telomere maintenance as an important explanation for the onset of marrow failure, relapse and clonal evolution after IST, in some patients with AA. The finding of inherited mutations in the telomerase gene complex in patients with apparent acquired AA has important implications for clinical management. Hematopoietic stem cell transplantation (HSCT) for acquired AA, whether from an HLA identical sibling or an unrelated donor, provides an excellent chance of long term cure. Current issues with HSCT include graft rejection, chronic GVHD and poor outcome in older patients. The lack of a suitable bone marrow donor for all patients who need a transplant, illustrates the need for novel transplant procedures, such as cord blood transplantation.