Analysis of Cytokine Profiling in Hepatitis-Associated Aplastic Anemia.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1036-1036
Author(s):  
Jun Lu ◽  
Susan Wong ◽  
Atanu Basu ◽  
Neal S. Young ◽  
Kevin E. Brown
Keyword(s):  
Bone Marrow ◽  
Liver Transplantation ◽  
Hepatitis B ◽  
Aplastic Anemia ◽  
Bone Marrow Failure ◽  
Rnase Protection ◽  
Rna Transcripts ◽  
Tnf Α ◽  
Significant Difference ◽  
Ifn Γ

Abstract In hepatitis-associated aplastic anemia (HAA), bone marrow failure follows an acute attack of seronegative hepatitis. Aplastic anemia (AA) is also associated with orthotopic liver transplantation for non-A, non-B, non-C hepatitis in young patients. Although the etiology of HAA is unknown, a clinical response to immunosuppressive therapy is observed in most clinical cases. The liver is the potential initial target organ of the immune response. We have previously determined the T-cell repertoire in the liver and peripheral blood lymphocytes in patients with HAA (Lu et al. Blood. 2004 Jun 15;103(12):4588–93). An antigen-driven T-cell expansion was implicated in the acute stage before bone marrow failure. In the present study, we measured serum cytokine levels in 22 HAA serum samples. Only interleukin (IL)-10 levels showed a significant increase compared to healthy controls (p<0.02); IFN-γ , TNF-α and IL-1, 2, 4, 5, 6, 8, 12 levels showed no significant difference. To further characterize the immune response in the liver, we performed RNAse protection assays and realtime PCR for RNA transcripts. In 4 HAA liver samples, 2 demonstrated increased IFN-γ by RNAse protection; TNF-α , IL-2, 3, 4, 5, 10, 15 transcripts were not different compared to transcript levels in 4 hepatitis B and/or C livers and 2 fulminant hepatitis (FH) samples. To confirm our preliminary data, we characterized the cytokine profile including CD4, CD8, CD69, TNF-α , IFN-γ and interleukins and compared them among the hepatitis B/C, FH and HAA liver samples by quantitative PCR for RNA transcripts. In 10 HAA liver samples, CD8 transcripts were increased and the CD4/CD8 ratio was decreased, but there was no significant difference between HAA and hepatitis B/C (p>0.05), confirming the important role of cellular immunity in HAA. IFN-γ transcripts were significantly overexpressed in both HAA and FH samples compared to hepatitis B/C samples (p<0.05), but without a difference between HAA and FH. CD69 transcripts were reduced in HAA but not significantly lower than in hepatitis B/C (p<0.05). There were no difference of TNF-α transcripts among HAA, hepatitis and FH samples (p>0.05). In summary, our data confirm that enhanced cellular immunity (increased IFN-γ level) is implicated in the pathogenesis of HAA, similar to that observed in the bone marrow of idiopathic AA, and in contrast to other liver diseases such as chronic hepatitis B/C. Quantitation of IFN-γ levels in acute hepatitis samples might be a predictive marker for the development of bone marrow failure after liver transplantation following seronegative hepatitis and FH.

scholarly journals The Effectiveness of Rapamycin Combined with Eltrombopag in Murine Models of Immune-Mediated Bone Marrow Failure

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 10-11
Author(s):  
Rong Fu ◽  
Shaoxue Ding ◽  
Xiaowei Liang ◽  
Tian Zhang ◽  
Zonghong Shao
Keyword(s):  
Bone Marrow ◽  
Nk Cells ◽  
Standard Dose ◽  
Bone Marrow Failure ◽  
Statistical Significance ◽  
Murine Models ◽  
Immune Mediated ◽  
Tnf Α ◽  
Significant Difference ◽  
Ifn Γ

Recent research has found that Rapamycin (Rapa) was an effective therapy in mouse models of immune-mediated bone marrow failure. However, it has not achieved satisfactory effect in clinical application. At present, many studies have confirmed that Eltrombopag (ELT) combined with IST can improve the curative effect of AA patients. Then whether Rapa combined with Elt in the treatment of AA will be better than single drug application. In this study, we tested efficacy of Rapa combined with Elt as a new treatment in mouse models of immune-mediated bone marrow failure. Compared with AA group, the whole blood cell count of Rapa+Elt group increased significantly (Figure 1A) (P<0.05). Survival of mice of Rapa+Elt group was significantly higher than that in the Rapa group (p <0.01)(Figure 1B).There was no obvious difference in the numbers of NK cells and their subsets were noted in Rapa group,CsA group and Rapa+Elt group.The expression of NKG2D on peripheral functional NK cells was up-regulated in CsA group, Rapa group and Rapa+Elt group compared with AA group (P<0.05). But there was no significant difference between effect of Rapa and CsA on the function of NK cells (Figure 1C).When Rapa combined with Elt, the expression of CD80 and CD86 are down-regulated more compared to Rapa group, but there is no statistical significance. Although these results suggested that Rapa+Elt had no statistical significance effect on numbers of mDC and expression of its functional molecule CD80 and CD86, the combined therapy still indicated that there is a potential synergy with immunosuppressant on AA mice to improve its outcome (Figure 1D).The results showed that CD4+/CD8+ ratio in CsA group, Rapa group, Rapa + Elt group had an obvious elevation than AA group (all P<0.05). But there were no significant difference among the three groups on the CD4+/CD8+ ratio (Figure 1E,1F). As for INF-gamma, Rapa can reduce the secretion of IFN-γ from CD8+T cells with efficacy similar to that of the standard dose of CsA, and had a better outcome when combined with Elt in bone marrow failure mice (Figure 1E,1G).CsA group, Elt group, Rapa group, Rapa + Elt group showed notable increased ratio of Tregs compared with AA group, among which there were only Rapa group, Rapa + Elt group showed statistical significance(P<0.05). for IL-10/Tregs ratio, Rapa group and Rapa +Elt group were superior to than CsA group(P<0.05) (Figure 1H,1I).Rapa+Elt group and Rapa showed more lower level of IFN-γ compared with CsA group, and there was significant difference in Rapa+Elt group(P<0.05). As for IL-10, IL-12p70, IL-2, IL-6, KC/GRO and TNF-α, the Rapa+Elt group showed more significant effect than Rapa or Elt alone(Figure1J). Thus, Rapa+Elt significantly down-regulated cytokines related to Th1 immune responses, such as IFN-γ, and upregulated cytokines related to Th2 immune responses, such as IL-10. To some extent, Rapa combined with Elt has a synergistic effect with CsA and Rapa alone in AA treatment. Conclusions In this study, Although Rapa combined with Elt had no significant improvement effect on the number and function of NK cells and their subsets, mDCs, and CD4+/CD8+ ratio in AA mice compared with Rapa alone, the Rapa+Elt can increase the secretion of IL-10 of Tregs and the number of Tregs, but has no significant effect on the number of Treg cells compared to with Rapa alone. Compared with AA group, the level of plasma IFN-γ, IL-2 and TNF-α decreased significantly (P<0.05), but IL-10, IL-4, IL-5 and IL-1β increased significantly in Rapa group(P<0.05). As for IL-10, IL-12p70, IL-2, IL-6, KC/GRO and TNF-α, the Rapa+Elt group showed more significant effect than Rapa alone. intervention treatment with Rapa in combination Elt in the AA mouse model more obviously ameliorated pancytopenia, improved bone marrow cellularity, and extended animal survival in a manner comparable to the standard dose of CsA and Rapa alone. Combination therapy support potential clinical utility in aplastic anemia treatment, which may further improve the efficacy of AA patients. Keywords: Rapamycin, Eltrombopag, murine models, bone marrow failure Figure 1 Disclosures No relevant conflicts of interest to declare.

scholarly journals Macrophage TNF-α licenses donor T cells in murine bone marrow failure and can be implicated in human aplastic anemia

Blood ◽  
2018 ◽  
Vol 132 (26) ◽  
pp. 2730-2743 ◽  
Author(s):  
Wanling Sun ◽  
Zhijie Wu ◽  
Zenghua Lin ◽  
Maile Hollinger ◽  
Jichun Chen ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Aplastic Anemia ◽  
Bone Marrow Failure ◽  
Murine Bone Marrow ◽  
Immune Mediated ◽  
Tnf Α ◽  
Donor T Cells ◽  
Ifn Γ

Abstract Interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) have been implicated historically in the immune pathophysiology of aplastic anemia (AA) and other bone marrow (BM) failure syndromes. We recently defined the essential roles of IFN-γ produced by donor T cells and the IFN-γ receptor in the host in murine immune-mediated BM failure models. TNF-α has been assumed to function similarly to IFN-γ. We used our murine models and mice genetically deficient in TNF-α or TNF-α receptors (TNF-αRs) to establish an analogous mechanism. Unexpectedly, infusion of TNF-α−/− donor lymph node (LN) cells into CByB6F1 recipients or injection of FVB LN cells into TNF-αR−/− recipients both induced BM failure, with concurrent marked increases in plasma IFN-γ and TNF-α levels. Surprisingly, in TNF-α−/− recipients, BM damage was attenuated, suggesting that TNF-α of host origin was essential for immune destruction of hematopoiesis. Depletion of host macrophages before LN injection reduced T-cell IFN-γ levels and reduced BM damage, whereas injection of recombinant TNF-α into FVB-LN cell-infused TNF-α−/− recipients increased T-cell IFN-γ expression and accelerated BM damage. Furthermore, infusion of TNF-αR−/− donor LN cells into CByB6F1 recipients reduced BM T-cell infiltration, suppressed T-cell IFN-γ production, and alleviated BM destruction. Thus, TNF-α from host macrophages and TNF-αR expressed on donor effector T cells were critical in the pathogenesis of murine immune-mediated BM failure, acting by modulation of IFN-γ secretion. In AA patients, TNF-α–producing macrophages in the BM were more frequent than in healthy controls, suggesting the involvement of this cytokine and these cells in human disease.

SAP (SH2D1A), the Immunomodulator Deficient in X-Linked Lymphoproliferative Syndrome (XLP), Is Profoundly Decreased in Aplastic Anemia: An Immunologic Link between Constitutional and Acquired Bone Marrow Failure.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1141-1141
Author(s):  
Elena E. Solomou ◽  
Valeria Visconte ◽  
Federica Gibellini ◽  
Neal S. Young
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Aplastic Anemia ◽  
T Cell Activation ◽  
Cell Activation ◽  
Bone Marrow Failure ◽  
Protein Levels ◽  
Ifn Γ ◽  
Th1 Polarization

Abstract Ligation of the signaling lymphocyte activation molecule (SLAM), a member of the immunoglobulin superfamily expressed in T and B cells, results in T cell activation and Th1 cytokine production. SAP is a small cytoplasmic protein expressed in T- and NK cells that controls the activation signals mediated by SLAM. On T cell activation, SAP binds to Fyn kinase; Fyn is activated and phosphorylates tyrosine residues on SLAM; phosphorylation results in the formation of a complex that selectively down-regulates co-stimulatory signals in activated T cells, resulting in inhibition of IFN-γ production. Thus SAP acts as a natural suppressor of SLAM-mediated T cell activation, and, in the absence of SAP, T cells are constitutively activated and overproduce IFN-γ. Mutations in the SAP gene lead to abnormal T cell activation and enhanced Th1 cytokine production in mouse models and in humans: about half of patients with X-linked lympoproliferative disease (XLP) have functionally disabling SAP mutations. Acquired aplastic anemia (AA) is a bone marrow failure syndrome in which hematopoietic cell destruction is effected by cytotoxic T cells and type 1 cytokines. We have recently shown that T cells from patients with AA have increased protein levels of T-bet, resulting in IFN-γ overproduction (Solomou EE et al, Blood2006; 107:3983). IFN-γ inhibits hematopoietic stem cell proliferation and induces Fas-mediated apoptosis; stem cell depletion results in marrow hypoplasia and peripheral blood pancytopenia. We examined SAP expression as an explanation for aberrant T cell activation and extreme Th1 polarization. SAP protein expression on immunoblot was very low to absent in unstimulated T cells from 16 of 20 AA patients examined, as compared to normal levels of expression in equivalent numbers of healthy donors (p<0.001). No significant differences were detected in Fyn and SLAM protein levels between AA and controls. SAP mRNA levels were also significantly decreased in T cells from those AA patients with low SAP protein levels, as determined by RT-PCR. Peripheral blood DNA samples from 18 patients with AA were analyzed for SAP mutations: three novel intronic mutations, not present in controls, were identified among 7 unrelated patients: one mutation was in the promoter region of SAP (position 106, C to T; 3 patients), and two mutations in the intron-exon junction between exons 1 and 2 (position 38975, C toT; 3 patients) and 3 and 4 (position 62771, C to A; 1 patient). IFN-γ, as measured by ELISA, in three patients with undetectable SAP protein levels was significantly increased compared to healthy controls (n=5, p<0.001). Increased IFN-γ levels and Th1 polarization in AA can in part be explained by functional SAP deficiency. SAP-deficient T cells in AA would be unable to block co-stimulatory signals, leading to an activated T cell phenotype and ultimately hematopoietic cell destruction and bone marrow failure. The SAP-deficient phenotype in T cells from patients with aplastic anemia may be secondary to subtle genetic alteration in the gene’s regulation (abnormal promoter binding sites or epigenetic modulation due to mutations in introns) or as yet unidentified aberrant upstream pathways (Ets-1 and Ets-2, the transcription factors that regulate SAP expression).

Study on Mechanism of Human Marrow Mesenchymal Stem Cell in Treating Patients with Aplastic Anemia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4099-4099
Author(s):  
Zhenhua Qiao ◽  
Xiujuan Zhao
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cells ◽  
Aplastic Anemia ◽  
Hematopoietic Cell ◽  
Control Group ◽  
Rt Pcr ◽  
Tnf Α ◽  
Ifn Γ ◽  
Tgf Β1

Abstract Objective: To explore mechanism of human marrow mesenchymal stem cells (MSCs) in treating patients with aplastic anemia(AA). Methods: MSCs in patients with aplastic anemia(AA) and the control group were separated with Percoll(1.073g/m L) and cultured in low glucose DMEM. Then, observed their morphologies,checked their molecule surface antigen by flow cytometry and examined the process of adipogenic differention. The mononuclear cells (MNC)of marrow in patients with AA were enriched based 1.077g/L density centrifuge and cultured in the 1640 medium. (1)MSC in control group and MNC in AA group were co-cultured with or without cytokines. The function of supporting hematopoiesis for MSC was to be observed in single confluence layer after plating by counting the total cells and the clones in every well every week. Then analyzed the dynamics of proliferation. T cells were harvested by using nylon column. MSC in control group and T cells in AA group were co-cultured. The proliferation of T cell was measured by MTT method. The CD25,CD69,CD4,CD8,Annexin-V expression rates of CD3+T cells were analyzed by flow cytometry .The gene and protein of IL-2, IL-4,IL-10,TNF-α,IFN-γ,TGF-β1 were examined by RT-PCR and ELISA respectively. MSC treated to the model of AA, by the examination of peripheral hemogram, bone marrow biopsy, pathological section of spleen. Results: There was no significant difference between control group MSC and AA-MSC in morphologies but adipogenic differentiation in AA patients is earlier than controls. The clones of CFU-GM in group(MSC)(78.46±3.58)/2×105 cells, after 14 days cultured was significantly higher than(9.21±4.32)/2×105 cells in group(CK + DMEM medium), while lower than (99.32±4.34)/2×105 cells in group(MSC+CK). (1)the Treg cells (TCD4+CD25+) in AA group (2.01±1.21)/ 2×105 was significantly lower than (4.43±1.67)/2×105 cells in control group, while(5.43±2.31) / 2×105 in group (MSC+AAT) was no more than (4.43±1.67)/2×105 cells in control group. (2) MSCs significantly inhibited T cell proliferation (P< 0. O5)by MTT. (3) RT-PCR and ELISA analysis showed that MSCs induced the expression of IL-4, IL-10, TGF-β1 and decreased significantly the expression of IL-2, TNF-α, IFN -γ in T cells of AA. the model of AA treated by MSCs showed improvements in 3 blood components greatly(p<0.05), Bone marrow proliferated and restored to the normal level, hematopoietic cell increased obviously (hematopoietic cell capacity was more than 40%), and atrophied spleen restore to normality. Conclusions: morphologies of AA’MSC had no evident different with the control but was more easy adipogenic differention. aplastic anemia belongs to autoimmune diseases in which T cells effect organ-specific destruction. The fundamental mechanism of MSC in treating AA should be potential to promote hematopoietic cell proliferation by adjusting immunity.

scholarly journals A multicenter trial of antithymocyte globulin in aplastic anemia and related diseases

Blood ◽  
1988 ◽  
Vol 72 (6) ◽  
pp. 1861-1869
Author(s):  
N Young ◽  
P Griffith ◽  
E Brittain ◽  
G Elfenbein ◽  
F Gardner ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Aplastic Anemia ◽  
Antithymocyte Globulin ◽  
Bone Marrow Failure ◽  
Multicenter Trial ◽  
Severe Aplastic Anemia ◽  
Transfusion Independence ◽  
Group I ◽  
Significant Difference ◽  
To Receive

One hundred fifty patients with bone marrow failure were treated in three groups with antithymocyte globulin (ATG; Upjohn, Kalamazoo, MI) in a multicenter trial. Patients were assessed at 3, 6, and 12 months after initiation of treatment by three criteria: transfusion independence, clinical improvement, and blood counts. Group I consisted of 77 patients with acute severe aplastic anemia, randomized to receive either ten or 28 days of ATG. There was no significant difference between the two arms of this protocol: 47% of all patients were clinically improved and 31% were transfusion independent at 3 months. Of the severely affected patients, 27% died before 3 months; most deaths occurred early in treatment. Factors associated with survival in severely affected patients included male sex, age less than 40 years, absolute neutrophil count greater than 200/microL, and idiopathic etiology. Neutrophil counts generally increased by 8 weeks after treatment, but patients continued to show improvement to 1 year posttreatment. In Group II, 44 patients with moderate or chronic severe aplastic anemia were randomized to receive either ten days of ATG or 3 months of high-dose nandrolone decanoate. No patient initially treated with androgens recovered, but 28% of ATG-treated cases achieved transfusion independence at 3 months. Group III consisted of patients with a variety of bone marrow failure syndromes. Patients with pancytopenia and cellular bone marrow showed response rates similar to those of patients with chronic or moderate aplastic anemia.

Disrupted Lymphocyte Homeostasis in Hepatitis-Associated Acquired Aplastic Anemia Is Associated with Very Low Telomere Lengths

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2408-2408
Author(s):  
Anne-Laure Grignon ◽  
Daria V. Babushok ◽  
Li Yimei ◽  
Helge Hartung ◽  
Ho-Sun Lam ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Aplastic Anemia ◽  
Telomere Length ◽  
Disease Severity ◽  
Bone Marrow Failure ◽  
Genetic Defect ◽  
Telomere Maintenance ◽  
Wilcoxon Test ◽  
Significant Difference ◽  
Lymphocyte Homeostasis

Abstract Acquired aplastic anemia (AA) is a hematologic disorder characterized by low blood counts and a hypocellular bone marrow, caused by autoimmune destruction of early hematopoietic cells. The diagnosis of AA is made by excluding other disorders that can present with bone marrow failure (BMF). Such disorders include dyskeratosis congenita (DC), a multisystem BMF syndrome, caused by an inherited defect in telomere maintenance. Although classical DC presents in childhood with stereotypical mucocutaneous changes, milder forms of telomere dysfunction associated with mutations in TERT and TERC genes can present with non-syndromic bone marrow failure clinically indistinguishable from AA. In clinical practice, lymphocyte telomere length measurements are used as a first-line screen for inherited telomeropathies before initiating treatment for AA. In our BMF center, we have observed that several patients with features of hepatitis-associated AA (HAA) had lymphocyte telomere lengths at diagnosis at or below the first percentile of age-matched controls, in the range similar to inherited telomere disorders. To confirm our initial observation, we performed a retrospective analysis of telomere lengths of consecutively enrolled HAA patients with non-hepatitis associated AA patients in our institution. A total of 30 patients with AA were included in this study: 10 had HAA and 20 had other AA (Table 1). The median age at telomere testing was 8.0 years (range 1-19 years). There was no significant difference in age or disease severity between the two groups (p=0.827). The patients' median lymphocyte telomere length (TL) was significantly lower in the HAA patients compared to AA (7.4kb versus 9.1kb, P= 0.021); the difference remained significant after adjusting for patient age (p<0.001). Strikingly, 5 of 10 HAA patients had telomeres at or below the 1st percentile of age-matched normal controls, within the diagnostic range for telomeropathies (Figure 1A). None of these 5 patients had clinical features of DC. As a comparison, TL measurements of genetically-confirmed DC patients (Figure 1) demonstrated TL below the 1st percentile for age-matched controls, and within a similar range to that seen in the HAA patients. To ensure that the significantly lower telomere lengths in the HAA patients were not caused by an occult TERT or TERC gene mutation, the five HAA patients with TL below the 1st percentile were screened for germline mutations in TERT and TERC. A known heterozygous polymorphism, Ala1062Thr was found in one patient, a known variant with no known telomere defect and no effect on telomere length. Because differences in lymphocyte activation and subset composition are known to impact telomerase activity, we hypothesized that alterations in lymphocyte populations caused by the unique inflammatory state of HAA could partly account for significantly shorter TL in this population. HAA patients exhibited significantly lower absolute lymphocyte counts and lower lymphocyte subsets across the board, as well as the decreased CD4/CD8 ratio compared to non-hepatitis AA patients (Figure 2). The median telomere length in the two groups was significantly correlated with lymphocyte counts (Pearson correlation coefficient 0.52, p=0.003). An altered lymphocyte homeostasis such as the one characteristic for HAA limits the specificity of telomere measurements as a screening method to identify patients with AA due to a genetic defect in telomere maintaining genes. As such, short telomeres in HAA in the absence of other features suggestive for DC does not necessarily warrant genetic testing for telomere length. Longitudinal studies of telomeres and study of clonal hematopoiesis in this population is ongoing. Table 1. Overall (n = 30) AA (n=20) HAA (n=10) P value* Patient Characteristic Gender, female n (%) 12 (40) 9 (45) 3 (30) 0.694 Gender, male n (%) 18 (60) 11 (55) 7 (70) Age at diagnosis, y, median (range) 8.0 (1-19) 8.3 (1-19) 7.5 (3-17) 0.827 Disease Severity, n (%) 0.999 Moderate 6 (20) 4 (20) 2 (20) Severe 22 (73) 15 (75) 7 (70) Very Severe 2 (7) 1 (5) 1 (10) Median Lymphocyte Telomere Length, kb (range) 8.9 (5.9-11.3) 9.1 (7.5-11.3) 7.4 (5.9-9.8) 0.021 ≤ 1st percentile of age-matched controls 5 0 5 1-10th percentile of age-matched controls 5 5 0 > 10th percentile of age-matched controls 20 15 5 *P-values are obtained by Fisher's exact test for gender and disease severity and by Wilcoxon test for age and telomere length. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures No relevant conflicts of interest to declare.

scholarly journals The Effectiveness of Rapamycin Combined with Eltrombopag in Murine Models of Immune-Mediated Bone Marrow Failure

2020 ◽  
Vol 2020 ◽  
pp. 1-10
Author(s):  
Shaoxue Ding ◽  
Xiaowei Liang ◽  
Tian Zhang ◽  
Rong Fu
Keyword(s):  
Bone Marrow ◽  
Combination Therapy ◽  
Mouse Model ◽  
Mouse Models ◽  
Bone Marrow Failure ◽  
Murine Models ◽  
Curative Effect ◽  
Immune Mediated ◽  
Tnf Α ◽  
Significant Difference

Severe aplastic anemia (SAA) is a rare disease characterized by severe pancytopenia and bone marrow failure. Most patients with AA respond to immunosuppressive therapy (IST), usually as antithymocyte globulin (ATG) and cyclosporine (CsA), but some relapse on CsA withdrawal or require long-term administration of CsA to maintain blood counts. Recent research has found that rapamycin (Rapa) was an effective therapy in mouse models of immune-mediated bone marrow failure. However, it has not achieved a satisfactory effect in clinical application. At present, many studies have confirmed that eltrombopag (ELT) combined with IST can improve the curative effect of AA patients. Then, whether Rapa combined Elt in the treatment of AA will acquire better efficacy than a single drug application remains unclear. In this study, an immune attack-mediated AA mouse model was constructed by total body irradiation (TBI) and allo-lymphocyte infusion. In our study, we tested the efficacy of Rapa combined with Elt as a new treatment in mouse models of immune-mediated bone marrow failure. It showed that treatment with Rapa in combination Elt in the AA mouse model ameliorated pancytopenia and extended animal survival in a manner comparable to the standard dose of CsA and Rapa alone. However, there was no significant improvement effect on the number and function of NK cells and their subsets, mDCs, and CD4+/CD8+ ratio in AA mice after the therapy of Rapa combined with Elt compared with Rapa alone. Furthermore, the secretion of IL-10 of Tregs in AA mice increased significantly after the therapy of Rapa combined with Elt, but there was no significant difference in the number of Treg cells. We did not observe the difference in the curative effect of the Rapa group and CsA group, but for IL-10/Tregs ratio, the Rapa group was superior to the CsA group. And the IFN-r secretion of CD8+T cells in AA mice decreased significantly after the combination therapy of Rapa and Elt than Rapa alone. Compared with the AA group, the level of plasma IFN-γ, IL-2, and TNF-α decreased significantly ( P < 0.05 ), but IL-10, IL-4, IL-5, and IL-1β increased significantly in the Rapa group ( P < 0.05 ). As for IL-10, IL-12p70, IL-2, IL-6, KC/GRO, and TNF-α, the therapy of Rapa combined with Elt showed a more significant effect than Rapa alone in AA mice. To some extent, this study had shown a relatively better synergistic effect in murine models of immune-mediated bone marrow failure after the combination therapy of Rapa and Elt, which was a promising clinical utility in SAA treatment.

Experiment Therapy of Aplastic Anemia Using Cyclosporin A Combined with IDO Agonist.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1695-1695
Author(s):  
Lifang Huang ◽  
Min Dai ◽  
Wenli Liu ◽  
Jianfeng Zhou ◽  
Hanying Sun ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Cyclosporin A ◽  
Aplastic Anemia ◽  
Immune Tolerance ◽  
Blood Cells ◽  
Bone Marrow Failure ◽  
Combination Group ◽  
Ifn Γ

Abstract Aplastic anemia(AA) is an unusual hematologic disease and the paradigm of the human bone marrow failure syndromes. Abnormal immunological responses have been found in most aplastic patients. Activated type 1 cytotoxic T cells may be the main contributing factor of pathogenesis. IDO is a rate-limiting enzyme in Tryptophan(Trp) metabolism. The proliferation of Th1 cells is specifically inhibited by the over-expression of IDO, which can degrade the Trp in local environment, hamper the conductioin of the activating signal in T cell and induce immune tolerance. 3,4-DAA is an IDO agonist and can activate IDO. In our experiment, Balb/c mice were irradiated (5.0Gy of 60Co), and then infused with 5×106 lymph node(LN) cells from DBA/2 mice in 4 hours. Blood count was monitored and marrow damage was assessed by histogical study. Concentrations of serum IFN-γ were measured by ELISA. The levels of Tryptophan and kynurenine were evaluated by high performance liquid chromatography (HPLC). CD4+CD25+ T cells in spleen were analyzed by flow cytometry. The level of Foxp3 in CD4+CD25+ T cell was measured by RT-PCR. Irradiation and infusion of LN cells led to rapid development of severe pancytopenia and bone marrow hypoplasia. Bone marrow of affected mice showed lymphocyte infiltration. Serum IFN-γ concentration increased 3.7 fold at d6 post infusion. The recipient mice were divided into 4 different treating groups as follow: 0.9% Sodium Chloride as control; Cyclosporin A (CsA) (50ug/g/d ×5d, peritoneal injection); 3,4-DAA(5mg/d, orally daily); the combination of CsA and 3,4-DAA. The effects of CsA, IDO agonist and CsA combined IDO agonist were analyzed at day 6,10,14,21,24 and 28 after LN cells infusion. The white cell and the platelet recovered to near normal, respectively (4.2±0.32)×109, (937±190.47)×1012 at d21 in the combination group. Early stage treatment with CsA can improve periphery blood cells and BM nucleated cells, but long term effect was not remarkable. In contrast, the 3,4-DAA group exhibited slow and gradually enhanced role. Periphery blood cells and BM nucleated cells were improved remarkably in the combination group. The number and function of CD4+CD25+T cells also increased remarkably. In the treatment of AA, abnormal immune response in bone marrow was inhibited by CsA, meanwhile immune tolerance was induced through up-regulating the regulatory T cells(Treg) by 3,4-DAA. In this way, the balance of immune in bone marrow could be reestablished quickly. CsA combined IDO agonist could provide a new strategy for the management of AA.

Bone Marrow Cellular Composition and Inflammatory Cytokine Expression in Patients with Inherited Bone Marrow Failure Syndromes

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4401-4401
Author(s):  
Neelam Giri ◽  
Ken Matsui ◽  
Blanche P Alter ◽  
Sharon A Savage ◽  
Yuanji Pan ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
B Cells ◽  
Mononuclear Cells ◽  
Bone Marrow Failure ◽  
Somatic Mosaicism ◽  
Bone Marrow Failure Syndromes ◽  
Cd34 Cells ◽  
Tnf Α ◽  
Ifn Γ

Abstract Abstract 4401 Proinflammatory cytokines, TNF-α and IFN-γ, are potent inhibitors of hematopoiesis, and may be relevant in the pathogenesis of bone marrow failure in inherited bone marrow failure syndromes (IBMFS). Increased levels of these cytokines in sera and in bone marrow CD3+ cells have been reported in Fanconi anemia (FA) patients. However, our study did not find increased TNF-α or IFN-γ in sera, or supernatants from phytohemagglutinin-stimulated peripheral blood mononuclear cells from IBMFS patients. To assess whether production of these cytokines is dysregulated in BM of these patients, we examined intracellular expression of TNF-α and IFN-γ in BM mononuclear cells from 16 FA, 20 dyskeratosis congenita (DC), 21 Diamond-Blackfan anemia (DBA) and 7 Shwachman-Diamond syndrome (SDS) patients by flow cytometry; 14 healthy adults were studied as controls. To detect intracellular TNF-α and IFN-γ, BM lymphocytes and monocytes were stimulated with phorbol 12-myristate 13-acetate plus ionomycin (P+I), or lipopolysaccharide (LPS), respectively. Separately, unstimulated cells were stained with antibodies to CD45, CD3, CD19, CD14, and CD34 to determine the proportion of cellular subsets. Percentages of T cells in patients with IBMFS were comparable to the controls, while DC patients had lower proportion of B cells (p=0.02). The percentages of monocytes were lower in FA (p=0.04), DC (p=0.009), and DBA (p<0.001) patients. The proportions of CD34+ cells were also lower in IBMFS patients (≤0.02 for all) except for those with DBA, who had similar proportions as the controls. When we compared the effect of cytopenia (counts below normal for age), only the proportion of CD34+ cells in DC patients was significantly affected. DC patients with cytopenia (n=15) had lower numbers of CD34+ cells (p=0.007) compared with those without (n=5). We also analyzed the effect of somatic mosaicism in FA because it may correct the hematopoietic defect in these patients. FA patients without mosaicism (n=11) had lower proportions of CD19+, CD14+, and CD34+ cells than those with mosaicism (n=5), while the CD3+ cell numbers were unaffected. We detected both intracellular TNF-α and IFN-γ in T cells, but only TNF-α in B cells in response to P+I, while LPS stimulation led to TNF-α production only in monocytes. Percentages of cytokine-producing T and B cells were significantly lower for patients with DBA when compared with healthy adult controls (p<0.006 for T cells and p=0.001 for B cells). There were no significant differences in the other syndromes. Comparison of intracellular cytokines between cytopenic and non-cytopenic patients showed that TNF-α-producing T cells were affected in FA (p=0.03), where the cytopenic patients had a higher proportion of TNF-α-positive T cells. For the LPS-stimulated monocytes, FA (p=0.01) and DBA (p=0.05) patients had significantly lower proportions of TNF-α-producing cells than the controls, and this was independent of cytopenia. There was no effect of mosaicism on cytokine production. Contrary to previous reports, we did not find an increase in intracellular TNF-α or IFN-γ in T cells from FA patients. However, the number of TNF-α-producing monocytes in FA was lower than that in healthy adult controls. This is consistent with reported dysregulation of monocytes in FA patients. We also identified reduced cytokine expression in lymphocytes and monocytes from DBA patients, but not from DC or SDS. As expected, we found reduced proportions of CD34+ cells in FA, DC and SDS, syndromes associated with multilineage cytopenia, and not in DBA which is associated with pure red cell aplasia. And, we ascertained that FA patients with somatic mosaicism had significantly higher percentages of cells including CD34+, suggesting that the corrected stem cell pool in FA mosaics is able to maintain hematopoiesis in contrast to non-mosaic FA patients who develop progressive cytopenia over time. Overall, the effect of cytopenia on cytokine production was mild; however, this may be related to the small sample size. In conclusion, our results suggest that mechanisms other than an excess of inflammatory cytokines may be responsible for bone marrow failure in IBMFS, and this area of research deserves a further attention in larger studies. Disclosures: No relevant conflicts of interest to declare.

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