Immuno-related Pancytopenia Research Articles

Exclusive diagnosis of aplastic anemia

As an independent disease, aplastic anemia (AA) has been recognized for more than a century. When AA is diagnosed, other non-AA bone marrow failures should be excluded. It is termed as exclusive diagnosis of AA. The exclusive diagnosis of AA is helplessly based on that there is no parameter by which AA can be sensitively and specifically diagnosed now. So further searching for the meaningful diagnostic parameters of AA should be carried on to establish a direct diagnostic protocol of this disease and make it possible to differentiate it clearly from other bone marrow failure disease such as congenenital bone marrow failure, hypoplastic myelodysplastic syndromes, AA-paroxysmal nocturnal hemoglobinuria syndromes, large granules lymphocyte leukemia, clonal cytopenia of undetermined significance, immunorelated pancytopenia, acute hemopoietic arresting and idiopathic cytopenia of undetermined significance. The new markers and technologies being helpful for distinguishing AA from other bone marrow failures should be used in diagnosing AA. Correct understanding and application of exclusive diagnosis is not only related to the correctness of diagnosis and treatment of excluded diseases, but also to the quality of AA diagnosis, treatment and research.

Antibodies specific to ferritin light chain polypeptide are frequently detected in patients with immune‑related pancytopenia.

Immuno-related pancytopenia (IRP) is characterized by pancytopenia resulting from bone marrow suppression or destruction mediated by auto-antibodies. In our previous study, a K562 cDNA library was established, which was used to screen for seven possible auto-antigens produced by hematopoietic cells in patients with IRP, including ferritin light chain (FTL). In the present study, FTL was expressed and purified, and the levels of the auto-antibodies specific to FTL were measured. Through ELISA, it was shown that the titer of anti-FTL antibodies was higher in patients with IRP without treatment compared with those who had recovered from IRP, those with severe aplastic anemia (SAA), those with myelodysplastic syndrome (MDS) and the healthy controls. Furthermore, the expression levels of FTL-mRNA were upregulated in patients with IRP without treatment compared with those who had recovered from IRP, those with MDS and the normal controls. The results suggest that FTL antibody expression is upregulated in patients with IRP. Detecting FTL antibodies may therefore have certain clinical value in differentiating between IRP, SAA and MDS. Furthermore, in specific patients with IRP, FTL as an auto-antigen may induce immune attack on hematopoietic stem cells.

Roles of immune responses in the pathogenesis of immunorelated pancytopenia.

Some patients with pancytopenia do not conform to any diagnostic criteria of known haematological or non-haematological diseases; however, they respond well to corticosteroid, high-dose intravenous immunoglobulin and rituximab treatment. This abnormality is termed immunorelated pancytopenia (IRP). Later studies indicated that IRP might be a kind of autoimmune disease in which T helper (Th) type 2 cell function is enhanced, resulting in the hyperfunction of B lymphocytes, which then produce excess autoantibodies that attack the bone marrow (BM) and cause cytopenia. Hypofunction of regulatory T (Treg) cells and enhanced Th17 cell function, an elevated percentage of plasmacytoid dendritic cells (pDCs) and a decreased percentage of natural killer (NK) cells help to promote the process. Moreover, increased expression of a synergistic stimulator of B lymphocytes, CD70 and the reactive overexpression of the BCR inhibitory coreceptor CD22 also support this claim. Candidate autoantigens targeted by autoantibodies on haematopoietic cell membranes have also been reported in IRP. This review is focused on studies that demonstrate the role of immune responses in the pathogenesis of IRP. Current diagnostic criteria and treatments for IRP are also referenced to provide a thorough understanding. Distinguishing IRP from idiopathic cytopenias of undetermined significance (ICUS) and other haematological disorders, for example myelodysplastic syndrome (MDS), aplastic anaemia (AA), paroxysmal nocturnal hemoglobinuria (PNH) and Evans syndrome, may help patients with pancytopenia benefit from proper treatment. Further studies are required to achieve new insight into the pathophysiology of IRP with regard to the immune system, which will be instrumental for the development of novel therapies for inhibiting disease initiation and/or progression.

Effects of Epstein-Barr Virus Infection on CD19+ B Lymphocytes in Patients with Immunorelated Pancytopenia.

Objectives To explore effects of Epstein-Barr virus (EBV) infection on CD19+ B lymphocytes in patients with immunorelated pancytopenia (IRP). Methods An enzyme-linked immunosorbent assay (ELISA) in vitro diagnostic kit was used to detect EBV capsid antigen- (CA-) IgG and VCA-IgM antibodies in the serum. We analyzed the EBV-DNA copies of CD19+ B lymphocyte by using real-time quantitative polymerase chain reaction (RT-qPCR). CD21, CD23, CD5, CD80, and CD86 receptors on the surfaces of CD19+ B cells were detected by flow cytometry (FCM). The correlation between these receptors and EBV-DNA copies were evaluated. Results The results revealed that the positive rate of EBVCA-IgM and CD19+ B lymphocyte EBV-DNA copy in the IRP group were significantly higher than those in the control group (P < 0.05). CD19+ B lymphocyte EBV-DNA copies were also more abundant in IRP patients than in control subjects (P < 0.05). CD19+ B lymphocyte EBV-DNA copies were also more abundant in IRP patients than in control subjects (P < 0.05). CD19+ B lymphocyte EBV-DNA copies were also more abundant in IRP patients than in control subjects (Conclusions EBV infection may activate CD19+ B lymphocytes and further disrupt bone marrow hematopoiesis in IRP patients.

Screening novel autoantigens targeted by serum IgG autoantibodies in immunorelated pancytopenia by SEREX.

Immunorelated pancytopenia (IRP) is characterized by pancytopenia caused by autoantibody-mediated destruction or suppression of bone marrow. However, the autoantigens targeted by autoantibodies in IRP remain unclear. In the present study, we screened novel autoantigens in IRP by serological analysis of recombinant cDNA expression libraries and compared anti-UQCR10 antibody levels between IRP and normal controls detected by immunoblotting. Our results indicate that we successfully constructed the K562 cDNA library, which we used to screen seven candidate autoantigens expressed in haematopoietic cells of IRP: ferritin, light polypeptide, ubiquinol-cytochrome c reductase, complex III subunit X (UQCR10), multifunctional methyl-transferase subunit TRM112-like protein isoform 1 (TRMT112), hemoglobin gamma-G, stathmin 1 (STMN1), transcript variant 3, phosphoglycerate kinase 1 (PGK1), and trafficking protein particle complex subunit 4 (TRAPPC4). Six of 17 (35.29%) IRP patients exhibited positive reactivity to UQCR10 antigen, while only one of 10 (10%) of normal controls reacted to UQCR10 antigen. The IRP patients with positive reactivity to UQCR10 antigen exhibited significantly improved total efficiency (6/6) compared with those with negative reactivity (5/11). Thus, UQCR10 may be implicated as one of the autoantigens involved in development of IRP.

Single Nucleotide Polymorphism of Mitochondrial DNA D-LOOP Region in Peripheral Blood Lymphocytes of Immuno-related Pancytopenia Patients

To explore the single nucleotide polymorphism(SNP) of mitochondrial DNA (mtDNA) D-LOOP region in peripheral blood lymphocytes of immuno-related pancytopenia (IRP) patients and its correlation with immune parameters. The D-LOOP region in mitochondrial DNA of lymphocytes in peripheral blood mononuclear cells from 43 patients with untreated IRP was detected by polymerase chain reaction(PCR). The PCR products were sequenced by the pros and cons direct sequencing methods. The sequencing results were compared with the revised Cambridge reference sequence (rCRS) and the Polymorphic Sites of Human Mitochondrial Genome Database. Among total of 110 variant positions of D-LOOP region in 43 patients, 62 was SNP sites and 48 was mutation sites, of which 14 were the new mutation sites not yet registered in the database, 516 base variations were observed at 110 positions, the most common variations were base substitutions, among them T/C and A/G was 184/410 and 113/410 respectively. In the 110 variant positions, the high frequency variation sites were 73 and 263 for 43/43,311 for 32/43,310 and 16 224 for 27/43,16 519 for 25/43, 489 and 16 362 for 24/43. By the analysis of mitochondrial DNA D-LOOP polymorphism and related clinical immunology indicators of the patient's lymphocytes, it was found that D-loop in adult patients (age≥ 18 years old) significantly correlated with CD15 IgM, GLYCoA+ Cells IgM, CD34+ CellsIgG, CD34+ Cells IgM correlation. The high frequency of polymorphism exists in mitochondrial DNA D-loop region of lymphocytes in IRPA patients, and was significantly correlates with the autoantibodies in bone marrow mononuclear cells in adult patients, which may be associated with the IRP occurrence.

Auto-Antigens Targeted By Auto-Antibodies IgG on the Bone Marrow Hematopoietic Cells of the Patients with Immuno-Related Pancytopenia

Objective: To screen and identify the auto-antigens targeted by auto-antibodies IgG on the bone marrow hematopoietic cells of the patients with immuno-related pancytopenia(IRP).Background: Immune-related pancytopenia (IRP) is characterized by pancytopenia caused by autoantibody-mediated bone marrow destruction or suppression.. Our previous studies have demonstrated that DC2 increased in the bone marrow of IRP patients, which promote Th0 cells to polarize to Th2 cells and cause the over-function of B lymphocytes.These B cells produce auto-antibodies that mediated BM destruction.. But the antigens that induced DC2 elevated are not clear at present, our group found several target antigens by western blot method before, including G protein coupled receptor (GPCR) 156 variants, P chain of red blood cells with band three proteins, lactoferrin and WD repeat sequences of proteins.Autoimmune diseases often have several or even dozens of target antigens, so this experiment adopts the method of cDNA expression library of serological analysis (SEREX) to sceen antigens targeted by IRP autoantibodies and further verify their antigenicity.Materials and Methods: We established the recombinant cDNA expression libraries derived from K562 cells. We performed immunoblotting analysis of this library with sera from patients and normal healthy controls. The presumptive phage expressed autoantigen proteins were initially proposed based on the differential affinity to the sera antibodies of patients versus normal healthy controls,the plagues which were positive with IRP serum but negative with normal control serum were positive screened plagues. The insert fragment of each positive plague was sequenced. After the gene sequence confirmation, the sequence of FTL gene, TRMT112 gene and TRAPPC gene were amplified by polymerase chain reaction (PCR) then inserted into Peasy-E1 vector separately. Each recombinant plasmid was transformed into E.coli BL-21 and was identified by PCR and sequencing. After gene identification, the right recombinant plasmid was transformed into E.coli BL21 and the bacteria was induced by isopropyl-β-d-thiogalactoside (IPTG) to express. The expected protein was identified by Western blot and purified by ProteinIso Ni-NTA Resin. Then we coated ELISA plate with purified FTL protein and EPOR protein respectively, and detected the titer of the serum antibody of IRP patients and normal controls by indirect ELISA. Finally, the clinical index of 121 IRP patients were analysed.Results: 1.We established the recombinant cDNA expression libraries derived from K562 cells successfully. The library has good capacity, ligation efficiency and recombination efficiency. 2. We screend 11 positive plagues by immunoblotting, the seven of them were sequenced and blasted successfully. 3.3 fusion protein was expressed and confirmed by Western blot. The purified protein was obtained by ProteinIso Ni-NTA Resin. 4. The titer of antibody against the FTL and EPOR proteins was higher in untreated IRP than in recovered IRP patients and in normal control.Conclusion: We screened 7 proteins that maybe autoantigens of IRP by SEREX. Furthermore, we expressed and purified 3 fusion proteins. Finally we identified the antigenicity of the FTL and epor proteins. DisclosuresNo relevant conflicts of interest to declare.

Hypomethylation and overexpression of CD70 in CD4+ T cells of the patients with immuno-related pancytopenia

目的研究免疫相关性血细胞减少症(IRP)患者外周血CD4+ T淋巴细胞CD70 mRNA和蛋白的表达水平及CD70启动子甲基化水平，探讨CD70在IRP发病中的作用。方法选取35例IRP患者(初治组16例，恢复组19例)及15名正常对照，应用密度梯度离心法分离其外周血单个核细胞，免疫磁珠分选CD4+ T淋巴细胞，应用实时定量PCR (RT-PCR)检测CD70 mRNA相对表达水平，采用流式细胞术检测CD4+CD70+/CD4+细胞比例，采用甲基化特异性PCR (MSP)法检测CD4+ T淋巴细胞CD70基因启动子区域甲基化水平。结果IRP患者外周血CD4+CD70+/CD4+细胞比例初治组[(7.46±1.51)%]高于恢复组[(5.95±1.34)%]和正常对照组[(1.83±0.60)%]，恢复组高于正常对照组(P值均<0.05)；IRP初治组、恢复组及正常对照组CD70 mRNA的表达水分别为2.314(0.200~6.084)、1.021(0.135~3.434)及0.353(0.008~2.258)，两两比较差异均有统计学意义(P<0.05)。IRP患者CD70启动子甲基化水平低于正常对照组(P<0.05)。IRP患者CD70表达率与CD5+ B淋巴细胞比例呈正相关(r=0.533，P<0.01)。结论CD4+ T淋巴细胞CD70启动子区域低甲基化水平导致CD70的过表达可引起其免疫紊乱，可能在IRP的发病中发挥重要作用。

Erythropoietin Receptors and IgG Autoantibody Expression on Nucleated Erythrocytes in Some Cases of Immuno-Related Pancytopenia.

In recent years, we have detected autoantibodies in bone marrow (BM) hemopoietic cells in some patients with idiopathic cytopenia of undetermined significance (ICUS), termed immunorelated pancytopenia (IRP). However, we know little about the targets of these autoantibodies. Twenty-six newly diagnosed IRP patients with IgG autoantibody on nucleated erythrocytes and 20 healthy donors as controls were enrolled in this study. The serum erythropoietin (EPO) level was examined by ELISA. Expression of EPO receptor (EPOR) and IgG autoantibody on the membrane of nucleated erythrocytes were detected by flow cytometry before and after autoantibody stripping. The serum EPO level of the untreated patients was 199.9 ± 106.4 mIU/mL, which was significantly higher than that of normal controls (13.2 ± 8.41 mIU/mL, p < 0.01). EPOR expression on nucleated erythrocytes in the patients was 1.38 ± 0.73%, lower than that of normal controls (2.33 ± 1.73%), but there was no significant difference; EPOR in these patients was inversely correlated with IgG autoantibody on erythrocytes (r = -0.479, p = 0.013). The regression equation was Y = 0.116-0.479X; EPOR expression on the membrane increased significiantly (5.63 ± 4.99%, p < 0.01) after stripping the autoantibodies. After immunosuppressive treatment, median hemoglobin increased from 72 g/L to 98 g/L, and median reticulocytes increased from 1.46% to 3.56%. IgG autoantibodies might block or competitively inhibit EPOR on nucleated erythrocytes in some cases of ICUS.

Hypomethylation and Overexpression of CD70 in CD4+ T Cells of the Patients with Immuno-Related Pancytopenia

Objective To study the expression of CD 70 and the methylation level of CD70 promoter in immuno-related pancytopenia (IRP) patients, and explore the role of CD70 in the pathogenesis of IRP.Methods Thirty-five IRP patients and fifteen healthy donors were enrolled in this study. Peripheral blood mononuclear cells were isolated from venous blood by density gradient centrifugation and CD4+ T cells were isolated by immunomagnetic beads.The mRNA level and the percentage of CD70 of CD4+ T cells were measured by real-time quantitative polymerase chain reaction (RT-PCR) and flow cytometry respectively. Methylation-Specific PCR (MSP) was performed to determine the methylation level of CD70 promoter.Results The percentage of CD70 expression on CD4+ T cells of untreated IRP patients[(7.46±1.51)%] was significantly higher than that of recovered IRP patients [(5.95±1.34) %] and normal controls [(1.83±0.6)%] and the result of recovered IRP patients was significantly higher than that of normal controls (P<0.05).The relative expressions of CD70 mRNA in CD4+ T cells were [2.314(0.200~6.084)]A[1.021 (0.135~3.434)]A [0.353 (0.008~2.258)] in three groups respectively. The differences between untreated IRP patients,recovered IRP patients and normal controls were significant (P<0.05). The CD70 promoter methylation level in CD4+ T cells of all IRP patients was significantly lower than that of normal controls (p<0.05). The expression of CD70 positively correlated to the ratio of CD5+ B cells (r=0.533, p<0.001).Conclusions The overexpression of CD70 may lead to immunologic disarrangement and patients and it may play important role in the pathogenesis of IRP. DisclosuresNo relevant conflicts of interest to declare.

Telomere length of peripheral lymphocytes in patients with immuno-related pancytopenia

To investigate the changes of relative telomere length (RTL) of peripheral blood (PB) CD3⁺, CD3⁺CD4⁺, CD3⁺CD8⁺T lymphocytes, CD19⁺B lymphocytes and bone marrow (BM) CD34⁺ cells and its association with disease severity in untreated patients with immuno-related pancytopenia (IRP). The PB CD3⁺ , CD3⁺ CD4⁺ , CD3⁺ CD8⁺ T lymphocytes, CD19⁺ B lymphocytes, and BM CD34⁺ cells were purified by magnetic activated cell sorting (MACS), and RTL were measured with flow-fluorescence in situ hybridization (FLOW-FISH). The RTL of CD3⁺, CD3⁺CD4⁺ , and CD3⁺CD8⁺T lymphocytes in untreated IRP patients were (27.754 ± 16.323)%, (7.526 ± 3.745)% and (25.854 ± 14.789)%, respectivly, which were significantly shorter than those in healthy-controls (54.555 ± 19.782)%, (12.096 ± 2.805)%, and (38.367 ± 4.626)% (P<0.05). The RTL of CD19⁺ lymphocytes in untreated IRP patients was (22.136 ± 16.142)%, which was significantly shorter than that in healthy controls (42.846 ± 16.353)% (P<0.01). There was no significant difference of BM CD34⁺ cells RTL between the untreated IRP patients (22.528 ± 21.601)% and the healthy controls (23.936 ± 19.822)% (P>0.05). There were significantly positive correlations between the RTL of B lymphocytes and the count of white blood cell (r=0.706, P=0.015). There were negative correlations between RTL of B lymphocytes and the clinical symptoms (r=-0.613, P=0.045) and positive correlations with therapeutic effect (r=0.775, P=0.005). The shorter RTL of CD3⁺, CD3⁺CD4⁺, CD3⁺CD8⁺, CD19⁺ lymphocytes, and the normal RTL of BM CD34⁺ cells in untreated IRP patients were identified, which might imply that IRP is a type of acquired autoimmune diseases.

The Mechanisms Of Damage To Bone Marrow By Iron Overload In Patients With Immuno-Related Pancytopenia

ObjectiveTo investigate the mechanisms of iron overloading bone marrow damage in patients with immuno-related pancytopenia. MethodsForty-seven IRP patients (21 iron overloading IRP patients) and 10 normal controls were enrolled in this study. The expression of ROS, apoptosis, Bcl-2 and Caspase-3 of the bone marrow mononuclear cells (BMMNC) were analyzed by flow cytometry. Antioxidants were added to the iron overloading IRP BMMNC, and then the changes were detected by FCM. The number and apoptosis of the T lymphocytes of IRP patients were detected. ResultsThe ROS and apoptosis of the BMMNC, myelocytes, erythrocytes and stem cells in the bone marrow were significantly higher than those of non iron overloading IRP patients and normal controls(P<0.05). The expression of Bcl-2 in BMMNC, erythrocytes and stem cells of the iron overloading IRP patients were significantly lower than those of non iron overloading IRP patients (P<0.05). The levels of Caspase-3 in myelocytes, erythrocytes and stem cells of the iron overloading IRP patients were significantly higher than those of non iron overloading IRP patients or normal controls (P<0.05). After being treated with antioxidants, the expression of ROS, Caspase-3 and apoptosis of the iron overloading IRP BMMNC significantly decreased, the opposite of the Bcl-2. The percentage of the CD4+ lymphocytes (40.86±8.74)% and CD4+/CD8+ (1.44±0.36) in PB of the iron overloading IRP patients were significantly higher than those of non iron overloading IRP patients (35.96±7.03)% and (1.13±0.37)and normal controls(28.00±6.73)% and (0.79±0.21) (P<0.05), the opposite of CD8+ lymphocytes (P<0.05). The apoptosis of CD8+ lymphocytes (27.35±10.76)% and the ratio of CD8+ apoptosis /CD4+ apoptosis (2.51±0.80) in BM were significantly higher than those of the non iron overloading IRP patients (15.47±8.99)% and (1.39±0.47) (P<0.05). The apoptosis of the erythrocytes and stem cells coated with auto-antibodies in BM of the iron overloading IRP patients were significantly higher than those of the non iron overloading IRP patients. ConclusionIron overload damaging the bone marrow hematopoiesis might be through the following mechanisms. 1. The increased ROS induced by iron overload affected the expression of Caspase-3 and Bcl-2, which caused the higher BMMNC apoptosis; 2. The abnormal number and ratio of T lymphocytes caused by iron overload aggravate the abnormality of immunity of IRP; 3. Iron overload may increase the damage to erythrocytes and stem cells coated with auto-antibodies. Disclosures:No relevant conflicts of interest to declare.

Preliminary study on the quantity and function of T follicular helper cells in the cytopenic patients with positive BMMNC-Coombs test

To study the quantity and function of bone marrow (BM) T follicular helper (Tfh) cells of the cytopenia patients with positive bone marrow mononuclear cells (BMMNC)- Coombs test (also known as immuno-related pancytopenia, IRP), and explore the role of Tfh cells in the pathogenesis of IRP. Forty- three untreated IRP patients, 47 recovered IRP patients and 25 healthy donors were enrolled in this study. The percentages of Tfh cells, Tfh-related molecules ICOS, CD40L, IL-21 and Bcl-6 in BM were investigated by flow cytometry and semiquantitive RT-PCR. The ratio of CD4⁺CXCR5⁺/CD4⁺ cells of untreated IRP patients [(28.79 ± 19.70)%] was significantly higher than that of recovered IRP patients [(21.15 ± 12.81)% ] and normal controls ([ 13.42 ± 6.72)% ](P<0.05). The ratio of CD4⁺CXCR5⁺ICOS⁺/CD4⁺CXCR5⁺ cells of untreated IRP patients [(5.05 ± 4.71)% ] was significantly higher than that of recovered IRP patients [(2.96 ± 2.89)% ] and normal controls [(2.99 ± 2.23)% ] (P<0.05). The ratio of CD4⁺CXCR5⁺CD40L⁺/CD4⁺CXCR5⁺ cells of untreated IRP patients [(5.87 ± 4.14)%] and recovered IRP patients [(6.52±5.47)%] were significantly higher than that of normal controls [(2.93 ± 2.92)%] (P<0.05). The ratio of intracytoplasmic CD4⁺CXCR5⁺IL-21⁺/CD4⁺CXCR5⁺ cells of untreated IRP patients [(8.20 ± 7.41)% ] and recovered IRP patients [(6.30 ± 6.03)% ] were significantly higher than that of normal controls [(3.43 ± 3.40)%] (P<0.05). The relative expressions of Bcl-6 mRNA in BMMNC were 0.625 ± 0.248, 0.485 ± 0.253, 0.306 ± 0.210 in three groups, respectively. The differences between untreated IRP patients, recovered IRP patients and normal controls were significant (P<0.05). There exists increased quantity and hyperfunction of Tfh cells in the IRP patients, they may play important role in the pathogenesis of IRP. Tfh cells and their related effector molecules could be a potential therapeutic target for the disease.

Detection and analysis of autoantigens targeted by autoantibodies in immunorelated pancytopenia.

Previously, we described a group of patients with hemocytopenia who did not conform to diagnostic criteria of known hematological and nonhematological diseases. Most patients responded well to adrenocortical hormone and/or high-dose intravenous immunoglobulin treatment, indicating that cytopenia might be mediated by autoantibodies. Autoantibodies were detected on the membrane of various bone marrow (BM) hemopoietic cells by bone marrow mononuclear-cell-Coombs test or flow cytometric analysis. Thus, the hemocytopenia was termed “Immunorelated Pancytopenia” (IRP) to distinguish it from other pancytopenias. Autoantigens in IRP were investigated by membrane protein extraction from BM hemopoietic cells and BM supernatant from IRP patients. Autoantibody IgG was detected in the BM supernatant of 75% of patients (15/20), which was significantly higher than that in aplastic anemia, myelodysplastic syndrome, or autoimmune hemolytic anemia patients (0%) and normal healthy controls (0%) (P < 0.01). Autoantigens had approximate molecular weights of 25, 30, 47.5, 60, 65, 70, and 80 kDa, some of which were further identified by mass fingerprinting. This study identified that a G-protein-coupled receptor 156 variant and chain P, a crystal structure of the cytoplasmic domain of human erythrocyte band-3 protein, were autoantigens in IRP. Further studies are needed to confirm the antigenicity of these autoantigens.

Preliminary study of autoantigens on the membrane of erythropoietic cells of the patients with BMMNC-Coomb's test(+) hemocytopenia

To observe the relationship between erythropoietin receptor (EPOR) and autoantibodies-IgG/IgM (auto-Ab) on the membrane of erythropoietic cells of the patients with bone marrow mononuclear cells (BMMNC)-Coomb's test(+) hemocytopenia (immunorelated pancytopenia (IRP)) and explore the probable autoantigens of auto-Ab in IRP. A total of 46 newly diagnosed IRP patients (15 with auto-Ab on erythropoietic cells and 31 without) and 18 healthy controls were enrolled. The EPOR expressions on their nuclear erythrocytes were tested with flow cytometry (FCM) to observe the relationship between EPOR and auto-Ab. EPOR mRNA was detected by reverse transcription (RT)-PCR. Stat5 and P-Stat5 proteins in nucleated erythrocytes were measured by Western blot. EPOR expressions on nucleated erythrocytes membrane were re-tested after stripping autoantibodies with glycine buffer. (1) EPOR of auto-Ab(+) group (1.6% ± 0.9%)was significantly lower than that of auto-Ab(-) group (4.6% ± 4.1%, P < 0.01)and the latter was significantly higher than that of normal controls (2.3% ± 1.8%, P < 0.05). EPOR of IRP patients was inversely correlated with their auto-Ab (r = -0.543, P = 0.000). (2) EPOR mRNA of auto-Ab(+) group (0.68 ± 0.14)was significantly higher than that of auto-Ab(-) group (0.55 ± 0.12, P < 0.01) and normal controls (0.58 ± 0.12, P < 0.05). (3) Protein Stat5 of auto-Ab(+) group (1.45 ± 0.94) was significantly higher than that of normal controls (0.54 ± 0.36, P < 0.05). While P-Stat5 of auto-Ab(+) group (0.42 ± 0.18)was significantly lower than that of normal controls (0.85 ± 0.38, P < 0.05). (4) EPOR expression increased significantly after auto-Ab stripping. The auto-Ab of some IRP patients blocks or competitively inhibits EPOR on the membrane of erythropoietic cells. And EPOR may be one of autoantigens in IRP.

Detection and Analysis of Autoantigens on the Membrane of Bone Marrow Cells In the Patients with BMMNC- Coomb's Test(+) Hemocytopenia

AbstractAbstract 4434 ObjectiveTo investigate the autoantigens on the membrane of bone marrow cells in the patients with bone marrow mononuclear cell Coomb's(BMMNC-Coomb's) test(+) Immuno-related pancytopenia(IRP),for further purification and cloning. Methods20 newly diagnosed IRP patients, 10 AA patients, 10 MDS patients and 10 healthy donors as controls were enrolled in this study.The level of autoantibody-IgG in bone marrow was measured by Western blot; The autoantigens targeted by autoantibody IgG in IRP were identified from membrane protein extracts by SDS-PAGE and Western blot. ResultsThe level of autoantibody IgG in bone marrow of untreated IRP patients was 75% (15/20), which was significantly higher than that of AA or MDS patients (0%) and normal controls(10%)(P<0.01). Autoantibody IgG in IRP could react with several autoantigens with approximate MWs of 25–30kDa, 60–65 kDa, 73 kDa and 83 kDa. Positive rates of individual autoantigens were 35%,50%,20% and 10%,respectively. ConclusionAutoantibody IgG reacted with membrane antigens could be found in bone marrow in IRP patients. The molecular weights of the autoantigens were mainly 25–30kDa, 60–65 kDa, 73 kDa and 83 kDa. The result showed that autoantibody IgG played an important role in IRP. Disclosures:No relevant conflicts of interest to declare.

Preliminary Study of the Autoantigens on the Membrane of Erythropoietic Cells of the Patients with BMMNC- Coomb's Test(+) Hemocytopenia

AbstractAbstract 4435 ObjectiveTo observe the relationship between EPO receptor(EPOR) and autoantibodies-IgG/IgM (auto-Ab) on the membrane of erythropoietic cells of the patients with bone marrow mononuclear cell Coomb's (BMMNC-Coomb's) test(+) immuno-related pancytopenia(IRP), and then explore the probable autoantigens of auto-Ab in IRP. Methods46 newly diagnosed IRP patients (15 with auto-Ab on erythropoietic cells and 31 without auto-Ab on erythropoietic cells) and 18 healthy donors as controls were enrolled in this study. EPOR expression on their nuclear erythrocytes were tested with flow cytometry to observe the relationship between EPOR and auto-Ab; After sorting erythropoietic cells in bone marrow, EPOR mRNA and protein Stat5,P-Stat5 were investigated by RT-PCR and Western blot to observe the production of EPOR and EPO/EPOR signal transduction; Finally, EPOR expression on the membrane were tested again after stripping auto-Ab with glycine buffer. Results(1) EPOR of auto-Ab(+) arm(1.59±0.87)% was significantly lower than that of auto-Ab (-) arm(4.58±4.09)%(P<0.01), and the latter was significantly higher than that of normal controls (2.27±1.76)%(P<0.05); EPOR of IRP patients was negatively correlated with their auto-Ab (r=-0.543,P=0.000) and its regression equation was Y(EPOR)=0.040-0.335X(auto-Ab);(2)EPOR mRNA of auto-Ab(+) arm(0.685±0.136)was significantly higher than that of auto-Ab (-) arm(0.554±0.116)(P<0.01)and normal controls (0.580±0.119)(P<0.05);(3)Protein Stat5 of auto-Ab(+) arm(1.45±0.94) was significantly higher than that of normal controls (0.54±0.36)(P<0.05); While P-Stat5 of auto-Ab(+) arm(0.42±0.18) was significantly lower than that of normal controls (0.85±0.38)(P<0.05); (4) EPOR expression became higher while auto-Ab became lower after stripping with glycine buffer. ConclusionThe auto-Ab of some IRP patients might block or competitively inhibit the EPOR on the membrane of erythropoietic cells. EPOR was one of autoantigens in IRP. Disclosures:No relevant conflicts of interest to declare.

Preliminary Study of the Relationship Between EPO Receptor and Autoantibody on the Membrane of Erythropoietic Cells of the Patients with BMMNC- Coomb's Test(+) Hemocytopenia

AbstractAbstract 4436 ObjectiveTo observe the relationship between EPO receptor (EPOR) and autoantibody(IgG) on the membrane of erythropoietic cells of the patients with bone marrow mononuclear cell Coomb's(BMMNC-Coomb's) test(+) immuno-related pancytopenia(IRP),and then explore the probable autoantigens of autoantibodies in IRP. Methods42 newly diagnosed IRP patients (14 with IgG autoantibody on erythropoietic cells and 28 without this autoantibody) and 11 healthy donors as controls were enrolled in this study. EPOR on their nuclear erythrocyte were tested with flow cytometry. Their clinical data were also analyzed. Results(1) The level of EPOR of IgG(+) arm(1.59±0.87)% was significantly lower than that of IgG(-) arm (4.58±4.09)%(P<0.01), and the level of EPOR of IgG(-) arm was significantly higher than that of normal controls(2.27±1.76)%(P<0.05) G The level of EPOR of IRP patients was negatively correlated with their IgG autoantibody(r=-0.543,P=0.000) and its regression equation was Y(EPOR)=0.040-0.335X(IgG); (2) Reticulocyte percentage of IgG(+) arm (1.55±0.64)% was significantly lower than that of IgG(-) arm (2.41±1.42)%(P<0.05). The level of EPOR of IRP patients was positively correlated with reticulocyte percentage(r=0.346,P=0.029); (3)The serum level of indirect bilirubin(IDBIL) of IgG(+) arm was normal. While there were six patients (21.4%) whose serum level of IDBIL were higher than those of normal controls in IgG(-) arm. ConclusionThe autoantibody(IgG) of some IRP patients might block or competitively inhibit the EPOR on the membrane of erythropoietic cells. It could inhibit the proliferation and differentiation of erythropoiesis in early phase. Disclosures:No relevant conflicts of interest to declare.

CD5+B Lymphocytes in the Bone Marrow of Immunic Hemocytopenia Patients and Its Clinical Significancy.

Background A subpopulation of B lymphocytes expressing CD5 antigen is involved in several autoimmune diseases through the release of autoantibodies. We investigate the quantitative of CD5+ B lymphocytes in the bone marrow of the patients with autoimmunic hemocytopenia and study the relationship between quantity of CD5+B cells and clinical or laboratorial parameters of these patients.Methods Quantities of CD5+B lymphocytes in the bone marrow(BM)of 14 patients with autoimmune hemolytic anemia(AIHA) or Evans syndrome, 22 immunorelated pancytopenia (IRP) patients and 10 normal controls were assayed by FACS. The correlation between their clinical or laboratory parameters and CD5+B lymphocytes was analyzed.Results The qutantity of CD5+B lymphocytes of AIHA/Evans syndrome ((34.64±19.81)% or IRP((35.81±16.83)%) patients was significantly higher than that of normal controls((12.0±1.97)%)(p<0.05). There was no significant difference between those of AIHA/Evans syndrome and IRP patients(p>0.05). The quantity of CD5+B lymphocytes in the bone marrow of all cytopenic patients showed negative correlation with complement C3(r=−0.416, p<0.05). In AIHA/Evans syndrome patients, the quantity of BM CD5+ B lymphocytes showed positive correlation with indirect bilirubin(IBIL) (r=1.00, p<0.05); In Evans syndrome patients, the quantity of CD5+B lymphocytes in their bone marrow showed positive correlation with PAIgG (r=0.761, p<0.05) and PAIgM(r=0.925, p<0.05). The qutantity of CD5+B lymphocytes in the bone marrow of all cytopenic patients showed negative correlation with treatment response (correlation coefficient tau-b=−0.289, p<0.05), but no relation with the yields of CFU-E, BFU-E,CFU-F and CFU-GM BM in vitro.Conclusion BM CD5+B lymphocytes of the patients with autoimmunic hemocytopenias significantly increased and was related to the disease severity and clinical response. It was suggested that CD5+B lymphocytes might play an important role in the pathogenesis of autoimmunic hemocytopenia.