Regulatory role of ceRNA network in B lymphocytes of patients with immune thrombocytopenia.

OBJECTIVE: High-throughput sequencing was used to screen expressing differences of miRNA, lncRNA, and mRNA in CD19+ B peripheral blood samples of newly diagnosed immune thrombocytopenia (ITP) patients and healthy controls. The study aimed to explore the regulatory role of ceRNA network in the pathogenesis of dysfunctional CD19 + B lymphocytes of ITP patients. METHODS: CD19+ B lymphocytes were isolated from peripheral blood samples of ITP patients and their healthy counterparts. High-throughput sequencing was used to screen for the expression of miRNA, lncRNA, and mRNA of ITP patients and healthy controls, which were analysed by the ceRNA network. Moreover, qPCR was used to verify the differential expression of miRNA, lncRNA, and mRNA in ITP patients and healthy controls. The correlation between differentially expressed miRNA, lncRNA, mRNA, and B lymphocyte subsets was also analysed. RESULTS: The CD19+ B lymphocytes of 4 newly diagnosed ITP patients and 4 healthy controls were sequenced and analysed. There were 65 differentially expressed lncRNA and 149 mRNA forming a ceRNA network showed that 12 lncRNA and 136 differentially expressed mRNA were closely associated. Similarly, miR-144-3p, miR-374c-3p, and miR-451a were highly expressed in ITP patients, as confirmed by qPCR, which was consistent with the high-throughput sequence results. LOC102724852 and CCL20 were highly expressed in ITP patients, while LOC105378901, LOC112268311, ALAS2, and TBC1D3F were not as compared to healthy controls, which was consistent with the high-throughput sequence results. In addition, the expression of miR-374c-3p, LOC112268311, LOC105378901, and CXCL3 were correlated with the percentage of B lymphocyte subsets. CONCLUSIONS: The ceRNA network of miRNA, lncRNA, and mRNA in peripheral CD19 + B lymphocytes plays an essential role in the pathogenesis of ITP.

Interleukin 6 exacerbates the progression of warm autoimmune hemolytic anemia by influencing the activity and function of B cells.

To explore the effect of IL-6 on the activity and secretory function of B cells and analyze its effect on clinical indicators and efficacy in wAIHA patients. This study included 25 hemolytic wAIHA patients, 13 remission patients, and 10 HCs. Plasma levels of various cytokines were detected using CBA. PBMCs were extracted from 12 hemolytic wAIHA patients and divided into three wells, stimulation with IL-6 and IL-6 + tocilizumab, the blank control wells were also set. After 48 h of in vitro cell culture, percentage of CD5+CD80+, CD5-CD80+,CD5+CD86+,CD5-CD86+,CD5+IL-10+,CD5-IL-10+B cells were determined by flow-cytometry. Plasma levels of IL-6 and IL-10 in hemolytic episode group were significantly higher than that in HCs group (p = 0.0243; p = 0.0214). RBC and Hb levels were negatively correlated with IL-6 levels in wAIHA patients, while LDH levels were positively correlated.Therapeutic effects of glucocorticoid and duration of efficacy were also significantly correlated with IL-6 levels in wAIHA patients. After 48 h in vitro cell culture, percentages of CD80+/CD5+CD19+and CD80+/CD5-CD19+ cells in the IL-6 stimulation group were higher than those in blank control group (p = 0.0019; p = 0.0004), while CD86+/CD5+ CD19+ and CD86+/CD5-CD19+ cells were not statistically different before and after IL-6 stimulation. Percentage of IL-10+/CD5+ CD19+ cells in IL-6 stimulation group was lower than that in blank control (p = 0.0017) and IL-6 + toc (p = 0.0117) group. Percentage of IL-10+/CD5- CD19+cells in the IL-6 stimulation group was lower than that in the blank control group (p = 0.0223). Plasma levels of IL-6 were significantly elevated in hemolytic wAIHA patients and correlated with clinical indicators and efficacy. IL-6 promotes the activation of B cells. Although the results were not statistically significant, IL-6R antagonist tocilizumab may hopefully become a targeted therapy for wAIHA patients.

Genome-wide DNA methylation profiles analysis in primary warm autoimmune hemolytic anemia patients.

BACKGROUND: Autoimmune hemolytic anemia (AIHA) is caused by auto-antibodies, secreted by overactivated B cells, directed against self-red blood cells, resulting in hemolysis. It found that aberrant DNA methylation in B cells can induce the production of autoantibodies. Therefore, we attempted to explore if similar aberrant DNA methylation occur in AIHA patients. METHODS: A 49-year-old female wAIHA patient and a 47-year-old female healthy control (HC) were enrolled. Peripheral blood (PB) B cells DNA was extracted. After constructing genomic libraries, bisulfite genomic sequencing (BSP) and DNA methylation profiles were analyzed. BSP was verified using PB B cells from 10 patients with hemolysis, 10 patients with hemolytic remission, and 10 healthy controls (HCs) by Methylation-specific PCR. RESULTS: Total DNA methylation of whole-genome C bases (4.8%) and CG type bases (76.8%) in wAIHA patient were lower than those in the HC (5.3 and 82.5%, respectively) (p = 0.022 and p < 0.001). DNA methylation of C bases and CG type bases in whole-genome regulatory elements, such as coding sequence, up2Kb and down2Kb in the patient were also lower than those in the HC (p = 0.041, p = 0.038, and p = 0.029). 30,180 DNA-methylated regions (DMRs) on all 23 chromosomes were identified. DMR-related genes were mainly involved in the Rap1, phospholipase D, HIF-1, calcium, vascular endothelial growth factor (VEGF) and Ras signaling pathways. CONCLUSION: The DNA methylation spectrum of B cells in AIHA patients is different from that of HC, and the proportion of hypo-methylation regions is higher than that of HC. DMR-related genes are mainly related to some signaling pathways.

Iron metabolism abnormalities in autoimmune hemolytic anemia and Jianpishengxue keli can ameliorate hemolysis and improve iron metabolism in AIHA mouse models.

OBJECTIVE: Autoimmune hemolytic anemia (AIHA) is rare heterogeneous disorder characterized by red blood cell (RBC) destruction via auto-antibodies, and after RBC is destroyed, proinflammatory danger-associated molecular patterns including extracellular hemoglobin, heme, and iron which causing cell injury. And oxidative stress represents one of the most significant effects of chronic hemolysis. Jianpishengxue keli can improve the symptoms of anemia patients with kidney disease and tumors and are beneficial in promoting recovery from chronic inflammation. Therefore, it is presumed that Jianpishengxue keli can improve the symptoms of AIHA. We aimed to investigate iron metabolism in AIHA and effects of Jianpishengxue keli on AIHA murine model. METHODS: Nineteen hemolytic episode AIHA patients, 10 remission patients and 10 healthy controls (HCs) were enrolled in this study. Serum hepcidin, ferritin and other related indicators of iron metabolism were measured. Mouse models of AIHA were established and received high, medium, or low doses of Jianpishengxue keli by gavage daily for 14 and 28 days respectively. The level of RBCs, Hb, bilirubin, LDH, hepcidin, and the expression level of hepcidin mRNA, and hepatic ferroportin 1(FPN1) protein were evaluated. RESULTS: Serum hepcidin in hemolytic episode AIHA patients and remission patients were significantly higher than that in HCs (p = 0.0083 and p = 0.0473, respectively). Serum ferritin in hemolytic AIHA patients was significantly higher than that in HCs (p = 0.008). Serum transferrin saturation levels are increased in patients with AIHA[ (57.21 ± 8.96) %]. EPO in hemolytic group was higher than that in healthy control (p＜0.05). In AIHA mouse models, IBIL decreased after 14 days of high dose drug intervention. After 28 days, TBIL and IBIL both significantly decreased in all dose groups and LDH significantly decreased in the medium-and high-dose groups. Body weight improved, and the level of RBCs, Hb and hepcidin in the high-dose group returned to normal. After 14 and 28 days of intervention, hepatic hepcidin mRNA in all dose group significantly decreased. Hepatic FPN1 protein which were significantly lower in the AIHA mouse models, increased in all dose groups after drug intervention for 28 days. CONCLUSION: Iron metabolism abnormalities exists in AIHA patients and Jianpishengxue keli can ameliorate hemolysis and improve iron metabolism in AIHA mouse models.KEY MESSAGESIron metabolism abnormalities exists in hemolytic episode AIHA patients. Hepcidin and ferritin levels significantly elevated and also correlated with the severity of AIHA patients. Jianpishengxue keli can ameliorate hemolysis and prompt the recovery of AIHA.

Retroversion of the hemipelvis rather than hypoplastic posterior wall decreases acetabular anteversion in hips affected by Perthes disease.

The acetabular retroversion has a moderate incidence of 31-60% in all patients of the Perthes disease. It might be caused by posterior wall dysplasia based on recent animal researches. However, some studies support that hemipelvic retroversion is the main factor for the acetabular retroversion. The primary pathological factor of increasing retroversion angle is still controversial anatomically. This study aimed to identify whether there is acetabular retroversion in children with Perthes disease,and to find a method to distinguish version types. Forty children with unilateral Perthes disease who were admitted to our hospital from January 1, 2012 to December 31, 2018 were enrolled, and 40 controls were matched based on sex and age. The acetabular anteversion angle (AAA), internal wall anteversion angle (IWAA), anterior wall height of the acetabulum (A), acetabular posterior wall height (P), and acetabular width (W) were assessed on computed tomography (CT) at the level of the femoral head center. The acetabular wall difference index (AWDI; AWDI = P-A)/W*100) was calculated. The mean AAA was significantly lower in Perthes disease hips (10.59 (8.05-12.46)) than in contralateral hips (12.04 (9.02-13.33)) (p = 0.002) but did not differ from control hips (9.68 ± 3.76) (p = 0.465). The mean IWAA was significantly lower in Perthes hips (9.16 ± 3.89) than in contralateral hips (11.31 ± 4.04) (p = 0.000) but did not differ from control hips (9.43 ± 3.82) (p = 0.753). The mean AWDI did not differ between Perthes hips (0.41 ± 4.94) and contralateral hips (- 1.12 (- 4.50, 2.17)) (p = 0.06) or control hips (- 0.49 ± 5.46) (p = 0.437). The mean W was significantly higher in Perthes hips (44.61 ± 5.06) than in contralateral hips (43.36 ± 4.38) (p = 0.000) but did not differ from control hips (45.02 ± 5.01) (p = 0.719). The mean A and P did not differ between Perthes hips and contralateral hips or control hips. Correlation analysis of all hip joints revealed a significant correlation between AAAs and IWAAs (r = 0.772; r = 0.643; r = 0.608; and r = 0.540). Linear regression analysis revealed that AAAs increased with IWAAs. Multiple linear regression showed that IWAAs and AWDIs have good predictive value for AAAs in both Perthes and control hips (R2 = 0.842, R2 = 0.869). In patients with unilateral Perthes disease, the affected acetabulum is more retroverted than that on the contralateral side, which may be caused by hemipelvic retroversion. The measurements in this study could distinguish the form of acetabular retroversion. IWAAs and AWDIs can be used as new observations in future studies of acetabular version.

Aberrant microRNA expression in B lymphocytes from patients with primary warm autoimmune haemolytic anaemia.

OBJECTIVE: To screen and analyze the micro-Ribonucleic Acid (miRNA) expression profile in B lymphocytes from patients with autoimmune haemolytic anaemia (AIHA) using high-throughput sequencing. METHODS: Twelve patients with warm autoimmune haemolytic anaemia (wAIHA) and twelve healthy controls (HCs) were enrolled. CD19+ B lymphocytes were isolated and purified using magnetic activated cell sorting (MACS). RNA was subsequently extracted from these cells and a small RNA library was created. The miRNA expression profile was analyzed using Beijing Genomics Institute Sequencing 500 (BGISEQ-500), and stem-loop real-time quantitative PCR (stem-loop qRT-PCR) was used to verify the sequencing results. Downstream target genes of the differentially expressed miRNAs were predicted using miRanda and TargetScan online software, and GO functional enrichment and pathway enrichment analyses were performed on these genes. RESULTS: Compared with HCs, 178 upregulated and 143 downregulated miRNAs were identified in wAIHA patients, and stem-loop qRT-PCR of four randomly selected differentially expressed miRNAs verified the sequencing results. Ninety-five significantly enriched GO terms and eighty-five significantly enriched pathways were identified. Genes targeted by differentially expressed miRNAs were found to be mainly involved in the regulation of signal transduction, metabolic processes, immune reactions, and neoplastic disease development. CONCLUSION: The expression of miRNAs in B lymphocytes from patients with primary wAIHA was deregulated, and this phenomenon may be involved in the pathogenesis of wAIHA.

The clinical characteristics of autoimmune haemolytic anaemia/Evans syndrome patients with clonal immunoglobulin/T cell receptor gene rearrangement.

OBJECTIVE: To investigate the clinical features of AIHA/ES patients with clonal Ig/TCR gene rearrangement. METHODS: Ig/TCR gene rearrangements were measured by BIOMED-2 PCR. 44 primary AIHA/ES patients were enrolled in the study. Clinical characteristics were analyzed and compared between patients with and without clonal Ig/TCR gene rearrangement. RESULTS: Clonal Ig/TCR rearrangements were identified in 34.09% (15/44) patients including 18.18% (8/44) clonal Ig rearrangement and 15.91% (7/44) clonal TCR rearrangement. 11.37% (5/44) patients showed TCR γ rearrangement, and 2.27% (1/44) patient showed ß rearrangement. 2.27% (1/44) patient showed both γ and ß chain rearrangement. The median ages of patients with Ig/TCR clonality (8 male and 7 female) and without Ig/TCR clonality (10 male and 19 female) were 60 (16 ∼ 81) and 53 (17 ∼ 78) years old, respectively. No significant differences were found in age or gender between the two groups (p = .26, p = .378). Hb and RBC of patients with Ig/TCR clonality [(64.31 ± 5.72) g/L, (1.78 ± 0.22) × 1012/L] were significantly lower than those of patients without Ig/TCR clonality (p = .0053 and p = .0189, respectively). The percentage of reticulocytes of Ig/TCR clonality group was obviously higher than that of patients without Ig/TCR clonality (p = .0248). No significant differences were found in levels of TBIL, IBIL, LDH, FHb, Hp, IgG, IgA, IgM, IgE, C3, C4, CD5+CD19+/CD19+ ratio, and CD3+CD4+/CD3+CD8+ ratio between the two groups. Treatment response of Ig/TCR clonality group occurred significantly later than that of the non-clonality group (p = .0016). There were no differences in relapse rate, time to recurrence, and duration of remission between two groups (p = .083, p = .72, and p = .61, respectively). CONCLUSION: AIHA/ES patients with clonal Ig/TCR gene rearrangement presented more severe haemolysis and anaemia. Longer treatment is needed for these patients to obtain remission.

Characteristics of patients with autoimmune haemolytic anaemia secondary to lymphoproliferative disorder: A single-centre retrospective analysis.

Autoimmune haemolytic anaemia (AIHA) is a kind of autoimmune diseases characterized by autoantibodies which produced and secreted by abnormal activated B lymphocytes directed against red blood cells (RBC). Study reveals that about 50% AIHA mainly occurs secondary to lymphoproliferative disorders (LPD) and autoimmune diseases. In this study, we aim to explore the characteristics of patients with AIHA secondary to LPD. Fifteen patients with AIHA secondary to LPD (secondary group) and 60 with primary AIHA (primary group) were retrospectively included. Patients in the secondary group [(59.40 ± 4.74) y] were older than those in the primary group [(47.53 ± 2.30) y] (p = 0.024). Reticulocyte counts were lower for the secondary group [(134.55 ± 20.67) × 109/L] than for the primary group [(193.88 ± 27.32) × 109/L] (p = 0.09). Haptoglobin was higher in the secondary (0.75 ± 0.19) g/L than in the primary group (0.34 ± 0.05) g/L (p = 0.004). The ratio of CD3+CD4+/CD3+CD8+ was higher in the secondary (1.81 ± 0.41) than in the primary (1.05 ± 0.12) group (p = 0.025). Duration of remission was shorter in the secondary [(23.52 ± 5.20) months] than in the primary [(40.87 ± 3.92) months] group (p = 0.013). Relapse rate was higher for the secondary (33.3%) than for the primary (8.3%) group (p = 0.003). Mortality rate was higher in the secondary (33.3%) than in the primary (8.3%) group (p = 0.003). Progression-free survival was shorter in the secondary than in the primary group (p = 0.021). In conclusion, patients with AIHA secondary to LPD showed higher age at diagnosis, shorter remission time, and higher recurrence and mortality rates than did those with primary AIHA.

Expression of BTK/p-BTK is different between CD5+ and CD5- B lymphocytes from Autoimmune Hemolytic Anemia/Evans syndromes.

Objective: To explore the activity of B subsets from Autoimmune Hemolytic Anemia/Evans syndrome (AIHA/ES) patients. Methods: The expression of Bruton's tyrosine kinase (BTK) and phosphorylated BTK (p-BTK) on CD5+CD19+B and CD5-CD19+B lymphocytes were detected using flow cytometry in AIHA/ES patients with different disease states, healthy controls (HCs) and chronic lymphocytic leukemia (CLL) patients. The correlations of expressed BTK and p-BTK with clinical variables were analyzed. Results: Thirty six AIHA/ES patients (16 hemolytic, 20 remission), 11 CLL patients, and 15 HCs were enrolled. The expression levels of BTK and p-BTK on CD5+B lymphocytes in AIHA/ES patients were higher than those in HCs and CLL patients. The latter two groups had no significant difference, and were positively correlated with the quantity of IgE. The ratio of p-BTK to BTK on CD5+B lymphocytes of the hemolytic and remission groups was obviously higher than that on CD5-B lymphocytes (74.62 ± 6.42% and 29.63 ± 10.19%, respectively; P = 0.001 versus 77.95 ± 9.57% and 26.29 ± 6.86%, respectively; P = 0.006). The ratio of p-BTK to BTK on CD5+B lymphocytes (54.89 ± 9.56%) and CD5-B lymphocytes (30.86 ± 12.47%) did not differ significantly in HCs (P = 0.109). BTK did not differ significantly between CD5+ and CD5-B lymphocytes in AIHA/ES, but p-BTK on CD5+B lymphocytes was significantly higher than that on CD5-B lymphocytes in AIHA/ES patients. Conclusions: CD5+B lymphocytes are the major B subtype that is activated in AIHA/ES patients and it positively correlates with IgE.

A new approach for surgical treatment of chronic Monteggia fracture in children.

PURPOSE: The aim of the study was to investigate the clinical outcomes of a combined anterior and posterior approach for the surgical treatment of chronic Monteggia fractures in children. MATERIALS AND METHODS: From November 2010 to January 2018, 33 patients (27 boys and 6 girls) with chronic Monteggia fracture who were treated surgically by one surgeon of our department were retrospectively analyzed. In the surgical procedure, open reduction and excision of fibrous scar were performed with the anterior Henry's approach, while ulnar osteotomy was carried out with a posterior approach. In cases of unstable radial head reduction, a trans-capitellar K wire was applied. Repair or reconstruction of the annular ligament (ALR) was not undertaken. RESULTS: The average follow-up of the patients was 33.8 months (range 8-87 months). At the last follow-up, Mayor Score and function of flexion and extension showed significant improvement compared to preoperative condition (p < 0.05). Two patients with palsy of the deep branch of the radial nerve with neurolysis recovered to normal over a 3-month follow-up. Redislocation occurred in two patients while subluxation occurred in one. One patient suffered a mild ischemic contracture but gradually recovered. Other severe complications, nerve injuries, heterotopic ossification, or synostosis, were not noted in the follow-up. CONCLUSION: A combined anterior and posterior approach for surgery resulted in a satisfactory outcome due to the advantages of better exposure, more convenient intraoperative management, and facilitate for radial nerve exploration. Our study provided a new approach for the surgery of chronic Monteggia fractures.

CD5+ B lymphocytes secrete IL-10 rather than TGF-ß1 which control the immune response in autoimmune haemolytic anaemia/Evans syndrome.

OBJECTIVE: To investigate the quantity and secretion function of cytokines-secreted CD5+ B lymphocytes in Autoimmune Haemolytic Anaemia (AIHA)/Evans syndrome (ES) patients. METHODS: Twenty-five untreated AIHA/ES patients, 28 remission AIHA/ES patients and 25 healthy controls (HCs) were enrolled in this study. The quantity of CD5+B lymphocytes which produce interleukin-10 (IL-10) (CD5+IL-10+) and transforming growth factor (TGF-ß1) (CD5+TGF-ß1+) were detected by flow cytometry (FCM). CD5+ B lymphocytes were sorted from peripheral blood (PB) by FCM and the expression of IL-10 and TGF-ß1 mRNA in CD5+ B cells were measured by real-time PCR (RT-PCR). RESULTS: The percentage of CD5+IL-10+B cells in CD5+ B lymphocytes in newly diagnosed patients was 82.18 ± 14.78%, which being significantly higher than that of remission AIHA/ES patients (56.68 ± 24.39%) and HCs (51.90 ± 22.95%) (p < .05). The percentage of CD5+IL-10+ B cells in CD5+ B lymphocytes in newly diagnosed patients was negatively correlated with haemoglobin (Hb), complement 3 (C3) (p < .05) and positively correlated with lactate dehydrogenase (LDH), total bilirubin (TBIL) and indirect unconjugated bilirubin (IBIL) (p < .05). The expression level of IL-10 mRNA in CD5+ B lymphocytes of newly diagnosed patients (49.34 ± 22.84) was higher than that of remission patients (3.97 ± 3.83) and HCs (1.78 ± 1.66) (p < .05). There was no significant difference among three groups with the proportion of CD5+TGF-ß1+ B lymphocytes and the expression level of TGF-ß1 mRNA in CD5+B lymphocytes (p > .05). CONCLUSIONS: CD5+ B lymphocytes could secrete IL-10 rather than TGF- ß1 which control the immune response in AIHA/ES.

miR-150 regulates B lymphocyte in autoimmune hemolytic anemia/Evans syndrome by c-Myb.

The objective of the study was to study the regulation of B lymphocytes in patients with autoimmune hemolytic anemia (AIHA)/Evans syndrome. From October 2015 to May 2016, 35 patients with AIHA/Evans in the Department of Hematology, Tianjin Medical University General Hospital were enrolled into this study. c-Myb mRNA and miR-150 expression in B lymphocytes were measured using real-time PCR (RT-PCR). Correlation between c-Myb and miR-150 and clinical parameters in patients with AIHA/Evans were analyzed. c-Myb mRNA expression in hemolysis patients was significantly higher than in remission patients and CLL patients, negatively correlated with hemoglobin (Hb) level and complement 3(C3) levels, and positively correlated with total bilirubin (TBIL) concentration and indirect bilirubin (IBIL) concentration. miR-150 expression in hemolysis patients was significantly lower than in patients in remission and CLL patients. miR-150 was negatively correlated with TBIL and IBIL, and positively correlated with Hb, C3. c-Myb mRNA expression levels in hemolytic episode patients were negatively correlated with the expression levels of miR-150. The expression of c-Myb, a regulatory factor of B lymphocytes, is increased in B lymphocytes of AIHA/Evans patients, while miR-150 expression is decreased. c-Myb was negatively correlated with miR-150.