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.

Bone marrow free immune checkpoints as a potential biomarker for differential diagnosis of acquired bone marrow failures.

OBJECTIVE: Clinically, to make a definite diagnosis of aplastic anemia (AA), idiopathic cytopenia of undetermined significance (ICUS) or myelodysplastic syndrome (MDS), they should be distinguished from each other. AA and ICUS have some incidence to transform into MDS. Immunosuppressive therapy (IST) is effective in AA and partial ICUS patients, while other ICUSs are more likely to progress to MDS without response to IST. To date, we neither found a technical method that could easily identify AA from hypoproliferative MDS, nor a simple parameter that could indicate ICUS with a response to IST. Here, we detected the concentration of free immune checkpoints in bone marrow supernatant of AA, ICUS, and MDS patients, analyzed the differences of immune status among these three diseases, to try to find a way to predict the response to IST in ICUSs. METHODS: Seventy-four novel patients were enrolled with newly diagnosed acquired bone marrow failure (including 29 AA patients, 11 ICUS patients, and 34 MDS patients), bone marrow supernatants were collected. Luminex liquid suspension array technology was used to measure the concentrations of 17 immune checkpoints to analyze the differences of immune status among these three diseases. RESULTS: The levels of 17 free immune checkpoints were elevated in MDS and showed a strong correlation with each other, followed by ICUS, and with the weakest in AA. By drawing the ROC curve, we found eight immune checkpoints, including sCD40, sCD86/B7-2, sCTLA-4, sGITR, sHVEM, sPD-1, sTIM-3, and sTLR-2, could easily distinguish AA from hypoproliferative MDS. ICUSs with lower concentrations of these eight free immune checkpoints predicted a better IST response. CONCLUSION: In conclusion, we found that there were notable differences in the immune status of AA, ICUS, and MDS. The concentrations of sCD40, sCD86/B7-2, sCTLA-4, sGITR, sHVEM, sPD-1, sTIM-3, and sTLR-2 could be used to identify AA and hypoproliferative MDS patients, as well as to distinguish ICUS patients who could benefit from IST.

Identification of potential pathogenic genes for severe aplastic anemia by whole-exome sequencing.

BACKGROUND: Severe aplastic anemia (SAA) is a syndrome of severe bone marrow failure due to hyperfunction of CD8+ T cells. While, the genetic background of SAA is still unknown. In this study, we tried to explore the possible genetic variants in CD8+ T cells of SAA patients. METHODS: We performed whole-exome sequencing (WES) in CD8+ T cells of 4 SAA patients and 7 normal controls. The mutations that existed in SAA but not in NCs were identified as candidate genes. Then, we compared them with genes in the enriched KEGG pathway of differently expressed genes (DEGs) from previous RNA-seq. After analyzing the types of mutations, we identified possible pathogenic genes and validated them by RT-PCR. Finally, we compared them with the autoimmune disease-related genes in DisGeNET database to select the most possible pathogenic genes. RESULTS: We found 95 candidate mutant genes in which, 4 possible pathogenic genes were identified: PRSS1, KCNJ18, PRSS2, and DGKK. RT-PCR results showed that compared with NCs, PRSS1 and KCNJ18 mRNA expression was significantly increased in SAA patients (p < 0.05), PRSS2 was also increased in SAA patients but without statistical difference, and DGKK gene could not be detected by RT-PCR in SAA patients. In addition, PRSS1 was associated with autoimmune diseases from the DisGeNET database. CONCLUSION: The mutations of PRSS1, KCNJ18, PRSS2, and DGKK, especially PRSS1 in CD8+T cells, may be involved in the immune pathogenesis of SAA.

Chemical oxidation of p-hydroxyphenylpyruvic acid in aqueous solution by capillary electrophoresis with an electrochemiluminescence detection system.

A system of capillary electrophoresis with electrochemiluminescence detection (CE-ECL) together with UV spectroscopic and electrochemical methods were used to study the chemical oxidation of p-hydroxyphenylpyruvic acid (pHPP) by dissolved oxygen in aqueous solution. The pHPP was observed to be readily oxidized by dissolved oxygen in alkaline solution and yielded a compound that strongly enhanced the electrochemiluminescence of Ru(bpy)23+. This compound was separated and detected by a new CE-ECL system and revealed to be oxalate by being compared with an authentic sample of oxalate. The chemical oxidation mechanism of pHPP by dissolved oxygen was discussed in this paper.