The novel HLA-A*69:01:04 allele identified by Sanger dideoxy nucleotide sequencing in a Chinese individual.

HLA-A*69:01:04 differs from HLA-A*69:01:01:01 in exon 2 at position 336 by a single synonymous mutation.

[The Establishment and Identification of Acute Myeloid Leukemia NOD-SCID-IL2rg-/-Mice Model by Using Luciferase-Expressing KG1a Cells].

OBJECTIVE: To establish the in vivo traceable acute myeloid leukemia mice model with Luciferase-Expressing KG1a Cells. METHODS: KG1a cells with stable luciferase gene expression (called as KG1a-Luc cells) were constructed by lentivirus transfection, then sifted out by puromycin. Eighteen male NOD-SCID-IL2rg-/-mice aged 8 to 12 weeks were randomly and equally divided into two groups: the control group and the KG1a-Luc group. The mice in KG1a-Luc group were injected with 200 µl PBS containing 5×106 KG1a-Luc cells through tail veins, and the mice in control group were injected with 200 µl PBS only. The bioluminescence imaging technology was used to monitor the tumor burden in vivo. The peripheral blood of the mice in both groups was analyzed by flow cytometry. After the mice were sacrificed, there were pathologic evaluations: bone marrow and spleens made into smears, and livers sliced to get paraffin sections. The survival time of the mice in the two groups was recorded and compared. RESULTS: KG1a cells expressing luciferase stably were successfully obtained. The tumor luminescence wildly spread at day 17 captured by in vivo imaging. The KG1a-Luc tumor cells could be detected in the peripheral blood of the mice, with the average percentage of (16.27±6.66)%. The morphology and pathology result showed that KG1a-Luc cells infiltrate was detected in bone marrow, spleens and livers. The survival time of the KG1a-Luc mice was notably shorter as compared with those in the control group, the median survival time was 30.5 days (95%CI: 0.008-0.260). CONCLUSION: The acute myeloid leukemia NOD-SCID-IL2rg-/-mouse model was successfully established by tail vein injection of 5×106 KG1a-Luc cells.

[Expression and Significance of BTLA and Its Ligand HVEM in Patients with Chronic Myelomonocytic Leukemia].

OBJECTIVE: To investigate the expression and significance of B and T lymphocyte weakening factor (BTLA) in patients with chronic myelomonocytic leukemia (CMML). METHODS: Real-time PCR was used to detect the expression of BTLA and its ligand HVEM mRNA in 11 patients with chronic myelomonocytic leukemia and 11 normal donors. Flow cytometry was used to detect expression of BTLA and its HVEM on the cell surface of peripheral blood T lymphocytes and γδ T cells. RESULTS: The median values of BTLA and its ligand HVEM mRNA expression in peripheral blood of patients with CMML were 0.009% and 559.4%, respectively, which were significantly lower than those of normal controls (0.053% and 1031%)(P<0.001). The expression level of BTLA and HVEM on cell surface of peripheral lymphocytes was not significantly different from that in normal controls (P=0.3031 and 0.2576), however, the proportion of peripheral blood T lymphocytes in patients with CMML (median: 37.73%) was significantly lower than that in controls (median 69.23%)(P=0.0005). The expression of BTLA on the surface of γδ T cells in peripheral blood of patients with CMML (median: 23.26%) was significantly lower than that of the controls (median: 52.64%) (P<0.05), and there was no significant abnormality in HVEM expression (P=0.2791). CONCLUSION: The expression of BTLA and its ligand HVEM, the proportion of T lymphocytes and the expression of BTLA on the surface of γδ T cells in patients with CMML are reduced. The effects of these abnormalities on T cell function and prognosis and efficacy of patients need to be further observed.

[Regulatory Effect of Exosomes Derived from Human Umbiilcal Cord Mesenchymal Stem Cells on Treg and TH17 Cells].

OBJECTIVE: To investigate the effects of exosomes from human umbilical cord mesenchymal stem cells on the development of Treg and TH17 cells. METHODS: Exosomes from the serum-free-culture supernatants of hUC-MSC were harvested by ultracentrifugation. The electron microscopy, nanoparticle tracking analysis and western blot were used to identify the hUC-MSC-exosomes, such as the morphology, the paticle chameter, and the protein content. The PBMC stimulated with anti-CD3/CD28 were incubated with the exosomes for five days, and then the percentage changes of Treg and TH17 cells were analyzed by using flow cytometry. RESULTS: The hUC MSC-derived exosomes were saucer-like in morphology the averge diameter was approximately 142 nm. They were identified as positive for CD9 and CD63. Flow cytometry showed that the proportion of CD4+CD25+Foxp3+ Treg cells in the PBMC were significantly higher, but the proportion of CD4+IL17A+ T cells in the hUC-MSC-exosome group was obviously lower than that in the group without the hUC-MSC-exosom (control group) (P<0.05). CONCLUSION: The hUC-MSC-exosomes have an immunomodulatory effect on T cells in vitro by increasing the ratio of Treg and reducing the ratio of TH17 cells, expecting the hUC-MSC-exosom as a novel cell-free target for immunotherapy.

[Effectiveness of CLAT Protocol for Treating Patients with Refractory Acute Myeloid Leukemia].

OBJECTIVE: To explore the clinical efficacy and toxicity of CLAT protocol (cladribine, cytarabine and topotecan) for treating patients with refractory acute myeloid leukemia (R-AML). METHODS: A total of 18 patients with R-AML (median age 37 years, range 18 to 58 years; male n = 16, female n = 2) were treated with CLAT protocol, which consisted of cladribine 5 mg/m(2)/d, i.v. on days 1-5, cytarabine 1.5 g/m(2)/d, i.v. on days 1-5, topotecan 1.25 mg/m(2)/d, i.v. on days 1-5 and G-CSF 300 µg/d subcutaneous injection on day 6 until neutrophile granulocyte recovery. RESULTS: Out of 18 patients 2 died of severe infection before the assessment. Among 16 evaluated patients, 10 (55.6%) achieved complete remission (CR), and 2 (11.1%) achieved partial remission (PR), the overall response rate was 66.7%, the rest 4 patients did not respond (NR). The median overall survival time and DFS for the CR patients was 9.5 months (95%CI: 6.7-16.64) and 9.5 months (95%CI: 6.1-16.7) respectively. The 1 year OS and DFS rates were 45% and 46.9%, respectively. All patients developed grade 4 of granulocytopenia and thrombocytopenia, the median duration was 13 (range 2 to 21) days and 12 days (range 2 to 21), respectively, all patients developed infection, 2 patients died of severe infection. The most common non-hematological side effects included nausea, vomiting, diarrhoea, rash, aminotransferase or bilirubin elevation and were grade 1 to 2. CONCLUSION: The CLAT protocol seems to have promising for the treatment of refractory AML patients, and patients well tolerated. This CLAT protocol offers an alternative treatment for R-AML patients who received severe intensive treatment, especially with anthracycline-containing chemotherapy.

[A method for induction of mouse CD8α(+);CD11b(+);jagged2(high);regulatory dendritic cells with mesenchymal stem cells in vitro].

AIM: To develop a method for induction of the mouse bone marrow-derived CD8α(+);CD11b(+);jagged2(high);regulatory dendritic cells (DCregs) using mesenchymal stem cells (MSCs) in vitro. METHODS: BALB/c(H-2(d);)mouse bone marrow cells (BMCs) were isolated and induced to CD8α(+);CD11c(+);CD11b(+);DCs (DCs) by 4 cytokines in vitro for 3 d. The DCs were selected by flow cytometry (FCM), and co-cultured with MSCs for 10 d to get DCregs. Immunophenotypes, cell cycle, Jagged1 and Jagged2 ligands of the Notch pathway of DCs were analyzed by FCM before and after co-culture. RESULTS: The novel DCs were transformed into DCregs successfully. The expressions of CD86, CD80, CD40, and MHC-II significantly decreased (P<0.05), while those of CD205, Jagged1 and Jagged2 obviously increased on DCregs (P<0.05). Meanwhile, the percentage of treated MSCs cells went up in G2 and S phases. CONCLUSION: MSCs co-cultured with DCs can induce the development of DCregs, which have immune tolerance-associated phenotypes and higher proliferation ability. This mechanism might be related to the up-regulated Jagged1 and Jagged2 expressions and the T-cell Notch pathway activation.

[Detection of methylation levels of multi-genes by real-time PCR in patients with myelodysplastic syndrome].

OBJECTIVE: To analyze the promoter methylation levels of p15, CDH1, DAPK and HICI genes of patients with myelodysplastic syndrome (MDS) and explore the relationship between the level of methylation and clinical features. METHODS: DNA methylation levels of p15, CDH1, DAPK and HICI in peripheral blood (PB) or bone marrow (BM) samples from 52 MDS patients were detected by real-time quantitative PCR. The correlation of the methylation level with clinical features and hematological findings was analyzed. 38 de novo AML patients and 46 normal individuals served as controls. RESULTS: The methylation levels of p15, CDH1, DAPK and HICI were 16.23 ± 21.69, 6.59 ± 9.39, 0.14 ± 0.11 and 7.81 ± 9.70 in BM, and 14.96 ± 20.16, 6.00 ± 9.26, 0.12 ± 0.14 and 6.74 ± 9.72 in PB, respectively from 18 MDS patients, and the difference between BM and PB was not statistically significant (P > 0.05). The methylation levels of p15 (14.70 ± 18.17) and CDH1 (6.61 ± 8.79) genes in high risk (RAEBI/II) MDS were significantly higher than in low risk (RCMD/RARS/5q-, p15: 1.99 ± 1.59, CDH1: 1.23 ± 1.14 and RCMD, p15: 3.02 ± 3.42, CDH1:1.53 ± 2.06) MDS or control (p15: 1.69 ± 1.82, CDH1: 1.01 ± 1.12) (P < 0.05). The methylation levels of DAPK gene had no difference among subtypes of MDS, and that of HIC1 gene only differed between RAEB I/II (9.16 ± 11.95) and control (2.49 ± 2.26) (P = 0.042). The difference of methylation levels of p15, CDH1, DAPK and HICI in BM was statistically significant among subtypes of MDS (P = 0.001, 0.003, 0.039, 0.023, respectively). And so did of p15 and DAPK in PB (P = 0.013, 0.006, respectively). The methylation level of p15 and CDH1 was significantly correlated with IPSS classification and blasts percentage in BM. CONCLUSIONS: p15 and CDH1 genes are special hypermethylation genes in MDS. Methylation level of HIC1 gene showed an upward tendency from low risk to high risk MDS.