[Expression and Significance of PD-1 and ICOS in Patients with Primary Immune Thrombocytopenia].

OBJECTIVE: To investigate the expression of programmed death receptor-1 (PD-1) and inducible costimulator (ICOS) on the surface of CD8+ T cells in peripheral blood of patients with primary immune thrombocytopenia (ITP), and explore the roles of PD-1 and ICOS in the occurrence and development of ITP. METHODS: A total of 28 ITP patients treated in Tianjin Medical University General Hospital from September to December 2020 were selected, including 13 patients with newly diagnosed ITP, 15 patients with chronic ITP, and 22 healthy volunteers were recruited as control group. Flow cytometry was used to detect the expression levels of PD-1 and ICOS, and evaluate their correlation with clinical indicators. RESULTS: The percentage of CD8 + T cells in ITP patients of chronic group was higher than that of the newly diagnosed group and the control group (P<0.05). The expression level of PD-1 on CD8+ T cells in ITP patients of newly diagnosed group and chronic group were significantly lower than that of the control group (P<0.05), while the expression level of ICOS were significantly higher (P<0.05). In ITP patients, PD-1 was negatively correlated with platelet count (r=-0.4942, P<0.01), but positively with ICOS (r=0.4342). PD-1 and ICOS were both negatively correlated with lymphocyte count (rPD-1=-0.4374; rICOS=-0.4492). CONCLUSION: In ITP patients, the unbalanced expression of PD-1 and ICOS may interfere with the immune homeostasis of the body, which can be used as a therapeutic target for ITP patients.

[Expression and Significance of IL-9 and IL-6 in Patients with BCR-ABL- MPN].

OBJECTIVE: To investigate the expression of IL-9 and IL-6 in patients with BCR-ABL- bone marrow proli- ferative tumor (MPN), and to explore its role in the occurrence and development of MPN. METHODS: A total of 71 newly diagnosis MPN patients treated in Tianjin Medical University General Hospital from 2018 to 2019 were selected, including 32 patients with polycythemia vera (PV) and 22 patients with primary thrombocytosis (ET), and 17 patients with primary myelofibrosis (PMF). Then 58 patients who retestine after treatment were selected as therapy group，and 20 healthy volunteers were recruited as control group. ELISA was used to detect the expression level of IL-6 and IL-9 in bone marrow supernatant, and the relative expression level of IL-6 and IL-9 mRNA in BMMNC was detected by real-time PCR. The proportion of Th9 cells in peripheral blood were detected by flow cytometry (FCM). The expression level of IL-6 mRNA and IL-9 mRNA of BMMNC and clinical indicators were analyzed, and the correlation between JAK2 gene mutation load and IL-9 level was further analyzed. RESULT: The level of IL-6 in bone marrow supernatant and the expression of IL-6 mRNA in BMMNC were higher in the newly diagnosed group as compared with those in the treated group and the control group (P<0.001). The expression level of IL-9 in bone marrow supernatant and the expression of IL-9 mRNA in BMMNC were lower in the newly diagnosed group as compared with those in the treated group and the control group (P<0.05). The proportion of Th9 cells in peripheral blood was lower in the newly diagnosed group as compared with that in the treated group and the control group (P<0.001). The level of IL-6 in bone marrow supernatant and the expression of IL-6 mRNA in BMMNC in JAK2+ group were higher than those in JAK2- group (P<0.05). The expression level of IL-9 in bone marrow supernatant and the expression of IL-9 mRNA in BMMNC were lower in JAK2+ group as compared with those in JAK2- group (P<0.05). The expression of IL-6 and IL-9 in the patient group showed correlation with the number of lymphocytes (IL-6: r=-0.49, P<0.01; IL-9: r=0.53, P<0.001), and also related with Hb in PV patients (IL-6: r= 0.87, P<0.001; IL-9: r=-0.54, P<0.01), and platelets in ET patients (IL-6: r=0.64, P<0.05; IL-9: r=-0.46, P<0.05). CONCLUSION: The increased expression of IL-6 in MPN and hyperfunction may promote the progression of BCR-ABL- MPN disease. The expression of IL-9 in MPN decreases, and it negatively correlates with the mutation load of JAK2 gene, which may be related with the decrease of tumor environmental antitumor immune effect.

[Relationship of Peripheral Blood IL-37 Expression with T Lymphocytes Subsets and NK Cells in Patients with Primary Immune Thrombocytopenia].

OBJECTIVE: To study the correlation of IL-37 with T lymphocytes subsets and NK cells in ITP patients, and to explore its possible mechanisms involved in the pathogenesis of ITP. METHODS: Forty-five patients with newly diagnosed ITP(newly diagnosed group), 32 patients of complete remission (remission group) and 22 healthy persons(control group) were selected. The serum level of IL-37 in 3 groups was determined by enzyme linked immunosorbent assay (ELISA). The mRNA expression of IL-37, IL-17 and IL-18 in peripheral blood mononuclear cells（PBMNC） in 3 groups was measured by real-time fluorescence quantitative polymerase chain reaction (PCR). The number of IL-18Rα+CD4+ T cells and Tim-3+NK cells in the peripheral blood in 3 groups was detected by flow cytometry (FCM). RESULTS: The serum level of IL-37 in the peripheral blood of ITP patients in the newly diagnosed group was significantly higher than that in the control group and the remission group(P＜0.01) . The expression level of IL-37 in PBMNC of the ITP patients in newly diagnosed group was higher than that in the control group and the remission group(P＜0. 05). The expression level of IL-17 and IL-18 in PBMNC of the ITP patients in newly diagnosed group was higher than that in the control group and the remission group(P＜0. 01); the expression of IL-18Rα in CD4+ T cells in newly diagnosed group was significantly higher than that in both the control and the remission group(P＜0.01).The expression of Tim-3 in NK cells in ITP patients was significantly lower than that in the control group (P＜0. 01). In ITP patients, the serum IL-37 level and IL-18Rα+CD4+T cells ratio both negatively correlated with Plt count (r=-0.58, r=-0.48) moreo-ver the serum IL-37 level also negatively correlated with amount of CD4+ T cells and NK cells (r=-0.29, r=-0.28), but positively correlated with amount of CD8+ T cells (r=0.329). CONCLUSION: The IL-37 and its receptors may play an immunoregulatory role in CD4+ T cells and NK cells, the IL-37 may be a therapeutic target for ITP patients.

Polycythemia vera transforming to myelofibrosis and then biphenotype acute leukemia.

BACKGROUND: A case with 17-year detailed illness history including evolution of polycythemia vera (PV) to myelofibrosis (MF) and then biphenotype acute leukemia (BAL) was reported. Ten years of PV followed by seven years of MF and then BAL, the patient experienced a classical "complete course" of myeloproliferative neoplasm (MPN). High WBC counts as well as low Hb and platelet counts in MF phase, long disease course, older than 50 years age, and positive JAK2 were her high risk factors of transformation from MPN to leukemia. Pancytopenia in her secondary MF phase responded well to the therapy of corticosteroids, which indicated that the immune mechanism was involved in the pathogenesis of MF. Progression of PV to MF and then BAL might be related to discontinuation of interferon-alpha because of poor tolerance.

[Telomere length and gene expression of shelterin in CD3(+) T cell of severe aplastic anemia patients].

OBJECTIVE: To explore the changes in telomere length and gene expression of complex shelterin (composed of 6 core components: TRF1, TRF2, POT1, TIN2, TPP1 and RAP1) in severe aplastic anemia (SAA). METHODS: Bone marrow samples were obtained from 20 SAA patients and 10 normal controls. CD3(+)T cells were sorted by immunomagnetic separation. Telomere length was tested by Southern blot and the gene expressions of TRF1, TRF2, POT1, TIN2, TPP1 and RAP1 were detected by reverse transcription-PCR(RT-PCR). RESULTS: Telomeres of CD3(+)T cells were found significantly shorter in SAA untreated ((4.4 ± 1.1) kb, n = 9) and recovering groups((5.8 ± 1.0) kb, n = 11) than control group ((9.2 ± 3.3) kb, P < 0.05). Telomere length of CD3(+)T cells shortened with TH/S decreasing (r = 0.564, P = 0.029). The mRNA expression of POT1 decreased in untreated SAA patients (0.16(0.02-0.29)) and over-expressed in recovering patients (1.17(0.82-1.86), P < 0.05). The mRNA expression of RAP1 was significantly higher in untreated patients (4.14 (1.93-6.92)) than that in recovering group (0.87 (0.30-1.73) ) and controls (0.62 (0.45-4.07) , both P < 0.05). CONCLUSION: Changes in telomere length and shelterin gene expression occur in CD3(+)T cells of SAA patients and may be correlated with disease severity.

Histone deacetylase inhibitor valproic acid inhibits proliferation and induces apoptosis in KM3 cells via downregulating VEGF receptor.

The expression of vascular endothelial growth factor receptor 1(VEGFR-1) in human multiple myeloma KM3 cells in vitro, effects of valproic acid (VPA), as a histone deacetylase inhibitor, on cell proliferation and apoptosis and the underlying molecular mechanism were investigated. The effects of VPA on the growth of KM3 cells were studied by MTT assay. The apoptosis rate was determined with flow cytometry. The mRNA level of VEGFR was determined by RT-PCR; and immunocytochemistry was used to detect the protein level of ac-H4 and VEGFR. VPA inhibited proliferation of KM3 cells in a time- and dose-dependent manner. Treatment with VPA (4, 2, 1 and 0.5 mmol/L) for 48h, the apoptosis rates of KM3 cells were (13.27+/-3.54)%, (22.13+/-1.20)%, (24.41+/-2.23)% and(40.62+/-4.28)% respectively. The expression of VEGFR-1 in KM3 cells were decreased in VPA-treated group by the immunochemistry and RT-PCR, whereas the acetylated histone H4(ac-H4) accumulated. It suggested VPA could decrease the expression of VEGFR-1 in KM3 cells, and it might play an important role in regulating the proliferation and apoptosis of multiple myeloma cell line KM3 cells. These results provide the framework for clinical trials.