Interleukin 27 inhibits cytotoxic T-lymphocyte-mediated platelet destruction in primary immune thrombocytopenia.

Cytotoxic T-lymphocyte (CTL)-mediated platelet destruction and aberrant cytokine profiles play important roles in the pathogenesis of primary immune thrombocytopenia (ITP). Interleukin-27 (IL-27) has pleiotropic immunomodulatory effects. However, the effect of IL-27 on CTL activity in ITP has not been reported. In the present study, platelets from ITP patients were cultured with autologous CTLs in the presence of IL-27. We found that IL-27 could inhibit CTL-mediated platelet destruction. In these IL-27-treated CTLs, granzyme B and T-bet expression decreased significantly, whereas granzyme A, perforin, and eomesodermin were not affected. To further investigate the role of granzyme B in CTL-mediated platelet destruction, granzyme B inhibitor was added and platelet apoptosis was significantly inhibited. These results suggest that IL-27 negatively regulates CTL cytotoxicity toward platelets in ITP by decreasing granzyme B expression, which is associated with reduced T-bet expression. IL-27 may have a therapeutic role in treating ITP patients.

[Relationship of TGF-ß and IL-4R gene polymorphisms with risk of classical Hodgkin lymphoma].

OBJECTIVE: This study was aimed to analyze the relationship between single nucleotide polymorphisms of transforming growth factor-ß1 G-800A and C-509T, interleukin-4 receptor V75I and susceptibility of CHL in adults. METHODS: Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was applied to analyze the expressed alleles of the selected SNP loca. The relationship between genomic polymorphisms of TGF-ß1 and IL-4R and susceptibility of CHL were coupled with clinical data. RESULTS: TGF-ß1G-800A and TGF-ß1C-509T had obvious linkage equilibrium (D' = 0.879, r(2) = 0.83, P = 0.020). GT haplotype distribution frequencies in mixed cellularity Hodgkin lymphoma cases and control group were of 53.1% and 34.2%, respectively, with statistically significant (OR = 2.35, P = 0.000); distribution frequencies of mutant gene T/T in disease and control groups were of 38.8% and 15.3%, respectively, also with statistically significant (OR = 3.654, P = 0.000); frequencies of nodular sclerosis CHL patients with IL-4R V75I mutant gene A/A in disease and control groups were of 19.2% and 41.75%, respectively, also with statistically significant (OR = 3.156, P = 0.000). CONCLUSION: Single nucleotide polymorphisms of TGF-ß1 G-800A, C-509T and IL-4R V75I has a significant correlation with Chinese susceptibility to classical Hodgkin lymphoma.

Rapid detection of BCR-ABL fusion genes using a novel combined LUX primer, in-cell RT-PCR and flow cytometric method.

Currently, quantitative and semiquantitative assays for minimal residual disease detection include fluorescence in situ hybridisation, multiparameter flow cytometric immunophenotyping and real-time quantitative polymerase chain reaction (RQ-PCR). We have developed a new approach to detect hybrid breakpoint cluster region and Abelson proto-oncogene (BCR-ABL) transcripts inside suspension cells using in situ RT-PCR and light upon extension (LUX) primer, followed by rapid quantitative analysis with flow cytometry. After cellular permeabilization and fixation of single cell suspension, the neoplastic mRNA was reverse transcribed and amplified by PCR with LUX primer. The results demonstrated that a strong positive yellow-green signal was observed in 99-100% cells of K562 cell line, only the red nucleus was detected in NB4 cell line and normal controls. The technique has been utilised to study 12 patients with chronic myeloid leukemia, and the results were compared with those of BCR-ABL fusion mRNA by RT-PCR and BCR-ABL fusion gene of the interphase cells by fluorescence in situ hybridization (FISH). In the five diagnosed patients, 90-98% cells were strongly positive. Four patients, including three patients treated with interferon-alpha and hydroxyurea and one patient treated with imatinib mesylate, had 26-82.5% positive cells. Three patients treated with imatinib mesylate were negative. The in situ RT-PCR results demonstrated complete concordance with the results of I-FISH and RT-PCR. A fluorescence signal was detectable at 1/10(4) cells and became negative below this threshold with flow cytometry. The results of the present study suggest that (1) LUX primers can be used to efficiently detect BCR-ABL fusion mRNA by in-cell RT-PCR; (2) the novel technique is a specific and sensitive way of detecting fusion gene with potential clinical usefulness.

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.

[The effect of selectively inhibiting the nuclear factor kappaB activation on survivin expression in leukemic cells].

OBJECTIVE: To investigate the highly specific proteasomal inhibitor MG132-induced apoptosis and its effect on nuclear factor (NF)-kappaB activation and survivin expression in leukemic K562 cell line. METHODS: leukemic cells of the line K562 were cultured and divided into 2 groups: treatment group, undergoing co-incubation with MG132 of the concentrations of 2, 4, 6, and 8 micromol/L respectively for 24 hours, and control group without treatment of MG132. Apoptosis was detected by examination of cell morphology and flow cytometry. Survivin expression and NF-kappaB activation were analyzed by immunocytochemistry and Western blotting. RESULTS: MG132 induced apoptosis of the K562 cells dose-dependently. Both survivin and NF-kappaB were highly expressed in the K562 cells. Compared with the control group, K562 cell treated with MG132 at the concentrations of 2, 4, 6, and 8 micromol/L for 24 hours showed the decrease of NF-kappaB activation to 75.0% +/- 3.7%, 59.9% +/- 5.3%, 45.4% +/- 5.7%, and 25.0% +/- 4.2% respectively, and decrease of survivin expression to 90.9% +/- 10.1%, 66.7% +/- 5.2%, 45.4% +/- 5.7%, and 30.3% +/- 6.6% respectively. Downregulation of survivin expression was closely correlated with the inhibition of NF-kappaB activation (Pearson correlation coefficient = 0.989, P < 0.01). CONCLUSION: MG132 induces apoptosis of leukemic cells, and effectively inhibits the NF-kappaB activation accompanied by the downregulation of survivin expression.

[Mechanism of cell-mediated lysis of autologous platelets in chronic idiopathic thrombocytopenic purpura].

OBJECTIVE: To investigate the contribution and mechanism of cell-mediated cytotoxity to the pathogenesis of idiopathic thrombocytopenic purpura (ITP). METHODS: (1) Mononuclear cells and platelets were prepared from the peripheral blood of 14 ITP patients and 10 healthy controls. Separately, CD 8(+) T cells and NK cells (CD 3(-)CD 16(+)CD 56(+)) were positively selected with magnetic microbeads. (2) As the target cells, the autologous platelets were cultured with CD 8(+) T cells or NK cells for 4 hours and then stained with annexin V. Ratio of platelets expressing annexin V was determined by flow cytometry. (3) The fraction of CD 8(+) T cells expressing FasL, TNFalpha and TRAIL were determined by flow cytometry. (4) The expression levels of perforin and granzyme B mRNA in CD 8(+) T cells were measured by semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) procedure. RESULTS: (1) When the platelets were incubated alone the annexin V positive platelet ratio of the ITP patients was 3.1% +/- 0.9%, not significantly different from that of the controls (3.2% +/- 1.1%, P > 0.05); (2) When the platelets were co-incubated with CD 8(+) cells the annexin V positive platelet ratio of the ITP group (7.6% +/- 2.8%), significantly higher than that of the control group (3.6% +/- 0.9%, P < 0.05); (3) When NK cells were used as effector cells, the annexin V positive platelet ratio of the ITP group was 3.5% +/- 1.1%), not significantly different from that of the control group (3.6% +/- 1.0%, P > 0.05); (4) The expression rates of FasL and TNFalpha on CD 8(+) T cells of the ITP group were 17.5% +/- 4.4% and 11.9% +/- 4.9% respectively, both significantly higher than those of the control group (8.9% +/- 1.5% and 6.4% +/- 2.1% respectively, both P < 0.05), while the expression rate of TRAIL of the ITP group was 16.1% +/- 3.8%, not significantly different from that of the control group (14.0% +/- 3.2%, P > 0.05); (5) The mRNA levels of granzyme B and perforin in the CD 8(+) T cells of the ITP group were 2.20% +/- 0.15% and 2.47% +/- 0.39% respectively, both significantly higher than those of the control group (1.63% +/- 0.22% and 1.80% +/- 0.31% respectively, both P < 0.05). CONCLUSION: Cytotoxic T lymphocyte (CTL) are activated in ITP and might be involved in the pathogenesis of ITP, while the NK cells have no direct cytotoxic effect on platelets. Apoptosis and perforin/granzyme-mediated cytotoxicity are two important pathways through which CTL destruct auto-platelets.

[Dendritic cells present particulate E7 protein of human papillomavirus and induce strong immunity].

OBJECTIVE: To investigate the feasibility of using dendritic cell (DC)-beads-antigen (Ag) as novel cancer vaccine form with E7 as the target antigen. METHODS: C57BL/6 mouse was killed and the femora were taken out. Marrow cells were isolated and cultured with IL-3 and granulocyte-macrophage growth stimulating factor (GM-CSF) so as to prepare DCs. Flow cytometry was conducted to analyze the phenotype. FITC-labeled polystyrene beads-ovalbumin (OVA) or polystyrene beads-human papillomavirus (HPV) E7 protein were added into the culture fluid to be co-cultured with the DCs for 3 hours. Flow cytometry was conducted to analyze the up-taking rates of polystyrene beads-OVA and of polystyrene beads-HPV E7 protein by the DCs and B3Z T cell hybridoma cells were co-cultured and then beads-OVA or beads-SIIFEKL polypeptide was added into the culture fluid. The absorbance was read. Twelve mice were injected with DC-beads-OVA, DC-beads-E7, DC-OVA antigen epitope (SIIFEKL), or DC-E7 epitope (RAHYNIVTF) into the plantae and killed in 36 hours to take out the iliac lymph nodes. Flow cytometry was conducted to analyze the phenotypes of the bead-positive cells. Mice were killed 10 days after immunization and their heart blood was collected. Enzyme linked immunosorbent assay was used to detect the levels of antibodies. Immunized and non-immunized mice were killed and their spleens were taken out. ELISPOT was used to detect the number of cells secreting interferon (IFN)gamma. RESULTS: DCs were seen in the culture fluid of mice marrow cells 5 days after culture with IL-3 and GM-CSF. The bead-positive rates of bead-E7 and bead-OVA were 64% +/- 18% and 58% +/- 16% respectively (P > 0.05) in the IL-3DCs. The CD40 expression rate in the bead-positive cells in the graining iliac lymph nodes was significantly higher after feeding by beads-E7 in comparison with that before the feeding (P < 0.05), the NLDC145, and MHC-II expression rates were increased to a certain degree, however the F4/80 expression rate was decreased. The DCs fed with bead-OVA or with OVA antigen epitope SIIFEKL, especially the former, significantly activated the B3Z cells. The serum IgG level in the mice immunized by beads-E7 was significantly increases, the IgM level was increased slightly, however, the IgA level almost remained unchanged. The numbers of IFNgamma SFU in the splenic cells of the mice immunized by DC-bead-OVA and DC-bead-E7 were significantly higher than that in the unimmunized mice, especially those immunized by DC-bead-OVA. CONCLUSION: DCs fed with beads-E7 can migrate to the draining lymph nodes and induce high level humeral and cellular immunity. DC-beads-Ag seems better than DC-epitope according to the strength of induced immunity. DC-beads-Ag may be an ideal vaccine form and can be used to develop other cancer vaccine.

[Study on the T lymphocytes early activation and soluble tumor necrosis factor receptor in patients with aplastic anemia].

OBJECTIVE: To investigate the expression of T cell early activation marker (CD(69)) on peripheral CD(4)(+) and CD(8)(+) lymphocytes and serum levels of soluble tumor necrosis factor receptor 1 and 2 (sTNF-R1 and sTNF-R2) in serum and bone marrow in patients with aplastic anemia (AA) and their pathophysiological significance. METHODS: In vitro activation of T lymphocytes was carried out by whole blood cell culture containing PHA (20 micro g/ml). The CD(69) expressions on CD(4)(+) and CD(8)(+) lymphocytes at 0 h and 4 h after PHA exposure were analyzed by two-color flow cytometry. The levels of sTNF-R1 and sTNF-R2 in serum and bone marrow were measured by ELISA. RESULTS: The CD(69) expression rates of CD(4)(+) and of CD(8)(+) cells in SAA patients were (8.96 +/- 7.23)% and (10.67 +/- 7.58)%, respectively, and that of CD(8)(+) cells in CAA patients was (7.36 +/- 5.49)% before PHA stimulation. The CD(69) expression rates of CD(4)(+) and of CD(8)(+) cells in SAA patients were (71.73 +/- 11.91)% and (61.74 +/- 13.44)% and in CAA (59.35 +/- 10.15)% and (48.78 +/- 8.25)% respectively, and were significantly elevated after PHA stimulation. CD(69) expression on CD(4)(+) cells was much higher than that on CD(8)(+) cells after stimulation. The levels of the two sTNF-R (sTNF-R1 and sTNF-R2) in peripheral blood and bone marrow of SAA patients were elevated and in the bone marrow of CAA patients were also increased. The serum levels of sTNF-R2 were positively related to the CD(69) expression rates of CD(8)(+) cells before PHA stimulation. CONCLUSION: Increased early activation and activated potentials of T lymphocytes, along with abnormally elevated immunologically active molecules might play a major role in the pathogenesis of AA.

[A study on early activation of T lymphocytes and soluble tumor necrosis factor receptor in patients with myelodysplastic syndrome].

OBJECTIVE: To investigate the expression of T cell early activation marker (CD(69)) on CD(4)(+) and CD(8)(+) lymphocytes in peripheral blood and the levels of soluble tumor necrosis factor receptor 1 (sTNF-R1) and sTNF-R2 in serum and bone marrow in patients with myelodysplastic syndrome (MDS). METHODS: Whole blood cell culture procedure was applied to activate T lymphocytes with phytohemagglutinin (PHA) (20 mg/L) in vitro. The expression rates of CD(69) on CD(4)(+) and CD(8)(+) lymphocytes at 0 h and 4 h after culture were analyzed with two-color flow cytometry. The levels of sTNF-R1 and sTNF-R2 in serum and bone marrow were measured with ELISA. RESULTS: There was an increase in the expression rates of CD(69) on CD(4)(+) and CD(8)(+) cells in RA and RAS patients (8.32% and 9.88% respectively) and in the expression rate of CD(69) on CD(8)(+) cells in RAEB and RAEB-T patients (7.92%) before PHA stimulation. CD(69) expression on CD(4)(+) and CD(8)(+) cells were significantly elevated in MDS patients after PHA stimulation (53.46% and 51.63% in RA + RAS; 42.93% and 41.96% in RAEB and RAEB-T) and the expression rate on CD(4)(+) cells was similar to that on CD(8)(+) cells. The levels of the two sTNF-R in MDS patients were elevated. sTNF-R1 in RA and RAS (1.58 +/- 0.68) micro g/L (PB), (2.10 +/- 0.26) micro g/L (BM); sTNF-R2 in RA and RAS (1.41 +/- 0.50) micro g/L (PB), (1.95 +/- 0.64) micro g/L (BM); sTNF-R1 in RAEB and RAEB-T (2.62 +/- 2.55) micro g/L (PB), (3.12 +/- 0.67) micro g/L (BM); sTNF-R2 in RAEB and RAEB-T (1.96 +/- 0.56) micro g/L (PB), (3.09 +/- 0.62) micro g/L (BM). The levels of sTNF-R2 in serum positively correlated to the expression rate of CD(69) on CD(8)(+) cells before PHA stimulation. CONCLUSION: It is indicate that the increased early activation and activated potentials of T lymphocytes, along with abnormally elevated immunologically active molecules play an important role in immune pathogenesis of patients with MDS.