Effect of high-dose dexamethasone on BAFF and Tregs in patients with immune thrombocytopenic purpura / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the change of B-cell activating factor of the TNF family (BAFF) and regulatory T-cells (Tregs) before and after high-dose dexamethasone(HD-DXM) therapy and assess the effect of BAFF on Treg cells in immune thrombocytopenic purpura (ITP).</p><p><b>METHODS</b>The plasma BAFF concentration was measured by ELISA, and Treg cell numbers by flow cytometry.</p><p><b>RESULTS</b>The plasma BAFF level \[(599.70 +/- 199.40) pg/ml\] was significantly increased (P < 0.05), and the percentage of Treg cells \[(1.56 +/- 0.73)%\] was significantly decreased (P < 0.01) in ITP patients before treatment as compared with that in controls \[(454.5 +/- 132.5) pg/ml and (4.08 +/- 1.08)%, respectively\]. After treatment with HD-DXM, the plasma BAFF level \[(296.9 +/- 119.7) pg/ml\] was significantly decreased (P < 0.01), and the percentage of Treg cells \[(5.94 +/- 2.22)%\] was significantly increased (P < 0.01). The BAFF level and Treg proportion had no significant correlation with platelets count (P > 0.05). In in vitro assays, no difference was found in the number of Treg cells between rhBAFF0 group and rhBAFF20 group \[(1.53 +/- 0.69)%, (1.49 +/- 0.67)%, P = 0.89)\].</p><p><b>CONCLUSION</b>BAFF level was increased and Treg cells decreased in ITP patients. HD-DXM might play a role in ITP treatment by down-regulating BAFF expression and up-regulating Treg cells number. BAFF had no influence on the number of Treg cells.</p>

Study of a humanized anti-platelet GP VI single chain Fv phage antibody / 中华血液学杂志

<p><b>OBJECTIVE</b>To study the anti-platelet GPVI single chain Fv phage antibody which can inhibit the aggregation function of platelet by using phage antibody library technology.</p><p><b>METHODS</b>ITP patients with anti-platelet GPVI autoantibody that could inhibit the aggregation function of platelet were screened by MAIPA assay and platelet aggregation test. The gene fragments of heavy chain and light chain variable region (VH and VL) of immunoglobulin were amplified by RT-PCR from peripheral blood lymphocytes mRNA of the screened patients. The VH and and VL fragments were linked through a DNA linker encoding the peptide (Gly4Ser)3 to construct single chain Fv (ScFv) gene. The ScFv gene was digested with SfiI/NotI restriction enzymes and cloned into the pHEN2 phage display vector, then electrically transformed to E. coli TG1. The TG1 containing ScFv-pHEN2 was rescued by helper phage M13K07 to produce ScFv phage antibody. The anti-platelet GPVI phage ScFv antibody was enriched and purified. The effect of the phage antibody on platelet aggregation function was studied.</p><p><b>RESULTS</b>Of 806 chronic ITP patients, 11 (1.36%) were positive for anti-platelet GPVI autoantibody and 2 (0.24%) patients'plasma significantly inhibited the collagen induced platelet aggregation. The length of VH and VL fragments was about 380 to 400 bp, and were successfully formed ScFv fragments of about 800 bp by DNA linker. After cloning ScFv to phagemid vector pHEN2 and transforming ScFv-pHEN2 to TG1, 4.1x10(7) clones were obtained. After M13K07 rescue, 2.62x10(10) cfu/ml ScFv phage antibodies were produced. The purified anti-platelet GPVI ScFv phage antibody inhibited the collagen induced platelet aggregation.</p><p><b>CONCLUSION</b>Anti-platelet GPVI ScFv phage antibody produced by phage antibody library technology can inhibit the aggregation function of platelet.</p>

The impact of platelet membrane autoantibodies on high-dose dexamethasone therapy in patients with idiopathic thrombocytopenic purpura / 中华血液学杂志

<p><b>OBJECTIVE</b>To evaluate the impact of platelet membrane glycoprotein (GP)-specific autoantibodies on high-dose dexamethasone therapy in patients with idiopathic thrombocytopenic purpura (ITP).</p><p><b>METHODS</b>Modified direct monoclonal antibody immobilization of platelet antigen assay (MAIPA) was used to detect platelet GPIIb/IIIa and/or GPI b specific autoantibodies. All patients received oral dexamethasone 40 mg/d for 4 days.</p><p><b>RESULTS</b>The response rate of high-dose dexamethasone in GPIIb/IIIa and/or GPIb specific autoantibody-negative patients was significantly different from that of antibody-positive patients (P<0.05). The response rate of GPIIb/IIIa specific autoantibody-positive patients was lower than that of antibody-negative patients (P<0.05). GPIb specific autoantibody had no significant impact on the efficacy of high-dose dexamethasone (P>0.05).</p><p><b>CONCLUSION</b>Platelet membrane GPIIb/IIIa-specific autoantibody can be a potential negative indicator for ITP patients'response to high-dose oral dexamethasone.</p>

The role of interleukin-18 and interleukin-18 receptor in predominant Th1 immune response of patients with immune thrombocytopenia / 中华血液学杂志

<p><b>OBJECTIVE</b>To evaluate the role of interleukin (IL)-18 and IL-18 receptor (IL-18R) in the predominant Th1 type cytokine response in patients with immune thrombocytopenia (ITP).</p><p><b>METHODS</b>Fifteen patients with active phase ITP, eighteen in remission and thirteen healthy controls were enrolled in this study. T-bet and GATA-3 mRNA levels in peripheral blood mononucleated cells (PBMNC) were measured by reverse transcriptase polymerase chain reaction (RT-PCR); the plasma IL-18 level by enzyme linked immunosorbent assay (ELISA), the expression of IL-18R on CD3(+) lymphocytes and total lymphocytes by flow cytometry(FCM).</p><p><b>RESULTS</b>The T-bet mRNA levels in patients with active phase ITP was 3.572 fold as much as that in the controls (P < 0.05), while the GATA-3 mRNA levels were 0.378 fold of that in controls (P < 0.05). The levels of plasma IL-18 and IL-18R on CD3(+) lymphocytes were significantly increased in active phase ITP than in remission phase and controls. There was no difference in ratio of T-bet/GATA-3 between remitted ITP and controls and so was for T-bet mRNA, GATA-3 mRNA, plasma IL-18 and IL-18R on CD3(+) lymphocytes.</p><p><b>CONCLUSION</b>ITP as a disease of Th1-dominant response there is an unbalance between T-bet and GATA-3 in its active phase; IL-18 and IL-18R being upregulated.</p>

The immunoreactivity of IgG and its fragments from ITP patients and their effects on platelet aggregation function / 中华血液学杂志

<p><b>OBJECTIVE</b>To prepare ITP plasma IgG and its F(ab')2 fragments and investigate their immunoreactivity to platelet GPIIb/IIIa and/or GPIb/IX and their effects on platelet aggregation function.</p><p><b>METHODS</b>The ITP patients having inhibitory autoantibody to the platelet aggregation were selected by modified MAIPA and platelet aggregation test with turbidimetry. Plasma IgG and its F(ab')2 fragments were prepared by streptococcal protein A affinity column and pepsin digestion. The immunoreactivity and the effects on platelet aggregation function of the whole antibody and its fragments were detected by modified MAIPA and platelet aggregation test, respectively.</p><p><b>RESULTS</b>(1) Anti-platelet GPIIb/IIIa and/or GPIb/IX autoantibodies were detected in 34 of 68 (53.6%) ITP patients' plasmas and that from 5 patients significantly inhibited the platelet aggregation induced by ADP or ristocetin. (2) By using protein A column combined with protease digestion, pure IgG and its F(ab')2 fragments were successfully obtained. (3) The purified IgG and its F(ab')2 fragments retained the ability to bind to their respective glycoproteins and inhibited the platelet aggregation function, whereas the IgG depleted plasma lost the ability of binding to the platelet GPs.</p><p><b>CONCLUSIONS</b>F(ab')2 fragment of the IgG antibody is a functional fragment, which not only has the binding ability to the platelet GPs but also inhibits the platelet aggregation function in a dose-dependent manner.</p>