Comprehensive geriatric assessment in newly diagnosed older myeloma patients: a multicentre, prospective, non-interventional study.

BACKGROUND: Multiple myeloma is a disease of the older people, whose prognoses are highly heterogeneous. The International Myeloma Working Group (IMWG) proposed a geriatric assessment (GA) based on age, functional status and comorbidities to discriminate between fit and frail patients. Given the multidimensional nature of frailty and the relatively recent exploration of frailty in the field of MM, reaching a consensus on the measurement of frailty in MM patients remains challenging. OBJECTIVE: We sought to assess the feasibility of performing a comprehensive GA (CGA) in older MM patients in a real-world and multicentre setting and to evaluate their baseline CGA profiles. RESULTS: We studied 349 older patients with newly diagnosed MM (age range, 65-86 years). Our results showed that a CGA is feasible for older MM patients. Using the IMWG-GA criteria, we identified significantly more frail patients in our cohort comparing to in the IMWG cohort (43% vs 30%, P = 0.002). In the IMWG-GA 'fit' group, risk of malnutrition, depression and cognitive impairment remains. The median follow-up time was 26 months (range 1-38). The median overall survival (OS) was 34.7 months, and the estimated 3-year OS rate was 50%. A high MNA-SF score (MNA-SF ≥ 12), low GDS score (GDS ≤ 5) and high CCI score (CCI ≥ 2) can be used to predict the OS of older patients with newly diagnosed MM. This study is registered at www.clinicaltrials.gov (NCT03122327). CONCLUSIONS: Our study justifies the need for a CGA in older patients with newly diagnosed MM.

A multicenter randomized open-label study of rituximab plus rhTPO vs rituximab in corticosteroid-resistant or relapsed ITP.

This study aimed to compare the efficacy and safety of rituximab (RTX) plus recombinant human thrombopoietin (rhTPO) with RTX alone in patients with immune thrombocytopenia (ITP) who had failed to respond to corticosteroids or relapsed. Recruited patients were randomized at a ratio of 2:1 into 2 groups: the combination group (RTX + rhTPO, n = 77) and the monotherapy group (RTX, n = 38). Overall response was achieved in 79.2% of patients in the combination group vs 71.1% in the monotherapy group (P = .36), and the complete response (CR) rate was 45.4% in the combination group compared with 23.7% in the monotherapy group (P = .026). The combination group had significantly shorter time to response (TTR; median and range, 7 and 4-28 days) compared with the monotherapy group (28 and 4-90 days) (P < .01). There was no difference between these 2 groups in terms of the long-term response (P = .12). Our findings demonstrated that the combination of RTX and rhTPO significantly increased the CR rate and shortened TTR compared with RTX monotherapy in the treatment of corticosteroid-resistant or relapsed ITP but failed to show a beneficial effect on the long-lasting response. This study is registered at www.clinicaltrials.gov as #NCT01525836.

[Multicenter Prospective Study of Different Induction Regimens of Azacytidine in Treatment of Elderly Patients with Acute Myeloid Leukemia].

OBJECTIVE: To observe the efficacy and safety of different induction regimens of same total dosage of azacitidine (Aza), including standard dose (standard dose group) and low-dose long-term (adjusted dose group), in the treatment of elderly acute myeloid leukemia (AML). METHODS: A total of 103 elderly patients with AML (non-acute promyelocytic leukemia) from January 2020 to June 2021 were enrolled. Aza was administered at the standard dose of 75 mg/(m2·d) for 7 days in the standard dose group (50 cases), while at 100 mg/d for 7-12 days in the adjusted dose group (53 cases). The administration days in adjusted dose group was calculated based on the total standard dose of the patient's single course of treatment. The efficacy and safety between standard dose group and adjusted dose group were compared. Subgroup analysis were performed in the two groups for Aza alone, Aza combined with BCL-2 inhibitor, and Aza combined with low-dose chemotherapy for efficacy and safety. RESULTS: There were no significant differences in overall response rate (ORR), incidence of adverse reaction, and 1-year overall survival (OS) rate between standard dose group and adjusted dose group (P >0.05). The ORR of combination was higher than that of Aza alone (P < 0.05), while there was no significant difference in ORR between Aza combined with BCL-2 inhibitor and Aza combined with low-dose chemotherapy (P >0.05). The combination of BCL-2 inhibitor did not increase the incidence of adverse reactions compared wtih Aza alone. There was a higher risk of myelosuppression and pulmonary infection with a combination of low-dose chemotherapy than with a combination of BCL-2 inhibitor and Aza alone (P <0.05). No significant difference was observed in 1-year OS between Aza alone, Aza combined with BCL-2 inhibitor, and Aza combined with low-dose chemotherapy (P >0.05). CONCLUSIONS: Both two induction regimens can be used in elderly AML patients who cannot tolerate intensive chemotherapy with similar overall effectiveness and safety. Aza combined with low-dose chemotherapy may result in increased ORR and an increased incidence of serious adverse reactions, and may not result in longer survival compared with Aza alone. Aza combined with BCL-2 inhibitor not only has similar effect in complete remission, objective response rate, and OS compared with Aza combined with low-dose chemotherapy, but also has higher safety.

A global study for acute myeloid leukemia with RARG rearrangement.

Acute myeloid leukemia (AML) with retinoic acid receptor γ (RARG) rearrangement has clinical, morphologic, and immunophenotypic features similar to classic acute promyelocytic leukemia. However, AML with RARG rearrangement is insensitive to alltrans retinoic acid (ATRA) and arsenic trioxide (ATO) and carries a poor prognosis. We initiated a global cooperative study to define the clinicopathological features, genomic and transcriptomic landscape, and outcomes of AML with RARG rearrangements collected from 29 study groups/institutions worldwide. Thirty-four patients with AML with RARG rearrangements were identified. Bleeding or ecchymosis was present in 18 (54.5%) patients. Morphology diagnosed as M3 and M3v accounted for 73.5% and 26.5% of the cases, respectively. Immunophenotyping showed the following characteristics: positive for CD33, CD13, and MPO but negative for CD38, CD11b, CD34, and HLA-DR. Cytogenetics showed normal karyotype in 38% and t(11;12) in 26% of patients. The partner genes of RARG were diverse and included CPSF6, NUP98, HNRNPc, HNRNPm, PML, and NPM1. WT1- and NRAS/KRAS-mutations were common comutations. None of the 34 patients responded to ATRA and/or ATO. Death within 45 days from diagnosis occurred in 10 patients (∼29%). At the last follow-up, 23 patients had died, and the estimated 2-year cumulative incidence of relapse, event-free survival, and overall survival were 68.7%, 26.7%, and 33.5%, respectively. Unsupervised hierarchical clustering using RNA sequencing data from 201 patients with AML showed that 81.8% of the RARG fusion samples clustered together, suggesting a new molecular subtype. RARG rearrangement is a novel entity of AML that confers a poor prognosis. This study is registered with the Chinese Clinical Trial Registry (ChiCTR2200055810).

Treatment and outcome patterns of patients with Waldenström's macroglobulinemia: a large, multicenter retrospective review in China.

In this study, we aimed to investigate treatment options and the prognosis of patients with WM in China. This retrospective study included 1141 patients diagnosed with symptomatic WM between January 2003 and December 2019 at 35 tertiary hospitals in 22 provinces of China. Fifty-four patients (7.3%) received monotherapy, 264 (36.0%) received chemoimmunotherapy, 395 (53.8%) received other combination regimens without rituximab, and 21 (2.9%) received ibrutinib. Using a multivariable Cox regression model, age > 65 years old, platelets <100 × 109/L, serum albumin <3.5 g/dl, ß2 microglobulin concentration ≥4 mg/L and LDH ≥250 IU/L predicted poor OS. In summary, our study showed that frontline treatment choices for WM are widely heterogeneous. We validated most of the established prognostic factors in the rIPSS (age >65 years, LDH ≥250 IU/L, ALB <3.5 g/dl and ß2 microglobulin ≥4 mg/L) together with PLT ≤ 100 × 109/L indicate a poor prognosis for patients with WM.

[Analysis of the Effect of TET2 Mutation and SNP on Clinical Characteristics and Prognosis of AML Patients].

OBJECTIVE: To evaluate the clinical significance of TET2 mutation(TET2mut) and SNP in adult acute myeloid leukemia (AML) and its effect on prognosis. METHODS: A total of 24 genes, including TET2, FLT3-ITD and NPM1, were detected in 124 adult AML patients using second-generation sequencing technology, and their clinical characteristics and effect on prognosis of patients were analyzed. RESULTS: A total of 25 TET2 gene mutations were detected in 124 AML patients, the mutation rate was 20.2%, there were 75 cases of TET2 single nucleotide polymorphisms(SNP), accounting for 60.5%. There were 47 cases of SNPrs2454206(G>A), accounting for 37.9%. Compared with TET2 wild-type(TET2wt) patients, TET2mut patients were mostly elderly. TET2SNP was commonly seen in male, with statistically significant differences (P＜0.05). SNP rs2454206 had no significant correlation with sex and age (P＞0.05). However, the analysis found that the complete remission rate of 1 course and the total complete rate (CR) of patients with TET2AG/GG were both obviously superior to those with TET2AA (P＜0.05). In the univariate analysis, the overall survival(OS) rate of the patients with TET2mut was lower than that of patients with TET2wt, and the event free survival (EFS) rate was higher than that of TET2wt patients, but the difference was not statistically significant (P＞0.05).The 1-year OS rate and EFS rate of the patients with TET2AA were significantly lower than those of the patients with TET2AG/GG (P＜0.05). Multivariate analysis showed that TET2AA was an independent risk factor for OS and EFS in AML patients. CONCLUSION: TET2 SNPrs2454206(G＞A) commonly appears in patients with acute myeloid leukemia, and these patients have the better response to chemotherapy and a better prognosis.

[Proventive and Therapentic Effects of Endothelial Progenitor Cells on Monocrotaline-Induced Hepatic Vein Occlusion Disease].

OBJECTIVE: To investigate the preventive and therapeutic effects of endothelial progenitor cells on monocrotaline-induced hepatic vein occlusion disease in mice. METHODS: C57BL/6 mice were randomly divided into 3 groups: saline group (n=15), monocrotaline group (n=15), and endothelial progenitor cell infusion group (n=15). Liver function (TBIL, ALT, AST), liver index, and serum levels of TNF-α and IL-6 were measured on the 8th day after intragastric administration. Hepatic sinusoidal endothelial cells, hepatic central venous endothelial cells and hepatocytes were observed by both HE and immunohistochemical staining. Hepatic fibrosis was observed by Masson's trichrome staining. RESULTS: By the light microscopy, the liver of the monocrotaline group showed moderate to the severe injuries of hepatic sinusoidal and central venous endothelial cells, and hepatic venous congestion. Masson staining showed moderate to severe hepatic fibrosis of central vein and hepatic sinus. In the endothelial progenitor cell group, hepatic sinusoidal and central venous endothelial cell injuries, and the fibrosis of central hepatic vein and hepatic sinus were mild to moderate. Hepatic venous congestion was reduced in comparison with that in the mice of the monocrotaline group. Compared with the endothelial progenitor cell group, the liver index was higher, the liver function was more abnormal, and the serum expression levels of TNF-α and IL-6 were higher in the monocrotaline group. CONCLUSION: The monocrotaline-induced damage of hepatic sinusoidal and central venous endothelial cells is an linitiating factor for hepatic vein occlusive disease. Infusion of endothelial progenitor cells can play a role in preventing and treating hepatic vein occlusion.

[Rituximab treatment for adults with steroid-resistant idiopathic thrombocytopenic purpura].

OBJECTIVE: To investigate the efficacy and safety as well as the effects of rituximab on B-lymphocytes and anti-platelet glycoprotein-specific antibodies, in patients with steroid-resistant idiopathic thrombocytopenic purpura (ITP). METHODS: Twelve steroid-resistant ITP patients, 16 to 54 years old, received intravenous rituximab at the dose of 375 mg/m2 once--weekly for 4 weeks. Lab studies included CBC, serum concentrations of IgG, IgM and IgA. CD3+, CD4+, CD8+, CD19+, CD20+ cell numbers were assayed by flow cytometry and anti-platelet glycoprotein-specific antibodies (GP IIb/IIa, GP Ib/IX) were assayed by monoclonal antibody-specific immobilisation of platelet antigens prior to and following rituximab therapy. RESULTS: A complete response (platelet counts > or = 100 x 10(9)/L) was observed in 4 cases, a partial response (platelet counts between 50 and 100 x 10(9)/L) in 3 cases, a minor response (platelet counts between 30 and 50 x 10(9)/L) in 2 cases, and nonresponse (platelet counts < 30 x 10(9)/L) in 3 cases. Responses were sustained 0.5 to 12 months (median 5 months). After 4 weeks of rituximab therapy, anti-platelet glycoprotein-specific antibodies (GP IIb/IIIa, GP Ib/IX) disappeared except one NR patient and CD19+/ CD20+ cells were almost depleted in all patients (295.0 +/- 86.4) x 10(6)/L vs (4.1 +/- 2.2) x 10(6)/L (P < 0.01). As expected, the T cell counts, and the serum concentrations of IgG, IgM and IgA were not changed after therapy. No severe side effects were observed. CONCLUSION: Rituximab may be an effective and safe treatment for adults with steroid-resistant ITP.

[Combination therapy of thymosin alpha1 and high dose dexamethasone in patients with chronic idiopathic thrombocytopenic purpura].

OBJECTIVE: To study the efficacy and mechanism of thymosin alpha(1) (Talpha(1)) combined with high dose dexamethasone (HD-Dex) in patients with chronic idiopathic thrombocytopenic purpura. METHODS: (1) Out of sixty-six newly diagnosed patients with chronic idiopathic thrombocytopenic purpura, 27 patients received oral HD-Dex at single daily doses of 40 mg for 4 consecutive days, 39 patients received HD-Dex plus Talpha(1) 1.6 mg subcutaneously thrice weekly for 4 weeks. (2) The plasma levels of Talpha(1), IFNgamma, IL-2, IL-4, IL-10 and TGF-beta(1) of the 66 patients and 20 healthy controls were detected with ELISA. RESULTS: (1) Twelve patients (44.4%) in HD-Dex treatment group and thirty patients (76.9%) in HD-Dex plus Talpha(1) treatment group achieved complete response respectively (P < 0.05). After a follow-up period of 6 months, HD-Dex plus Talpha(1) treatment group showed a significantly greater rate of sustained response (24/39, 61.5%) and a lower replacing rate (15/39, 38.5%) than HD-Dex treatment group (9/26, 34.6%; 17/26, 65.4%) (P < 0.05). (2) After treatment, a remarkable decrease of Talpha(1) levels was seen HD-Dex plus Talpha(1) treatment group [(2.43 +/- 1.47), (1.83 +/- 1.22)] microg/L (P < 0.05). (3) In HD-Dex plus Talpha(1) treatment group, the plasma levels of both IFNgamma and IL-2 were significantly higher [(22.71 +/- 7.98), (28.42 +/- 11.27)] ng/L than those in controls [(10.23 +/- 3.97), (8.73 +/- 8.22)] ng/L (P < 0.01). The levels of both IL-4 and IL-10 were significantly lower [(5.93 +/- 3.85), (3.24 +/- 1.36)] ng/L after treatment as compared with those in the controls [(14.39 +/- 8.03), (8.67 +/- 3.04)] ng/L (P < 0.01). After treatment, IFNgamma and IL-2 decreased [(11.57 +/- 4.33), (14.56 +/- 10.76)] ng/L (P < 0.01) and IL-4 and IL-10 increased greatly [(9.87 +/- 4.82), (7.90 +/- 2.71)] ng/L (P < 0.05). (4) TGF-beta(1) in HD-Dex plus Talpha(1) treatment group significantly increased from [(1.31 +/- 0.71), (4.19 +/- 1.80)] microg/L after treatment (P < 0.01). (5) There was a significantly positive correlation between Talpha(1) and TGF-beta(1) (r = 0.6028, P < 0.05). CONCLUSIONS: (1) Combination therapy of Talpha(1) and HD-Dex seems to be effective and safe in newly diagnosed patients with ITP. (2) Talpha(1) may balance the Th1/Th2/Th3 subgroups and induce a physiologic immunosuppressive effect of NK cells and autoimmune tolerance.

[Identification of human platelet specific functional antibody and its fragments].

OBJECTIVE: To study the immunoreactivity of the specific anti-platelet glycoprotein (GP) IgG antibody and its F(ab')2 fragments from patients with chronic idiopathic thrombocytopenic purpura (ITP) and to investigate their effects on platelet aggregation function. METHODS: Peripheral blood samples were collected from 84 patients with ITP. Modified monoclonal antibody immobilization of platelet antigen assays was used to detect the IgG antibodies specific for GP I b/II a, GP I b/IX and GP VI. The IgG antibody and its F(ab')2 fragments in the positive plasma inhibiting platelet aggregation function were prepared and purified. Plate-rich Peripheral blood sample was collected from a normal person with O type blood and platelet-rich plasma (PRP) and platelet-poor plasma (PPP) were prepared. Plasma pf ITP patient or purified IgG or F(ab')2 fragments of different concentrations were added into PRP, and then inducers of platelet aggregation ADP, ristocetin, or collagen were added. The platelet aggregation was measured. Platelet GP II b/III a specific human-rat chimeric antibody 7E3 and GP I b specific antibody SZ2 were used as positive controls and PBS was used as negative control. RESULTS: GP II b/III a and/or GP I b/IX and/or GP VI specific antibodies were found in 48 (57.1%) patients. The plasma, purified IgG and F(ab')2 fragments of 7 of these 48 patients (14.6%) with positive autoantibody showed significant activity against GP II b/III a (4 patients), GP I b/IX (2 patients), or GP VI (one patient). The purified IgG and F(ab')2 fragments of 2 patients positive in GP II b/ III a autoantibody out of the 7 patients significantly inhibited the platelet aggregation induced by ADP, the purified IgG and F(ab')2 fragments of 1 patients positive in GP I b/IX out of the 7 patients significantly inhibited the platelet aggregation induced by ristocetin, and the purified IgG and F(ab')2 fragments of 1 patients positive in GP VI out of the 7 patients significantly inhibited the platelet aggregation induced by collagen. CONCLUSION: A functional fragment, F(ab')2 portion of IgG is responsible for the autoantibody interaction with platelet GPs in ITP, and some of them also affect the platelet function. It can be used to develop completely humanized anti-GP small molecular phage antibody.

[Research Progress on role of Abnormal Tryptophan Metabolism in Immune Thrombocytopenia].

Immune thrombocytopenia (ITP) is a common acquired autoimmune hematological disorders. Platelet autoantibodies lead to the decrease of platelet production and (or) increase of its destruction. The latest researches showed that the abnormal tryptophan metabolism mediated by indoleamine-2, 3-dioxygenase(IDO) is related with the pathogenesis of ITP. The patients with ITP show less expression of IDO, reduction of Treg cells and increase of autoreactive T cells and autoantibodies. CTLA-4-Ig can improve the expression of IDO in the patients with ITP, which also can inhibit the proliferation and activation of self-reactive T cells. Thus, clarifying the abnormal tryptophan metabolism mediated by IDO may provide a new idea for improving the understand of the pathogenesis and treatment of ITP. This review focuses on reasearch progress of the tryptophan metabolism mediated by IDO and ITP.

[Role of dendritic cells and their Toll-like receptor 4 in the pathogenesis of idiopathic thrombocytopenic purpura].

OBJECTIVE: To study the surface antigen of the dendritic cells (DC) and their Toll-like receptor 4 (TLR4) expression in patients with idiopathic thrombocytopenic purpura (ITP), and to explore their role in ITP pathogenesis. METHODS: The peripheral blood mononuclear cells isolated from complete remission patients (CR), non-complete remission patients (n-CR) and normal controls were stimulated by rhGM-CSF and rhIL-4. The surface antigen of the DC was analyzed by flow cytometry. The level of IL-12p70 in the supernatant was detected by enzyme linked immunosorbent assay. The expression of TLR4 mRNA of DC was detected by real time PCR. RESULTS: In the 21 CR ITP patients, the expression of both CD80 and CD86 in DC was significantly increased compared with that in normal controls \[(51.60 ± 13.47)% vs (36.03 ± 15.43)%, (61.50 ± 15.93)% vs (40.28 ± 11.49)%, respectively\] (P < 0.01). The expression of CD80 and CD86 in n-CR group was also significantly increased \[(53.29 ± 19.49)% and (62.91 ± 18.43)%, respectively\] (P < 0.01). After HD-DXM treatment, both CD80 and CD86 in CR patients were decreased (P < 0.01). There was no difference between the DXM treatment patients and the normal controls. In n-CR group, there was no difference in CD80 and CD86 expression before and after DXM therapy \[(52.30 ± 20.98% and (49.79 ± 20.28)%, respectively\] (P > 0.05). CD80 was still higher than normal (P < 0.05), while CD86 was not changed. The level of IL-12p70 in CR ITP patients before treatment was significantly higher \[(67.52 ± 14.43) pg/ml\] than that of the controls \[(39.78 ± 10.03) pg/ml\](P < 0.01), and after treatment, was significantly decreased to (43.90 ± 8.49) pg/ml, being no difference from that in control. In n-CR group, IL-12p70 was lower after treatment \[(48.45 ± 9.68) pg/ml\] than that before treatment \[(65.35 ± 12.52) pg/ml\] (P < 0.01), but still higher than that in control (P < 0.05). The TLR4 mRNA level in DCs of CR ITP patients before treatment were significantly higher 0.69 ± 0.17 than that of controls (0.31 ± 0.09) (P < 0.01) and after treatment, was reduced to 0.35 ± 0.11, being no difference from that in control. In n-CR group, TLR4 mRNA was decreased from 0.65 ± 0.09 to 0.52 ± 0.21 after treatment (P < 0.01), but still higher than normal (P < 0.01). CONCLUSION: DC may play an important role in ITP by their Toll-like receptor and cytokine secretion.

Dexamethasone inhibits immunoreactivity of dendritic cells in patients with chronic idiopathic thrombocytopenic purpura.

The objective of this study was to investigate the possible effects of dexamethasone treatment on the immunoreactivity of dendritic cells in patients with chronic idiopathic thrombocytopenic purpura (ITP). Thirty-six newly diagnosed patients with chronic ITP received an oral high dose of dexamethasone (HD-DXM) at single daily doses of 40 mg for 4 consecutive days. The CD14 leukocytes isolated from the 21 remission patients and 10 normal controls were stimulated by recombinant human granulocyte-macrophage colony-stimulating factor and rhIL-4. The surface antigens of the dendritic cells were analyzed by flow cytometry and the level of IL-12p70 in the supernatant was detected by enzyme-linked immunosorbent assay. In ITP patients, the expression of both CD80 and CD86 in dendritic cells were significantly increased compared with those of the normal controls (51.60 +/- 13.47 vs. 36.03 +/- 15.43%, 61.50 +/- 15.93 vs. 40.28 +/- 11.49%, respectively; P < 0.05). After HD-DXM treatment, both CD80 and CD86 were decreased to levels comparable to normal controls (P > 0.05). The level of IL-12p70 in ITP patients was significantly higher (67.52 +/- 14.43 pg/ml) than the controls (39.78 +/- 10.03 pg/ml, P < 0.05). After treatment, IL-12p70 was reduced to 43.90 +/- 8.49 pg/ml with no significant differences between ITP group and control (P > 0.05). Dendritic cells and their cytokine secretion play important roles in ITP, and DXM may achieve its therapeutic effect on ITP by inhibiting immune responses through suppressing the function of dendritic cells.

[Study of a humanized anti-platelet GP VI single chain Fv phage antibody].

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: Anti-platelet GPVI ScFv phage antibody produced by phage antibody library technology can inhibit the aggregation function of platelet.

[Advances in the studies of platelet glycoprotein VI (GPVI): review].

Platelet glycoprotein VI (GPVI) is a major receptor for collagen on the platelet surface. It mediates the initial platelet contact with collagen, generates intracellular signals, increases the affinity of integrin receptor, and causes platelet aggregation and thrombosis. Suppression of GPVI function can significantly inhibit collagen-induced platelet adhesion, aggregation and thrombosis, so GPVI has become a novel target for antiplatelet therapy. Within the last few years, major advances have been made in understanding platelet-collagen interactions. In this paper, the advances of study on GPVI, including composition of GPVI, functions of GPVI, factors related with functions of GPVI, GPVI and clinic were summarized.

Effects of IgG and its F(ab')2 fragments of some patients with idiopathic thrombocytopenic purpura on platelet aggregation.

OBJECTIVES: To make humanized monoclonal antibodies by phage surface display technology, we screened out the specific anti-platelet glycoproteins (GPs) IgG antibody from patients with chronic idiopathic thrombocytopenic purpura (ITP), which can inhibit platelet aggregation. METHODS: We studied plasmas from 68 patients with ITP for the presence of IgG antibodies specific for GPIIb/IIIa and/or GPIb/IX using modified monoclonal antibody immobilization of platelet antigen assays. The IgG antibody and its F(ab')(2) fragments of the positive plasmas which could inhibit platelet aggregation function were prepared and purified. Their immunoreactivity to platelet GPs and effects on platelet function were further analyzed. RESULTS: GPIIb/IIIa- and GPIb/IX-specific antibodies were found in 21 and 19 patients, respectively. Six of them had antibodies against both GP complexes. Among the 34 positive plasmas, four with positive anti-GPIIb/IIIa autoantibody showed significant inhibition of platelet aggregation induced by adenosine diphosphate (ADP), whereas one with GPIb/IX-specific antibody inhibited ristocetin-induced platelet aggregation. The purified IgG and its F(ab')(2) fragments from two patients not only retained the ability to bind to platelet GPs but also impaired the in vitro ADP-induced platelet aggregation. CONCLUSIONS: F(ab')(2) portion of the IgG is a functional fragment, which is responsible for the autoantibody interaction with platelet GPs in ITP, and some of them also affect platelet function, which can be used to develop completely humanized anti-GPIIb/IIIa small molecular phage antibody.

[The immunoreactivity of IgG and its fragments from ITP patients and their effects on platelet aggregation function].

OBJECTIVE: 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. METHODS: 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. RESULTS: (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. CONCLUSIONS: 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.

[Advances in the studies of humanized anti-platelet antibody--review].

Platelet plays an important role in bleeding and thrombotic diseases. Humanized anti-platelet antibodies have great clinical effects in treatment of ITP and preventing thrombosis. The important role of platelet in bleeding and thrombotic diseases, the present status of development on study of humanized anti-platelet antibody and its application in treatment of bleeding and thrombotic diseases were summarized in this review.