扁桃体灭活菌株对IgA肾病扁桃体CD4+ CD25+细胞和J链产生的影响

目的:以扁桃体隐窝内甲型链球菌(HS)灭活菌株刺激IgA肾病(IgAN)患者和非肾炎患者扁桃体淋巴细胞,观察未刺激及刺激后CD4 CD25 细胞和分泌J链IgA细胞数量,探讨IgAN的发病机制。方法:(1)收集37例IgAN患者及37例非肾炎慢性扁桃体炎患者手术摘除的扁桃体;(2)分离鉴定两组患者扁桃体隐窝内细菌及分离培养扁桃体淋巴细胞;(3)以分离最多的灭活菌株HS体外刺激扁桃体淋巴细胞72h;(4)以流式细胞仪检测扁桃体淋巴细胞CD4 CD25 细胞数,以原位杂交技术检测J链mRNA表达,以免疫荧光及荧光原位杂交技术同步分析分泌J链IgA细胞。结果:(1)两组患者均有甲型链球菌,且甲型链球菌在分离的细菌中是最多的。两组患者的细菌谱和细菌量无统计学差异。(2)未刺激、非肾炎患者HS(HS-controls)、IgAN患者HS(HS-IgAN)刺激后CD4 CD25 细胞数[(0.98±0.204)% vs (3.58±0.554)%,P<0.05,(1.37±0.214)% vs (5.78±0.562)%,P<0.05,and(1.43±0.202)% vs (6.05±0.521)%,P<0.05],IgAN组与非肾炎组比较,前者均显著低于后者。HS对IgAN组CD4 CD25 细胞的刺激指数(stimulation index,SI)显著低于非肾炎组(P均<0.05)。(3)未刺激、HS-controls、HS-IgAN刺激后J链mRNA阳性IgA细胞[(11.9±3.1)% vs (6.5±1.5)%,P<0.05,(33.5±5.7)% vs (13.9±1.2)%,P<0.05,and(35.1±6.2)% vs (13.9±1.2)%,P<0.01],IgAN组与非肾炎组比较,前者均显著高于后者。HS对IgAN组J链mRNA阳性IgA细胞的SI显著高于非肾炎组(P均<0.01)。(4)HS对CD4 CD25 细胞的SI与对分泌J链IgA细胞的SI呈显著性负相关(P均<0.01)。结论:IgAN患者扁桃体CD4 CD25 细胞减少和分泌J链IgA细胞增多可能与IgAN的发病机制有关。     

Monoclonal antibody therapy for B-cell lymphoma: clinical trials of an anti-CD20 monoclonal antibody for B-cell lymphoma in Japan

Twelve patients with relapsed CD20+ B-cell non-Hodgkin's lymphoma (B-NHL) were enrolled in a phase I study of rituximab; 4 received rituximab 250 mg/m2 and 8 received rituximab 375 mg/m2 once weekly for 4 weeks. Grade 1 or 2 infusion-related toxicity was observed. Of the 11 eligible patients, 2 achieved complete responses and 5 achieved partial responses. The elimination half-life (T1/2) of rituximab was 445 +/- 361 hours, and serum rituximab levels were detectable at 3 months. Thereafter, 90 relapse patients with indolent B-NHL or mantle cell lymphoma (MCL) were enrolled in a phase II study and treated with rituximab at 375 mg/m2 per infusion in 4 weekly infusions. Sixteen patients were ineligible in protocol compatible analyses. The overall response rates (ORR) in indolent B-NHL and MCL were 61% (37 of 61 patients) and 46% (6 of 13 patients), respectively. Factors affecting response and progression-free survival (PFS) were analyzed for 77 patients whose histopathology was centrally confirmed as indolent B-NHL or MCL. The ORR in patients receiving 1 prior chemotherapy regimen was higher than the ORR in those receiving > or = 2 regimens (P < .05). The median PFS was shorter in MCL patients, in those with extranodal disease, and in those receiving > or = 2 prior chemotherapy regimens (P < .01). The PFS of patients with higher serum rituximab levels (> or = 70 microg/mL) immediately before the third infusion was longer than that of other patients (P < .01). Several pretreatment factors and serum rituximab levels are useful for predicting the efficacy of rituximab monotherapy. Rituximab re-treatment was well tolerated in 13 patients with no grade 3 or 4 nonhematological toxicities. A partial response was observed in 5 patients (38%), and the median PFS after re-treatment was 5.1 months. In conclusion, rituximab is a highly effective agent in relapsed indolent and aggressive B-NHL and MCL and has acceptable toxicities.     

小鼠感染后不同时间血清对日本血吸虫童虫细胞介导的细胞毒作用

用抗体依赖、细胞介导的细胞毒试验(ADCC)观察了小鼠感染后不同时间血清在体外杀伤系统中对日本血吸虫童虫的作用规律。未加补体时,嗜酸粒细胞或巨噬细胞介导的童虫杀伤作用于感染后4wk出现明显作用,分别于5—7wk和6—8wk达高峰,然后下降,至11wk时降至4wk时的水平;而未加补体时无明显的中性粒细胞介导的杀伤作用。在补体参与下,这三种细胞均能介导对童虫的杀伤作用,且作用增强。证实ADCC是日本血吸虫获得性抵抗力的一个重要组分,嗜酸粒细胞、巨噬细胞介导的对童虫的细胞毒作用是不依赖补体的,而中性粒细胞介导的作用是依赖补体的。结果对选择免疫预防适宜时间和评价候选疫苗效果有参考意义。     

Potentiation of lysis of leukaemia cells by a bispecific antibody to CD33 and CD16 (FcγRIII) expressed by human natural killer (NK) cells

Bispecific antibodies recognizing tumour-associated antigens and trigger molecules expressed on immune effector cells have been shown to redirect cytotoxicity of several types of peripheral blood cells against relevant tumour targets. Among various effector cells, natural killer (NK) cells appear to play a role in defence against leukaemia. Here we report the successful chemical conjugation of monoclonal antibodies to CD33 and CD16 to create a bispecific antibody (BsAb 251x3G8). This bispecific antibody is capable of augmenting the killing of otherwise resistant leukaemia cells by peripheral blood lymphocytes (PBL), purified resting NK (R-NK) cells, and activated NK (A-NK) cells. BsAb 251x3G8 may play a role in the therapy of acute myeloid leukaemia (AML) through redirecting the cytotoxic activity of endogenous or adoptively transferred NK cells.     

Antibody-directed superantigen-mediated T-cell killing of myeloid leukaemic cell line cells

Abstract: Bacterial superantigens (SAgs) bound to MHC class II molecules on target cells are efficient activators of cytotoxic T cells expressing certain T cell receptor (TCR) Vβ regions. We described earlier that the specificity of the SAg Staphylococcus enterotoxin A (SEA) can be changed by introducing a D227A point mutation in the major MHC class II binding site and by genetically fusing the SEA mutant (SEAm) to protein A (PA). This SEAm-PA fusion protein can then be used to direct cytotoxic T cells to tumour cells coated with monoclonal antibodies (mAbs). In this communication, we tested the PA-SEAm fusion protein together with mAbs against the myeloid cell surface antigens CD13, CD15 and CD33. A SEA-reactive T cell line was used as effector cells against 10 different myeloid leukaemic cell lines. Optimal lysis of antigen positive leukaemic cells was obtained at a PA-SEAm concentration of 1 ng/ml and effector: target cell ratios of 15 : 1. No correlation between target cell sensitivity and the level of surface antigen expression could be seen. The 6 acute myeloid leukaemia (AML) cell lines tested appeared to be more sensitive than the 4 chronic myeloid leukaemia (CML) cell lines. The sensitivity of the AML cell line HL-60 could be improved further by stimulation with TNFα. This was accompanied by increased surface ICAM-1 expression whereas specific target molecule expression (CD13, CD33) was unchanged. This suggests that sensitivity to lysis is related to the leukaemic subtype and ICAM-1 expression but not to the tumour antigen density. Our results show that it is possible to direct cytotoxic T cells to myeloid leukaemia cells by using SAgs linked to mAbs, and encourage the construction and testing of a recombinant direct SAg-mAb fusion protein as a candidate drug for therapy of myeloid leukaemias.     

Rituximab and alemtuzumab induce a nonclassic,caspase-independent apoptotic pathway in B-lymphoid cell lines and in chronic lymphocytic leukemia cells

The monoclonal antibodies (MoAbs) alemtuzumab (anti-CD52) and rituximab (anti-CD20) produce objective clinical responses in patients with chronic lymphocytic leukemia (CLL). However, their mechanisms of action are not fully understood. Therefore, we investigated the mechanisms of lymphoma and CLL cell killing by two anti-CD20 antibodies (rituximab, B1) and by alemtuzumab. All antibodies induced complement-independent cell death in B-lymphoid cell lines Raji, Ramos, and Mec-1. The efficiency of cell killing was increased by the addition of human complement in Raji but not Ramos cells. Both alemtuzumab and rituximab also killed freshly isolated CLL cells, with a much stronger response for alemtuzumab (from eight of eight patients) compared to rituximab (from two of six patients). Cell morphology and Western blot analyses revealed that the antibody-induced cell death lacked some typical features of apoptosis such as chromatin condensation or poly-ADP-ribose polymerase (PARP) cleavage. Taken together, the results suggest that the tumor killing activity of these MoAbs is not only mediated by complement-mediated cytotoxicity (CDC) or antibody-dependent cytotoxicity (ADCC), but also by a nonclassic, caspase-independent apoptotic pathway.     

A CD33-specific single-chain immunotoxin mediates potent apoptosis of cultured human myeloid leukaemia cells

A novel single-chain immunotoxin was constructed by combining a CD33-specific single chain Fv (scFv) antibody fragment with an engineered variant of Pseudomonas exotoxin A (ETA). The variant toxin carries the KDEL peptide at its C-terminus, a cellular peptide mediating improved retrograde transport to the endoplasmic reticulum. The purified recombinant fusion protein induced potent apoptosis of the human myeloid cell lines U937, HL-60 and THP-1. Up to 98% of U937 cells were eliminated after treatment for 72 h with a single dose of 500 ng/ml (c. 7 nmol/l). Killing was antigen-specific and occurred by apoptosis. A control protein, consisting of a CD19-specific scFv antibody fragment fused to the ETA-KDEL toxin, failed to induce death of the CD19-negative cell lines U937, HL-60 and THP-1. The CD33-ETA toxin also mediated apoptosis of fresh patient-derived acute myeloid leukaemia cells from bone marrow and peripheral blood. The pronounced antigen-restricted cytotoxicity of the novel fusion protein makes it a candidate for further evaluation of its therapeutic potential.     

Subcutaneous injections of low-dose veltuzumab (humanized anti-CD20 antibody) are safe and active in patients with indolent non-Hodgkin's lymphoma

Background

Subcutaneous injections of anti-CD20 antibodies may offer benefits to both patients and the healthcare system for treatment of B-cell malignancies.

Design and Methods

A pilot study was undertaken to evaluate the potential for subcutaneous dosing with 2^nd generation anti-CD20 antibody veltuzumab in patients with CD20⁺ indolent non-Hodgkin’s lymphoma. Patients with previously untreated or relapsed disease received 4 doses of 80, 160, or 320 mg veltuzumab injected subcutaneously every two weeks. Responses were assessed by computed tomography scans, with other evaluations including adverse events, safety laboratories, B-cell blood levels, serum veltuzumab levels, and human anti-veltuzumab antibody (HAHA) titers.

Results

Seventeen patients (14 follicular lymphoma; 13 stage III or IV disease; 5 treatment-naive) completed treatment with only occasional, mild-moderate, transient injection reactions and no other safety issues. Subcutaneous veltuzumab demonstrated a slow release pattern over several days, achieving a mean Cmax of 19, 25 and 63 μg/mL at 80, 160, and 320 mg doses for a total of 4 administrations, respectively. Depletion of circulating B cells occurred after the first injection. The objective response rate (partial responses plus complete responses plus complete responses unconfirmed) was 47% (8/17) with a complete response/complete response unconfirmed rate of 24% (4/17); 4 of 8 objective responses continued for 60 weeks or more. All serum samples evaluated for human anti-veltuzumab antibody were negative.

Conclusions

Subcutaneous injections of low-dose veltuzumab are convenient, well tolerated, and capable of achieving sustained serum levels, B-cell depletion, and durable objective responses in indolent non-Hodgkin’s lymphoma. (Clinicaltrials.gov identifier: {"type":"clinical-trial","attrs":{"text":"NCT00546793","term_id":"NCT00546793"}}NCT00546793)     

HuMab-7D8, a monoclonal antibody directed against the membrane-proximal small loop epitope of CD20 can effectively eliminate CD20low expressing tumor cells that resist rituximab-mediated lysis

Background

Incorporation of the chimeric CD20 monoclonal antibody rituximab in the treatment schedule of patients with non-Hodgkin’s lymphoma has significantly improved outcome. Despite this success, about half of the patients do not respond to treatment or suffer from a relapse and additional therapy is required. A low CD20-expression level may in part be responsible for resistance against rituximab. We therefore investigated whether the CD20-expression level related resistance to rituximab could be overcome by a new group of CD20 mAbs (HuMab-7D8 and ofatumumab) targeting a unique membrane-proximal epitope on the CD20 molecule.

Design and Methods

By retroviral transduction of the CD20 gene into CD20-negative cells and clonal selection of transduced cells a system was developed in which the CD20-expression level is the only variable. These CD20 transduced cells were used to study the impact of rituximab and HuMab-7D8 mediated complement-dependent cytotoxicity. To study the in vivo efficacy of these mAbs an in vivo imaging system was generated by retroviral expression of the luciferase gene in the CD20-positive cells.

Results

We show that HuMab-7D8 efficiently killed CD20^low cells that are not susceptible to rituximab-induced killing in vitro. In a mouse xenograft model, we observed a comparable increase in survival time between HuMab-7D8 and rituximab-treated mice. Most significantly, however, HuMab-7D8 eradicated all CD20-expressing cells both in the periphery as well as in the bone marrow whereas after rituximab treatment CD20^low cells survived.

Conclusions

Cells that are insensitive to in vitro and in vivo killing by rituximab as the result of their low CD20-expression profile may be efficiently killed by an antibody against the membrane-proximal epitope on CD20. Such antibodies should, therefore, be explored to overcome rituximab resistance in the clinic.     

Susceptibility to opportunistic infections in HIV-infected patients with increased CD4 T-cell counts on antiretroviral therapy may be predicted by markers of dysfunctional effector memory CD4 T cells and B cells

OBJECTIVES: HIV-infected patients responding to combination antiretroviral therapy (ART) after experiencing severe immunodeficiency may exhibit persistent immune defects and occasionally experience opportunistic infections (OIs) despite increased CD4 T-cell counts. The investigation of immune defects in such patients was examined in this study. METHODS: CD4 effector memory T-cell (T(em)-cell) function [assessed by blood cytomegalovirus (CMV) interferon-gamma (IFN-gamma) enzyme-linked immunosorbent spot-forming cell assay (ELISPOT) counts] and B-cell dysregulation [assessed by serum immunoglobulin A (IgA) and IgE levels] were examined in 27 patients with increased CD4 T-cell counts after receiving ART for over 2 years. Two of these patients and one other had developed OIs on ART and are described in detail. RESULTS: Serum levels of IgA and IgE were higher than reference intervals (P<0.001) and CMV IFN-gamma ELISPOT counts were lower than those in non-HIV-infected controls (P<0.001) in the HIV-infected patients. Low CMV IFN-gamma ELISPOT counts were associated with high IgA levels (r=-0.5, P=0.01, Spearman's correlation test) and segregated with high IgE levels (P=0.06, Fisher's test). CMV IFN-gamma ELISPOT counts and serum IgA and IgE levels did not change significantly over a median time of 35 (range 8-60) months after the first measurement, whereas CD4 T-cell counts increased. All three patients who experienced OIs had repeatedly low CMV IFN-gamma ELISPOT counts and increased serum levels of IgA and/or IgE. CONCLUSION: Low CD4 T(em)-cell function and B-cell dysregulation are immune defects that may persist independently of changes in the CD4 T-cell count in HIV-1-infected patients responding to ART and are associated with an increased risk of developing an OI.     

Remission of demyelinating polyneuropathy with immunoadsorption, low dose corticosteroids and anti-CD20 monoclonal antibody

Demyelinating polyneuropathy with anti-myelin associated glycoprotein (anti-MAG) antibodies is an immune mediated disorder characterized by proximal and distal symmetric weakness. Electrophysiological measurements depict features characteristic for demyelination, including prolonged distal latency, retarded conduction velocity, delayed or absent F-waves, and, rarely, partial conduction block. We report on a 65-year-old patient who was diagnosed with demyelinating polyneuropathy and anti-MAG antibodies five years before admission. Despite immunosuppressive agents and extracorporeal therapy (plasmapheresis) the disease progressed as assessed by clinical symptoms and neurological tests. Laboratory results showed an increase of serum immunoglobulin M and anti-MAG antibodies over time. Because of progressive disease we decided to treat the patient with immunoadsorption followed by application of the anti-CD20 antibody, rituximab. Six cycles of selective immunoadsorption were performed over three-weekly intervals with a repetitively used column (Globaffin); each cycle consisted of four consecutive daily treatments. Starting at cycle 4 the anti-CD20 antibody rituximab was administered with 375 mg/m(2) after immunoadsorption. The pretreatment anti-MAG antibody level of 10,000 U/mL, indicating disease activity, initially increased during treatment to a maximum of 30,559 U/mL. However, after completion of the six cycles, the anti-MAG level had decreased to 2348 U/mL; 16 months after the last immunoadsorption cycle the anti-MAG level had increased to 4134 U/mL, while the conduction velocity and compound motor action potentials remained stabile. Immunoadsorption in combination with a monoclonal anti-CD20 antibody in patients with demyelinating polyneuropathy with anti-MAG is effective and can be used an alternative treatment option in patients with progressive disease.     

A glycoengineered anti-CD19 antibody with potent antibody-dependent cellular cytotoxicity activity in vitro and lymphoma growth inhibition in vivo

Human cluster of differentiation (CD) antigen 19 is a B cell-specific surface antigen and an attractive target for therapeutic monoclonal antibody (mAb) approaches to treat malignancies of B cell origin. MEDI-551 is an affinity-optimized and afucosylated CD19 mAb with enhanced antibody-dependent cellular cytotoxicity (ADCC). The results from in vitro ADCC assays with Natural Killer cells as effector cells, demonstrate that MEDI-551 is effective at lower mAb doses than rituximab with multiple cell lines as well as primary chronic lymphocytic leukaemia and acute lymphoblastic leukaemia samples. Targeting CD19 with MEDI-551 was also effective in several severe combined immunodeficiency lymphoma models. Furthermore, the combination of MEDI-551 with rituximab resulted in prolonged suppression of tumour growth, demonstrating that therapeutic mAbs with overlapping effector function can be combined for greater tumour growth inhibition. Together, the data demonstrate that MEDI-551 has potent antitumour activity in preclinical models of B cell malignancies. The results also suggest that the combination of the ADCC-enhanced CD19 mAb with an anti-CD20 mAb could be a novel approach for the treatment of B cell lymphomas.     

Treatment of relapsed idiopathic thrombocytopenic purpura with the anti-CD20 monoclonal antibody rituximab: a pilot study

We performed a prospective pilot study on 12 patients to evaluate the efficacy of the anti-CD20 monoclonal antibody rituximab in relapsed idiopathic thrombocytopenic purpura (ITP). Inclusion criteria were relapse of ITP with a thrombocyte count <20 000 micro L-1 and unsuccessful corticosteroid treatment. Eleven patients had a previous splenectomy, five patients had unsuccessful cytotoxic treatment, and six patients were refractory to intravenous immunoglobulins before rituximab therapy. Response criteria were as follows. Complete remission (CR): normalization of thrombocyte count for at least 30 d. Partial remission (PR): an increase of thrombocytes to above 30 000 microL(-1) for at least 30 d. Minor response (MR): any increase above 30 000 microL(-1) for less than 30 d but more than 10 d. No response (NR): failure to achieve any of the above responses. Treatment plan: We administered 375 mg m(-2) of rituximab once weekly on up to four consecutive weeks, unless there was early CR. Five patients (41%) achieved CR, two patients (17%) PR, and two patients MR (overall response rate 75%, median follow-up of responders 320 d). Four CR patients are ongoing; one CR patient relapsed after 6 months. Adverse events included excessive thrombocytosis in one patient as well as minor infusion-related (grade I) toxicities in four patients. We conclude that rituximab is a promising agent in the treatment of relapsed ITP.     

Dendritic cells fused with core binding factor-beta positive acute myeloid leukaemia blast cells induce activation of cytotoxic lymphocytes

Several reports have described various strategies of dendritic cell (DC) vaccination to induce specific T-cell responses in patients with acute myeloid leukaemia (AML). About 50-60% of AML cases blasts have chromosomal abnormalities, such as inv(16) or t(8,21), which could encode for leukaemia-specific antigenic peptide sequences, possibly presented in the context of self-major histocompatibility complex (MHC) molecules. As the co-culture of AML blasts with T lymphocytes seldom resulted in T-cell stimulation, we fused AML blasts with autologous DC to enhance this effect. The fusion cells expressed MHC class I and II, CD40, B7-1, B7-2, CD209 and several adhesion molecules. In a mixed lymphocyte hybrid reaction, the fusion cells induced the proliferation of autologous T cells. Moreover, in the special case of fusion cells established from AML blasts with the chromosomal abnormality inv(16), the autologous T lymphocytes could be primed to induce cytotoxicity against up to 70% autologous AML blasts in a effector:target ratio of 20:1. Blocking assays demonstrated that the lysis was chiefly mediated by CD8(+), CCR7(-) T lymphocytes, which could be further expanded in the form of effector memory CD8(+) T cells by repeated co-cultures with the autologous fusion cells.     

酶联免疫吸附试验检测弓形虫体IgA抗体及其临床应用的初步研究

试用弓形虫膜抗原ELISA对先天性弓形虫感染的畸形围产儿和后天获得性弓形虫感染者进行了特导性IgA抗体检测。11例先天性弓形虫宫内感染畸形围产儿的15份标本,6份检出IgA抗体,7份同时检出IgA;IgM抗体,两份仅检出IgM抗体。8份弓形虫抗体阳性的孕妇血清皆检出较强的IgA抗体。后天获得性弓形虫近期感染者32份血清,IgA抗体阳性3份,IgA、IgM抗体阳性8份,IgM抗体阳性21份;30份慢性感染者血清未有测到IgA抗体。对照组30份抗体阴性的孕妇血清及其围产儿的脐血检测结果抗体皆为阴性。IgA抗体是弓形虫感染早期的一个重要标志物,与IgM、IgG抗体同时检测,对临床急性弓形虫感染的诊断和疗效观察有着极其重大意义。     

A multicenter phase I/II trial of rituximab for refractory systemic lupus erythematosus

Although corticosteroids and immunosuppressants are widely used for the treatments of systemic lupus erythematosus (SLE), safer and more effective therapies are prerequisite. We and others have reported that anti-CD20 antibody rituximab targeting B cells are effective for refractory SLE and, therefore, safety and clinical efficacy of rituximab in SLE was evaluated by a multicenter phase I/II clinical trial. An open-label, multicenter study of 15 patients with active and refractory SLE (total British Isles Lupus Assessment Group [BILAG] score 8 to 17) was conducted. Rituximab was administered to 5 SLE patients as 4 infusions of 500 mg/body every week and to 10 SLE patients as 2 infusions of 1000 mg/body every other week. Assessment of safety, infusion reactions and adverse effects was used as the primary outcome for clinical tolerability and was evaluated by 28 weeks. Rituximab was well tolerated, with most experiencing no significant adverse effects. B cells rapidly reduced in all patients and remained low until 6 months post-treatment. Four patients developed human antichimeric antibodies without affecting efficacy of rituximab. Changes in routine safety laboratory tests clearly related to rituximab were not observed. Nine among 14 evaluable patients achieved the major or partial clinical response of BILAG score and prednisolone dose significantly decreased at the 28 weeks. Rituximab therapy appears to be safe for the treatment of active SLE patients and holds significant therapeutic promise, at least for the majority of patients experiencing profound B-cell depletion.