HPMA copolymer-bound doxorubicin targeted to tumor-specific antigen of BCL1 mouse B cell leukemia.

N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymer carrier containing the anticancer drug doxorubicin and targeted with B1 monoclonal antibody (mAb) to BCL1 leukemia cells was synthesised and tested in vitro and in vivo. BCL1 leukemia growing in syngenic Balb/c mice was selected as a tumor model system. B1 mAb recognising the idiotype of surface IgM on BCL1 cells was used as a targeting moiety. Both B1 mAb and doxorubicin were conjugated to HPMA copolymer carrier by aminolysis through a tetrapeptidic Gly-Phe(D,L)-Leu-Gly spacer to ensure the intracellular delivery and controlled release of the drug. B1 mAb-targeted conjugate was shown to possess strictly tumor-specific binding capacity to target BCL1 cells in vitro. A similar conjugate, but containing human nonspecific Ig (HuIg) instead of B1 mAb, failed to bind to BCL1 cells. In vitro, B1 mAb-targeted conjugate demonstrated 40-fold higher cytotoxic effect than nontargeted or human nonspecific Ig-containing HPMA copolymer-bound doxorubicin. Conjugate targeted with B1 mAb was also shown to bind to target BCL1 cells in vivo. B1 mAb-targeted conjugate was shown to be more efficient in the treatment of established BCL1 leukemia than free doxorubicin, nontargeted and human nonspecific Ig-containing conjugate. Antibody-targeted polymeric drugs are thus promising conjugates for cancer treatment.     

Targeting tumor angiogenic vasculature using polymer-RGD conjugates.

Sites of neovascular angiogenesis are important chemotherapy targets. In this study, the synthesis, characterization, in-vivo imaging and biodistribution of a technetium-99m labeled, water-soluble, N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer carrying doubly cyclized Arg-Gly-Asp motifs (HPMA copolymer-RGD4C conjugate) are reported. In vitro endothelial cell adhesion assays indicated that HPMA copolymer-RGD4C conjugates inhibited alphaVbeta3-mediated endothelial cell adhesion while HPMA copolymer Arg-Gly-Glu control conjugates (HPMA copolymer-RGE4C conjugate) and hydrolyzed HPMA copolymer precursor (HPMA copolymer) showed no activity. The scintigraphic images of prostate tumor bearing SCID mice obtained 24 h post-i.v. injection indicated greater tumor localization of HPMA copolymer-RGD4C conjugate than the control, HPMA copolymer-RGE4C conjugate. The 24-h necropsy radioactivity data showed that HPMA copolymer-RGD4C conjugate had significantly higher (p<0.001) tumor localization compared to HPMA copolymer-RGE4C conjugate and HPMA copolymer. Also, HPMA copolymer-RGD4C conjugates had sustained tumor retention over 72 h and reasonably efficient clearance from the background organs. These results suggest that specific tumor angiogenesis targeting is possible with HPMA copolymer-RGD4C conjugates. This construct provides a foundation that should support targeted delivery of radionuclides and drugs to solid tumors for diagnostic and therapeutic applications.     

Time- and concentration-dependent apoptosis and necrosis induced by free and HPMA copolymer-bound doxorubicin in human ovarian carcinoma cells.

A2780 sensitive and A2780/AD doxorubicin (DOX) resistant human ovarian carcinoma cells were exposed to different concentrations (0.25, 0.5, 1, 5 and 10xIC(50)) of free and HPMA copolymer-bound DOX for 12, 24, 36, 48, 60 and 72 h. Apoptosis and necrosis were evaluated using the FITC-conjugated annexin V and propidium iodide staining. The data obtained showed that the induction of apoptosis and necrosis by both free DOX and HPMA copolymer-bound DOX were time- and concentration-dependent. The data also showed significant differences between the drugs. It was found that: (i) under the action of HPMA copolymer-bound doxorubicin the alterations in the plasma membrane permeability preceded disturbances in cellular metabolism; (ii) HPMA copolymer-bound doxorubicin kills the cells mainly by necrosis; (iii) HPMA copolymer-bound doxorubicin is a more effective anticancer drug than free doxorubicin.     

Polymeric drugs based on conjugates of synthetic and natural macromolecules. II. Anti-cancer activity of antibody or (Fab')(2)-targeted conjugates and combined therapy with immunomodulators.

We provide data on in vivo targeting of the Thy 1.2 (CDw90) cell surface receptor expressed on neoplastic T cells, mouse EL4 T cell lymphoma. The targeting antibody and the anticancer drug, doxorubicin (DOX) were conjugated to a water-soluble copolymer based on N-(2-hydroxypropyl)methacrylamide (HPMA) acting as a carrier responsible for controlled intracellular release of the conjugated drug. The in vivo therapeutic efficacy of HPMA copolymer-bound DOX targeted with anti-EL4 antibody, polyclonal anti-thymocyte globulin (ATG), monoclonal anti-Thy 1.2 antibody or its F(ab')(2) fragment was compared with the efficacy of DOX conjugated to HPMA copolymer containing nonspecific IgG or bovine serum albumin (BSA). Anti-EL4 antibody-targeted conjugate caused a significant retardation of tumor growth and an extension of the life span of treated mice. The effect was comparable with that of HPMA copolymer-bound DOX targeted with ATG, anti-Thy 1.2 antibody or its F(ab')(2) fragment. However, considerable antitumor effect was seen also in conjugates targeted instead of specific antibodies with syngeneic nonspecific IgG or BSA. Patients with advanced cancer are often immunocompromised due to dysfunction of their immune system induced by cancer and cytotoxic drugs. A significant decrease of unwanted side-effects of targeted drugs against a number of vital organs was already documented. In this study we have compared immunotoxic effects of free DOX with those of its antibody-targeted form on NK cells and cytolytic T lymphocytes (CTLs) isolated from C57BL/10 mice bearing EL4 T cell lymphoma. In the same model we have tested the combination therapy with immunomodulators (beta-glucan or AM-2) injected together with targeted daunomycin. We have observed a significant protective effect of targeted DOX against NK cells and CTLs. Moreover, the data revealed that combination therapy considerably enhances antitumor efficacy of the targeted anticancer drug.     

Optimal timing for the collection and in vitro expansion of cytotoxic CD56(+) lymphocytes from patients undergoing autologous peripheral blood stem cell transplantation

To identify the optimal time for the collection of CD56(+) cytotoxic lymphocytes for adoptive immunotherapy in patients undergoing high-dose chemotherapy (HDCT) and peripheral blood stem cell (PBSC) transplantation, 18 breast cancer patients receiving either three cycles of epirubicin/paclitaxel (CT x 3) followed by HDCT and PBSC transplantation (n = 12) or CTx6 (n = 6) were studied. Blood samples were obtained before each CT/HDCT cycle, from PBSC collections, and repeatedly after autografting for up to 12 months. The number of CD56(+)3(-) and CD56(+)3(+) lymphocytes, their in vitro expandability with interleukin-2, and their cytotoxicity against MCF-7 and Daudi cells were analyzed. Six healthy females served as controls. CD56(+) cell counts in both treatment groups were subnormal but stable during the observation period. The cytotoxicity of the expanded CD56(+) cells was normal and unaffected by the treatment. The in vitro CD56(+) cell expandability (controls, 100 +/- 31-fold, mean +/- SEM) was normal before CT1 and CT2, but reduced in PBSC harvests performed after CT2 and application of G-CSF (21 +/- 6-fold; p < 0.01). After PBSC harvesting, the CD56(+) cell expandability increased to 185 +/- 74-fold and 170 +/- 69-fold (before CT3 and HDCT). This increase was not observed in those patients who did not undergo PBSC mobilization. Two weeks after autografting, the CD56(+) cell expandability was minimal (6 +/- 1-fold), and recovered to 34 +/- 6-fold. Thus, CT, HDCT and autografting do not alter the frequency and inducible cytotoxicity of CD56(+) cells in breast cancer patients. However, the proliferative capacity of CD56(+) cells obtained from PBSC harvests and after autografting is impaired. Therefore, instead of the PBSC graft, maximally expandable CD56(+) cells obtained at least 1 week after PBSC collection should be considered for adoptive immunotherapy after PBSC autografting.     

细胞因子联合激活人骨髓和外周血免疫细胞的比较研究

在本研究中比较了某些细胞因子在体外联合激活骨髓和外周血免疫细胞后二免疫细胞数量、形态、细胞化学染色、免疫表型和细胞毒变化的差异。在体外加入INF－γ，rIL-1，rIL-2和McAb-CD3分别激培养骨髓笔外周血单个核细胞，培养过程中观察两组免疫细胞增数量及形态的变化，培养前后两组取样进行细胞化学染色和有型检测，用MTT法检测培养后两组的细胞毒情况。研究结果发现，两组的细胞数量在激活培养后均较培养前明显增加，但外周血组增加倍数更多（P＜0．05）；细胞化学染色可见两组培养以后髓过氧化物酶积分均较培养前减少，过碘酸雪夫染色见两组含较多粗颗粒的淋巴样细胞明显较培养前多，两组CD3^ ，CD56^＋和CD38^ 细胞较培养前明显增加（P＜0．05），但两组增殖无明显差别；骨髓组培养后CD3^ CD56^ 细胞无明显增加，而外周血组培养后激活增加显（P＜0．05）；两组激活培养后细胞的细胞毒无明显差异，结论：rIL-γ，rIL-1，rIL-2和CD3单抗联合激活骨髓和外周血单个核细胞可使二细胞数量和细胞毒明显增加，在临床上可利用激活的不同来源的细胞因子诱导的杀伤细胞进行细胞免疫治疗。     

Inverse correlation between CD4+ CD25high CD127low/- regulatory T-cells and serum immunoglobulin A in patients with new-onset ankylosing spondylitis

This study investigated the role of regulatory T (T(reg)) cells in patients with new-onset, treatment-na?ve ankylosing spondylitis (AS). Levels of CD4(+)CD25(high)CD127(low/-) T(reg) cells in the peripheral blood of 14 AS patients and 18 age-matched healthy volunteers were investigated by flow cytometry and correlations with serum levels of immunoglobulin A (IgA) and AS activity, as assessed by the Bath AS Disease Activity Index (BASDAI), were analysed. The number of peripheral blood CD4(+)CD25(high)CD127(low/-) T(reg) cells in AS patients was found to be significantly lower than in healthy controls and was inversely correlated with serum IgA levels. There was no significant correlation between CD4(+)CD25(high)CD127(low/-) T(reg) cell numbers and BASDAI scores. It is concluded that CD4(+)CD25(high)CD127(low/-) T(reg) cells may play a role in the pathogenesis and activity of AS.     

异基因造血干细胞移植后免疫重建临床研究

为探讨异基因外周血造血干细胞移植（allo—PBSCT）后12个月内患者免疫重建特点及其与供受者年龄、供受者间HLA相容性、移植物抗宿主病（GVHD）及病毒感染的关系，随访37例异基因造血干细胞移植患者，用流式细胞术测定移植后1、3、6及12个月的T细胞亚群（CD3^＋、CD4^＋、CD8^＋）、B细胞（CD19^＋）及NK（CD16^＋CD56^＋）细胞，用散射比浊法检测免疫球蛋白IgG、IgA及IgM水平。结果显示，移植后1个月CD3^＋细胞的百分比为（47．5±23．2）％，3个月为（75．1±6．4）％，6个月为（69．7±12）％，12个月为（71．7±4．2）％。移植后1个月CD4^＋细胞百分比为（13．3±6．4）％，3个月为（20．2±11．4）％，6个月为（18．4±9．3）％，12个月为（29．1±8．7）％；移植后1个月CD8^＋细胞百分比为（43．1±17．4）％，3个月为（42．6±16．9）％，6个月为（46．9±10．3）％，12个月为（47±5．6）％；移植后1个月CD16^＋CD56^＋细胞百分比为（14．4±8．4）％，3个月为（15．9±7．6）％，6个月为（14．7±6．6）％，12个月为（13．6±3．4）％；移植后1个月CD19^＋细胞百分比为（6．4±5．6）％；3个月为（11．7±2．4）％，6个月为（13．3±7．3）％，12个月为（16．7±5．7）％。血清免疫球蛋白检测结果显示：移植后1个月IgA为（0．37±0．14）g／L，3个月为（0．28±0．21）g／L，6个月为（0．42±0．18）g／L，12个月为（0．53±0．34）g／L；移植后1个月IgG为（12．7±3．8）g/L，3个月为（16．3±5．2）g／L，6个月为（14．3±6．2）g/L，12个月为（15．4±6．9）g／L。移植后1个月IgM为（0．56±0．24）g／L，3个月为（0．64±0．16）g／L，6个月为（1．1±0．35）g／L，12个月为（1．2±0．28）g／L。大于等于45岁的患者T细胞亚群检测和血清免疫球蛋白与小于45岁患者比较无差异。发生慢性GVHD的患者移植后12?     

HPMA copolymers containing doxorubicin bound by a proteolytically or hydrolytically cleavable bond: comparison of biological properties in vitro.

N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymer carrier containing the anticancer drug doxorubicin bound either by a proteolytically degradable bond (non-targeted PK1 or targeted with alpha-CD71 mAb) or by a hydrolytically degradable bond were synthesised and tested in vivo for various biological properties. Mouse 38C13 B-cell lympoma was used as a well established and defined cell line for this study. 38C13 cells are sensitive to free doxorubicin and IC50 was very low, about 0.014 microM. PK1 showed a strongly decreased cytostatic effect, IC50 being 12.6 microM. alpha-CD71 targeted conjugate, which can be considered as an antibody-targeted form of PK1, had IC50 0.358 microM. HPMA copolymer with doxorubicin bound via a hydrolytically sensitive bond (HYD conjugate) showed a high cytostatic effect with IC50 about 0.052 microM. We demonstrated that HYD conjugate inhibited DNA synthesis and induced p21(Waf1/Cip1) protein expression (p21(Waf1/Cip1) is cyclin-dependent kinase inhibitor which blocks cell cycle progression) as quickly as free doxorubicin, whereas PK1 acted much more slowly. Similarly, apoptosis induction measured by Annexin V binding and Caspase 3 activity was detected later after incubation of cells with PK1 or alpha-CD71 targeted conjugate. Apoptosis was manifested by elevation of bax and bad mRNA levels, which was much more rapid and intense in the case of free doxorubicin and HYD conjugate. Expression of antiapoptotic genes as well as cyclin-dependent kinases was surprisingly not affected.     

Mobilization of dendritic cells and NK cells in non-Hodgkin's lymphoma patients mobilized with different growth factors

Conflicting results have been reported regarding the effect of various growth factors on the mobilization of natural killer (NK) cells and dendritic cells in patients undergoing stem cell mobilization for autotransplantation. We compared the extent of mobilization of NK cells and dendritic cells in non-Hodgkin's (NHL) patients undergoing mobilization with granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage (GM)-CSF, or GM-CSF followed by G-CSF. Overall, 35 patients were studied. NK cells and dendritic were quantitated by flow cytometry. NK cells were defined as the sum of CD56(+) cells and CD56/CD16(+) cells. Dendritic cells were defined as the sum of CD80(+) and CD80(+)/CD14(+) cells. NK activity was determined by by microcytotoxicity assay. NK activity correlated well with the total amount of CD56(+) cells mobilized to the peripheral blood. Patients in the three arms of the study mobilized similar amounts of NK cells and NK activity, and patients who lacked NK activity in the peripheral blood, before mobilization, lacked NK activity in their apheresis collections. In contrast to NK cell mobilization, mobilization of dendritic cells/kg was three- to five-fold higher in patients mobilized with GM-CSF-containing regimens compared to patients mobilized with G-CSF alone. We conclude that GM-CSF-containing mobilization regimens are superior for dendritic cell mobilization but similar in the mobilization of NK cells. Therefore, we recommend using GM-CSF-containing regimens for patients undergoing ex vivo or in vivo manipulation of dendritic cells.     

Which has the least immunity depression during postoperative analgesia--morphine, tramadol, or tramadol with lornoxicam?

BACKGROUND: Analgesics are commonly used to provide pain relief after surgery. These drugs produce some extended depression of immunity. A prospective randomized controlled trial was designed to observe expressions of T-lymphocyte subsets (CD3(+), CD3(+)CD4(+) and CD3(+)CD8(+)), natural-killer cells (CD3(-)CD16(+)CD56(+)), and activated T-lymphocytes (CD3(+)CD25(+)) of patients undergoing gastric cancer surgeries and receiving patient-controlled intravenous analgesia (PCIA). METHODS: Forty-five patients undergoing elective gastric cancer surgeries under general anesthesia were randomly allocated into 3 groups. Group I received PCIA using morphine after surgery, group II using tramadol, and group III using tramadol with lornoxicam. The analgesic efficacy was evaluated by visual analog scale (VAS) and Bruggrmann comfort scale (BCS). Expressions of CD3(+), CD3(+)CD4(+), CD3(+)CD8(+), CD3(-)CD16(+)CD56(+), and CD3(+)CD25(+) were measured as percentages of total lymphocytes by flow cytometer at 5 time points. RESULTS: There was no significant difference in analgesic efficacy and the baselines of CD3(+), CD3(+)CD4(+), CD3(+)CD8(+), CD3(-)CD16(+)CD56(+), and CD3(+)CD25(+) in all groups. Compared with the baseline, CD3(+)CD8(+) had no changes in all groups at any time point. Ninety minutes after incision, CD3(+), CD3(+)CD4(+), CD3(-)CD16(+)CD56(+), and CD3(+)CD25(+) were lower in all groups (P<0.05). 24 h after surgery, CD3(+), CD3(+)CD4(+), CD3(-)CD16(+)CD56(+), and CD3(+)CD25(+) were lower in group I and group II (P<0.05); meanwhile CD3(+), CD3(+)CD4(+), and CD3(+)CD25(+) returned to the baseline but CD3(-)CD16(+)CD56(+) was still low (P<0.05) in group III. 48 h after surgery, CD3(+), CD3(+)CD4(+), CD3(-)CD16(+)CD56(+), and CD3(+)CD25(+) returned to the baseline in group II and group III, but not in group I (P<0.05). 72 h after surgery, CD3(+), CD3(+)CD4(+), CD3(+)CD4(+)/CD3(+)CD8(+) returned to the baseline, but CD3(+)CD25(+) and CD3(-)CD16(+)CD56(+) were still low in group I (P<0.05). CONCLUSION: PCIA using lornoxicam with tramadol has the same good analgesic efficacy and less immunity depression than PCIA using morphine or tramadol.     

低剂量rhG-CSF对56例非血缘供者外周造血干细胞动员

本研究观察低剂量人重组粒细胞集落刺激因子(rhG-CSF)对非血缘健康供者的影响,探讨用于中华造血干细胞捐赠者资料库提供的非血缘健康供者外周造血干细胞动员方案。56例非血缘健康供者接受rhG-CSF 5μg/(kg.d)皮下注射,在动员第4、5两天或第5、6两天采集干细胞,观察动员效果及不良反应,检测动员前后血常规指标、CD3+、CD4+、CD8+和CD20+细胞比例;对采集物进行单个核细胞(MNC)和CD34+细胞计数;对所有供者随访至2006年5月31日。结果显示:在rhG-CSF动员过程中出现1级毒副作用(按WHO分级标准):腰背酸痛17.9%(10/56)、焦虑失眠8.9%(5/56)、疲乏4.5%(3/56)等,无需特殊处理,无需终止动员。第4、5两天采集和第5、6两天采集所得的MNC分别是(5.95±1.52)×108/kg和(7.19±2.12)×108/kg;CD34+细胞分别是(3.03±1.09)×106/kg和(7.92±2.50)×106/kg。血红蛋白水平、血小板量、CD3、CD4、CD8、CD20百分比动员前后无变化。结论:5μg/(kg.d)rhG-CSF用于非血缘健康供者的动员是安全而有效的。     

HPMA copolymer conjugates with reduced anti-CD20 antibody for cell-specific drug targeting. I. Synthesis and in vitro evaluation of binding efficacy and cytostatic activity

Synthesis, physicochemical and biological properties and preliminary anticancer activity of new star-shaped polymer–doxorubicin (DOX) conjugates targeted with anti-CD20 monoclonal antibody were investigated. Mild reduction of antibody (Ab) with dithiothreitol (DTT) resulted in introduction of thiol groups into Ab. Polymer precursors used for the synthesis of the conjugates were based on N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers with a functional group at the polymer chain end. The copolymers were linked to the thiol groups of the reduced Ab via one-point attachment forming a star-shaped structure with central antibody surrounded by hydrophilic polymer chains. Neither reduction nor polymer modification of Ab influenced binding activity of the Ab to its specific cancer cell membrane antigen as it was confirmed in vitro by standard flow cytometry. The anticancer drug DOX was attached to the HPMA copolymer chain in an Ab–polymer system via a pH–labile hydrazone linkage or via an oligopeptide sequence degradable by lysosomal enzymes. Such Ab–polymer–DOX conjugates were fairly stable in aqueous solution at pH 7.4 and the drug was readily released in mildly acid environment at pH 5–5.5 by hydrolysis of hydrazone bond or more slowly by enzymolysis with lysosomal enzymes. The cytostatic activity of the anti-CD20 monoclonal Ab-targeted conjugates tested on several CD20-positive or negative human and mouse cancer cell lines confirmed considerable targeting capacity of the monoclonal Ab after its binding to the polymer carrier. New method of synthesis of star antibody-targeted polymer–drug conjugates with pH-controlled drug release described in this paper opens new perspectives for development of new therapeutics intended for cancer therapy.     

Dysregulated expression of T cell immunoglobulin and mucin domain 3 is associated with the disease severity and the outcome of patients with spontaneous intracerebral hemorrhage

Objectives

We aimed to investigate the expression of T cell immunoglobulin and mucin domain 3 (Tim-3) on peripheral blood cells in spontaneous intracerebral hemorrhage (ICH) patients and to analyze its clinical significance.

Design and methods

Tim-3 expression on peripheral immunocytes from ICH patients and healthy volunteers was measured by flow cytometry. The correlation between Tim-3 expression and the clinical indices was estimated using linear regression.

Results

Tim-3 expressions on peripheral CD3⁺ T cells and CD8⁺ T cells in ICH patients are significantly downregulated, while Tim-3 expressions on CD14⁺ monocytes and CD16⁺CD56⁺ NK cells are increased. Furthermore, Tim-3 expression on peripheral CD8⁺ T cells was negatively correlated with the inflammatory response, the disease severity and the outcome of ICH patients. However, there was no relationship between Tim-3 expression and blood glucose concentration.

Conclusions

Altered expression of Tim-3 might play an important role in the pathogenesis of ICH, demonstrating that Tim-3 might be a novel candidate molecule for prognosis evaluation of ICH patients.     

外周血CD3+CD45+T细胞在原发性肝癌患者中的表达及意义

目的探讨原发性肝癌（Hepatocellular Carcinoma，HCC）患者外周血中CD3^＋CD56^＋T细胞、CD3^＋CD56^-T细胞、CD3^＋CD56^＋NK细胞在（HCC）细胞免疫中所发挥的作用。方法采用流式细胞技术对14例HCC患者及30例健康对照人群外周血中的CD3^＋CD56^＋T细胞、CD3^＋CD56^-T细胞、CD3^-CD56^＋NK细胞进行检测。结果14例HCC患者与30例正常对照组外周血测定结果比较发现：HCC患者外周血中CD3^＋CD56^-明显降低，CD3^＋CD56^＋则明显升高，与正常对照组比较差异具有极显著性（P均小于0．005）。HCC患者CD3^-CD56^＋（NK）较正常对照组下降，两组比较差异具有显著性（P小于0．05）。结论CD3^＋CD56^-T细胞在HCC患者外周血中增生活跃，提示高表达的CD3^＋CD56^-T细胞在肝癌免疫应答中具有重要意义。     

Targetable polymeric prodrugs

N-(2-Hydroxypropyl) methacrylamide (HPMA) copolymers containing oligopeptide side-chains terminating in anticancer drugs (daunomycin, adriamycin) have been synthesized. The bond between the drug and the carrier was stable in the bloodstream, but was cleaved intracellularly on exposure to the lysosomal cysteine proteinases. HPMA copolymers have also been modified with targeting moieties: galactosamine, which targets the conjugate to hepatocytes; anti θ antibodies recognizing θ alloantigen expressed on immunocompetent lymphocytes; and fucosylamine, since there is a receptor on mouse leukemia L1210 cells that recognizes and binds this carbohydrate moiety.In vitro and in vivo experiments demonstrated preferential interaction of modified HPMA copolymers with the respective target cells. Subsequent experiments were performed to test the pharmacological activity of anticancer polymeric prodrugs in vivo against L1210 leukemia in DBA₂ mice. Two localizations of tumor were chosen — intraperitoneal and subcutaneous. In both cases experimental animals were treated intraperitoneally with free drug or drug-HPMA copolymer conjugates. HPMA copolymers containing anticancer drugs have shown therapeutic effect only when the oligopeptide sequence between the drug and the polymeric carrier was biodegradable.Polymeric products produced increased life span and an increased number of long term survivors depending on the structure of the conjugate (i.e. presence of biodegradable side-chains or targeting moieties), timing of administration and number of doses. From the data presented it can be concluded that targetable anticancer polymeric prodrugs may be useful clinically.     

CD4^＋CD25^highT细胞在特发性血小板减少性紫癜发病机制中的作用

本研究探讨特发性血小板减少性紫癜（ITP）治疗前后患者外周血中CD4^＋CD25^highT细胞（CD25-PE染色阳性〉10^2为CD4^＋CD25^highT细胞）的表达情况及其可能的作用机制。应用免疫荧光标记和流式细胞术检测20例治疗前ITP患者、20例治疗后患者及14名正常人外周血中CD4^＋CD25^highT细胞的表达。结果表明：ITP患者治疗前外周血T细胞表面CD4^＋CD25^highT细胞表达水平明显低于治疗后组及正常对照组（P均〈0．01）；ITP治疗后患者组与正常对照组相比无显著性差异（P〉0．05）。结论：ITP治疗前患者外周血中CD4^＋CD25^highT细胞水平较正常对照组明显降低，而在治疗有效后有所升高。外周血CD4^＋CD25^highT细胞表达率与血小板数量呈正相关。CD4^＋CD25^highT细胞水平的检测可作为对ITP患者预后判断的一个有效指标，并可以作为免疫治疗的靶点进行进一步研究。     

中剂量rhG-CSF动员对供者外周血免疫细胞组成的影响

本研究观察12例健康供者在使用10μg/(kg·day)rhGCSF动员前后白细胞总数变化。应用外周血涂片瑞氏染色对白细胞进行形态学分类,使用流式细胞术分析动员前后外周血单个核细胞中T细胞、B细胞、NK细胞和单核细胞比例的变化。结果发现,动员前1天外周血白细胞计数中位数为6.25(4.7-7.8)×109/L,其中淋巴细胞中位数为2.07(1.63-3.1)×109/L,单核细胞中位数为0.163(0.078-0.414)×109/L;动员第5天外周血白细胞计数中位数为37.47(24-72.57)×109/L,其中淋巴细胞中位数为3.22(1.46-5.31)×109/L,单核细胞中位数为1.2(0.706-3.627)×109/L。供者外周血白细胞的增加为动员前的6.26±2.14倍(P<0.01),其中淋巴细胞的增加为动员前的1.45±0.76倍(P<0.05),单核细胞数增加为动员前的7.48±4.41倍(P<0.01)。流式细胞术分析发现,动员前CD3 T淋巴细胞占外周血单个核细胞(PBMNC)比例的中位数为46.96%[(32.36-57.45)%],动员后为40.94%[(25.31-48.9)%];动员前CD4 /CD8 淋巴细胞比例为1.27±0.46,动员后为1.36±0.51;动员前CD4 CD8 T淋巴细胞占PBMNC比例的中位数为0.41%[(0.16-1.51)%],动员后为0.49%[(0.09-2.0)%];动员前CD16 CD56 NK细胞占PBMNC比例的中位数为13.98%[(4.08-25.08)%],动员后为16.65%[(12.06-33.05)%];动员前CD3 CD16 CD56 NK-T细胞占PBMNC比例的中位数为2.75%[(0.37-6.38)%],动员后为3.13%[(0.46-5.95)%];动员前CD20 B淋巴细胞占PBMNC比例的中位数为9.28%[(5.97-16.33)%],动员后为9.94%[(7.36-20.41)%];动员前CD14 单核细胞占PBMNC比例的中位数为12.48%[(3.54-19.35)%],动员后为29.52%[(16.51-36.76)%]。动员后CD14 单核细胞在PBMNC中的比例比动员前增加2.87±1.51倍(P<0.05);动员前后T淋巴细胞、NK细胞、NK-T细胞、B淋巴细胞在PBMNC中的比例以及动员前后CD4 /CD8 淋巴细胞比均无显著变化(P>0.10)。结论:rhGCSF动员引起的单核细胞增加可能在异基因外周血造血干/祖细胞移植的相关事件中发挥着重要作用。     

Cytotoxic and cytostatic effects of anti-Thy 1.2 targeted doxorubicin and cyclosporin A

Recently we have developed a new generation of antibody-targeted immunosuppressive (cyclosporin A, CyA) and cytostatic (doxorubicin, Dox) drugs effective in vitro and in vivo. The drugs and the targeting antibody (polyclonal and monoclonal anti-Thy 1.2) are conjugated to the oligopeptidic side chains of a water-soluble synthetic carrier, copolymer of N-(2-hydroxypropyl)methacrylamide (HPMA). In this study we investigated the effects of such drugs on cell proliferation and cytokine release by EL-4 mouse thymoma and X63Ag8-653 (IL-2) and X63Ag8-653 (IL-3) mouse myeloma transfected with IL-2 and IL-3 gene, respectively. The Thy 1.2⁺ EL-4 mouse thymoma cell line strongly reacted with the anti-Thy 1.2-targeted polymer bound CyA and Dox as assayed by cell fluorometry, whereas Thy 1.2⁻ X63Ag8-653 (IL-2) and X63Ag8-653 (IL-3) mouse myelomas bound anti-Thy 1.2 targeted drugs to a considerably lower extent. Anti-Thy 1.2 targeted doxorubicin and cyclosporin A effectively inhibit proliferation and IL-2 release by EL-4 mouse thymoma. More importantly, low sensitivity of EL-4 mouse thymoma to the anti-proliferative effect of free doxorubicin is partly overcome when anti-Thy 1.2 targeted form of the drug is used. Proliferation of both X63Ag8-653 mouse myelomas and their lymphokine (IL-2 or IL-3) release is inhibited only in the presence of a very high concentration of antibody-targeted conjugates with no difference between the efficacy of non-targeted and anti-Thy 1.2 targeted form. Preincubation of mouse T-cell lymphoma EL-4 with free anti-Thy 1.2 antibody considerably decreases the cytotoxic effects of anti-Thy 1.2 targeted doxorubicin.     

沙利度胺对多发性骨髓瘤患者CD4^＋CD25^＋调节性T细胞作用的临床研究

本研究探讨沙利度胺治疗前后多发性骨髓瘤（MM）患者CD4^＋CD25^＋调节性T细胞的比例及变化规律，为有效的免疫治疗提供理论依据。采用流式细胞术检测MM患者外周血CD3、CD4、CD8、NK及CD4^＋CD25^＋Treg细胞的比例；采用成组设计的两样本均数比较的t检验进行统计学分析，以P〈0．05为检验水准。结果表明：MM患者治疗前CD4^＋CD25^＋highT比例较正常人明显升高（P〉0．01），沙利度胺治疗有效患者的CD4^＋CD25^＋highT比例较治疗前明显降低（P〈0．01），治疗无效者Treg比例无显著变化（P〉0．05）。16例经化疗完全缓解，CD4^＋CD25^＋highT比例为6．91±1．12％，较治疗前升高，但无显著性差异（P〉0．05）。沙利度胺治疗有效者CD3^＋T、CD4^＋T、NK细胞比例及CD4／CD8比值较治疗前明显升高（P〉0．05或0．01），CD8^＋T治疗前后无显著变化（P〉0．05）。结论：CD4^＋CD25^＋Treg细胞的免疫抑制作用可能是MM免疫逃逸的重要机制，下调MM患者CD4^＋CD25^＋Treg可能是沙利度胺治疗MM有效的机制之一。