Acrylic microspheres in vivo V: Immunological properties of immobilized asparaginase in microparticles

L-Asparaginase was immobilized in microparticles of polyacrylamide. Such particles were then injected by intramuscular/subcutaneous, intraperitoneal, or intravenous routes into mice to investigate the immunological consequences of the immobilization. Entrapment of L-asparaginase in microparticles did not prevent the formation of antibodies in intensively treated animals. Intraperitoneal and intravenous injections of particles produced significantly higher antibody levels than soluble L-asparaginase. Antigen administered intramuscularly/subcutaneously in microparticles elicited, however, a weak immune response. Dependent on the route of administration, the particles may thus function as an adjuvant. A modified Arthus reaction in the foot pads of immunized mice indicated that antigenicity decreased when L-asparaginase was immobilized in microparticles. Injection of free L-asparaginase, intramuscularly/subcutaneously (2 x 20 IU) in the preimmunized mice produced no effects on the serum level of L-asparagine, whereas intramuscular/subcutaneous injection of L-asparaginase in microparticles produced a depression of the serum concentration. It is concluded that the intramuscular/subcutaneous injection of L-asparaginase in microparticles is the choice route of administration with respect to duration and the immunological reaction.     

Polyethylene glycol-L-asparaginase and L-asparaginase studies in rabbits

Injections of polyethylene glycol (PEG)-L-asparaginase or L-asparaginase were given to two rabbits each at doses of 40 units/kg. Ten min following injection of either enzyme preparation, the plasma enzyme concentration was approximately 1 unit/ml. This level decreased steadily in the rabbits given L-asparaginase, with a t1/2 of approximately 20 hr. In contrast, the enzyme level in rabbits given PEG-L-asparaginase decreased much more slowly, with a t1/2 of approximately 144 hr. The slower disappearance rate of PEG-L-asparaginase resulted in greater values for the area under the concentration versus time curve and smaller values for total clearance. Immediately following the enzyme injections, no L-asparagine could be detected in the plasma, and a transient elevation of L-aspartic acid levels was noted. By 4 hr, the L-aspartic acid level in all of the rabbits returned to near normal. The L-asparagine, however, was not measurable as long as plasma enzyme was detectable. Levels of L-asparagine returned to normal 4 days after L-asparaginase administration, and 27 days elapsed before L-asparagine was detected in rabbits given PEG-L-asparaginase.     

Monitoring of Erwinia asparaginase therapy in childhood ALL in the Nordic countries

AIMS: Evaluation of L-asparaginase therapy in the NOPHO-92 ALL-protocol (treatment protocol of acute lymphoblastic leukaemia of the Nordic Society of Paediatric Haematology and Oncology, initiated in 1992) after intravenous and intramuscular administration of Erwinia asparaginase during induction and re-induction therapy. METHODS: Forty children with newly diagnosed acute lymphoblastic leukaemia received Erwinia asparaginase (30 000 IU/m2 i.v. or i.m.) during induction therapy (every day for 10 days), and 19 children received Erwinia asparaginase (30 000 IU/m2 i.v. or i.m.) during re-induction therapy (twice a week for 2 weeks). Within the treatment periods asparaginase trough activity (using a spectrophotometric assay) was determined on specific days. The goal of therapy is complete L-asparagine depletion, which asparaginase activities above 100 IU l(-1) have been shown to ensure. Therefore determination of L-asparagine (using a h.p.l.c. method) was performed only in plasma samples with asparaginase activities below 100 IU l(-1). RESULTS: During induction therapy 92.2% of the trough enzyme activities were above 500 IU l(-1) for the i.v.-treated patients, and 92.4% of the trough enzyme activities were above 500 IU l(-1) for the i.m.-treated patients. During re-induction therapy 64.7% of the trough enzyme activities were below 100 IU l(-1) in the i.v.-treated group, and 73.3% of the trough enzyme activities were below 100 IU l(-1) in the i.m.-treated group. For trough enzyme activities below 100 IU l(-1) L-asparagine depletion was complete in two thirds of the samples. CONCLUSIONS: In the NOPHO-92 ALL-protocol L-asparaginase treatment during induction therapy was unnecessarily intense, but during the re-induction phase it appeared inadequate.     

Affinity of intraperitoneally injected activated carbon particles adsorbing mitomycin C to tumor surface of Yoshida sarcoma

A new dosage form of mitomycin C (MMC-CH) comprising 0.75 mg/ml of activated carbon particles adsorbing mitomycin C at 124 micrograms/mg and 7 micrograms/ml of mitomycin C in a free state was injected intraperitoneally to male rats of Donryu strain transplanted intraperitoneally with 10(7) cells/rat of Yoshida sarcoma 4 days before injection. The rats were subjected to autopsy within 60 min after injection. MMC-CH adhered selectively to the tumor surface of Yoshida sarcoma growing intraperitoneally rather than to the surface of organs such as small intestines which the surface cancer did not affect. Within 120 min after injection, mitomycin C concentration in the tissue samples was bioassayed. Intraperitoneally injected MMC-CH distributed high levels of mitomycin C to the tumor rather than to the unaffected organ located intraperitoneally. Animals killed by cancer after the treatment of MMC-CH were autopsied. Histological effects of MMC-CH were studied microscopically. Mitomycin C adsorbed on activated carbon particles induced degenerative changes in the tissues of tumors to which the activated carbon particles had adhered.     

Acute systemic toxicity of pure dimercaprol and trimercaptopropane.

Dimercaprol, free of 1,2,3-trimercaptopropane (TMP), TMP, and mixtures of these two compounds were injected intramuscularly and intraperitoneally into fasted rats and mice, respectively. The LD50 values for dimercaprol and TMP in rats were 87 and 19 mg/kg. The corresponding LD50 values in mice were 90 and 20 mg/kg. The potency ratio of TMP as compared to dimercaprol was 4.5 in both animal groups. Mixtures of pure dimercaprol and TMP, prepared to give 0, 2.5, 5, 10 and 20% TMP, relative to the total amount of dimercaprol and TMP, were injected intramuscularly into rats. A potency ratio of 4.5 was again found. These results indicated that the TMP in dimercaprol did not cause synergistic toxicity. Animal reaction showed that the injection of TMP was much more painful and produced more severe convulsions than injections of dimercaprol.     

Antimetastatic activity of acteoside,a phenylethanoid glycoside

We examined the antimetastatic effect of acteoside, a phenylethanoid glycoside widely distributed in the plant kingdom, on lung metastasis using a mouse model injected with B16 melanoma cells intravenously. Male C57BL/6 mice were injected intravenously with 2 x 10(5) of B16 melanoma cells, while acteoside at a dose of 50 mg/kg was administered intraperitoneally every other day from 13 d before B16 melanoma cell injection until all mice had succumbed to the metastatic tumor burden in the lung. Administration of acteoside prolonged survival time significantly and the average survival time was 63.3 +/- 3.4d compared with 52.1 +/- 2.5d in control mice. This result suggests that acteoside showed suppressive effect on lung metastasis of B16 melanoma cells.     

复方苦参注射液联合环磷酰胺对小鼠Lewis 肺癌抑瘤作用的实验研究

目的探讨复方苦参注射液联合环磷酰胺对Lewis肺癌小鼠移植瘤生长、转移及血管新生的抑制作用及相关机制。方法建立C57BL·6^-1小鼠Lewis肺癌移植瘤模型,随机分为对照组、复方苦参组、CTX组和联合治疗组,每组各10只小鼠,分别腹腔注射生理盐水、复方苦参注射液、环磷酰胺、联合复方苦参注射液和环磷酰胺,定期检测皮下肿瘤体积,接种后12d处死小鼠,检测比较两组小鼠瘤体质量和体积、肺转移瘤结节数,同时以免疫组化法检测小鼠肿瘤组织中微血管密度（MVP）以及VEGF水平。结果复方苦参注射液联合环磷酰胺可显著减少瘤体质量和体积,减轻肿瘤转移,下调VEGF表达,抑制血管生成,与对照组、复方苦参组和CTX组比较差异均有统计学意义（P〈0．05或0．01）。结论复方苦参注射液和CTX可协同拮抗肿瘤新生血管生成,抑制肿瘤组织生长与转移,效果优于两者单独使用。     

The parenteral controlled release of liposome encapsulated chloroquine in mice

Free (0.6 mg), and liposome encapsulated chloroquine (0.6, 3 mg), were injected intraperitoneally, intramuscularly and subcutaneously in mice. Intraperitoneal administration of liposome-encapsulated chloroquine resulted in high and long lasting concentrations of chloroquine in the blood compared with intraperitoneal administration of free chloroquine. After administration of the liposome-encapsulated chloroquine the concentrations in the spleen were also higher, indicating that chloroquine liposomes reached the blood compartment intact after intraperitoneal administration. After intramuscular and subcutaneous administration the chloroquine liposomes acted as a local depot, giving a slower release from the subcutaneous fat layer than from the muscle depot. After the 0.6 mg dose a burst effect was found at about 7 h in most of the animals; this was not found after the 3 mg dose. This finding and the slower release after the 3 mg dose than after the 0.6 mg dose could be explained by the formation of aggregates after the injection.     

Manganese chloride enhances natural cell-mediated immune effector cell function: effects on macrophages

A single intramuscular injection of MnC12 in mice caused an increase in macrophage functional activity. Spleen cell antibody-dependent cell-mediated cytotoxicity against both chicken erythrocytes and P815 tumor cell targets was enhanced 24 h following a single injection of MnC12. Enhanced antibody-dependent cell-mediated cytotoxicity activity following MnC12 treatment was not associated with a change in spleen cellularities compared with saline-injected mice. Resident peritoneal macrophages from mice injected intramuscularly with MnC12 displayed enhanced phagocytic activity for chicken erythrocytes in the presence or absence of opsonizing antibody. Enhanced cytolytic activity against P815 mastocytoma target cells and enhanced cytostatic activity against MBL-2 lymphoma target cells was also observed for nonelicited resident peritoneal macrophages from mice injected intramuscularly with MnC12. There were no differences in the cellularity or relative number of adherent cells obtained from the peritoneal cavity of saline or MnC12-injected mice. These enhanced macrophage functions were associated with the induction of increased interferon levels in mice injected with MnC12.     

血必净对免疫抑制脓毒症模型的保护作用

目的 观察血必净对免疫抑制脓毒症小鼠的影响。方法 使用随机数字表法将152只小鼠分为对照组（Control）、免疫抑制组（IM）、免疫抑制脓毒症模型组（ISM）、血必净治疗组（XT）,每组38只小鼠。IM组沿下腹正中线旁边腹腔注射环孢素A免疫抑制,25 mg/kg,隔日1次,共3次。ISM组免疫抑制后沿下腹正中线旁边腹腔注射300 μL浓度为1×109 CFU /mL的大肠杆菌44102;XT组免疫抑制脓毒症造模后30 min,沿下腹正中线旁边腹腔注射血必净4mL/kg,30 min后按相同剂量重复注射1次;Control组注射等体积的生理盐水。（1）造模8 h,各组取4只小鼠,进行血细菌培养。（2）造模12 h,各组取10只小鼠,使用流式细胞法检测外周血CD3+CD4+和CD3+CD8+。（3）造模12 h,各组取10 只小鼠,采用酶联免疫吸附试验（ELISA）检测外周血肿瘤坏死因子（TNF）-α、白细胞介素（IL）-6。（4）造模12 h,各组取4只小鼠,采用蛋白免疫印迹法测定高迁移率蛋白（HMGB1）。（5）造模12 h,各组取10只小鼠,使用全自动分析仪检测外周血丙氨酸转氨酶（ALT）、天冬氨酸转氨酶（AST）、尿素氮（BUN）、血肌酐（CR）。结果 与ISM组相比,XT组的CD3+CD4+/CD3+CD8+的比值明显升高,血细菌培养数量明显降低;肝肾功能指标ALT、AST、CR、BUN和炎症指标TNF-α、IL-6、HMGB1均明显下降。结论 血必净能够明显调节免疫抑制脓毒症小鼠的免疫系统,对抗细菌,抑制炎症反应,并对重要器官有保护作用。     

Reduced pulmonary toxicity of peplomycin in a new drug-delivery system

Pulmonary toxicity was examined by means of Matsuda and Takahashi's procedure in peplomycin solution, and two types of a new dosage form of peplomycin (PEP-CH), which has peplomycin adsorbed on to activated carbon particles. One was PEP-CH IP, and is designed for intraperitoneal administration. The other was PEP-CH IM, for intramuscular administration. Male mice of ICR strain received a bolus injection of 50 mg kg-1 peplomycin in the form of aqueous solutions PEP-CH IP and PEP-CH IM. The survival rate was 100% after 5 weeks in both groups that were administered PEP-CH. The rate was 50% for the group given peplomycin solution intraperitoneally, and 70% for the group given peplomycin solution intramuscularly. Five weeks after administration the mice were killed, and the grade and the incidence of pulmonary fibrosis were evaluated histologically. The grade and incidence of pulmonary fibrosis were zero in the two groups given PEP-CH. The grade was 0.33 and 1.76, and the incidence was 60% and 86%, respectively, in the group given peplomycin solution intraperitoneally and in the group given peplomycin solution intramuscularly.     

阿魏酸钠对甘油致小鼠肾脏氧化性损伤的拮抗效应阿魏酸钠对甘油致小鼠肾脏氧化性损伤的拮抗效应

目的从氧化应激角度,探讨阿魏酸钠对实验动物中毒性肾损害的防治作用。方法建立甘油所致小鼠急性肾小管损伤模型,观察不同剂量的阿魏酸钠对肾损伤小鼠肾功能指标、抗氧化指标和组织学的影响。结果在甘油注射后6和72 h,阿魏酸钠100～200 mg·kg^-1 ip能剂量依赖性的降低甘油所致血清尿素氮、肌酐和N-乙酰-β-葡糖苷酶的升高。阿魏酸钠200 mg·kg^-1减少肾组织丙二醛生成,提高肾脏谷胱甘肽含量及谷胱甘肽过氧化物酶、谷胱甘肽S-转移酶、过氧化氢酶和超氧化物歧化酶活性,且可明显减轻肾组织病理改变。结论阿魏酸钠对甘油致肾损伤有防治作用,其机制与增强肾脏抗氧化功能有关。     

Effect of ethanol on fatal carbon monoxide poisoning in awake rats

The effect of ethanol on fatal carbon monoxide (CO) poisoning was investigated in mice injected intraperitoneally with ethanol. Ethanol (1.5 and 3.0 g/kg) was injected 15 min prior to exposure to gas containing 6.6% CO. The survival period was significantly lengthened with ethanol in proportion to the doses injected, although the carboxyhemoglobin (CO-Hb) saturation level in postmortem blood was almost the same in all groups. On the other hand, the CO-Hb level in the blood of mice injected with ethanol was significantly lower than that of control mice during the early exposure period when all mice were still alive.

Our results showed that the acute ethanol injection did not influence the CO-Hb saturation level in blood at death, but did affect the duration of survival, probably because of ethanol's ability to decrease blood flow and CO intake.     

Changes in serum levels of cytokines in mice injected with an immunostimulator C3bgp isolated from Cuscuta europea

The effect of C3 binding glycoprotein (C3bgp), isolated from Cuscuta europea seeds on induction of in vivo cytokine synthesis was investigated. Different groups of mice were stimulated with 30 microg C3bgp per mouse, injected intraperitoneally. The quantitative determination of IL-1alpha, IL-2, IL-4, IL-6, IL-10, TNF-alpha and IFN-gamma was performed in mouse sera by ELISA. The quantities of these cytokines were measured at different hours: 1, 2, 3, 4, 5, 6, 7, and 24 h after injection. No significant changes in serum level of IL-2, IL-4 and TNF-alpha in experimental animal groups were found. A little increase of IL-1alpha, moderate elevation of IL-10 and IFN-gamma (5- to 6-fold more) and strong release, more than 10-fold of IL-6 in sera of C3bgp-treated mice were detected. The results obtained from C3bgp stimulated cultures of mouse peritoneal macrophages and mouse splenocytes suggest that C3bgp binds to mouse peritoneal macrophages and induces production mainly of IL-6, followed by IFN-gamma and in a very low degree of IL-1alpha and IL-10. Based on the results presented, we conclude that the increased level of IL-6 was the basic after injection of C3bgp and that the mouse macrophages were the major cell targets for the C3bgp effect.     

The relationship between morphine, aspartic acid and L-asparaginase in rats

Morphine and aspartic acid were administered separately and in combination to 80 rats divided into 8 groups. Ten and 20 min following the injections, brain, liver and kidney L-asparaginase activity was determined. Morphine decreased brain and liver L-asparaginase activity and increased that of kidney. Aspartic acid completely antagonized the effect of morphine. Additionally 500 IU/kg L-asparaginase and 5 or 10 mg/kg morphine were i.v. injected into 56 rats divided into 5 groups. L-Asparaginase, which, in turn, increased motor activity, antagonized the morphine-induced hypoactivity and analgesia. These results support our previous findings.     

Caspase抑制剂F1013对刀豆蛋白A诱导小鼠急性肝损伤的治疗作用

目的研究Caspase抑制剂F1013对刀豆蛋白A(Co-nA)所致小鼠急性肝损伤模型的治疗作用,并对其机制进行初步探讨。方法 60只♂BALB/c小鼠随机分成对照组、模型组、阳性药组(NAC 155 mg.kg-1)和F1013(5,2.5,1.25 mg.kg-1)给药组。除对照组外,其余组小鼠均尾静脉注射ConA建立小鼠急性肝损伤模型,造模后2 h,阳性药组腹腔注射NAC,F1013给药组和模型组分别皮下注射F1013和溶媒。造模后8 h留取血清检测丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)、总胆红素(T-Bil)含量和TNF-α水平,肝组织进行HE染色,并检测肝细胞凋亡率。结果在Co-nA诱导小鼠急性肝损伤模型,F1013组均明显改善肝脏病理组织损伤;明显降低血清ALT、AST、T-Bil和TNF-α水平及肝细胞凋亡率(P<0.01或P<0.05)。结论 F1013对Co-nA所致小鼠急性肝损伤具有较好的治疗作用,其机制可能与减少TNF-α的产生及抗肝细胞凋亡有关。     

螺旋藻多糖对小鼠移植性肿瘤化疗后造血恢复及相关细胞因子的影响

目的：研究螺旋藻多糖（polysaccharide from spirulina platensis,PSP)对小鼠移植性肿瘤化疗后造血恢复及相关细胞因子的影响。方法：通过右前肢皮下接种肿瘤细胞形成小鼠移植性实体瘤，给予PSP边疆灌胃10d,d4给予环磷酰胺（CTX）腹腔注射连续3d。于d11，分别了外周细胞，骨髓有核细胞及CFU－S计数，用紫外分光光度计检测骨髓DNA含量，用双抗体夹心ELISA法检测血清中IL-1,IL-3,GM-CSF,TNF含量。结果：CTX可造成明显骨髓抑制，而PSP则提升了外周血细胞，增加了骨髓中有核细胞计数和DNA含量以及促进了CFU－S形成。此外，PSP还增加了血清中IL－1，IL－3，GM－CSF，TNF含量。结论：PSP可能通过促进内源性细胞因子的分泌来实现其促进小鼠移植性肿瘤化疗损伤后的造血恢复。     

Anti-tumoral effect of native and immobilized bovine serum amine oxidase in a mouse melanoma model

Bovine serum amine oxidase (BSAO) oxidatively deaminates polyamines containing primary amine groups, spermidine and spermine, to form the cytotoxic products hydrogen peroxide and aldehyde(s). Polyamines are present at elevated levels in many tumor tissues. The aims of the study were to evaluate the anti-tumoral activities of native and immobilized BSAO in mouse melanoma and also to determine the mechanism of tumor cell death. C57BL mice received a subcutaneous injection of B16 melanoma cells to induce formation of tumors, prior to antitumor treatments with native and immobilized BSAO. The enzyme was immobilized in a poly(ethylene glycol) (PEG) biocompatible matrix. Antitumor treatments consisted of a single injection of enzyme into the tumor. When immobilized BSAO (2.5mU) was injected into the tumor, there was a marked decrease of 70% of the tumor growth. This was compared with a decrease of only 32% of tumor size when the same amount of native BSAO was administered. The type of cell death was analysed in tumors that were treated with native or immobilized BSAO. When tumors were treated with immobilized BSAO, there was induction of a high level of apoptosis (around 70%), compared to less than 10% with the native enzyme. Apoptotic cell death was assessed by nuclear chromatin condensation using Hoechst staining and labelling of externalized phosphatidylserine using Annexin V. However, native BSAO, probably due to a burst of cytotoxic products, induced a high level of necrosis of about 40%, compared to less than 10% with immobilized BSAO. In conclusion, the advantage is that immobilized BSAO can act by allowing the slow release of cytotoxic products, which induces tumor cell death by apoptosis rather than necrosis.     

氧代赖氨酸的抗癌及药理实验研究

氧代赖氨酸系我所抗菌素室从辽宁省大连地区土壤中分离得到的新种玫瑰录褐链霉菌(Streptomyces roseoviridofuscus n sp)所产生的抗菌素。经动物实验证明对多种实体瘤有效,本文继续报道以不同给药途径和给药方案对脑瘤-22小鼠的治疗作用。用氧代赖氨酸的总量为1～1.5g/kg,于按种后不同时间以腹腔、尾静脉、肌肉、口服给药,对脑瘤-22均有明显治疗作用,其抑制率为47～78%。氧代赖氨酸200～400 mg/kg静脉注射对狗的血象、肝、肾功能,心电图影响不明显,100 mg/kg剂量对猴也无毒性反应。以氚标记的氧代赖氨酸对实验小鼠进行研究,表明口服吸收良好。静脉注射或口服200mg/kg,均以肝放射性为高,瘤中放射性虽不高,但能维持48小时以上。放射性排出以肾脏为主,静脉注射后24小时从尿中排出约为63%。     

Neoplastic transformation of Chinese hamster cells in vitro after treatment with flavoring agents

Chinese hamster B241 cells were treated with 5 nM allylisothiocyanate (AI) or 10 nM trans-cinnamaldehyde (CA) and surviving cells were cultivated for generations until the cells acquired the characteristics of transformed cells based on in vitro criteria: increase in (a) saturation density in monolayer culture, (b) plating efficiency at low serum level and (c) colony forming efficiency (CFE) in soft agar medium. When the values of CFE of the treated cells had become significantly high, anchorage-independent colonies were isolated, propagated and then subjected to an assay for neoplastic transformation. The anchorage-independent clonal cells (CH-AI-AG+-1 and CH-CA-AG+-2) were subcutaneously injected into a suprascapular site in nude mice. The mice were maintained in an SPF animal care facility and observed for tumor formation. Growth of neoplasm at the injection sites was observed in 6 out of 7 mice and in all of the 6 mice injected with CA- and AI-transformed cells, respectively, during 3 to 8 months after the injection, as compared with 1 out of 6 mice injected with untreated control cells. Subsequent transplantation of the tumor cells into new mice induced tumor production at the injection site in all the animals within a considerably shorter period of time than that following the initial inoculation. Malignancy of the neoplastic cells was ascertained by histological examination, and the cells were found to have karyotypes of the hamster cells after in vitro cultivation of the tumor cells. These experimental results suggest the transforming potency of the flavoring agents in Chinese hamster cells.