Adoptive immunotherapy of tumor in mice in combination with cyclophosphamide

Cyclophosphamide causes temporary regression of Dalton's lymphoma when injected at palpable stage. Dose-dependent response to cyclophosphamide was analyzed. Low dose was found to be most effective as compared to the high dose. Adoptive transfer of immunized splenocytes after cyclophosphamide therapy protects the animals from the danger of reappearance of tumor. Such combination therapy is proved to be effective in causing complete and permanent regression of tumor in majority of the treated animals.     

Detecting disseminated solid tumor cells in hematopoietic samples: methodological aspects

Detection of tumor cell dissemination in solid tumor patients recently became essential to determine the prognosis of the disease and to monitor response to the therapy. Accurate detection of disseminated tumor cells in hematological samples requires tumor-specific target molecules, which allow sensitive and specific assays and, further, enable the quantification of tumor cells. Currently, numerous applications are in use, including immunological and molecular biological approaches. Theoretically, both ways are sensitive enough to detect less than one tumor cell in 1 million hematopoietic cells. With the improved sensitivity, however, the likelihood that unspecific events will be amplified is also increased. Moreover, biological and analytical variables may fundamentally influence the findings in a particular case. Basic methods, significant pitfalls and the most recent developments in this field are discussed in this overview.     

Effects of rotational stress on the effectiveness of cyclophosphamide and razoxane in mice bearing Lewis lung carcinoma

The effects of conventional vs protected housing, and those caused by the periodic application of a psychological Stressor (rotational stress, spatial disorientation) on mice kept in a protected housing, with spontaneous tumor metastasis have been determined in mice implanted with Lewis lung carcinoma as a function of tumor inoculum size and response to treatment with cyclophosphamide and razoxane. With a reduced inoculum size, tumor takes do not occur in mice kept in the protected housing, but do occur with spatial disorientation. With a larger inoculum size, tumor takes occur in all untreated mice, and the weight of spontaneous lung metastasis is significantly increased by spatial disorientation. For mice in protected housing, cyclophosphamide results in the absence of macroscopically detectable tumors in all of the treated mice, whereas the use of spatial disorientation abolishes this therapeutic action. The antimetastatic effects of razoxane are also reduced by rotational stress. These results indicate that housing conditions and a psychological stressor can control tumor takes and metastasis formation. They also indicate that host's antitumor resistance effectors, which are susceptible to neuroendocrine modulation by environmental and psychological Stressors, participate to determine the effectiveness of the treatment with a cytotoxic (cyclophosphamide) and antimetastatic (razoxane) antitumor drug.     

Low-dose cyclophosphamide combined with IL-2 inhibits tumor growth by decreasing regulatory T cells and increasing CD8+ T cells and natural killer cells in mice

Interleukin-2 (IL-2) benefits some cancer patients by promoting the proliferation of cytotoxic effector T cells, but this process is limited by the expansion of regulatory T cells (Tregs). Low-dose cyclophosphamide (CTX) can inhibit the number and function of Tregs. We treated carcinoma-bearing mice with Vehicle, CTX, IL-2 and CTX + IL-2 to investigate the effects of low-dose CTX combined with IL-2 in antitumor treatment. In comparison to monotherapy, CTX + IL-2 significantly limited tumor growth, via tumor cell proliferation inhibition and increased apoptosis. The infiltration of CD8⁺ T cells in tumor tissues was significantly increased in the CTX + IL-2 group. CTX + IL-2 safely increased CD8⁺ T and natural killer cells in the spleen, lymph nodes and peripheral blood, and CTX attenuated the increase in Tregs induced by IL-2 in the spleen.     

Effect of cyclophosphamide on the clearance of IgG-sensitized red cells in mice

The effects of cyclophosphamide (Cy) on the clearance of IgG-sensitized erythrocytes (EA) were examined. The results clearly indicate that Cy treatment enhances the capacity of the mononuclear phagocytic system to remove antibody-coated cells from the circulation in normal and decomplemented mice. The enhanced rate of clearance is the consequence of an increased uptake of EA by the liver and spleen. We explored the possibility that the enhancement of Fc receptor-mediated clearance might be an important effect to be taken into account in the search for a more effective therapy of immune complexes diseases.     

Inhibition of adhesion and proliferation of peritoneally disseminated tumor cells by pegylated catalase

Hydrogen peroxide may aggravate the peritoneal dissemination of tumor cells by activating the expression of a variety of genes. In this study, we used pegylated catalase (PEG-catalase) to examine whether prolonged retention of catalase activity within the peritoneal cavity is effective in inhibiting peritoneal dissemination in mouse models. Murine B16-BL6 cells or colon 26 cells labeled with firefly luciferase gene were inoculated intraperitoneally into syngeneic mice. Compared with unmodified catalase, PEG-catalase was retained in the peritoneal cavity for a long period after intraperitoneal injection. A single injection of PEG-catalase just before tumor inoculation significantly reduced the number of the tumor cells at 1 and 7 days. The changes in the expression of molecules involved in the metastasis were evaluated by real time quantitative PCR analysis. Inoculation of the tumor cells increased the expression of intercellular adhesion molecule (ICAM)-1 in the greater omentum, which was inhibited by PEG-catalase. An injection of PEG-catalase at 3 days after tumor inoculation also reduced the number of the tumor cells, suggesting that processes other than the adhesion of tumor cells to peritoneal organs are also inhibited. Daily doses of PEG-catalase significantly prolonged the survival time of tumor-bearing mice. These results indicate that intraperitoneal injection of PEG-catalase inhibits the multiple processes of peritoneal dissemination of tumor cells by scavenging hydrogen peroxide in the peritoneal cavity.     

The production of pulmonary oedema in mice by cyclophosphamide and iodide

Mice given 400–450 mg/kg cyclophosphamide (a 30-day LD₅₀) and 500 mg/kg potassium or sodium iodide (30-day LD₅₀) died within a few hours with marked pulmonary oedema and pleural effusions. Among halides this effect was restricted to iodides, and among alkylating agents it was restricted to cyclophosphamide, and was not shared even by the close analogues isophosphamide or trophosphamide. Administration of mixed-function oxidase inhibitors delayed these acute deaths but did not alter their incidence at 24 hours, suggesting that the cyclophosphamide effect was due to its metabolites and not to the unchanged drug. As the optimum time for giving iodides was 2 hours after the cyclophosphamide, when the metabolism of the latter would have largely been completed, the effect of iodide was probably not due to modification of cyclophosphamide metabolism. Either cyclophosphamide or potassium iodide given alone in doses in the 24-hour LD range caused minor degrees of pulmonary oedema, shown by increases in lung weight. The rapid killing by smaller doses of these drugs given together therefore appears to be due to synergy in the production of pulmonary changes.     

Induction of micronuclei and toxic effects in embryos of pregnant rats treated before implantation with anticancer drugs: cyclophosphamide, cis-platinum, adriamycin

To evaluate a possible relationship of maternal exposure to anticancer drugs during the preimplantation period to blastopathies and postimplantation embryotoxicity, CD female rats were injected intraperitoneally on day 3 of pregnancy with 15 and 30 mg/kg of cyclophosphamide (CPA), 2 and 4 mg/kg of Adriamycin (ADR), 3 and 6 mg/kg of cis-platinum (Cis-Pt), or with 5 ml/kg of saline. Blastocysts were collected on day 5 of gestation and evaluated for gross morphology, cell number, and micronuclei. Some females were sacrificed on day 21 of pregnancy in order to evaluate postimplantation embryotoxicity. A reduction in cell number/blastocyst was observed only in animals exposed to Cis-Pt 6 mg/kg; vice versa, a dose-related increase of micronuclei and of blastocysts with micronuclei was found in all groups treated with the anticancer agents. A significant increase of postimplantation loss was recorded in the groups treated with high doses of Cis-Pt and ADR, but no clear signs of teratogenicity were observed.     

The influence of recombinant human tumor necrosis factor-alpha, alone and in combination with cyclophosphamide or methotrexate, on leukemia L1210 and normal hematopoiesis in mice.

We investigated the influence of rh-TNF administered as a single agent or in combination with CY or MTX on the survival time of mice inoculated with lymphoid leukemia L1210 and the effects of similar treatment on normal hematopoiesis in mice. The MST of rh-TNF--treated mice was longer than that of control animals. The longest survivals were observed in mice treated with 250 and 275 micrograms/kg of rh-TNF. Groups of mice receiving a combination of rh-TNF at doses of 225 or 250 micrograms/kg and MTX lived longer than animals treated with these agents separately. We observed the longest survival time of mice treated with combined administration of rh-TNF at a dose of 250 micrograms/kg and CY, but survival time was not significantly prolonged compared with mice receiving only CY. Additional studies were performed to examine the influence of rh-TNF administered as a single agent or in combination with toxic doses of CY or MTX on the number of granulocytes, lymphocytes, erythrocytes with hematocrit values and hemoglobin concentration, and platelets in peripheral blood, and the number of mononuclear cells as well as multipotential stem cells (CFU-GEMM) in bone marrow. Rh-TNF caused dose-dependent suppression of mononuclear cells and multipotential stem cells in bone marrow. The addition of MTX to rh-TNF caused no enhanced suppression of any of the above mentioned hematological parameters. In contrast, the addition of CY to rh-TNF suppressed erythrocytes and hematocrit values, as compared with rh-TNF alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

不同剂量环磷酰胺对小鼠免疫功能的影响

目的探索不同剂量环磷酰胺(CTX)对正常小鼠免疫功能的影响,为CTX临床应用提供实验数据。方法 C57BL/6小鼠24只,随机分为对照组、低剂量组和高剂量组,分别给生理盐水(NS)、低剂量和高剂量的CTX,检测血常规、Treg比率、T细胞和B细胞增殖功能及巨噬细胞吞噬功能、细胞增殖周期及凋亡比率。结果 RBC、WBC、PLT数量在高剂量组显著降低,对照组与低剂量组无差异;Treg占CD4+T细胞的比例在低剂量组显著下降,对照组与高剂量组无差异;T、B细胞增殖指数及巨噬细胞吞噬功能在低剂量组高于对照组和高剂量组,高剂量组低于对照组;细胞凋亡比率在高剂量组明显增加,对照组与低剂量组间无差异;高剂量组细胞阻滞在S期,对照组与低剂量组间无差异。结论低剂量CTX通过下调Treg、增强免疫细胞的功能,促进机体免疫;而高剂量CTX通过抑制DNA的复制、促进细胞凋亡和降低免疫细胞功能,使机体处于免疫抑制状态。相关数据可以为CTX的临床应用提供借鉴。     

Efficacy of intracavitary administration of cyclophosphamide, interleukin-2 and lymphokine activated killer cells against established intraperitoneal tumor

In an established intraperitoneal tumor modell combination of chemo- and immunotherapy was tested in mice and shown to be superior to either treatment modell alone. This combination treatment is shown to prolong survival significantly in animals massively inoculated with tumor cells.     

Effect of preliminary injection of allogeneic cells on transplantation immunity in mice receiving cyclophosphamide

Injection of 1×10⁸ C57BL/6 mouse spleen cells into CBA mice 1 day before treatment with cyclophosphamide (CP) was shown to promote survival of 2×10⁷ allogeneic or semiallogeneic cells injected later (3–6 h after CP). The criterion of survival was the ability of the donor cells to produce antibodies against sheep's red blood cells in recipients tolerant to this antigen. Injection of 1×10⁸ allogeneic cells 2 days before CP treatment had no protective effect. After intravenous immunization with allogeneic cells, killer cells began to appear in the recipient's spleen as early as on the 2nd day, and their number reached a maximum on the 5th day. The results suggest that CP eliminates the recipient's lymphocytes responding to transplantation antigens, but the killer cells already formed are resistant to the action of CP.Laboratory of Immunologic Tolerance, N. F. Gamaleya Institute of Epidemiology and Microbiology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR P. A. Vershilova.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 85, No. 2, pp. 192–194, February, 1978.     

Genotypic,sex, and age differences in the clastogenic action of cyclophosphamide in mice

Interstrain genotypic, sex, and age differences in the clastogenic action of cyclophosphamide in various doses are established for C57B1/6, MRL/1, and BALB/c mice. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 120, N ^o , pp. 387–390, October, 1995     

Delayed toxicity of cyclophosphamide in normal mice

Delayed toxicity of a single dose of 300 mg/kg cyclophosphamide (CP) was investigated in female DBA/2 mice. Lethality was low up to 30 days but increased markedly afterwards reaching a peak of 50% between 50-70 days with a total mortality of more than 80% by day 120 after CP. One week before death, the mice suffered a sharp loss of weight and showed typical signs of wasting disease. There was a decrease in the white cell count and lymphocyte neutrophil ratio was reversed as a result of lymphocyte depletion whereas neutrophil count remained similar to the controls. Profound lymphocyte depletion was also observed in light and electron microscopy preparations of thymus from mice with CP-induced wasting disease. Histochemical methods demonstrated increased activity of four lysosomal enzymes, acid phosphatase, beta-glucuronidase, E600 resistant esterase and n-acetyl-beta-glucosaminidase, in the thymus of treated mice. Acid phosphatase was notably active in thymus epithelial cells; the reaction product was localized in multiple primary Golgi lysosomes, Golgi cisternae, cisternae of the endoplasmic reticulum, and secondary lysosomes. The appearance of numerous cystic formations, as well as the activation of the lysosomal system and the presence of large areas of degradation support the assumption that CP-delayed toxicity is accompanied by thymus involution. Delayed mortality was partially prevented when syngenic bone marrow cells were injected as early as 24 h after CP injection. On the other hand thymus transplants were incapable of reducing delayed lethality. It is suggested that CP provokes a delayed wasting syndrome with thymic involution that is not caused by a direct effect on specific thymus structures but rather secondary to a primary injury to pre T cells in bone marrow.     

美法仑治愈荷瘤小鼠的过程与TNFα的关系

目的探讨单一剂量的美法仑治愈荷瘤野生型C57BL/6小鼠的过程与肿瘤坏死因子α(TNFα)的关系。方法以3种遗传背景相同、肿瘤坏死因子受体1(TNFR1)基因型不同的TNFR1 / 、TNFR1 /-和TNFR1-/-C57BL/6小鼠为实验动物,皮下接种数量相同的小鼠淋巴瘤EL4细胞。接种瘤细胞后12d,给基因型不同的各组荷瘤小鼠腹腔内单次注射7.5mg/kg的美法仑。以荷瘤野生型C57BL/6小鼠(TNFR1 / )为对照,观察美法仑对荷瘤TNFR1 /-C57BL/6小鼠和荷瘤TNFR1-/-C57BL/6小鼠的治疗效应。结果在美法仑(7.5mg/kg)治疗后的1周内,基因型不同的各组荷瘤小鼠肿瘤消退的速度基本相同。在随后的2月内,荷瘤TNFR1 / 和TNFR1 /-C57BL/6小鼠的肿瘤结节逐渐消退、肿瘤治愈;而多数荷瘤TNFR1-/-C57BL/6小鼠的肿瘤结节缩小后又再次出现并逐渐长大、肿瘤复发。结论TNFα与美法仑治愈肿瘤的过程密切相关,其中美法仑的抗肿瘤作用与荷瘤小鼠TNFR1的表达无关,但在美法仑治疗后,机体预防或避免肿瘤复发方面需要TNFR1在机体细胞的表达,而不是在肿瘤细胞的表达。