Suppressive effects of Lactobacillus casei cells,a bacterial immunostimulant,on the incidence of spontaneous thymic lymphoma in AKR mice

 The mean survival age of female AKR/J mice was significantly prolonged, the enlargement of thymus was markedly suppressed, and the proliferation of ecotropic and recombinant murine leukemia viruses (MuLV) was markedly inhibited when 8-week-old female AKR/J mice were injected intraperitoneally (i. p.) with heat-killed Lactobacillus casei cells twice weekly for 8 weeks. In contrast, such actions of heat-killed L. casei cells were not seen in 20-week-old female AKR/J mice. The leukemogenic activity of the cell-free extract of thymus from adult female AKR/J mice in newborn female AKR/J mice was drastically reduced by i. p. treatment with heat-killed L. casei cells. The difference in adjuvant effectiveness of heat-killed L. casei cells on 8- and 20-week-old animals may be dependent on the difference in the enhancing activity of the cell-mediated immune systems between the groups induced by heat-killed L. casei cells, and, as a result, on the difference in the degree of proliferation of ecotropic and recombinant MuLV in thymus, which consequently causes thymic lymphoma. Received: 13 February 1996 / Accepted: 18 April 1996     

Induction of immune resistance against L1210 lymphatic leukemia in mice after lethal irradiation and reconstruction with fetal liver cells.

The immunohematopoietic potential of syngeneic fetal liver cells (SFLC) was examined and compared with syngeneic bone marrow cells (SBMC). SFLC generated about 3 times less 12th-day spleen colonies (CFU-S) than adult SBMC did. To test the SFLC ability for reconstitution of the immune system, mice were lethally total body irradiated (TBI) and transplanted i.v. with 3 x 10(7) SFLC or 1 x 10(7) SBMC. Thus, injected hematopoietic cells contained the same number of CFU-S. On days 28, 35, 42, and 49 after transplantation the mice were injected i.p. with 10(6) immunogenic L1210-Maf cells (L1210 leukemia cells treated in vitro with mafosfamide for inhibition of their growth in vivo) to test the ability for generation of immune response against L1210 leukemia. On day 56 the animals were challenged with 10(3) L1210 leukemia cells. Strong resistance against the leukemia was induced in TBI + SFLC and TBI + SBMC mice, suggesting that the SFLC similarly as SBMC are able to reconstitute immune system of the TBI host.     

Effects of granulocyte-colony-stimulating factor and interleukin-2 on ascites formation and the survival time of nude mice bearing human ovarian cancer cells

 The aim of this study was to elucidate the effect of intraperitoneal (i.p.) instillations of granulocyte-colony-stimulating factor (G-CSF) and/or interleukin-2 (IL-2) on ascites formation and the survival time of nude mice with malignant ascites, produced by i.p. inoculation of human ovarian cancer cells. When the nude mice were treated with medium alone, ascites was observed in all mice 28 days after tumor inoculation. When the mice were treated with cis-diamminedichloroplatinum(II) (cisplatin) alone, G-CSF alone or IL-2 alone, it took 35 days for the ascites to form in all mice. When cisplatin was combined with G-CSF or IL-2, one of ten mice did not form ascites during the observation period. Surprisingly, when G-CSF and IL-2 were simultaneously administered, ascites formation was not observed in any mice. Although i.p. treatment with cisplatin alone significantly prolonged the survival time, compared to medium alone, the lytic activity of spleen cells against HRA cells was significantly suppressed. When G-CSF or IL-2 was combined with cisplatin, the suppression by cisplatin was eliminated and subsequently resulted in a prolongation of the survival time. When G-CSF was combined with IL-2, both the peritoneal and splenic macrophages/monocytes were stimulated and the splenic lytic activity was about double that following treatment with G-CSF alone on IL-2 alone, suggesting that complete inhibition of ascites formation results not only from a significant increase of the peritoneal macrophages but also from enhancement of the lytic activity. Two mice, died from dissemination of tumor in the abdominal cavity, but eight mice survived without tumor for more than 90 days. As confirmed by monitoring body weight and hematocrit, G-CSF and IL-2 seemed to have no adverse effect. From these results, we conclude that a combination therapy with G-CSF and IL-2 might be of clinical use for inhibiting large amounts of ascites, which may inhibit therapeutic effects for ovarian cancer patients. Received: 20 May 1996 / Accepted: 19 September 1996     

Potentiation of the efficacy of murine L1210 leukemia vaccine by a novel immunostimulator 7-thia-8-oxoguanosine: Increased survival after immunization with vaccine plus 7-thia-8-oxoguanosine

Summary We have investigated the ability of a novel immunopotentiator, 7-thia-8-oxoguanosine (7T8OG) to increase the efficacy of a weakly immunogenic murine L1210 leukemia vaccine. The vaccine was prepared by irradiating L1210 leukemia cells in a cesium source with a total of 6000-R dose. DBA/2 mice were treated with 150 mg/kg 7T8OG and/or with vaccine consisting of 10⁷ irradiated cells. In combination therapy, mice first received the vaccine and then were injected with 75 mg/kg 7T8OG 2 h and 4 h after vaccination. One week after the last treatment all mice were inoculated with 10⁴ live leukemia cells intraperitoneally. Control, untreated mice (n = 66) injected with 10⁴ live leukemia cells had a mean survival time ± standard error of 10.5±0.2 days. Treating mice (n = 66) with one, two or three doses of 7T8OG administered i.p. 1 week apart did not increase survival (mean survival time = 10.7 days). Mice immunized with one, two or three doses of vaccine had 14.5±1.1, 45.4±6.2 and 68.3±10.6 days mean survival, respectively. 7T8OG-stimulated vaccination increased the survival dramatically. The best survival was noted when the mice were treated with 2× (vaccine + 7T8OG). Immunization of mice (n = 30) with this treatment regimen increased the mean survival to 156±10.0 days. Over 90% of mice that were treated this way had a cumulative survival time greater than 160 days. In contrast, only 12% of the mice immunized twice with the leukemia vaccine alone survived over 160 days. These results suggest a rationale for the use of this immuno-potentiator with various vaccines for a more effective immunization.     

Thymosin α1 restores NK-cell activity and prevents tumor progression in mice immunosuppressed by cytostatics or X-rays

The effect of thymosin against tumor progression was examined in mice immunosuppressed by cytostatics or X-ray irradiation. When pretreated with cytostatic agents, such as 5-fluorouracil (5-FU) or BCNU, or by X-ray, and then inoculated with P388 or L1210 leukemias, mice died rapidly within a few days. In these systems, thymosin alpha 1 given concomitantly with the cytostatic agents or after X-irradiation prevented rapid death and extended survival, although the mice eventually died with leukemia like normal mice inoculated with cells of the same tumor. Rapid death in the 5-FU-treated mice was also prevented by adoptive transfer of spleen cells from the donor mice if these had been treated with 5-FU plus thymosin alpha 1, but not if they had received 5-FU alone. However, the restorative activity of the donor spleen cells was abrogated by treatment with anti-asialo GM1, but not by treatment with anti-Thy 1 or anti-mouse Ig serum, suggesting that the effector cells in the spleen are NK cells. In fact, thymosin alpha 1, when given concomitantly with 5-FU or after X-irradiation, maintained the NK activity of spleen, which was damaged by treatment with 5-FU or X-irradiation alone. The present study indicates that thymosin alpha 1 exerts a preventive activity against progression of leukemias at least in part through an effect on NK cells or their progenitor cells.     

In vitro induction of cell-mediated immunity to murine leukemia cells

Summary An adoptive chemoimmunotherapeutic model based on the use of chemotherapy and lymphocytes specifically sensitized against tumor cells in vitro was tested in mice transplanted with syngeneic leukemia cells. C57BL/6 and A strain mice were inoculated i.p. or i.v. (day 0) with lethal doses (1×10³–1×10⁵) of EL4 and YAC leukemia cells, respectively. Leukemic mice were subsequently treated (day 1 or day 3) with partially curative doses (80–140 mg/kg) of cyclophosphamide (Cy), followed by i.p. or i.v. administration of 1–3×10⁷ cytotoxic lymphocytes (CL) induced in macro-mixed leukocyte-tumor cell cultures (MLTC). The following results were obtained: untreated mice died with tumor within 20 days; mice receiving sensitized lymphocytes only showed a modest prolongation of survival and only 5–15% of the animals were cured; treatment with Cy alone or with Cy and normal lymphocytes prolonged survival considerably and cured 20–60% of the mice; mice subjected to Cy in conjunction with in vitro-sensitized lymphoid cells, either syngeneic or allogeneic, had survival rates of 80–100% (100 days). Under the conditions employed, no severe manifestations of clinical graft-versus-host (GVH) reaction were observed. These findings imply that in vitro-sensitized immunocytes and cytoreductive drugs can operate cumulatively.     

Microwave treatment of intracerebral l1210 leukemia: Schedule-dependent partial reversal of the effects of methotrexate

One-GHz microwave (MW) irradiation at a power density of 5 mW/cm² was combined with methotrexate (MTX) in an attempt to treat more effectively central nervous system (CNS) L1210 leukemia in DBA/2J mice. When mice with CNS leukemia were treated with the combination of MW and MTX, there was no improvement in survival compared with a group of animals treated with MTX alone; however, the group that received MTX before the MW exposure had a significantly reduced survival time compared with the group treated with MTX alone or with the group to which MTX was administered after MW.     

Development of host-dependent high-grade tumor-specific immunity through a novel mechanism triggered by the Lyt-2+ tumor-specific T cell clone (K7L) that induces temporal growth of L1210 leukemia-K7L-variant

When a murine leukemia L1210-specific Lyt-2+ T cell clone, K7L, was injected i.p. into CD2F1 mice together with L1210, the normal growth of L1210 in the peritoneal cavity of the mice at the early stage (days 0 to 5) was strongly inhibited, but L1210 grew progressively at the middle-stage (days 5 to 10), and then was rejected at the late stage (days 10 to 20). The mice thus survived for long times (more than 60 days), whereas the normal control injected with L1210 alone died within 14 days. The L1210 that grew at the middle stage in mice initially inoculated with L1210 together with K7L was a K7L-insensitive (K7L-) variant. All of eight tumor clones established from L1210-K7L- by limiting dilution was insensitive to the antitumor activity of K7L, and this property of tumor clones was stable after repeated in vitro passage. The initial depression of the L1210 growth by K7L followed by growth and rejection of the variant L1210-K7L- by the host T cell activity was then found to prepare a strong, long-lasting (more than 3 mo) immunity to protect mice against the high-dose (10(7) cells per mouse) challenge of original L1210. Corresponding to this result, definite tumor (L1210)-specific cytotoxic T lymphocyte (CTL) activity against both variant and original L1210 targets was developed by antigen (L1210) restimulation in the culture of spleen cells from these mice, but was not increased to a detectable level before L1210-K7L- variant started to grow. It was suggested that the 1210-K7L- variant and the original L1210 should have the common tumor-specific antigen that was independent of the K7L-reactive antigen, and that original L1210, whose growth was retarded by K7L, primed the host with the common antigen to be enormously boosted by the subsequently growing L1210-K7L- variant.     

THF-γ2, a thymic hormone,increases immunocompetence and survival in 5-fluorouracil-treated mice bearing MOPC-315 plasmacytoma

Summary The effect of the thymic hormone, THF-2, on the immunocompetence of 5-fluorouracil (5-FU)-treated BALB/c mice, bearing MOPC-315 tumor, was examined. Treatment of noninoculated or tumor-bearing mice with THF-2 after 5-FU injection, resulted in an increase in the antibody response to sheep red blood cells and in the allogeneic response in spleen cell cultures and had no effect on the concanavalin-A-induced interleukin-2 secretion beyond that caused by 5-FU alone. Treatment with either 5-FU alone or 5-FU and THF-2 resulted in restoration to normal values of Lytl- and L3T4-positive populations in tumor-bearing mice. THF-2 prolonged the survival time of mice bearing MOPC-315 tumor beyond that observed in mice treated with 5-FU alone.     

Cellular composition of spleen and peritoneal exudate in mice after injection of cyclophosphamide-treated L 1210 leukemia cells

The composition of peritoneal exudate and spleen cells of CD2F1 mice after fourfold i.p. administration of L 1210 leukemia cells treated with cyclophosphamide (L 1210-CY cells) were examined. The number of cells in peritoneal cavity did not increase, however, the spleen weight rose after administration of L 1210-CY cells. The per cent of lymphocytes T was increased 2.5 times but the content of macrophages and lymphocytes B was normal in the peritoneal cavity after L 1210-CY cells injections. In the spleen an 1.4 times increase of the per cent of lymphocytes B, but normal level of macrophages and lymphocytes T were observed.     

Kinetics and distribution of transplanted sarcoma JWS and leukemia L1210 cells by intravenous route

The aim of this work was to compare the distribution in organs of syngeneic mice sarcoma JWS and leukemia L-1210 cells previously labelled with sodium dichromate (Na2 51CrO4) and iododeoxyuridine (125IUDR) after i.v. transplantation. Also, the results obtained with both labels were compared. Both kinds of cells under study are trapped in lungs, but the number of trapped JWS cells is greater. L-1210 cells probably recirculate. The cells undergo extensive destruction during the first 3 days after transplantation. This kind of study requires the use of two markers: cytoplasmic and nuclear and careful analysis of radioactivity changes is also required to obtain proper conclusions.     

Acknowledgement

In the series of tetraalkylammonium ions from tetramethylammonium to tetra-n-heptylammonium, tetra-n-pentylammonium ion was a potent protector of both murine L1210 leukemia and bone marrow progenitor cells against mechlorethamine cytotoxicity. It was also a non-competitive inhibitor of choline uptake. Phosphonium analogs of the tetraalkylammonium ions were equal to their corresponding tetraalkylammonium ions in their protection against mechlorethamine cytotoxicity and in their inhibition of choline uptake. Treatment of tumor bearing L1210 leukemia mice with the combination of tetra-n-pentylammonium ion and mechlorethamine resulted in no major improvement in survival of the tumor-bearing mice compared to mechlorethamine treatment alone.     

Selective treatment of leukemia L1210 with combination of deoxycytidine and lethal doses of cytosine arabinoside]

Oral administration of deoxycytidine simultaneously with intraperitoneal injections of toxic doses of cytosine arabinosidetomice with advanced L1210 leukemia diminished the toxic effects preventing drug death of these mice. They developed a marked antitumor effect. The mean survival time of these mice was considerably extended as compared to that of untreated animals or those given one of these drugs alone. At the optimum schedule of treatment about 23% of the mice survived over 60 days. Deoxycytidine protection reduced the antileukemic effect of cytosine arabinoside administered in nontoxic doses. The deoxycytidine plus cytosine arabinose combination was ineffective in the treatment of transplantable myeloid leukemia in mice.     

5-Hydroxymethyl-2′-Deoxyuridine: Studies of Antileukemic Properties in Vitro and in Vivo

Abstract

5-Hydroxymethyl-2-deoxyuridine (5HmdUrd) prolonged the survival of mice carrying leukemia L1210 in a dose-dependent manner. Furthermore, a dose-dependent synergism/antagonism was observed when human leukemia cells were exposed simultaneously to Ara-C and 5HmdUrd in vitro.     

Disruption of DNA synthesis and structure of mouse L1210 leukemia cells, sensitive and resistant to 1-methyl-1 nitrosourea and 1,3-bis(2-chloroethyl)-1-nitrosourea in vivo

Leukemia L1210 cells with acquired resistance to 1-methyl-1-nitrosourea (MNU) (L1210/MNU) and 1.3-bis(2-chloroethyl)-1-nitrosourea (BCNU) (L1210/BCNU) were developed from leukemia L1210 cells sensitive to these drugs (L1210/0). The modal chromosome number of leukemia L1210/MNU and L1210/BCNU cells increases from 40 (L1210/0) to 41. It was shown that in leukemia L1210/MNU cells the inhibition of DNA synthesis after MNU administration in a therapeutic dose (80 mg/kg) is lasted within 24 hours, while that in leukemia L1210/0 cell--within 96 hours. After administration of BCNU (20 mg/kg) inhibition of DNA synthesis in leukemia L1210/BCNU cells reached of 50% of control in comparison with practically complete inhibition of DNA synthesis in leukemia L1210/0 cells. Centrifugation on alkaline sucrose density gradients revealed no differences in the rate of sedimentation of leukemia L1210/0, L1210/MNU and L1210/BCNU cell lysates. After 1 hour treatment with MNU of mice bearing L1210/MNU and L1210/0 leukemia cells single-strand breaks in DNA were determined. After 4 hours these strand-breaks retained in leukemia L1210/0 cells, but were eliminated in leukemia L1210/MNU cells. Administration of BCNU to mice with leukemia L1210/0 and L1210/BCNU cells resulted in both cases in the production of DNA aggregates. There is no complete cross-resistance between MNU and BCNU which allows a substitution of these drugs providing for the increase in their therapeutic efficiency.     

Toxicological and immunological evaluation of the MHC-non-restricted cytotoxic T cell line TALL-104

 The human MHC-non-restricted cytotoxic T cell line TALL-104 has been shown to display potent antitumor effects in several animal models with spontaneous and induced malignancies. In view of its potential future use in cancer therapy, we investigated the tolerability and target-organ toxicity of these cells in various animal species. The acute toxicity of TALL-104 cell administrations was evaluated in: (a) healthy immunocompetent mice and immunodeficient (SCID) mice bearing human tumors using multiple (up to 15) intraperitoneal (i.p.) injections, and (b) healthy dogs, tumor-bearing dogs, and healthy monkeys using multiple (up to 17) intravenous (i.v.) injections. TALL-104 cells were γ-irradiated (40 Gy) prior to administration to mice and dogs, but administered without irradiation in monkeys. Cell doses ranged from 5×10⁷/kg to 10¹⁰/kg for each injection. All regimens were well tolerated, the main clinical signs observed being transient gastrointestinal effects. Moderate and transient increases in liver transaminase levels were observed in all animal species. Discrete and transient leukocytosis with neutrophilia was also noted in dogs and monkeys after i.v injections of TALL-104 cells. Histological analysis revealed foci of hepatic necrosis with lympho-/mono-/granulocytic infiltration in immunocompetent mice injected i.p. with 5×10⁹ – 10¹⁰ cells/kg. In the same mice, the colon showed an increased number of muciparous cells and alterations in the villi structure: these alterations were completely reversed by 72 h after the last injection, while liver alterations reversed more slowly (1 week). No delayed or chronic toxicity was observed in any of the animals even when non-irradiated TALL-104 cells were administered: both immunocompetent mice and healthy dogs were found to be grossly and histopathologically normal when sacrificed (1 year and 1 month after the last TALL-104 injection respectively). TALL-104 cells did not persist in these hosts. In addition, monkeys showed no molecular signs of TALL-104-cell-induced leukemia in their blood 1 year after the last cell injection. Despite immunosuppression, most of the tumor-bearing dogs as well as the healthy dogs and monkeys developed both humoral and cellular immune responses against TALL-104 cells. The data derived from these preclinical studies suggest that administration of high doses of irradiated TALL-104 cells is well tolerated and would be unlikely to induce severe toxicity if applied in clinical trials to the treatment of patients with refractory cancer. Received: 8 September 1996 / Accepted: 28 January 1997     

Leukemia L 1210 cells induce depletion of Lyt 2+ thymocytes

The thymocytes were analyzed on the 7th day after i.p. inoculation of 10(6) leukemia L 1210 cells to syngeneic DBA/2 Wf mice. A three-fold decrease of the total number of thymocytes was found as well as 1.7-fold decrease of the per cent of thymocytes with Lyt 2+ phenotype, while the per cent of cells with phenotypes Thy 1.2+ and Lyt 1+ was unchanged.     

Rejection of reovirus-treated L1210 leukemia cells by mice

Summary L1210 leukemia cells were treated in vitro with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and reovirus to determine their interactive effects on rejection of these tumor cells by mice. The cells were treated with BCNU at concentrations of 0, 3, or 10 M, incubated for 48 h, then treated with reovirus at a multiplicity of infection of 0, 10, 30, or 100 for 2, 6, or 12 h. The survival of mice injected with cells treated with any amount of reovirus, regardless of BCNU treatment, was greater than that of mice injected with untreated cells. Exposure of the cells to reovirus for 6 or 12 h increased the survival of mice injected with these cells as compared with that of mice injected with cells exposed to reovirus for 2 h. Of the survivors, 76% were resistant to subsequent challenge with untreated L1210 cells. These results suggest that activities associated with reovirus replication may cause modifications of L1210 cells that enable them to induce an immune response, thus facilitating their rejection. A lack of correlation between differences in DNA synthesis (measured by ³H-thymidine uptake) by treated cells and the ability of those cells to kill recipient mice indicates that rejection of cells treated with reovirus or BCNU is not due to a decrease in their ability to proliferate or, presumably, to generate lethal tumors. The survival of mice injected with treated L1210 cell preparations containing as few as 2.9% reovirus-infected cells was enhanced to the same degree as that of mice injected with those containing as many as 14.6% infected cells, indicating that modification of only a minor component of the tumor cell population is sufficient to alter the ability of the cells to generate a lethal tumor.This work was supported by a research grant from the Miami University Faculty Research Committee and a Sigma Xi Grant-in-Aid of Research     

The Combination of a Low-Dose Chemotherapeutic Agent, 5-Fluorouracil,and an Adenoviral Tumor Vaccine Has a Synergistic Benefit on Survival in a Tumor Model System

Standard cancer therapies, particularly those involving chemotherapy, are in need of modifications that both reduce short-term and long-term side effects as well as improve the overall survival of cancer patients. Here we show that combining low-dose chemotherapy with a therapeutic vaccination using an adenovirus encoding a model tumor-associated antigen, ovalbumin (Ad5-OVA), had a synergistic impact on survival in tumor-challenged mice. Mice that received the combinatorial treatment of Ad5-OVA plus low-dose 5-fluorouracil (5-FU) had a 95% survival rate compared to 7% and 30% survival rates for Ad5-OVA alone and 5-FU alone respectively. The presence of 5-FU enhanced the levels of OVA-specific CD8⁺ T lymphocytes in the spleens and draining lymph nodes of Ad5-OVA-treated mice, a phenomenon that was dependent on the mice having been tumor-challenged. Thus 5-FU may have enhanced survival of Ad5-OVA-treated mice by enhancing the tumor-specific immune response combined with eliminating tumor bulk. We also investigated the possibility that the observed therapeutic benefit may have been derived from the capacity of 5-FU to deplete MDSC populations. The findings presented here promote the concept of combining adenoviral cancer vaccines with low-dose chemotherapy.     

In vivo antitumor effects of murine interferon- γ -inducing factor/interleukin-18 in mice bearing syngeneic Meth A sarcoma malignant ascites

Interferon-γ-inducing factor/interleukin-18 is a novel cytokine that reportedly augments natural killer (NK) activity in human and mouse peripheral blood mononuclear cell cultures in vitro and has recently been designated IL-18. In this study, IL-18 exhibited significant antitumor effects in BALB/c mice challenged intraperitoneally (i.p.) with syngeneic Meth A sarcoma when administered i.p. on days 1, 2 and 3 after challenge. Intravenous (i.v.) administration also induced antitumor effects in the tumor-bearing mice; however, subcutaneous (s.c.) administration did not. When mice were twice pretreated with 1 μg IL-18 3 days and 6 h before tumor challenge, all mice survived whereas control mice died within 3 weeks of challenge. Inhibitory effects on Meth A cell growth in vitro were not observed with either IL-18 or interferon γ. The effects of IL-18 pretreatment were abrogated by abolition of NK activity after mice had been injected with anti-asialo GM1 antibody 48 h before and, 24 h and 72 h after tumor challenge. Mice pretreated with IL-18 and surviving tumor challenge resisted rechallenge with Meth A cells but could not reject Ehrlich ascites carcinoma, and spleen cells from the resistant mice, but not control mice, exhibited cytotoxic activity against Meth A cells in vitro after restimulation with mitomycin C-treated Meth A cells for 5 days. The effector cells in the spleen cell preparations from resistant mice appear to be CD4⁺ cells because cytolytic activity was significantly inhibited after depletion of this subset by monoclonal antibodies and complement. In conclusion, IL-18 exhibits in vivo immunologically (primarily NK) mediated antitumor effects in mice challenged with syngeneic Meth A sarcoma and induces immunological memory and the generation of cytotoxic CD4⁺ cells. Received: 17 September 1996 / Accepted: 8 November 1996