Effects of N G-methyl-L-arginine,an inhibitor of nitric oxide synthesis,on interleukin-2-induced capillary leakage and antitumor responses in healthy and tumor-bearing mice

 We tested whether treatment with an inhibitor of nitric oxide synthesis (N ^G-methyl-L-arginine, MeArg) can ameliorate interleukin-2(IL-2)-therapy-induced capillary leak syndrome in healthy or tumor-bearing mice without compromising the antitumor effects of IL-2 therapy. Healthy or C3-L5-mammary-adenocarcinoma-bearing C3H/HeJ mice were treated with one or two rounds of various doses of IL-2 (ten injections, i. p., every 8 h) or MeArg (ten injections s. c., every 8 h) or their combination. In an additional experiment, MeArg was given chronically in the drinking water, rather than s. c. to healthy mice subjected to one round of therapy as above. Mice were killed 1 h after their last IL-2 injection to measure the water content of the lungs and pleural cavities (markers of capillary leakage), NO production (given by NO₂ ^– and NO₃ ^– levels in the serum and pleural effusion), as well as the effect of therapies on the primary tumor size and number of spontaneous lung metastatic nodules. Results revealed that all doses of IL-2 (7500 – 35 000 Cetus U/injection), as well as both rounds of IL-2 therapy, caused capillary leakage. However, no pleural effusion was seen after the second round in any of the IL-2-treated groups. MeArg therapy, given subcutaneously (5 – 20 mg kg^–1 injection^–1 in healthy and 20 mg kg^–1 injection^–1 in tumor-bearing mice), did not ameliorate IL-2-induced capillary leakage in either group of mice, and did not compromise antitumor effects of IL-2. However, subcutaneous MeArg therapy alone reduced the growth of the primary tumors, the occurrence of spontaneous lung metastases and the amount of tumor-induced pulmonary edema. When MeArg therapy was given orally (1 mg/ml drinking water), a substantial drop in NO production, as well as reduction in capillary leakage was noted in IL-2-treated healthy mice. These findings suggest that NO inhibitors could be a valuable adjunct to IL-2 therapy of cancer and infectious diseases. Received: 23 October 1995 / Accepted: 22 November 1995     

Levamisole regulates the proliferation of murine liver T cells through Kupffer-cell-derived cytokines

 We have previously shown that levamisole increases the cytotoxic, cytostatic, and proliferative activity of murine nonparenchymal liver cells (NPC) in vitro. We have also shown that the nonadherent subpopulation of NPC, which are composed predominantly of T lymphocytes, is very responsive to this agent when administered to mice. Kupffer cells or immigrant macrophages are also responsive to levamisole but to a lesser extent. These findings prompted us to investigate changes in cytokine production by NPC following-treatment of mice with levamisole (25 mg/kg, i.p.), which may help explain the observed alterations in the immune functions of these cells. We found that levamisole treatment of mice causes a threefold increase in production of interferon (IFN) α/β by adherent NPC (more than 80% – 90% Kupffer cells) in vitro. When IFN α/β was added to cultured cells, it decreased the proliferative capacity of liver T cells in a dose-dependent manner. In contrast, the addition of anti-IFNα/β was shown to augment levamisole-induced proliferation of unfractionated NPC and Kupffer cells. NPC production of interleukin 1 (IL-1) and interleukin-6 (IL-6) in vitro was also increased threefold following treatment of mice with levamisole. IL-6 added in vitro to cells significantly augmented levamisole-induced proliferation of liver T cells while anti-IL-6 reduced proliferative activity to control levels. These findings suggested that IFNα/β, IL-6, and IL-1 play important regulatory roles in controlling the proliferative response of murine liver-associated T lymphocytes to levamisole. Finally, the proliferation of bone marrow cells was increased in mice given 5-fluorouracil (5FU). On the other hand, the proliferation of NPC was dramatically suppressed when 5FU was administered. However, the proliferation of these cells was restored when levamisole was given after 5FU. Received: 27 November 1995 / Accepted: 16 October 1996     

Carbohydrate vaccines that induce antibodies against cancer. 1. Rationale

  Resistance to chemotherapy is a major cause for failure in the treatment of lung cancer. Compared to conventional cytotoxic drugs, immunotoxins act by different mechanisms and thus might be promising for the treatment of chemoresistant cancer. The monoclonal antibody MOC31 recognises the epithelial glycoprotein-2 (EGP-2), a cell-surface antigen associated with small-cell lung cancer (SCLC) and a major fraction of lung adenocarcinomas. An immunotoxin composed of MOC31 and a recombinant form of Pseudomonas exotoxin A lacking the cell-binding domain (ETA_{252 – 613}) was prepared, and its effect on lung cancer cell lines examined. MOC31-ETA_{252 – 613} was selectively cytotoxic to EGP-2-positive SCLC and adenocarcinoma cell lines inhibiting proliferation by 50% at concentrations ranging from 0.01 nM to 0.3 nM. Moreover, the immunotoxin reduced the numberof clonogenic tumour cells from cultures by factors of 10⁴ and 10⁵ during a 24-h and a 3-week exposure respectively. In athymic mice, the immunotoxin, which revealed a serum half-life of approximately 4 h, caused substantial regression of small (40 mm³) chemoresistant tumour xenografts and significantly delayed the growth of larger tumours (120 mm³). This finding indicates that MOC31-ETA_{252 – 613} may be useful for the treatment of lung cancer in the setting of chemoresistant minimal residual disease. Received: 31 October 1996 / Accepted: 5 December 1996     

Transpiration and drought resistance of Douglas-fir seedlings exposed to excess ammonium

 In a pot trial growth and transpiration of 3-year-old Douglas-fir seedlings on an acid, sandy soil was examined at a deficient (30 kg N ha^{–  1} year^{–  1}) and an excessive level (120 kg N ha^{–  1} year^{–  1}) of NH₄ application. Dissolved ammonium sulphate was applied to the pots weekly for two growing seasons. In half of the pots a complete set of other nutrients was applied in optimal proportions to the applied nitrogen. Water supply was optimal and transpiration was recorded. At the end of the second treatment season irrigation was stopped for 2 weeks during dry and sunny weather. Both high application of NH₄ and additional nutrients increased shoot growth and transpiration demand in the first treatment year. The root system was smaller at higher N level and this reduced water uptake accordingly. In the second year the combination of high NH₄ ⁺ and additional nutrients affected root functioning predominantly due to salinity effects and this seriously decreased water uptake capacity and shoot water potentials, finally resulting in tree death. Without addition of other nutrients the high NH₄ ⁺ application resulted in a high degree of soil acidification, which damaged the roots, that showed a decrease in water uptake capacity. At the low NH₄ supply level soil acidification was lower, and root functioning was not affected, and the trees recovered quickly from the imposed drought. Higher needle K and P status depressed transpiration rates at the low NH₄ application rate. Received: 9 January 1995 / Accepted: 18 September 1995     

1α ,25-Dihydroxyvitamin D3 activates T cells of tumor bearers through protein phosphatase 2A

 The sterol 1α,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] can inhibit T cell activation as well as restore the functional competence of suppressed T cells. The present studies determined whether 1,25(OH)₂D₃ had a differential effect on the activation of normal T cells or of suppressed T cells from mice bearing Lewis lung carcinoma tumors. Normal spleen cell proliferation in response to immobilized anti-CD3 was unaffected by the lower doses of 0.1 – 10 nM 1,25(OH)₂D₃, and was inhibited by the higher dose of 100 nM 1,25(OH)₂D₃. In contrast, 1,25(OH)₂D₃ increased proliferation and interferon γ secretion by T cells of tumor bearers in response to stimulation through T cell receptor/CD3. Assessment of mechanisms associated with the 1,25(OH)₂D₃ stimulation of tumor-bearer T cells implicated protein phosphatase 2A (PP-2A). First, PP-2A activity of spleen cells from tumor bearers was reduced compared to that of normal spleen cells but was increased by 1,25(OH)₂D₃. Second, 1,25(OH)₂D₃ stimulation of tumor-bearer T cell proliferation was dependent on this PP-2A activity as it was blocked by doses of okadaic acid that selectively inhibit PP-2A. These results suggest that 1,25(OH)₂D₃ preferentially enhances the responsiveness of immunosuppressed T cells from tumor bearers to TCR/CD3 stimulation by restoring PP-2A activity. Received: 7 November 1996 / Accepted: 2 January 1997     

Immunotoxin sensitivity of Chinese hamster ovary cells expressing human transferrin receptors with differing internalization rates

 Previous studies have shown that immunotoxin action is dependent upon selective binding to the target cell, internalization and then passage into the cytosol. It is important to define precisely how these critical steps are controlled so that the underlying relationship of each to high cytotoxic effectiveness is understood. In order to evaluate the contribution of internalization rate and receptor number on immunotoxin potency, the effects of an anti-(transferrin receptor, TfR)/ricin A chain immunotoxin, 7D3-A, were assessed on a parent Chinese hamster ovary cell line developed in our laboratory with no TfR (TfR^neg) and two lines transfected with either wild-type TfR (Tfr^wt) or an internalization-deficient (TfR^{δ7 – 58del}) mutated human TfR. Potent, receptor-mediated cytotoxicity resulted from the action of 7D3-A on TfR^wt cells (ID₅₀<1 nM) while both TfR^neg cells and TfR^{δ7 – 58del} were only minimally affected (ID₅₀>100 nM). Butyrate up-regulation substantially increased receptor expression on the TfR^wt and TfR^{δ7 – 58del} cells, but no corresponding rise in sensitivity to 7D3-A was observed. In contrast, immunotoxin potency was increased by co-treatment of TfR^wt cells with the carboxylic ionophore monensin and the effect was even more pronounced for TfR^{δ7 – 58del} cells. We conclude that internalization rate or intracellular destination is a much more important determinant of immunotoxin efficacy than receptor number. Received: 15 March 1996 / Accepted: 28 May 1996     

Induction of accessory cell function of human alveolar macrophages by inhalation of human natural interleukin-2

 Accessory function allows antigen-presenting cells to produce sufficient secondary signals for optimum T cell proliferation and interleukin-2 (IL-2) production. Alveolar macrophages are inferior accessory cells compared to monocytes (PBM). We report here that the accessory index (AI) of alveolar macrophages and PBM of patients with lung metastases of solid tumors treated with inhalations of human natural IL-2 (hnIL-2) increased following its administration (P<0.005). The accessory index was significantly elevated from baseline values after 2 weeks of inhalation of 300 000 IU hnIL-2/day (8.2±10.2 compared to 1.1±1; P<0.001). The inhalation of 150 000 IU also induced increases in the index (AI = 2.3±1.9), however, without reaching statistical significance. In addition at 300 000 IU IL-2/day a significant increase in the accessory index was observed for PBM (4±2.5; P<0.05). The indices of PBM and alveolar macrophages prior to inhalation showed a significant negative correlation with the age of the patients (r _s =  – 0.5; r _s =  – 0.8, respectively; P<0.03 for all comparisons). Our data demonstrate that the inhalational application of hnIL-2 enhances the accessory function of alveolar macrophages and, to lesser extent, the accessory index of PBM, indicating the occurrence of pharmacological immunostimulation. Received: 16 August 1995 / Accepted: 4 January 1996     

Immune modulation by interleukin-12 in tumor-bearing mice receiving vitamin D3 treatments to block induction of immunosuppressive granulocyte/macrophage progenitor cells

 Lewis lung carcinoma (LLC-LN7) tumors stimulate myelopoiesis and increase the presence of granulocyte/macrophage (GM) progenitor cells having natural suppressor activity. Treatment of these tumor-bearing mice with interleukin-12 (IL-12) resulted in minimal immune modulation. The objective of this study was to determine whether eliminating natural suppressor activity would allow for immune stimulation by IL-12. Treatment of LLC-LN7 tumor-bearing mice with vitamin D₃ eliminated natural suppressor activity. In mice that were first treated with vitamin D₃ and then also with IL-12, there was stimulation of splenic T cell proliferation in response to immobilized anti-CD3 plus IL-2. In addition, spleen and lymph node cells from vitamin-D₃/IL-12-treated tumor-bearing mice became stimulated in response to autologous tumor to produce interferon γ (IFNγ), although IL-2 production was not stimulated. A prominent effect of the combined vitamin-D₃/IL-12 treatment regimen was the synergistic augmentation of autologous tumor-specific cytolytic activity within the regional lymph nodes. The generation of these tumor-specific effector cells required the presence of the tumor mass since such activity was not elicited in the lymph nodes of mice from which the tumors had been surgically excised. The results of this study show that, after treatment of tumor bearers with vitamin D₃ to eliminate GM-suppressor cells, IL-12 can induce select regional antitumor immune responses, particularly IFNγ production and cytolysis by regional lymph node cells of autologous tumor. Received: 15 December 1995 / Accepted: 22 March 1996     

Selective Protection by Uridine of Growth Inhibition by 5-Fluorouracil (5FU) Mediated by 5FU Incorporation into RNA,But Not the Thymidylate Synthase Mediated Growth Inhibition by 5FU-Leucovorin

Fluorouracil (5FU) acts by RNA-incorporation and inhibition of thymidylate synthase; the first action is counteracted by uridine, and the second is enhanced by leucovorin (LV). Growth inhibition of C26-10 colon cancer cells by 5FU was enhanced by LV and rescued by uridine, but 5FU-LV was only partially rescued by uridine. In WiDr cells, 5FU sensitivity was not enhanced by LV, while both 5FU and 5FU-LV were rescued by uridine. Intermediate trends were found in SW948 and HT29 cells. Uridine rescue in mice allowed 1.5-fold increase in 5FU dose, leading to 2-fold increase in the antitumor effect and thymidylate synthase inhibition in resistant Colon-26 tumors. In the sensitive Colon-26-10 tumor, uridine rescue decreased 5FU-RNA incorporation > 10-fold, without affecting the antitumor activity. The use of LV and uridine can differentiate between two mechanisms of action of 5FU.     

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     

Effects of increasing saturation vapour pressure deficit on growth and ABA levels in black spruce and jack pine

 Plant responses to saturation vapour pressure deficit (SVPD) were studied by subjecting black spruce [Picea mariana (Mill) B.S.P.] and jack pine seedlings (Pinus banksiana Lamb.) to humid (0.3 – 0.8 kPa) or dry (2.0 – 2.5 kPa SVPD) regimes for 4 weeks using a computer-controlled environmental system to control diurnal variation in SVPD. Dry matter accumulation in needles was not altered by increasing SVPD. However, root growth declined by 60% which increased shoot to root ratio and reduced total seedling dry weight in both black spruce and jack pine. Relative growth rate of jack pine also declined to about half the rate of plants grown under humid conditions. In situ root marking studies showed that the decline in root growth of jack pine under the high SVPD was the result of reduced lateral root initiation, whereas root elongation was unaffected by humidity. A 4-week exposure to dry air increased abscisic acid (ABA) levels in needles, but not roots, of jack pine whereas ABA levels in black spruce were not altered. A short (3-day) exposure failed to increase needle ABA levels in either species. These results suggest that the responses of conifers to dry air were not the result of ABA accumulation. Received: 24 March 1996 / Accepted: 30 May 1996     

Influence of edaphic and climatic factors on dynamics of root colonization and spore density of vesicular-arbuscular mycorrhizal fungi in Acacia farnesiana Willd. and A. planifrons W.et.A.

 A study was conducted to assess the dynamics of vesicular-arbuscular mycorrhizal (VAM) fungi associated with Acacia farnesiana and A. planifrons in moderately fertile alkaline soils. The intensity of root colonization by VAM fungi and the distribution of VAM fungal structures varied with host species over a period of time. The occurrence of vesicles with varied morphology in the mycorrhizal roots indicates infection by different VAM fungal species. This was further confirmed from the presence of spores belonging to different VAM fungal species in the rhizosphere soils. Root colonization and spore number ranged from 56% – 72% and 5 – 14 g^{–  1}soil in A. farnesiana and from 60% – 73% and 5 – 15 g^{–  1} soil in A. planifrons. Per cent root colonization and VAM spore number in the rhizosphere soil were inversely related to each other in both the Acacia species. However, patterns of the occurrence of VAM fungal structures were erratic. Spores of Acaulospora foveata, Gigaspora albida, Glomus fasciculatum, G. geosporum and Sclerocystis sinuosa were isolated from the rhizosphere of A. farnesiana whereas A. scrobiculata, G. pustulatum, G. fasciculatum, G. geosporum and G. microcarpum were isolated from that of A. planifrons. The response of VAM status to fluctuating edaphic factors varied with host species. In A. farnesiana though soil nitrogen (N) was positively correlated with root colonization, soil moisture, potassium and air temperature were negatively correlated to both root colonization and spore number. Per cent root colonization and spore number in A. planifrons were negatively related to each other. Further, in A. planifrons as the soil phosphorus and N were negatively correlated with the density of VAM fungal spores, the same edaphic factors along with soil moisture negatively influenced root colonization. Received: 16 May 1995 / Accepted: 7 February 1996     

A novel glycoprotein obtained from Chlorella vulgaris strain CK22 shows antimetastatic immunopotentiation

 A glycoprotein extract (CVS), derived from the unicellular green alga Chlorella vulgaris, strain CK22, exhibited a pronounced antitumor effect against both spontaneous and experimentally induced metastasis in mice. Inhibition of tumor metastasis was enhanced when intratumor administration of CVS was followed by s.c. injection of CVS. Anti-metastatic immunopotentiation was observed in euthymic mice, but not in athymic nude mice. The antitumor activity of CVS was reflected in antigen-specific, T-cell-mediated immunity. Both CD4 and CD8 T cells contributed to the antimetastatic effects, as shown by in vivo depletion experiments with anti-T-cell subset antibodies. Furthermore, CVS caused the recruitment of T cells to the regional lymph nodes and their proliferation in these organs. The CD4-positive population, following CVS injection at the time of tumor rechallenge, displayed a pronounced increase in the proportion of T cells that were CD18 bright, CD44 bright, CD25⁺, CD54⁺, CD69⁺ or CD71⁺ in the lymph nodes. Thus, CVS induces T cell activation in peripheral lymph nodes in tumor-bearing mice. We conclude that CVS augments antimetastatic immunity through T cell activation in lymphoid organs and enhances recruitment of these cells to the tumor sites. Presurgical treatment with CVS might prevent metastasis or tumor progression. Received: 5 June 1997 / Accepted: 12 September 1997     

Isolation of Mhc class I cDNAs from the axolotl Ambystoma mexicanum

 Class I major histocompatibility complex (Mhc) cDNA clones were isolated from axolotl mRNA by polymerase chain reaction (PCR) and by screening a cDNA phage library. The nucleotide and predicted amino acid sequences show definite similarities to the Mhc class Iα molecules of higher vertebrates. Most of the amino acids in the peptide binding region that dock peptides at their N and C termini in mammals are conserved. Several amino acids considered to be important for the interaction of β₂-microglobulin with the Mhc α chain are also conserved in the axolotl sequence. The fact that axolotl class I A cDNAs are ubiquitously expressed and highly polymorphic in the α1 and α2 domains suggests the classical nature of axolotl class I A genes. Received: 3 June 1996 / Revised: 14 October 1996     

Sap flow rates of mangrove trees are not unusually low

 In contrast with previous reports, we observed high transpiration rates in mangrove trees. Maximum sap velocities and mean daytime sap flow rates were estimated from heat pulse velocity in entire, field grown trees of Avicennia cf. alba Blume and Rhizophora apiculata Blume. Results were within the range of values measured by identical techniques for trees in lowland dipterocarp and tropical heath forests with a similar climate in Brunei Darussalam (north Borneo). High stomatal conductance (400 mmol m^{–  2} s^{–  1}) was also measured for well insolated leaves of A. cf. alba, with midday water potentials reaching about  – 3 MPa in both species. Received: 11 September 1996 / Accepted: 27 January 1997     

UV-B induction of flavonoid biosynthesis in Scots pine (Pinus sylvestris L . ) seedlings

 Cultivation of Scots pine (Pinus sylvestris L.) seedlings under simulated global radiation including the UV-B band (280 – 320 nm; 220 mW m^–2 UV-B_BE) led to increased formation of the diacylated flavonol glucosides 3″,6″-di-p-coumaroyl-astragalin and 3″,6″-di-p-coumaroyl-isoquercitrin in primary and cotyledonary needles, respectively. 3″,6″-Di-p-coumaroyl-astragalin was also the main constitutive diacylated flavonol glucoside in both needle types. This compound predominantly accumulated in primary needles upon UV-B irradiation, and reached concentrations of 2.4 μmol g^–1 fresh weight (fw). Its concentration was only weakly affected in cotyledonary needles. 3″,6″-Di-p-coumaroyl-isoquercitrin was mainly induced in cotyledonary needles with maximum concentrations of 0.8 to 0.9 μmol g^–1 fw, but was virtually unaffected in primary needles under the same irradiation conditions. Pulse labelling with L-(U-¹⁴C)phenylalanine revealed that these metabolites were formed de novo. Phenylalanine ammonia-lyase (EC 4.3.1.5) and chalcone synthase (EC 2.3.1.74) were only slightly induced by the UV-B treatment. The results described here represent the first report on UV-B-induced flavonoid biosynthesis in a conifer species. Received: 5 December 1995 / Accepted: 20 March 1996     

Cortical microtubules rearrange during differentiation of vascular cambial derivatives, microfilaments do not

The plant cytoskeleton has been implicated in a variety of morphogenetic events in higher plants. Most of this work, however, has concentrated on epidermal cells or primary tissues. We have investigated the cortical microtubular (CMT) and microfilament (MF) components of the cytoskeleton in a secondary tissue  –  active vascular cambium of Aesculus hippocastanum L. (horse-chestnut)  –  and followed the changes in these components during the early stages of differentiation of fusiform cambial derivatives to axial elements of the secondary vascular system. A correlative approach was used employing indirect immunofluorescence microscopy of α-tubulin on 6 μm sections, and transmission electron microscopy of 60 nm sections. The study has demonstrated a rearrangement of the CMT cytoskeleton, from random to helical, as fusiform vascular cambial cells begin to differentiate as secondary phloem vascular tissue. A similar CMT rearrangement is seen as fusiform cambial cells begin to differentiate as secondary xylem fibres. This rearrangement is interpreted as evidence of determination of cambial derivatives towards vascular development. Axially-oriented MF bundles are present in fusiform cambial cells and their axial orientation is retained in the vascular derivatives at early stages of their development even though the CMTs have become rearranged. Received: 5 August 1996 /  Accepted: 23 September 1996     

Treatment of human lung carcinoma xenografts with a combination of 131I-labelled monoclonal antibody Po66 and doxorubicin

 Po66, a mouse monoclonal antibody, is directed against an intracytoplasmic antigen present in human lung squamous cell carcinoma cells. In previous work it was found that the co-administration of ¹²⁵I-radiolabelled Po66 and doxorubicin strongly enhanced the uptake of radioactivity by the tumour. The present-work was designed to evaluate, in a tumour-bearing mouse model of lung carcinoma, the ability of ¹³¹I-labelled Po66 to retard tumour growth when injected alone, or in combination with doxorubicin (8 mg kg ^– 1 at 1-week intervals). A single dose of 550 μCi ¹³¹I-Po66 alone had no effect on tumour growth, whereas three fractionated doses of 250 μCi ¹³¹I-Po66 decreased it over two doubling times from 14.5±1.5 days for untreated control mice to 24.8±2.7 days. Mice treated with doxorubicin alone had a double tumour doubling time of 22.6±4.9 days, compared to 35.2±2.9 days (1.55-fold increase) in mice treated with doxorubicin and a single dose of 550 μ Ci ¹³¹I-Po66. Doxorubicin combined with three fractionated doses of 250 μCi ¹³¹I-Po66 provoked a twofold decrease in tumour growth compared to mice treated with doxorubicin alone. The administration of fractionated doses of ¹³¹I-Po66 simultaneously with doxorubicin resulted in a highly delayed mortality, which was not observed when ¹³¹I-Po66 was administered after doxorubicin. Thus, in a non-small-cell lung tumour model, a ¹³¹I-radiolabelled monoclonal antibody, directed against an intracellular antigen, significantly potentiated the effect of chemotherapy. Such a therapeutic approach could be used as an adjuvant therapy and improve the effect of chemotherapy on distant small metastases. Received: 20 June 1996 / Accepted: 3 October 1996     

Interleukin-1α synergistic in vivo enhancement of cyclophosphamide- and carboplatin-mediated antitumor activity

 Interleukin-1α (IL-1α) has potent acute antitumor activity in vivo and can enhance the efficacy of chemotherapeutic drug-mediated antitumor responses. Studies were undertaken to examine the ability of IL-1α to enhance the activity of cyclophosphamide (CTX) administered in combination with carboplatin. To determine the in vivo effect of IL-1α, CTX and/or carboplatin, mice bearing 14-day RIF-1 tumors were treated on day 0 with a concurrent i.p. injection of varying doses of CTX (5–150 mg/kg), human IL-1α (125 μg/kg), and carboplatin (50 mg/kg) and examined 24 h later for the surviving fraction by the in vivo excision clonogenic-tumor-cell assay. Even at the lowest doses of CTX, IL-1α significantly enhanced the clonogenic tumor cell kill when compared to treatment with CTX alone. When carboplatin was added to the treatment schema, significantly greater clonogenic cell killing and tumor regrowth delay were observed as compared to any agent alone or a two-drug combination (CTX/IL-1α or CTX/carboplatin). Significant enhancement was observed even at low doses of CTX in combination with carboplatin and IL-1α. The interaction between the three-drug combination was found to be synergistic as determined by the median dose effect with significant dose reduction apparent for IL-1α and CTX when used in this combination. These results demonstrate that IL-1α can synergistically enhance the antitumor efficacy of CTX and the combination of CTX and carboplatin. Received: 11 September 1996 / Accepted: 20 May 1997     

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