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     

Increased microvascular permeability induced by prolonged interleukin-2 administration is attenuated by the oxygen-free-radical scavenger dimethylthiourea

 Effective use of interleukin (IL)-2 as an antineoplastic agent may be hindered by severe side-effects, in particular vascular leak syndrome, which leads to generalized, especially pulmonary, edema. The oxygen-free-radical scavenger dimethylthiourea (DMTU) was shown to attenuate IL-2-induced vascular leak syndrome in sheep receiving a single IL-2 injection. However, in the clinical setting multiple injections are necessary to gain a therapeutic effect. The present study tests whether DMTU attenuates IL-2-induced vascular leak syndrome following multiple IL-2 injections without affecting IL-2-induced cytotoxicity in peritoneal mononuclear cells. Mice were treated intraperitoneally with 1×10⁵ units IL-2 three times daily for four consecutive days. DMTU (10 mg/0.5 ml) was administered to the study group once daily, prior to the first IL-2 injection. Comparing the wet/dry weight ratio of lungs, liver, and spleen showed that IL-2 caused a significant (P<0.05) wet/dry increase in all three organs. DMTU attenuated the wet/dry increase in the lungs (P<0.05), in the spleen (P<0.05), and not at all in the liver. IL-2 induced a marked increase in peritoneal mononuclear cell counts, which was not attenuated by DMTU. The cytotoxic effect of IL-2-activated peritoneal mononuclear cells on target B16 cells was also unchanged in animals pretreated with DMTU. In conclusion, we have shown that DMTU ameliorates pulmonary permeability and vascular leak syndrome associated with multiple-dose IL-2 therapy, without eliciting an inhibitory effect on IL-2 induced-cytotoxicity. Received: 11 July 1996 / Accepted: 25 September 1996     

The tumor-bearing state induces augmented responses of organ-associated lymphocytes to high-dose interleukin-2 therapy in mice

 A high-dose bolus regimen for interleukin(IL)-2 administration to cancer patients frequently causes serious side-effects in which various organs are involved. In order to reveal the mechanism of toxicities associated with this regimen, we compared the augmenting effect of high-dose IL-2 on murine organ-associated lymphocytes between neoplastic and non-neoplastic states. Intraperitoneal administration of IL-2 at a dose of 10⁵ JRU (Japanese Reference Units) twice daily for 3 days led to the death of all the syngeneic MH134-hepatoma- or X5563-myeloma-bearing mice, whereas it had no lethal effect on non-tumor-bearing mice. Histological and morphometric analyses demonstrated that tumor-bearing mice displayed more extensive infiltration of large granular lymphocytes and agranular lymphocytes in the liver and lungs than did the non-tumor-bearing mice. Large granular lymphocytes had the ultrastructural characteristics of lymphokine-activated killer cells. Lymphocytes often underwent extravasation into the interstitial space and exhibited local proliferation without causing any direct injury to apposed parenchymal cells. Flow-cytometric analysis of hepatic mononuclear cells demonstrated that IL-2-receptor-β(IL-2Rβ)-bearing lymphocytes, i.e., natural killer cells and intermediate CD3 cells, were increased in number in the neoplastic state before the IL-2 injection. The present study indicates that the tumor-bearing state increases the number of organ-associated IL-2Rβ⁺ lymphocytes, which are then greatly amplified by the challenge of high-dose IL-2, leading to the functional disturbance of organs. We have further demonstrated here that an intermittent low-dose IL-2 regimen has a potential therapeutic effect on tumor regression without causing lethal side-effects. Received: 15 July 1996 / Accepted: 23 July 1997     

Autoantibody to p185 erbB2/neu oncoprotein by vaccination with xenogenic DNA

 The passive transfer of antibodies and vaccination procedures against p185, the erbB2/neu oncoprotein, are approaches being explored for treatment of human breast cancer. We now report the possibility of using the erbB2/neu gene as an immunogen. This study demonstrates that intramuscular or intradermal injections of rat neuNT full-length DNA into mice generate anti-p185 autoantibodies. Anti-p185 polyclonals were also shown to bind the homologous human receptor ErbB2 and to stain specimens of breast adenocarcinoma from both neu-transgenic mice and humans. Further, in vitro assays demonstrated that anti-p185 IgG (probably dependent on CD4⁺ Th1) were able to inhibit human SKBR3 tumour cell growth and to mediate their lysis by natural killer cells. The continuous presence of circulating neu autoantibodies in mice did not cause any discernible toxic effects on normal tissues expressing low levels of self-antigen, even after 1 year. Received: 29 August 1996 / Accepted: 31 October 1996     

Antimetastatic effect of recombinant human macrophage-colony-stimulating factor against lung and liver metastatic B16 melanoma

 We studied the effect of recombinant human macrophage-colony-stimulating factor (rhM-CSF) on the formation of lung and liver metastases following the i.v. injection of the B16 melanoma subline (B16 LiLu) into mice. When rhM-CSF was administered before the B16 inoculation, the number of tumor metastases decreased in the lung and liver. However, the administration of rhM-CSF after B16 inoculation did not produce an antimetastatic effect in the lung, but did in the liver. B16 cells labeled with 5-[¹²⁵I]-iodo-2′-deoxyuridine (¹²⁵I-dUrd) were injected and the arrest of tumor cell emboli was examined in the capillary beds of the lung and liver of mice treated with either vehicle or rhM-CSF. In both groups, there were the same numbers of B16 cells in both the lung and the liver 3 minutes after the B16 injection, and almost all tumor cells died within 24 h. However, the number of cells surviving in the lung was decreased in mice injected with rhM-CSF (37%). There was no difference in the number of cells in the livers of mice treated either with vehicle or rhM-CSF in the first 24 h after tumor cell injection. The administration of rhM-CSF increased NK 1.1⁺ cells in the mouse spleen and facilitated NK activity in vivo. At the same time, the administration of an anti-NK 1.1 antibody blocked the antimetastatic effect of rhM-CSF in the lung but not in the liver. The antibody was effective only when it was injected before the B16 inoculation. These results suggest that the antimetastatic effect of rhM-CSF in the lung was mediated by NK 1.1⁺ cells within 24 h of B16 injection. In contrast, the antimetastatic effect of rhM-CSF in the liver was mediated not only by NK 1.1⁺ cells but also by other antimetastatic systems such as macrophages. Received: 8 April 1996 / Accepted: 26 November 1996     

Inhibition of rat C6 glioblastoma tumor growth by expression of insulin-like growth factor I receptor antisense mRNA

 The expression of insulin-like growth factor I receptor (IGF-IR) antisense mRNA inhibits the growth of C6 rat glioblastoma cells both in vitro and in vivo [Cancer Res (1994) 54: 2218]. Moreover, the injection of C6 cells expressing an antisense mRNA to the IGF-IR into syngeneic rats prevents subsequent wild-type tumorigenesis and induces regression of established tumors. For the study of immune function in syngeneic rats, C6 cells expressing either IGF-IR sense or IGF-IR antisense mRNA were injected and splenic lymphocyte function analyzed in vitro after 2 weeks. Cytotoxic, CD8⁺ lymphocytes from animals injected with IGF-IR antisense cells, but not from those treated with IGF-IR sense cells, proliferated in vitro in response to wild-type C6 cells. Wild-type C6 cells or IGF-IR-sense-RNA-expressing cells rapidly formed tumors upon subcutaneous injection into athymic nude mice. IGF-IR antisense cells were weakly tumorigenic, exhibiting a six- to tenfold increase in tumor latency. Injection of IGF-IR antisense C6 cells mildly delayed the development of wild-type tumors, and did not induce the regression of established wild-type C6 tumors in athymic nude mice. Thus, these findings demonstrate the stimulation of a cellular immune response in rats following the injection of IGF-IR antisense cells. However, studies of athymic nude mice indicate that expression of IGF-IR antisense mRNA also inhibits C6 cells tumorigenicity by additional mechanisms. Received: 27 January 1995 / Accepted: 15 November 1995     

Prevention of superantigen-induced tolerance in vivo by interleukin-2 treatment

 Injection of the superantigen staphylococcal enterotoxin A (SEA) activates both CD4⁺ and CD8⁺ T cells expressing certain families of T cell receptor (TCR) variable-region β (V_β) chain. T cells respond with profound cytokine production and induction of cytotoxicity. Repeated injections, however, cause deletion and anergy of both CD4⁺ and CD8⁺ T cells, resulting in reduced frequency of SEA-responsive cells TCR-V_β11⁺ as well as reduced cytokine levels in serum upon challenge with SEA. Exogenous interleukin-2 (IL-2) in vivo rescued SEA-responsive CD4⁺ and CD8⁺ cells from SEA-induced deletion and/or increase expansion of SEA-primed cells as well as preventing down-regulation of endogenous IL-2 production in vivo. Combined treatment with SEA and IL-2 also superinduced production of important cytokines for the cytotoxic function of T cells, tumour necrosis factor α, interferon γ and IL-6, on a cellular level. These studies show that continuous stimulation with IL-2 in vivo could be useful for superantigen-based immunotherapy by induction of excessive T cell activation and by prevention of the development of T cell deletion and anergy. Received: 29 August 1996 / Accepted: 16 January 1997     

Therapeutic effectiveness of the immunity elicited by P815 tumor cells engineered to express the B7-2 costimulatory molecule

 It is well accepted that inoculation of B7-1-transfected tumor cells into normal mice leads to tumor rejection and subsequent resistance to challenge. However, the effectiveness of B7-2-transfected tumor cells in eliciting protective antitumor immunity is less clear. Here we show that B7-2-transfected P815 tumor cells (B7-2⁺) are as effective as B7-1-transfected P815 tumor cells (B7-1⁺) in eliciting protective immunity in normal DBA/2 mice. In addition, B7-2⁺ cells were found to be at least as effective as B7-1⁺ cells in retarding tumor progression when admixed with parental P815 tumor cells prior to inoculation into normal mice. Moreover, the B7-2⁺ cells and the B7-1⁺ cells were equivalent in their ability to retard tumor growth when administered peritumorally into mice bearing established (approx. 3 mm in diameter) parental P815 tumors. Finally, P815 tumor cells infected with a recombinant replication-defective adenovirus encoding the murine B7-2 gene were effective in retarding the growth of established parental P815 tumors. Thus, B7-1 and B7-2 are comparable in terms of their ability to stimulate the generation of tumor-eradicating immunity in normal mice as well as in mice bearing established parental tumors. Moreover, adenovirus vectors can be used to generate B7-2-expressing tumor cells effective in the immunotherapy of established parental tumors. Received: 10 January 1996 / Accepted: 23 February 1996     

Efficacy against lung metastasis with a tumor-targeting mutant of Salmonella typhimurium in immunocompetent mice

Salmonella typhimurium double leu-arg auxotrophs have been shown to be highly effective as antitumor agents in nude mouse models of human metastatic cancer. In order to proceed to clinical development of the S. typhimurium double auxotroph, termed A1-R, it is necessary to evaluate antitumor efficacy in immunocompetent mice. In the present study, we have observed the efficacy of A1-R on the Lewis lung (LLC) carcinoma in vitro as well as in C57BL/6 (C57) immunocompetent mice. In vitro, A1-R treatment of LLC began to induce cell death within one hour. Various doses and schedules of A1-R were administered to C57 mice implanted with LLC, including bolus single intravenous injection; medium dose with weekly intravenous administration and metronomic treatment with small intravenous doses twice a week. Bolus treatment was toxic to the immunocompetent host in contrast to nude mice. Lower-dose weekly doses and metronomic doses were well-tolerated by the immunocompetent host. Weekly intravenous injection with 2 × 10⁷ bacteria and twice a week intravenous injection with 10⁷ bacteria significantly inhibited metastasis formation, while bolus injection was toxic. Intrathoracic administration was performed with 10⁸ A1-R bacteria injected into Lewis lung-bearing C57 mice weekly for three weeks. Lung metastasis was significantly inhibited by intrathoracic bacterial administration without toxicity. The results in this report, demonstrating the anti-metastatic efficacy of S. typhimurium A1-R in immunocompetent mice, indicate the clinical potential of bacterial therapy of cancer.Key words: Salmonella typhimurium, amino acid auxotroph, selective tumor targeting, lung, metastasis, RFP, GFP, fluorescence imaging, confocal microscopy     

The helper T-cell repertoire of mice expressing class II major histocompatibility complex β chains in the absence of α chains

 Mutant mice generated by disrupting the H2-Aa ^b major histocompatibility complex (Mhc) gene are demonstrated here to express Aβ^b chains in the absence of α chains. These mice possess a CD4⁺ helper T cell (Th) repertoire that uses predominantly the Vβ7 T-cell antigen receptor (Tcr) segment for recognition of any protein antigen presented by the α-free Aβ molecule. As an alloantigen, the Aα-free Aβ molecule is recognized very poorly by T cells from a series of class II disparate mouse strains, indicating that it is grossly different from normal α/β heterodimers. Indeed, molecular modeling suggests a β/β homodimer arrangement with an altered geometry of the Tcr contact area. Interestingly, the mutant mice exhibit normal alloreactivity, without a restricted Vβ usage, toward a series of foreign α/β class II heterodimers, although their T cells developed in the absence of such heterodimers. Thus, the complementarity of Tcr to normal α/β heterodimers, and thereby also alloreactivity, appears to be an ontogeny independent (i. e., germline-encoded) feature. Received: 30 September 1996 / Revised: 18 October 1996     

Efficacy against lung metastasis with a tumor-targeting mutant of Salmonella typhimurium in immunocompetent mice

Salmonella typhimurium double leu-arg auxotrophs have been shown to be highly effective as antitumor agents in nude mouse models of human metastatic cancer. In order to proceed to clinical development of the S. typhimurium double auxotroph, termed A1-R, it is necessary to evaluate antitumor efficacy in immunocompetent mice. In the present study, we have observed the efficacy of A1-R on the Lewis lung (LLC) carcinoma in vitro as well as in C57BL/6 (C57) immunocompetent mice. In vitro, A1-R treatment of LLC began to induce cell death within one hour. Various doses and schedules of A1-R were administered to C57 mice implanted with LLC, including bolus single intravenous injection; medium dose with weekly intravenous administration and metronomic treatment with small intravenous doses twice a week. Bolus treatment was toxic to the immunocompetent host, in contrast to nude mice. Lower-dose weekly doses and metronomic doses were well tolerated by the immunocompetent host. Weekly intravenous injection with 2 x 10⁷ bacteria and twice a week intravenous injection with 10⁷ bacteria significantly inhibited metastasis formation, while bolus injection was toxic. Intra-thoracic administration was carried out with 10⁸ bacteria A1-R injected into Lewis lung-bearing C57 mice weekly for three weeks. Lung metastasis was significantly inhibited by intrathoracic bacterial administration, without toxicity. The results in this report, demonstrating the anti-metastatic efficacy of S. typhimurium A1-R in immunocompetent mice, indicate the clinical potential of bacterial therapy of cancer.     

Effects of lethal irradiation and cyclosporin A treatment on the growth and tumoricidal activity of a T cell clone potentially useful in cancer therapy

The TALL-104 cell line, originally derived from a patient with T cell leukemia, can be maintained indefinitely in culture in the presence of interleukin-2 (IL-2) and is endowed with a highly potent major-histocompatibilitycomplex (MHC)-non-restricted tumoricidal activity both in vitro and in animal models. The present study analyzes in detail the short- and long-term effects of irradiation and cyclosporin A (CsA) treatment on the growth and tumoricidal function of this T cell clone as compared to polyclonal lymphokine-activated killer (LAK) cell preparations from healthy donors. DNA and RNA syntheses by both TALL-104 and LAK cells were irreversibly arrested a few hours after irradiation with 40 Gy. However, 4-h⁵¹Cr-release assays, performed on different days (day 1 to day 7) after irradiation, showed that the cytotoxic efficiency of TALL-104 cells against hematopoietic and solid tumor targets was only modestly reduced, whereas that of LAK cells was severely inhibited. Moreover, the cytotoxic responses to recombinant human IL-2 and IL-12, measured 18 h after irradiation and cytokine addition, were normal in the case of TALL-104 cells but were abolished in the case of LAK cells. Co-culture of IL-2-or IL-12-preactivated TALL-104 cells with a tumor target for 5 days in the absence of cytokines resulted in a lower efficiency of lysis, as compared to the non-irradiated effectors, especially if the initial stimulus was IL-12. These findings suggest the requirement of multiple cytokine stimulation for optimal expression of tumoricidal activity by lethally irradiated TALL-104 cells. CsA, while abrogating TALL-104 cell proliferation at the low dose of 0.5 g/ml, inhibited their cytotoxic function marginally only at high doses (100 g/ml). By contrast, CsA reduced dose-dependently the cytotoxicity of LAK cells starting at very low doses (0.5 g/ml). CsA did not impair the ability of TALL-104 and LAK cells to produce interferon (IFN), tumor necrosis factor (TNF) , and granulocyte/macrophage-colony-stimulatory factor (GM-CSF) in response to IL-2, IL-12, or tumor targets. Irradiation reduced drastically IFN production by LAK, but not TALL-104 cells; release of TNF and GM-CSF by either type of effector was inhibited by 10%–50%, depending on the stimulus. The high resistance of the TALL-104 cells' tumoricidal function to irradiation and immunosuppressive drugs renders this immortal T cell clone a potentially safe and effective reagent for new adoptive-transfer approaches to cancer in MHC-incompatible recipients.     

Experimental Pathogenicity of a Clinical Isolate of <Emphasis Type="Italic">Trichosporon dermatis</Emphasis> in a Murine Model

The pathogenicity of Trichosporon dermatis isolated from skin lesions of a patient has been examined in mice. Balb/c mice were treated with two intraperitoneal injections of 100 mg/kg cyclophosphamide on days 4 and 1 and one subcutaneous injection of 10 mg/kg dexamethasone on day 1 pre-inoculation, and then challenged with 0.2 ml T. dermatis inoculum (1 × 10⁸ CFU/ml) by topical application on an abrasive wound in the dermabrasive group and by hypodermic injection in the subcutaneous group. In the intravenous group, 0.2 ml of high (1 × 10⁸ CFU/ml) or low (1 × 10⁷ CFU/ml) inoculum was injected into the tail vein. Histopathology and inverse fungal culture were performed on the skin lesion and viscera, and renal fungal burden was also determined. Inoculated sites developed localized infections after dermabrasive and subcutaneous challenge in all mice, but the maximum area of skin lesions, and number of positive cultures from the lesions, were higher for immunocompromised mice. In the intravenous group, all immunocompetent animals survived during the four-week period, whereas 100 and 70% of immunocompromised animals died by 3 and 5 days in the high and low-inoculum groups, respectively. The incidence of disseminated infection and the renal fungal burden of immunocompromised mice were higher than those of immunocompetent mice. Our results demonstrate that subcutaneous and intravenous injection of T. dermatis can successfully establish cutaneous and systemic infection models in immunocompromised mice, with the kidney and lung being most susceptible.     

Shake-flask culture of Laccaria laccata,an ectomycorrhizal basidiomycete

 Large-scale exploitation of the potential benefits of ectomycorrhizal fungi in improving plantation yields means that fermentation techniques for these fungi will be required. Starting with a base performance on a rich, complex medium, the effect of variations in some physicochemical culture parameters on biomass yield was studied. It was possible to reduce the amount of phosphate salts (to 1/9th) and other ingredients (to 1/3rd) in the medium. A shaking speed of either 100 rpm or 200 rpm in an orbital incubator was satisfactory and biomass yield responded to an increase in carbon substrate (glucose, from 10 g l^-1 and 20 g l^-1) though Y _x/s declined. An increase in inoculum size shortened culture time but decreased biomass yield. The upper limit of the incubation temperature was between 25°C and 30°C. Biomass yields were about 12 g l^-1 dry weight (Y _x/s=0.63) when 20 g l^-1 glucose was supplied, and about 7 g l^-1 (Y _x/s=0.74) when 10 g l^-1 glucose was supplied. Received: 9 October 1995/Accepted: 4 December 1995     

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     

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     

Interleukin-4 is effective in restoring cytotoxic T cell activity that declines during in vivo progression of a murine B lymphoma

 We previously reported [Chakrabarti et al. (1992) Cell Immunol 142:54; 144:455] that, in a murine B lymphoma model 2C3, idiotype (Id)-specific CD8⁺ cytotoxic T lymphocytes (CTL) are generated in mice following hyperimmunization with irradiated tumor cells, and that they are effective in tumor rejection. The present study reveals that 2C3-specific CTL are also induced in spleens during tumor progression, but are not sustained. At the early stage of tumor growth, the splenic T cells following a 5-day incubation in vitro with killed 2C3 tumor targets, produce high levels of cytokines, namely interleukin-4 (IL-4), IL-10 and interferon γ (IFNγ). Their cytotoxic T lymphocyte (CTL) activity and cytokine levels, except IL-2, sharply decline at the late stage when the mice are increasingly moribund. Although the decline in cytokine level is also evident with CD4⁺ T cells, a precipitous and concurrent decrease occurs primarily in the IL-4 level with both CD4⁺ and CD8⁺ T cells of late-tumor-bearing animals (TBA). Study with the unseparated splenocytes also reveals that sevenfold less IL-4 is produced at the late stage. Furthermore, the cytotoxicity of CTL from late TBA can be effectively restored by addition of supernatants from the splenocyte culture of early TBA, or by IL-4, but not by IFNγ and IL-10. In addition, only IL-4-activated CD8⁺ T cells from the late TBA are found, by Winn assay, to be protective in vivo. Thus it appears that IL-4, required to sustain antitumor CTL activity, is consumed by T and possibly other cells at the late stage of tumor growth, thereby compromising host immunity against the tumor. We contend that induction or maintenance of protective immunity depends not only on the tumor antigen but also on the specific cytokine milieu in a tumor-bearing host. Received: 8 February 1997 / Accepted: 24 April 1997     

Protective effect of an acidic glycoprotein obtained from culture of Chlorella vulgaris against myelosuppression by 5-fluorouracil

 An acidic glycoprotein prepared from a culture of Chlorella vulgaris (CVS) was examined for its protective effect on 5-fluorouracil(5FU)-induced myelosuppression and indigenous infection in mice. Subcutaneous administration of CVS greatly reduced the mortality of non-tumor-bearing mice given a high dose of 5FU, and could increase the LD₅₀ value of 5FU for these mice. After 5FU treatment, indigenous infection developed probably as a result of the impairment of the host defense system. CVS reduced the incidence of indigenous infections and this effect was attributable to the acceleration of recovery from 5FU-induced myelosuppression. Early recovery of hematopoietic stem cells, or cells responding to interleukin-3 or granulocyte/macrophage-colony-stimulating factor, was especially observed in the bone marrow of CVS-treated mice on days 4 – 9 after the injection of 5FU. When tumor-bearing mice were given CVS during treatment with 5FU, CVS prolonged the survival of mice without affecting the antitumor activity of 5FU. In addition, CVS was itself shown to exert an antitumor effect. These results suggested that CVS may be beneficial for the alleviation of side-effects in cancer chemotherapy without affecting the antitumor activity of the chemotherapeutic agent. Received: 15 August 1995 / Accepted: 23 April 1996     

Simultaneous utilization of pyridine and fructose by Rhodococcus opacus UFZ B 408 without an external nitrogen source

 A bacterium classified as Rhodococcus opacus, which is able to use pyridine (a potentially growth-inhibiting substrate) as its sole source of carbon, energy and nitrogen, was isolated. In a carbon-limited chemostat culture, the kinetics was determined for growth on both pyridine and a mixture of pyridine and fructose (9 mM/22.15 mM). With growth on pyridine, stable steady states were achieved up to dilution rates of about 0.1 h^-1. A further increase in the dilution rate resulted in the progressive accumulation of pyridine in the culture liquid and the cells were washed out. The maximum specific growth rate (μ_max = 0.23 h^-1) and the K _S value (0.22 mM) for growth on pyridine were determined from the residual pyridine concentrations measured within the range of stable steady states. With growth on the substrate mixture, the specific pyridine consumption rates and the residual pyridine concentrations were lower at similar dilution rates than with growth on pyridine alone, and stable steady states were established at dilution rates of up to 0.13 h^-1. The maximum pyridine degradation rate was enhanced to 270 mg pyridine l^-1 h^-1 compared to 210 mg pyridine l^-1 h^-1with growth on pyridine as a single substrate. An external nitrogen source did not need to be added in the case of growth on the substrate mixture. Fructose was assimilated by means of ammonium released from pyridine. Analysis of the nitrogen balance furnished proof that pyridine is an energy-deficient substrate; pyridine was assimilated and dissimilated at a ratio of 1 mol/0.67 mol respectively. The resulting yield coefficient was about 0.55 g dry weight/g pyridine. Moreover, it was demonstrated that, in regard to the biologically usable energy, 1 mol pyridine corresponds to 0.43 mol fructose. Received: 3 July 1995/Received revision: 19 October 1995/Accepted: 24 October 1995     

Human T lymphocyte activation in the presence of acute myelogenous leukaemia blasts: studies of allostimulated interferon-γ secretion

 Normal peripheral blood mononuclear cells (PBMC responders) were cultured together with non-irradiated allogeneic PBMC (more than 95% leukaemia blasts) derived from patients with acute leukaemia (referred to as leukaemic PBMC stimulators). Cytokine secretion was determined as cytokine concentrations in supernatants. Both normal PBMC and enriched CD4⁺ and CD8⁺ T cells responded to allostimulation with interferon (IFNγ) secretion. Interleukin-1 (IL-1) receptor antagonist and IL-2-neutralizing antibodies decreased IFNγ secretion. Exogenous IL-1β, IL-2 and IL-7 increased allostimulated IFNγ secretion, whereas decreased levels were seen in the presence of IL-6, IL-10 and granulocyte-colony-stimulating factor (G-CSF). During allorecognition IFNγ -neutralizing antibodies decreased acute myelogenous leukaemia (AML) blast secretion of G-CSF. We conclude that (i) both CD4⁺ and CD8⁺ T cells show allostimulated cytokine secretion in response to allogeneic stimulator cells containing a dominating population of native, cytokine-secreting leukaemia blasts, and (ii) IFNγ released during this response can modulate the function of allogeneic AML blasts. Received: 4 June 1996 / Accepted: 15 October 1996