Interleukin-15 effectively potentiates the in vitro tumor-specific activity and proliferation of peripheral blood γδT cells isolated from glioblastoma patients

γδT cells play a regulatory role in both primary and metastatic tumor growth in humans. The mechanisms responsible for the activation and proliferation of circulating γδT cells should be fully understood prior to their adoptive transfer to cancer patients. We have examined in vitro functional effects of interleukin-15 (IL-15) on highly purified γδT cells isolated from glioblastoma patients. γδT cells constitutively express the heterotrimeric IL-2 receptor (IL-2R) αβγ, but the levels of IL-2Rβ or γ expression were not increased by incubation with saturating amounts of IL-15. IL-15 was shown to induce a maximal γδT cell proliferation, although at much higher concentrations (at least 2000 U/ml) than IL-2 (100 U/ml). Submaximal concentrations of IL-15 plus low concentrations of IL-2 produced an additive proliferative response. In contrast to the IL-2-induced response, this activity was completely or partially abrogated by anti-IL-2Rβ, or anti-IL-2Rγ antibodies, but not by anti-IL-2Rα antibodies. Incubation of γδT cells in the presence of IL-15 resulted not only in the appearance of NK and LAK activity, but also in specific autologous tumor cell killing activity, an additive effect being seen with IL-15 and IL-2. This IL-15-induced tumor-specific activity could be significantly blocked by anti-IL-2Rγ and anti-IL-2R-β mAb, but not by anti-IL-2Rα mAb. Thus, in contrast to IL-2, IL-15 activates tumor-specific γδT cells through the components of IL-2Rβ and IL-2Rγ, but not IL-2Rα. These enhanced in vitro tumor-specific and proliferative responses of γδT cells seen with IL-15 suggest a rational adjuvant imunotherapeutic use of γδT cells in cancer patients. Received: 23 January 1998 / Accepted: 20 May 1998     

Evaluation of natural killer cell expansion and activation in vivo with daily subcutaneous low-dose interleukin-2 plus periodic intermediate-dose pulsing

 Natural killer (NK) cells may be expanded in vivo with a prolonged course of daily subcutaneous interleukin-2 (IL-2). However, cellular activation requires higher concentrations of IL-2 than are achieved with low-dose therapy. The objective of the current trial was to determine the toxicity and immunological effects of periodic subcutaneous intermediate-dose IL-2 pulses in patients receiving daily low-dose therapy. A group of 19 patients were treated with daily subcutaneous low-dose IL-2 at 1.25×10⁶ International Units (1.25 MIU) m^–2 day^–1. After 4–6 weeks, patients received escalating 3-day intermediate-dose IL-2 pulses administered as single daily subcutaneous injections, repeated at 2-week intervals. The maximum tolerated pulse dose was 15 MIU m^–2 day^–1, with transient hypotension, fatigue, and nausea/vomiting dose-limiting. Subcutaneous IL-2 resulted in in vivo expansion of CD56⁺ NK cells (796±210%) and CD56^bright natural killer (NK) cells (3247±1382%). Expanded NK cells coexpressed CD16, and showed lymphokine-activated killer activity and antibody-dependent cellular cytotoxicity in vitro. Intermediate-dose pulsing resulted in serum IL-2 concentrations above 100 pM. Cellular activation was suggested by rapid margination of NK cells following pulsing, coincident with peak IL-2 levels, with return to baseline by 24 h. In addition, interferon γ production in response to lipopolysaccharide was augmented. Subcutaneous daily low-dose IL-2 with intermediate-dose pulsing is a well-tolerated outpatient regimen that results in in vivo expansion and potential activation of NK cells, with possible application in the treatment of malignancy and immunodeficiency. Received: 31 December 1997 / Accepted: 20 April 1998     

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     

Adjuvant treatment of breast cancer: A pilot immunochemotherapy study with CMF, interleukin-2 and interferon alpha

Immune responses, including natural immunity (NI), potentiate the antitumor effects of chemotherapy. Since interferons and interleukin-2 (IL-2) augment NI, a pilot study was conducted to assess the tolerability and the effects on host immunity of adjuvant chemotherapy associated with IL-2 + interferon alpha (IFN) in breast cancer patients after surgery. Ten patients underwent alternating 28-day cycles of chemoimmunotherapy [cyclophosphamide + methotrexate + 5-fluorouracil (CMF, days 1, 8) + IL-2 (days 15–19) + IFN (day 22)] and chemotherapy alone (CMF). With this regimen each patient was considered to be a reasonable “control” of herself. Blood cell count and natural killer cell activity (NKA) were tested on days 1, 8, 15, 22, and 23. Preliminary in vitro studies indicated that IL-2 or IFN antagonized the severe inhibition of NKA induced by hydroxy-peroxy-cyclophosphamide (in vitro active derivative of cyclophosphamide), alone or associated with methotrexate + 5-fluorouracil. Nine patients completed all six alternating cycles, whereas one patient proved to have metastatic lesions after four cycles. The protocol was well tolerated, although leukopenia (CMF alone) and leukopenia with fever and moderate or minimal flu-like symptoms (CMF + IL-2 + IFN) were generally observed. Treatment with IL-2 facilitated complete recovery of white cell counts and NKA after the nadir on day 15. In conclusion, the present protocol appears to be well tolerated and amenable to administration on an outpatient basis. Therefore, further investigations should be performed to verify whether CMF + IL-2 + IFN would be superior to CMF alone for adjuvant treatment after surgery in breast cancer. Received: 9 April 1998 / Accepted: 16 July 1998     

Synergistic antitumor effects of interleukin-12 gene transfer and systemic administration of interleukin-18 in a mouse bladder cancer model

We introduced the interleukin-12 (IL-12) gene into the mouse bladder cancer cell line (MBT2) to establish sublines that secrete bioactive IL-12. IL-12-secreting MBT2 (MBT2/IL-12) sublines were completely rejected when subcutaneously implanted into immunocompetent syngeneic C3H mice. Although this antitumor effect did not change when IL-12-secreting cells were injected into immunodeficient mice whose CD8⁺ T or CD4⁺ T cells had been depleted by the corresponding antibody, it was abrogated when natural killer cells were depleted by anti-asialoGM1 antibody. In addition, when parental MBT2 cells mixed with MBT2/IL-12 cells were subcutaneously injected into mice, admixed MBT2/IL-12 inhibited the growth of the parental tumor. Furthermore, this antitumor effect was enhanced by systemic IL-18 administration. This synergism was abrogated when the mice were treated with interferon-γ-neutralizing antibody in vivo. In conclusion, local secretion of IL-12 led to effective antitumor activity that was enhanced by systemic administration of IL-18. Interferon-γ plays an important role in the synergism of IL-12 gene transduction and systemic administration of IL-18. Received: 7 May 1998 / Accepted: 27 May 1999     

126Gln is the residue of human IL-2 binding to IL-2R γ subunit

The 126Gln of human interleukin-2 (IL-2) is a conserved amino acid residue. After substitution of 126Gln with Asp, the binding abilities of this mutant to different composites of IL-2 receptor (R) subunits have been determined. Results show that 126AspIL-2 has higher affinity to IL-2R α β γ complex and normal affinity to IL-2R α β complex, but loses its binding ability to IL-2R β γ complex, demonstrating that the 126Gln is the residue of human IL-2 which binds to IL-2R γ subunit. Project supported by the “863” Project of China.     

Differential expression of type IV collagen isoforms, α5(IV) and α6(IV) chains, in basement membranes surrounding smooth muscle cells

 Smooth muscle is composed of cigar-shaped, non-striated cells, each of which is encapsulated by a basement membrane and forms the contractile portion of tubular organs such as the gastrointestinal tract, pulmonary tract, genitourinary tract, and vasculature, in which slow and sustained contractions are needed. We examined basement membranes produced by smooth muscle cells and, using α(IV) chain-specific monoclonal antibodies, analyzed type IV collagens in these organs. Detailed distribution analysis of the α chains in normal and Alport cases by use of specific antibodies indicated that there are at least three molecular forms of type IV collagen, [α1(IV)]₂α2(IV), α3(IV)α4(IV)α5(IV), and α5(IV)/α6(IV). Smooth muscle cells in the urinary bladder and uterus were enclosed by basement membranes composed of α1, α2, α5, and α6 chains. The same α chains were present around smooth muscle cells in the muscular layer of the fundus of the stomach, whereas those in the antrum and further distal side of the gastrointestinal tract expressed mostly α1 and α2 chains. In addition, immunostaining analysis of the vasculature also showed that most of the smooth muscle cells were positive for α1 and α2 chains; however, α5 and α6 chains were also expressed by smooth muscle cells in the aorta and some arteries where blood pressure changes significantly. These results suggest that the smooth muscle cells enclosed by α5/α6-containing basement membranes might have some particular function related to mechanical stress or tensile strength during the characteristic contractile activity of tubular organs. Accepted: 23 March 1998     

Manganese toxicity towards Saccharomyces cerevisiae : Dependence on intracellular and extracellular magnesium concentrations

Inhibition of the growth of Saccharomyces cerevisiae was evident at concentrations of 0.5 mM Mn²⁺ or higher, but a tolerance to lower Mn²⁺ concentrations was observed. The inhibitory effects of 2.0 mM Mn²⁺ were eliminated by supplementing the medium with excess Mg²⁺ (10 mM), whereas addition of excess Ca²⁺ and K⁺ had negligible effect on Mn²⁺ toxicity. Growth inhibition by Mn²⁺, in the absence of a Mg²⁺ supplement, was attributed to Mn²⁺ accumulation to toxic intracellular levels. Mn levels in S. cerevisiae grown in Mg²⁺-supplemented medium were severalfold lower than those of cells growing in unsupplemented medium. Mn²⁺ toxicity was also influenced by intracellular Mg, as Mn²⁺ toxicity was found to be more closely correlated with the cellular Mg:Mn ratio than with cellular Mn levels alone. Cells with low intracellular levels of Mg were more susceptible to Mn²⁺ toxicity than cells with high cellular Mg, even when sequestered Mn²⁺ levels were similar. A critical Mg:Mn ratio of 2.0 was identified below which Mn²⁺ toxicity became acute. The results demonstrate the importance of intracellular and extracellular competitive interactions in determining the toxicity of Mn²⁺. Received: 18 June 1997 / Received last revision: 10 January 1998 / Accepted: 24 January 1998     

A phase I study of a HER2/neu bispecific antibody with granulocyte-colony-stimulating factor in patients with metastatic breast cancer that overexpresses HER2/neu

A phase I study of escalating doses of humanized bispecific antibody (bsAb) MDX-H210 with granulocyte-colony-stimulating factor (G-CSF) was conducted in patients with metastatic breast cancer that overexpressed HER2/neu. The main objectives of the study were to define the maximal tolerated dose (MTD) of MDX-H210 when combined with G-CSF, to measure the pharmacokinetics of MDX-H210 when administered with G-CSF, and to determine the toxicity, biological effects and possible therapeutic effect of MDX-H210 with G-CSF. MDX-H210 is a F(ab)′ × F(ab)′ humanized bispecific murine antibody that binds to both HER2/neu and the FcγR1 receptor (CD64), and was administered intravenously weekly for three doses followed by a 2-week break and then three more weekly doses. A total of 23 patients were treated, and doses were escalated from 1 mg/m² to 40 mg/m² with no MTD reached. The toxicity of the bsAb + G-CSF combination was modest, with no dose-limiting toxicity noted: 19 patients had fevers, 7 patients had diarrhea, and 3 patients had allergic reactions that did not limit therapy. The β-elimination half-life varied from 4 h to 8 h at doses up to 20 mg/m². Significant release of cytokines interleukin-6, G-CSF, and tumor necrosis factor α was observed after administration of bsAb. Circulating monocytes disappeared within 1 h of bsAb infusion, which correlated with binding of bsAb, noted by flow-cytometric analysis. Significant levels of human anti-(bispecific antibody) were measured in the plasma of most patients by the third infusion. No objective clinical responses were seen in this group of heavily pre-treated patients. Received: 23 July 1998 / Accepted: 6 November 1998     

Effect of local interleukin-2 treatment on spontaneous tumours of different immunogenic strength

Transplantable tumour lines established from spontaneous tumours of BALB/c, CBA, and DBA/2 mice displayed different immunogenic strength. This report describes tumour susceptibility to interleukin-2 (IL-2) therapy in relation to tumour immunogenicity. The following tumour lines were used: X5, X6, and X9 mammary tumours of DBA/2, BALB/c, and CBA origin respectively, X7 carcinoma of BALB/c and X18 papilloma of CBA mice. Two spontaneous tumours of long transplantation history, SL2 lymphoma (SL2) of DBA/2 and Madison lung carcinoma M109 (M109) of BALB/c origin, were used as control systems. Experimental mice were transplanted with different inocula of tumour cells at day 0; treatment with IL-2 was initiated on days 1–3 or delayed until day 10 and consisted of daily injections of low doses of 5000 or 20 000 U/mouse given five times a week for a period of 3 weeks. Treatment of SL2 (2 × 10⁴ cells injected i.p.) consisted of i.p. injections of 5000 or 20 000 U IL-2/mouse given on days 10–14 after tumour transplantation. IL-2 therapy of SL2-bearing DBA/2JIco mice resulted in a significant proportion of cures; however, no response to IL-2 treatment was achieved in SL2-bearing DBA/2CrIiw mice. BALB/c mice with the i.p. transplant of M109 responded to IL-2 treatment with 40% increase in lifespan. The low-dose IL-2 therapy of the five spontaneous tumours resulted, in general, in transient growth inhibition of the i.m. transplants of lines X5, X6, and X7 provided that IL-2 was administered locally. The therapeutic effect depended on the number of transplanted tumour cells, the best results being achieved at cell numbers close to the dose-inducing tumour growth in 50% of animals. We found that the spontaneously arising tumours responding to IL-2 treatment were all slowly growing and immunogenic (X6 and X7) or might have viral association (X5) and, as such, might express foreign antigens. The data suggest a correlation between tumour immunogenicity and the therapeutic effect. However, IL-2 can still exert some effect against tumours with negligible immunogenicity. Received: 16 July 1998 / Accepted: 5 October 1998     

ImmTher, a lipophilic disaccharide derivative of muramyl dipeptide, up-regulates specific monocyte cytokine genes and activates monocyte-mediated tumoricidal activity

ImmTher, a liposome-encapsulated lipophilic disaccharide tripeptide derivative of muramyl dipeptide, previously showed activity against liver and lung colorectal metastases in a phase I trial. The purpose of the current studies was to investigate whether ImmTher could up-regulate specific cytokine gene expression and protein production, as well as activate the tumoricidal or cytostatic activity of human monocytes. ImmTher induced the expression and production of interleukin(IL)-1α IL-1β, IL-6, IL-8, IL-12, macrophage chemotactic and activating factor, and tumor necrosis factor α but not IL-2 or IL-10. Cytostatic or cytotoxic monocyte activity was stimulated against human Ewing's sarcoma, osteosarcoma, and melanoma cells but not breast cancer cells. Production and secretion of these cytokine proteins may play a role in the antitumor activity of ImmTher. Received: 15 December 1998 / Accepted: 18 March 1999     

The effect of T1 and T2 cytokines on the cytotoxic T cell response to mannan-MUC1

MUC1 is a mucin over-expressed in breast cancer and a proposed target for immunotherapy. By immunising mice with MUC1 conjugated to mannan (M-FP), CD8⁺ MHC-class-I restricted cytotoxic T lymphocytes (CTL), of high CTL precursor (CTLp) frequency (1/8000) and with significant tumour protection, can be induced. The effect of various cytokines [interleukin-2 (IL-2), IL-4, IL-6, IL-7, interferon γ (IFNγ), and granulocyte/macrophage-colony-stimulating factor (GM-CSF)] on the MUC1 CTL immune response was investigated (a) by measuring the frequencies of CTLp in mice immunised with vaccinia virus constructs containing recombinant cytokines and M-FP, or (b) by immunising cytokine- or cytokine-receptor-knockout (−/−) mice with M-FP. Vaccinia virus (VV) constructs containing recombinant cytokines were used either individually or in combination in vivo with M-FP immunisation. M-FP immunisations combined with VV-IL-2, VV-IL-7 and VV-GM-CSF, and combinations of VV-IFNγ + VV-IL-2, VV-IFNγ + VV-IL-4 or VV-GM-CSF + VV-IL-7 increased CTLp frequencies up to threefold (1/17 666: M-FP + VV-GM-CSF + VV-IL-7) compared to M-FP (1/77 500) alone. By contrast, M-FP combined with VV-IL-4 decreased the CTLp frequency threefold whereas VV-IL-6 and VV-IFNγ had no effect. Studies in cytokine- and cytokine-receptor-gene-knockout (−/−) mice demonstrated that mice that are IL-2 −/− and IL-7 receptor −/− produce the same CTLp response to M-FP as do control mice, whereas responses in the IL-6 −/−, IL-10 −/− and IFNγ−/− mice were marginally improved and responses to M-FP in IL-4 −/− and tumour necrosis factor receptor 2 −/− mice were weaker. In spite of the increase in CTLp frequency, this was not reflected in an in vivo tumour model. Tumour challenges using MUC1⁺ P815 cells, demonstrated that the addition of cytokines had little additive effect on the already effective tumour-regression capabilities of M-FP alone. Received: 24 September 1998 / Accepted: 21 September 1999     

Phase I trial of interleukin-2 and high-dose arginine butyrate in metastatic colorectal cancer

Interleukin-2 (IL-2) and sodium butyrate allow rats to be cured of peritoneal carcinomatosis from colon cancer. We performed a phase I trial of IL-2 and high-dose arginine butyrate (ArgB) in patients with advanced metastatic colorectal cancer. Patients and methods: From April to July 1997, six patients were included in the trail; they had a median age of 52 years, four had a performance status of 0, two had a performance status of 1 with normal biological functions. All patients had received at least two prior lines of chemotherapy. A fixed dose of 18 MIU/m² IL-2,was administered by subcutaneous injection and ArgB was delivered via continuous intravenous infusion on days 1–6 with escalating doses starting at 2 g kg⁻¹ day⁻¹. Results: The planned dose escalation was not possible because of toxicities. A daily ArgB dose of 2 g/kg was delivered for nine cycles. Level 2 (4 g/kg) could not be delivered in three of the six patients because of liver toxicity. The dose-limiting toxicities were fatigue and liver function disturbances. The maximum tolerated dose for ArgB was 3 g kg⁻¹ day⁻¹, in combination with IL-2 at 12 MIU m² day⁻¹. No clinical response was seen. Pharmacokinetic analysis showed large intra- and interindividual variations. Conclusion: This schedule with a high dose of ArgB proved to be highly toxic with liver insufficiency. We will be running another trial with lower doses of ArgB calculated from the schedule used in the experimental model, starting at a dose of 20 mg kg⁻¹ day⁻¹ for ArgB and 200 000 UI kg⁻¹ day⁻¹ IL-2, every 8 h. Received: 13 May 1999 / Accepted: 28 October 1999     

Sequential administration of interferon γ and interleukin-2 in metastatic renal cell carcinoma: results of a phase II trial

Background: Because of the known efficacy of several cytokines in the treatment of advanced renal cell cancer (RCC), we have conducted a phase II trial of the efficacy and toxicity of subcutaneous interferon γ (IFNγ) and interleukin-2 (IL-2). Methods: 63 patients with progressive metastatic RCC were treated with 100 μg recombinant IFNγ1b administered three times weekly during weeks 1 and 2 and with 4.5 MU recombinant IL-2 administered on 4 consecutive days during weeks 3 and 4, every 6 weeks. Results: 11% of patients had an objective response (CR: 3%, PR: 8%), 33% had SD. Toxicity was generally mild. The median duration of remissions (CR + PR) was 9.6 months; the median duration of SD 8 months. A significant survival benefit was evident at a median observation time of 51 months for patients (44%) responding to therapy (P < 0.0001). Conclusions: we conclude that sequential treatment with IFNγ and IL-2 may prolong survival in patients with metastatic RCC responding to therapy. Received: 2 April 2000 / Accepted: 21 April 2000     

Distinct biological activities of recombinant forms of human interleukin-2 in vivo

 The biological activity of all recombinant forms of interleukin-2 (IL-2) is based upon an in vitro lymphocyte proliferation assay and measured in international units (IU). Numerous in vitro investigations have suggested that there may be different cellular effects of recombinant human IL-2 retaining the natural sequence (nIL-2) as compared to another recombinant form containing a serine substitution at amino acid position 125 ([Ser]IL-2). In the present study we investigated whether nIL-2 and [Ser]IL-2 cause similar patterns of systemic toxicities. C57BL/6 mice were treated with identical doses of either nIL-2 or [Ser]IL-2, as measured in IU, for 3 days and had blood and tissues removed for analysis of lymphocyte activation and organ dysfunction. The administration of nIL-2 had considerably greater effects on lymphocyte activation than did [Ser]IL-2, causing much greater up-regulation of the α subunit of the IL-2 receptor and the adhesion molecule lymphocyte function-associated antigen-1. Furthermore, nIL-2 induced more organ edema than did [Ser]IL-2 and caused hepatocellular injury, which was absent in mice treated with [Ser]IL-2. These data demonstrate that equivalent doses, measured in IU, of nIL-2 and [Ser]IL-2 have profoundly different effects on the induction of organ toxicity, suggesting that the IU standard may not be appropriate for the measurement of many in vivo biological activities. Received: 30 August 1996 / Accepted: 8 November 1996     

Antitumor response of regional lymph node lymphocytes in human lung cancer

 Monoclonal antibodies (mAb) are promising substances for the treatment of colorectal carcinoma, but the efficiency of this therapy still needs further improvement. We used a flow-cytometric cytotoxicity test to determine the efficacy of the cytokines interferon α (IFNα) and γ (IFNγ), interleukin-2 (IL-2), macrophage-colony-stimulating factor (M-CSF), granulocyte/macrophage-CSF (GM-CSF) and tumor necrosis factor α (TNFα) in enhancing the antibody-dependent cellular cytoxicity (ADCC) of the mAb 17-1A and the mAb BR55-2 against the colorectal carcinoma cell line HT29. In experiments performed at an effector to target ratio of 9:1, with peripheral blood mononuclear cells from five healthy volunteers as effector cells, we found that IFNα, IFNγ and IL-2 significantly augmented the ADCC of both mAb at concentrations between 3 ng/ml and 30 ng/ml. The other three cytokines were not effective. In further experiments we examined combinations of the three effective cytokines in different concentrations. The combination of IFNα and IL-2 proved to be optimal in enhancing ADCC of both mAb. Thus, the examination of ADCC by flow cytometry may reveal potentially useful combinations of cytokines and mAb for the treatment of colorectal carcinoma. Received: 11 September 1997 / Accepted: 19 February 1998     

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     

Evidence for Clostridium perfringens Enterotoxin (CPE) Inducing a Mitogenic and Cytokine Response In Vitro and a Cytokine Response In Vivo

We investigated some immunogenic properties of Clostridium perfringens enterotoxin (CPE) in vitro using murine J774A macrophages (MΦ) and in vivo using Swiss Webster (SW) mice. CPE was a potent mitogen in vitro, where cell proliferation increased with CPE concentration. CPE was nonmitogenic when MΦ were concurrently incubated with CPE and interferon gamma (IFN-γ). MΦ incubated in the presence of CPE induced the synthesis of interleukin-1 (IL-1), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α) and interferon-gamma (IFN-γ), but not interleukin-2 (IL-2). In vivo, CPE induced a pro-inflammatory cytokine response with striking production of IFN-γ, IL-1, and IL-6. Regardless of route of CPE entry, serum cytokine levels generally peaked within 1 h of administration and were maintained for 4–8 h. Although CPE engenders an intense immune response during toxicosis, the toxin does not appear to be a superantigen. Death from CPE-induced shock appears to result from various interrelating immunological mechanisms. Received: 25 April 1998 / Accepted: 2 September 1998     

Investigating the lysis of small-cell lung cancer cell lines by activated natural killer (NK) cells with a fluorometric assay for NK-cell-mediated cytotoxicity

Activation of natural killer (NK) cells with interleukin-2 (IL-2) and IL-12 leads to an enhanced lysis of tumour cells. We investigated the ability of NK cells, with or without prior activation, to lyse a variety of small-cell lung cancer (SCLC) target cells. Specific lysis was measured with a fluorometric assay for NK-cell-mediated cytotoxicity: target cells were labelled with 3,3′-dioctadecyloxacarbocyanine, a green membrane dye. After co-incubation with NK cells, dead target cells were stained with propidium iodide, a red DNA dye that only penetrates dead cells. Of all eight SCLC cell lines tested, three were susceptible to lysis by non-activated NK cells, three were only susceptible to lysis by NK cells activated with IL-2 and IL-12 and two were not even susceptible to lysis by activated NK cells. The differences in target cell susceptibility showed no correlation with the expression of MHC-I on the surface of the target cells or with the expression of the adhesion molecules CD50, CD54, CD58 or CD102. Comparing the kinetics of the lysis of one SCLC cell line sensitive to non-activated NK cells and one sensitive only to activated NK cells, we found that maximum lysis of the former was obtained after 1 h, whereas significant lysis of the latter was only obtained after 4 h of incubation. This might be due to different mechanisms engaged in target cell lysis. Received: 23 December 1998 / Accepted: 8 April 1999     

Fused organs in the adherent1 mutation in maize show altered epidermal walls with no perturbations in tissue identities

In the absence of wounding, the epidermis is only rarely involved in cell or organ fusion events; in fact, intact epidermal layers prevent graft unions. In Zea mays L. the mutation adherent1 (ad1) shows abnormal fusions between cells and organs. Fusions involve epidermal cells of vegetative and floral organs and occur early in the ontogeny of organs. Even so, epidermal cell types differentiate normally in the fused regions and internal tissue identities are maintained. In contrast, the extracellular matrix (cell wall and cuticle) of the epidermal cells is perturbed. Epidermal cell walls in adherent leaves are thicker than normal. Epicuticular wax particles appear reduced in size and number and altered in shape in mutant leaves. In addition, the outer epidermal cell walls of adherent leaves fluoresce when stained with aniline blue, a reagent that binds to callose. Immunolocalizations to specific cell wall epitopes suggest that pectins but not arabinogalactans may have a role in the fusion events. Taken together, these results suggest that the ad1 mutation results in cell-wall and epicuticular-wax defects similar to responses seen in wounding, pollination by incompatible pollen, or pathogen attack. Since cell wall components and epicuticular waxes are extracellular secreted products, the ad1 mutation may disrupt normal functioning and/or composition of the secretory pathway and its cargo. Received: 30 January 1998 / Accepted: 5 March 1998