A pathogenic role of Th2 cells and their cytokine products on the pulmonary metastasis of murine B16 melanoma

The role of Th2 cells and the cytokines produced by these cells on experimental pulmonary metastasis of B16 melanoma was investigated in a murine model implanted with high metastatic (B16F10) or low metastatic (B16F1) melanoma cells. An average of 250 colonies of metastasis in the lungs was counted in mice (BF10 mice) at 14 days after the inoculation of 2 x 10(5) B16F10 cells/mouse, while <20 colonies were detected in mice (BF1 mice) inoculated with the same number of B16F1 cells. CD4+ CD11b+ TCR-alphabeta+ T cells (BF10-Th2 cells) were produced in the spleens of BF10 mice, while these cells were not detected in BF1 mice. The BF10-Th2 cells produced IL-4 and IL-10 into culture fluids when stimulated in vitro with anti-CD3 mAb. However, IL-2 and IFN-gamma were not produced. The level of a pulmonary metastasis in BF1 mice increased to the level observed in BF10 mice, when BF10-Th2 cells were adoptively transferred to BF1 mice. Also, an increase in the number of pulmonary melanoma was demonstrated in BF1 mice treated with 10 microg/kg murine rIL-4. The level of pulmonary metastasis in BF10 mice or in BF1 mice inoculated with BF10-Th2 cells decreased to the level observed in BF1 mice when mice were treated with an anti-IL-4 mAb at a dose of 250 microg/kg on days 1, 3, and 5 after tumor inoculation. These results suggest that the severity of pulmonary metastasis in mice receiving B16 melanoma cells is strongly influenced by the IL-4 released from tumor-associated Th2 cells.     

Involvement of MHC class I molecule and ICAM-1 in the enhancement of adhesion and cytotoxic susceptibility to immune effector cells of tumor cells transfected with the interleukin (IL)-2, IL-4 or IL-6 gene

To investigate the molecular and cellular mechanisms involved in the reduced tumorigenicity and increased immunogenicity of interleukin-2 (IL-2)-, IL-4- or IL-6-gene-transfected B16 melanoma vaccine, we have analyzed the functional and phenotypic properties of these genetically engineered melanoma cells in the present study. The cytokine-gene-transfected B16 melanoma cells showed stronger adhesion to the lymphokine-activated killer (LAK) cells or cytotoxic T lymphocytes (CTL), and higher sensitivity to cytotoxicity of LAK cells or CTL. Using fluorescence-activated cell sorting analysis, we found that both MHC class I and ICAM-1 expression were increased after IL-2, IL-4 or IL-6 gene transfection. The increased level of MHC class I and ICAM-1 expression seems to be responsible for the high sensitivity of these gene-transfected B16 cells to LAK or CTL cytotoxicity because anti-(MHC class I) or anti-ICAM-1 mAb could inhibit the adhesion and cytotoxicity increment simultaneously. The CTL induction was partly inhibited by anti-ICAM-1 mAb and was completely blocked by anti-MHC class I mAb. These results suggested that the decreased tumorigenicity of IL-2-, IL- 4-, and IL-6-gene-transfected B16 melanoma cells may be partly due to the increased sensitivity to effector cell cytotoxicity mediated by increased expression of ICAM-1 or MHC class I molecules on the tumor cell surface after cytokine gene transfection.     

Structural characterization of the disialogangliosides of murine peritoneal macrophages

Sialoglycosphingolipids (gangliosides) have been increasingly implicated as regulators of membrane signaling events. Macrophage ganglioside patterns dramatically increase in complexity when murine peritoneal macrophages are stimulated in vivo with the appearance of the sialidase-sensitive monosialoganglioside GM1b (cisGM1) as a major component. Gangliosides from stimulated murine peritoneal macrophages were separated into monosialo and polysialo fractions and the polysialo fraction structurally characterized by enzymatic, chemical, and mass spectra methods. All detectable components of the polysialo fraction were determined to be disialogangliosides. Treatment of the polysialo fraction with Clostridium perfringens sialidase produced mostly the sialidase-resistant monosialoganglioside, GM1a, and a minor amount of asialoGM1. Periodate oxidation and mass spectrometry analyses demonstrated the lack of tandem disialo moieties which indicated the absence of GD1b or GD1c (GD1) entities. The combined data showed the major disialogangliosides consisted of GD1a entities comprising IV3-NeuAc,II3NeuAc-GgOse4Cer, IV3-NeuGc,II3NeuAc-GgOse4Cer, IV3NeuAc,II3NeuGc-GgOse4Cer, and IV3-NeuGc,II3NeuGc-GgOse4Cer. Minor components consisted of GD1alpha entities, IV3NeuAc, III6NeuAcGgOse4Cer, IV3NeuGc, III6NeuGc-GgOse4Cer, and also positional isomer(s) of GD1alpha(NeuAc, NeuGc). These isomeric components were identified by collision analysis and tandem mass spectrometry. Consistent with previous analyses, the ceramide portion of all polysialo (disialo) gangliosides contained solely C18 sphingosine with C16 and C24 fatty acid moieties. These results, combined with the previous characterization of macrophage monosialogangliosides, indicate normal murine macrophage ganglioside biosynthesis proceeds along the "a" ganglioside pathway, e.g., GM3-->GM2-->GM1a-->GD1a, and the proposed asialoganglioside or "alpha" pathway, asialoGM1-->GM1b-->GD1alpha. The presence of totally sialidase-sensitive gangliosides appears to be characteristic of functional murine peritoneal macrophages while they are reduced in genetically impaired cells.     

Chemical, metabolic and immunological characterization of gangliosides of human glioma cells

The patterns of ganglioside profiles were studied in 10 human glioma and one melanoma cell lines. Ganglio-series gangliosides, GM3 (NeuAc alpha2-3Gal beta1-4Glc beta1-Cer) and GM2 (GalNAc beta 1-4 (NeuAc alpha2-3)Gal beta1-4Glc beta 1-1Cer), and a neolacto-series ganglioside, sialylparagloboside (SPG) (NeuAc alpha 2-3Gal beta1-4GlcNAc beta1-3Gal beta1-4Glc beta1-1Cer), were the predominant constituents. The activities of the two key enzymes, GM3 synthetase and lactotriaosyl ceramide (Lc3Cer) synthetase, alone did not account for the ganglioside profile. Metabolic labeling with the use of [3H]glucosamine-HCl showed more pronounced difference in the synthetic rate of each ganglioside type, in which GM2 was the most strongly labeled in 7 out of the 10 glioma cell lines. On quantifying the chemical content of GM3 and GM2, the GM3/GM2 molar ratio of above 2.0 was arbitrarily classified into GM3 dominant type (KG-1C and Mewo); the ratio below 0.5 was designated as GM2 dominant type (H4, U138MG, U373MG, T98G and A172); and the ratio between 0.5 and 2.0 was regarded as GM3 and GM2-co-dominant type (U87MG, Hs683, SW1088 and U118MG). Subsequently, the capabilities of the antibody binding to these gangliosides were examined in native forms in the cell membrane and in chemically-isolated forms. The intensity of reaction against chemically isolated GM3 and GM2 gangliosides was dependent on the quantity, and GM2 was more reactive than GM3; however, the reactivities on the cell surface did not correlate with the chemical content indicating other factors to influence their immunoreactivities.     

Adenoviral-mediated herpes simplex virus-thymidine kinase gene transfer in vivo for treatment of experimental human melanoma

To assess the efficacy of an in vivo adenoviral-mediated cytotoxic gene therapy, human melanomas were established in nude mice and transduced with herpes simplex virus-thymidine kinase (tk) followed by treatment with ganciclovir (GCV). In initial experiments, adenovirus (adv) containing the beta-galactosidase reporter gene was employed to determine melanoma cell infectivity in vitro. In comparison to murine melanoma cell lines B16 and K1735-M2, human A375-SM cells exhibited up to a 10-fold greater susceptibility to adenoviral transduction, similar to the degree of infectivity found for human epidermal HaCaT cells. In addition, human A375-SM melanoma cells exhibited a greater sensitivity in vitro to the cytotoxic effects of transduction with tk-adv and treatment with GCV, which was mediated by a strong bystander effect. In vivo, intratumoral injection of relatively large human melanomas (160 mm3) with 1.2 X 109 pfu of tk-adv, followed by intraperitoneal GCV treatment (60 mg/kg twice daily) over 4 days, typically resulted in a 50% reduction in melanoma growth rate compared to mock or untreated controls. Moreover, histometrical analysis employing a rigorous computerized imaging system revealed that the residual viable tumor area in the tk-adv/GCV-treated group was only one-fifth that of solvent controls. These data show that adv is a highly efficient in vivo gene delivery system to treat experimental human melanomas. In comparison to a previous murine melanoma study, human melanomas appeared to exhibit a greater sensitivity to this cytotoxic treatment in vivo, which may hold significant promise for development of effective gene therapy modalities to treat melanoma in humans.     

Isolation perfusion in extracorporeal circulation with interleukin-2 and lymphokine-activated killer cells in the treatment of in-transit metastases from limb cutaneous melanoma

BACKGROUND: Therapies of advanced melanoma patients with interleukin-2 (IL-2) and cytotoxic lymphocytes have produced interesting results, but a larger diffusion of these treatments is limited by the severe side effects due to IL-2 systemic infusion. A strictly regional administration of IL-2 and cells by an isolation perfusion (IP) in extracorporeal circulation (ECC) for the treatment of regional melanoma metastases could improve tolerability and efficacy of this specific modality of immunotherapy. METHODS: Ten patients were submitted to adoptive immunotherapy with IL-2 and lymphokine-activated killer (LAK) cells by IP in ECC. The schedule of treatment included the first course of a 5-day systemic administration of IL-2 (Proleukin, EuroCetus 9-12 x 10(6) IU/m2/day continuous infusion); autologous LAK cells were obtained via leukapheresis and after in vitro activation were given (range 8-28 x 10(9)) along with IL-2 (120-2,400 IU/ml of perfusion priming) to the affected limb by IP; IL-2 (9-12 x 10(6) IU/m2/day) was also administered by systemic continuous infusion for 5 days starting on the day after IP. RESULTS: All patients concluded the treatment without any major local or systemic toxicities. Clinical responses included one complete and six partial remissions; three patients had stable disease. All patients are alive. Follow-up after IP ranged from 12 to 35 months (median: 22). The analysis of circulating lymphocytes revealed the rapid disappearance of LAK cells, suggesting their extravasation and/or endothelial adhesion in perfused tissues. CONCLUSIONS: IP with IL-2 and LAK cells is a new approach for the treatment of in-transit metastases due to cutaneous melanoma. The treatment appears to be feasible and reliable. Further biological and immunological studies should permit amelioration of the present modality of treatment.     

Ultrasound backscatter microscope analysis of mouse melanoma progression

The incidence and mortality rate of cutaneous melanoma continue to increase throughout the world, making the study of melanoma biology an important area of current research. While recent breakthroughs in transgenic mouse technology have led to promising mouse skin models of melanoma, there is presently no technique available for quantitatively studying subsurface melanoma progression, in vivo. We demonstrate the first application of an imaging method called ultrasound backscatter microscopy (UBM) for imaging early murine melanomas with spatial resolution of 30 microns axial and 60 microns lateral. Murine B16 F10 melanomas have been imaged from their earliest detection, over several days, until they are 2 to 5 mm in diameter. Melanoma dimensions measured by UBM were found to be in excellent agreement with those determined histopathologically on the excised tumours. The relative rms errors in UBM-determined melanoma height and width were found to be 8.7% and 4.2%, respectively. The mean rate of increase in tumour height of early murine melanoma was found to be 0.37 +/- 0.06 mm/day. Computer-generated volumetric renderings of melanomas have been produced from three-dimensional image data, allowing quantitative comparisons of tumour volumes to be made. Using a priori assumptions of ellipsoid tumour shape, the relative error in UBM-determined volume was shown to be less than 17%. These results should be of considerable interest to investigators studying melanoma biology using mouse skin models, and have implications in the use of high frequency ultrasound imaging for the clinical assessment of cutaneous melanoma.     

Does recurrent acute pancreatitis lead to chronic pancreatitis? Sequential morphological and biochemical studies

BACKGROUND: For the relatively nonimmunogenic B16-F10 murine melanoma, it has been found that genetically engineered expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) but not interleukin (IL)-2, IL-4, or interferon-gamma (IFN-gamma) resulted in a vaccine that could induce resistance to rechallenge. Because T cells from lymph nodes draining the sites of some progressive tumors can mediate tumor regression after in vitro activation, it seemed possible that even apparently nonimmunogenic melanoma cells might induce similar preeffector cells in the vaccine-draining lymph nodes (DLNs). METHODS: C57BL/6 mice were vaccinated with B16-F10 cells that were either unmodified or genetically modified to produce IL-2, IL-4, GM-CSF, or IFN-gamma. DLNs were harvested 10 days after vaccination for adoptive immunotherapy (AIT). The DLN cells were activated with bryostatin 1 and ionomycin (B/I), expanded for 10 days in culture, and transferred to mice with 3-day pulmonary metastases. Pulmonary nodules were counted 14 days after AIT. RESULTS: Adoptive transfer of expanded DLN lymphocytes sensitized by inoculation of WT B16-F10, or IL-4, GM-CSF, or IFN-gamma expressing cells significantly reduced pulmonary metastases. Despite the spontaneous regression of IL-2-transduced B16-F10 tumors, DLN from mice inoculated with IL-2 producing B16 cells had little or no antitumor activity. CONCLUSIONS: B16-F10 vaccination strategies that apparently do not induce systemic immunity can effectively sensitize DLN preeffector cells.     

Characterization of lymphokine-activated killing by human peripheral blood mononuclear cells stimulated with interleukin 2 (IL-2) analogs specific for the intermediate affinity IL-2 receptor

Interleukin 2-stimulated human peripheral blood mononuclear cells (PBMC) generate lymphokine-activated killing (LAK). Using the IL-2 analogs R38A and F42K, which interact primarily with the beta and gamma subunits of the IL-2 receptor, we assessed the roles of IL-2R beta gamma and the high-affinity IL-2 receptor complex in LAK activation. Although the kinetics of LAK activation were identical, lytic activity was approximately 30% lower and proliferation was up to 55% lower in those PBMC stimulated by R38A or F42K than in those exposed to wild-type IL-2. The percentage of cells expressing cell-surface markers such as CD3, CD4, CD8, and CD16 was not significantly different after treatment with wild-type IL-2, R38A, or F42K; however, the proportion of cells expressing IL-2R alpha increased dramatically in response to stimulation by F42K (30%) compared to stimulation by either rIL-2 or R38A (15%). In addition, by Day 7 the concentration of soluble IL-2R alpha in analog-stimulated LAK culture supernatants was 50-75% less than that from wild-type IL-2-cultured cells. These findings suggest that interaction of IL-2 with IL-2R beta gamma alone is sufficient for both proliferation and the generation of LAK, and that stimulation with subunit-specific IL-2 analogs results in differential regulation of the IL-2R alpha on human LAK cells.     

Abrogation of B16 melanoma metastases by long-term low-dose interleukin-6 therapy

We investigated the antitumor effects of human recombinant interleukin-6 (hrIL-6) on the highly metastatic B16 melanoma clone F10.9. These tumor cells were found to have very low levels of IL-6 receptors and in vitro IL-6 had no effect on cell proliferation or on the expression of MHC class I antigens. However, in vivo IL-6 was active against the metastatic growth of this tumor in mice, presumably through indirect immune effects. Low-dose IL-6 (1-10 micrograms/day), in three daily injections, 4 days a week, for 3 weeks, strongly inhibited the formation of experimental lung metastases following intravenous tumor cell inoculation. IL-6 therapy could be started even 10 days after tumor injection, when metastases are already established. Moreover, IL-6 treatment of mice bearing F10.9 tumors in the footpads resulted in complete protection against pulmonary spontaneous metastasis and in long-term survival. Histology confirmed the absence of micrometastases in most of the IL-6-treated animals. Analysis of the cytolytic activity of splenocytes at different times during therapy of tumor-bearing mice revealed significant lysis (up to 42%) of the melanoma F10.9 cells in the mice receiving IL-6 but not in the control mice.     

GD3, a prominent ganglioside of human melanoma. Detection and characterisation by mouse monoclonal antibody

Mouse monoclonal antibody AbR24 has a high degree of specificity for human melanoma cells when tested on viable cultured cells using the protein A mixed hemagglutinin serological assay. The antigen detected by this antibody has been isolated from melanoma cells and shown to be GD3 ganglioside by compositional and partial structural analysis and by comparison with authentic GD3 in thin layer chromatography (TLC). AbR24 reacts with authentic GD3, but not with any other ganglioside tested. Using TLC and reactivity with AbR24, a wide range of cells and tissues was examined for the presence of GD3. A new serological assay, termed glycolipid-mediated immune adherence, was devised for assaying the reactivity of AbR24 with gangliosides. Melanomas (cultured cells or tumor tissue) were shown to have GD3 and GM3 as major gangliosides. Other cells and tissues examined also contained GD3, but usually only in low amounts. Melanomas (and MOLT-4, a T cell line) were characterized by a simplified ganglioside profile with GD3 and GM3 as major components. The apparent discrepancy between the ubiquitous presence of GD3 and the serological specificity of AbR24 for melanoma cells can be explained in terms of localization and concentration of GD3 in different cells.     

A new mouse model of experimental melanoma for vaccine and lymphokine therapy

The annual incidence of malignant melanoma is estimated at 10-12 per 100,000 inhabitants in countries of central Europe and the United States, and alarmingly there has been a dramatic upward trend in that estimate. The B16 murine melanoma is a rapidly growing metastatic tumor of spontaneous origin, as are human malignant melanomas. Melanoma cells produce specific antigens which are uniquely different from normal cellular antigens, and the expression of such antigens is the cornerstone for preparation of anti-melanoma vaccines. One major problem in evaluating the effectiveness of vaccination and other biologic therapies is the variability of experimental tumor models. A new metastatic model of experimental melanoma which was developed in our laboratory imitates the major clinical stages of malignant metastatic melanoma: stage I, primary (local) tumor growth and bone marrow invasion; stage II, regional lymph node involvement; and stage III, metastasis to distant organs, such as the lungs. This model has been used successfully for screening vaccines constructed in our laboratory. Immunization with formalinized vaccines (of extracellular antigens, intact melanoma cells, or B700 antigen) or irradiated vaccines (of intact melanoma cells) partially inhibit primary melanoma tumor growth, reduce metastasis to regional lymph nodes and lungs, and significantly increase mean survival time. These anti-tumor effects were improved when polyvalent and monovalent vaccines were combined with IL-2 therapy. We also compared the immunogenic activity of vaccines made from B16 melanoma cells transfected with genes encoding murine IL-2 or GM-CSF, and effects on tumor bearing mice were compared with or without therapy using the corresponding lymphokines. In sum, comparison of antibody production, growth of primary melanoma tumors, number of surviving mice, mean survival time, and percent of mice with lung metastases, showed that the best course of immunotherapy involves vaccination of mice with irradiated B16 melanoma cells transfected to secrete GM-CSF, coupled with GM-CSF therapy.     

Effects of interferon-alpha and gamma on development of LAK activity from mononuclear cells in breast cancer patients

We examined the effect of recombinant IFN-alpha and IFN-gamma on induction of LAK cells from peripheral blood mononuclear cells (PBMNCs) in 7 pre-operative breast cancer patients and 4 healthy volunteers. Significant LAK activity was developed from PBMNCs of pre-operative breast cancer patients and healthy volunteers after incubation for 4 days with IL-2 (presence of IL-2 vs. absence of IL-2). Incubation of PBMNCs of pre-operative breast cancer patients with 1000 U/ml of IFN-alpha for 4 days suppressed the LAK activity significantly (P < 0.05). By contrast, incubation of PBMNCs of pre-operative patients with 1000 U/ml of IFN-gamma for 4 days increased the LAK activity significantly (P < 0.05). Significant cytotoxicity against MCF-7 cells (estrogen receptor positive human breast cancer cell line) was developed from PBMNCs of pre-operative breast cancer patients at 20:1 and 40:1 E/T ratios after incubation for 4 days with IL-2 (absence of IL-2 vs. 20:1 or 40:1, P < 0.05, P < 0.05), whereas PBMNCs of healthy volunteers did not. Stimulation of LAK cells with IFN-gamma produced a significant augmentation of cytotoxic activity against MCF-7 (P < 0.05), while IFN-alpha suppressed the cytotoxicity significantly (P < 0.05). These findings suggested that combined stimulation by IFN-gamma and IL-2 might be a reasonable treatment for breast cancer patients.     

Deletion of the putative effector region of Era, an essential GTP-binding protein in Escherichia coli, causes a dominant-negative phenotype

Gangliosides are highly immunosuppressive molecules but the mechanism(s) by which they act upon cells remains to be fully defined. Several metabolic products of exogenous gangliosides, including ceramide, have recently been suggested as second messengers in programmed cell death (PCD). Therefore, we have probed the role of gangliosides and ceramides in the induction of PCD and in the inhibition of in vitro lymphoproliferation. PCD was caused only by exogenous ceramides with short fatty acyl groups-d18:1-C2:0 (C2-ceramide, where d18:1 is sphingosine and C2:O is an acetyl group) and d18:1-C6:0 (C6-ceramide, where C6:O is a hexanoyl group). None of the gangliosides studied induced PCD, including naturally occurring GM3, synthetic d18:1-C18:0 GM3 (C18-Cer GM3, where C18:0 is a stearoyl group), or even d18:1-C2:0 GM3 (C2-Cer GM3), which itself contains a PCD-causing ceramide. However, these gangliosides were highly immunosuppressive, inhibiting antigen-induced lymphoproliferation at micromolar concentrations. We conclude that exogenous sphingolipids cause inhibition of lymphoproliferation and PCD by two separate and distinct mechanisms of action.     

Transformation of hematopoietic cell lines to growth-factor independence and induction of a fatal myelo- and lymphoproliferative disease in mice by retrovirally transduced TEL/JAK2 fusion genes

Recent reports have demonstrated fusion of the TEL gene on 12p13 to the JAK2 gene on 9p24 in human leukemias. Three variants have been identified that fuse the TEL pointed (PNT) domain to (i) the JAK2 JH1-kinase domain, (ii) part of and (iii) all of the JH2 pseudokinase domain. We report that all of the human TEL/JAK2 variants, and a human/mouse chimeric hTEL/mJAK2(JH1) fusion gene, transform the interleukin-3 (IL-3)-dependent murine hematopoietic cell line Ba/F3 to IL-3-independent growth. Transformation requires both the TEL PNT domain and JAK2 kinase activity. Furthermore, all TEL/JAK2 variants strongly activated STAT 5 by phosphotyrosine Western blots and by electrophoretic mobility shift assays (EMSA). Mice (n = 40) transplanted with bone marrow infected with the MSCV retrovirus containing either the hTEL/mJAK2(JH1) fusion or its human counterpart developed a fatal mixed myeloproliferative and T-cell lymphoproliferative disorder with a latency of 2-10 weeks. In contrast, mice transplanted with a TEL/JAK2 mutant lacking the TEL PNT domain (n = 10) or a kinase-inactive TEL/JAK2(JH1) mutant (n = 10) did not develop the disease. We conclude that all human TEL/JAK2 fusion variants are oncoproteins in vitro that strongly activate STAT 5, and cause lethal myelo- and lymphoproliferative syndromes in murine bone marrow transplant models of leukemia.     

L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol stimulates ganglioside biosynthesis, neurite outgrowth and synapse formation in cultured cortical neurons, and ameliorates memory deficits in ischemic rats

To address the role of brain gangliosides in synaptic plasticity, the synthetic ceramide analog, 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) was used to manipulate the biosynthesis of gangliosides in cultured cortical neurons. Spontaneous synchronized oscillatory activity of intracellular Ca2+ between the neurons, which represents synapse formation, was suppressed by the depletion of endogenous gangliosides by D-threo-PDMP, an inhibitor of glucosylceramide synthase. The decreased functional synapse formation was normalized by supplementation of GQ1b but not by the other gangliosides, suggesting that de novo synthesis of ganglioside GQ1b is essential for the synaptic activity (Mizutani A. et al., Biochem. Biophys. Res. Commun. 222, 494-498, 1996). On the other hand, the enantiomer of the inhibitor, L-threo-PDMP, could elevate cellular levels of glycosphingolipids including gangliosides. This paper presents our recent findings on the neurotrophic actions of L-threo-PDMP in vitro and in vivo. We found that L-PDMP could up-regulate neurite outgrowth, functional synapse formation and ganglioside biosynthesis through activating GM3, GD3 and GQ1b synthases. Simultaneously, the activity of p42 mitogen-activated protein kinase was also facilitated by L-PDMP. To evaluate the efficacy of this drug on long term memory, rats were trained for 2 weeks using an 8-arm radial maze task, and then forebrain ischemia was induced by 4-vessel occlusion (for 10 min x 2 with a 60 min interval). Repeated treatment of L-threo-PDMP (40 mg/kg, i.p. for 6 days, twice a day) starting 24 h after the ischemia, improved the deficit of the well-learned spatial memory, demonstrating the potential therapeutic use of the ceramide analog for treatment of neurodegenerative disorders.     

Phase II study of combined levamisole with recombinant interleukin-2 in patients with advanced malignant melanoma

Adoptive immunotherapy (AI) with interleukin-2 (IL-2) and lymphokine-activated killer cells (LAK) is an antineoplastic modality in which immune-activated cells are administered to a host with advanced cancer in an attempt to mediate tumor regression. Levamisole (LEV), an immune stimulant, has been suggested to have therapeutic effectiveness in a variety of cancers. After a phase I trial of recombinant IL-2 plus LEV, a phase II trial of this combination was conducted in patients with advanced malignant melanoma. Nineteen patients were entered in the trial. They received IL-2 at 3 x 10(6) U/m2 subcutaneously daily x 5 plus LEV 50 mg/ m2 orally three times daily (p.o. t.i.d.) x 5. Patients were reevaluated at four-week intervals. None of the patients achieved a partial or complete regression (PR, CR). The median time to treatment failure (refusal, progression, or off study due to toxicity) was 56 days. Grade IV toxicities included vomiting (3 patients), lethargy (1 patient), and musculoskellar pain (1 patient). This regimen is not recommended for further testing in patients with advanced malignant melanoma.