Permanent, lowered HLA class I expression using lentivirus vectors with shRNA constructs: Averting cytotoxicity by alloreactive T lymphocytes.

Transplantation of many tissues requires histocompatibility matching of human leukocyte antigens (HLA) to prevent graft rejection, to reduce the level of immunosuppression needed to maintain graft survival, and to minimize the risk of graft-versus-host disease, particularly in the case of bone marrow transplantation. However, recent advances in fields of gene delivery and genetic regulation technologies have opened the possibility of engineering grafts that display reduced levels of HLA expression. Suppression of HLA expression could help to overcome the limitations imposed by extensive HLA polymorphisms that restrict the availability of suitable donors, necessitate the maintenance of large donor registries, and complicate the logistics of procuring and delivering matched tissues and organs to the recipient. Accordingly, we investigated whether knockdown of HLA by RNA interference (RNAi), a ubiquitous regulatory system that can efficiently and selectively inhibit the expression of specific gene products, would enable allogeneic cells to evade immune recognition. For efficient and stable delivery of short hairpin-type RNAi constructs (shRNA), we employed lentivirus-based gene transfer vectors, which provide a delivery system that can achieve integration into genomic DNA, thereby permanently modifying transduced graft cells. Our results show that lentivirus-mediated delivery of shRNA targeting pan-Class I and allele-specific HLA can achieve efficient and dose-dependent reduction in surface expression of HLA in human cells, associated with enhanced resistance to alloreactive T lymphocyte-mediated cytotoxicity, while avoiding MHC-non-restricted killing. We hypothesize that RNAi-induced silencing of HLA expression has the potential to create histocompatibility-enhanced, and, eventually, perhaps "universally" compatible cellular grafts.

Secondary screening system for preclinical testing of human lung cancer therapies.

The National Cancer Institute has instituted a primary screening system for testing new agents against cultured cancer cell lines. The purpose of this study was to determine the feasibility of using a nude rat orthotopic (organ-specific) human lung cancer model system as an in vivo secondary screen for general evaluation of new anticancer agents and therapies active against lung cancer. To make this determination, we tested whether this system allows measurement of uptake and tumoricidal activity of anticancer therapies. Tumor-bearing lungs from 53 Rowett nude rats with orthotopically implanted human large-cell undifferentiated lung carcinoma (NCI-H460) were perfused ex vivo for 1 hour with or without each of two anticancer modalities. Lungs were perfused with blood-free perfusate alone (untreated control), perfusate with 100 micrograms/mL doxorubicin (treated positive control), or perfusate with lymphokine-activated killer cells plus human recombinant interleukin-2 (LAK/rIL-2). Weight gain during perfusion was the criterion used to quantitate lung injury. Treatment efficacy was measured by clonogenic assay after enzymatic disaggregation of the perfused tumors. Doxorubicin levels in the tumor and in the uninvolved lung were measured by high-performance liquid chromatography. Both treatment groups showed only slight increases in lung weight compared with that in the untreated control group, suggesting good lung tolerance of the procedure. Lung and tumor levels of doxorubicin were 320 +/- 21 ng/mg of tissue and 32 +/- 5 ng/mg of tissue (means +/- SE), respectively. Clonogenic assay demonstrated a fivefold to 10-fold reduction in the surviving fraction of tumor cells with doxorubicin but no change with LAK/rIL-2.(ABSTRACT TRUNCATED AT 250 WORDS)

Overexpression of a transmembrane isoform of neural cell adhesion molecule alters the invasiveness of rat CNS-1 glioma.

CNS-1 is a highly invasive neural cell adhesion molecule (NCAM)-positive rat glioma that exhibits similarities in its pattern of infiltration to human gliomas. To investigate whether increasing NCAM expression alters invasive behavior, retroviruses encoding human NCAM 140 and a cytoplasmic truncation of NCAM 140 were used to transduce a population of CNS-1 glioma cells that had a relatively low endogenous level of NCAM. Compared to cells transduced with a control virus, cells overexpressing either intact or truncated human NCAM 140 showed decreased invasion of a reconstituted basal lamina. Changes in growth rate or in key matrix metalloproteinase activities could not account for this result. In a migration assay on type IV collagen, cells exhibited a substrate concentration-dependent increase in the rate of migration; however, overexpression of NCAM 140 or truncated NCAM 140 inhibited motility at higher substrate concentrations. Consistent with these findings was the decreased spread of NCAM 140 overexpressers in vivo following instillation of cells into the right frontal cortex of rat brain. NCAM 140 overexpressers showed considerably more restricted perivascular and periventricular spread than cells transduced with a control virus. However, NCAM-140-overexpressing tumor exhibited a less cohesive pattern of growth near the site of tumor instillation and more individual cell infiltration of brain parenchyma with more pronounced perineuronal satellitosis. The stability of recombinant NCAM expression was confirmed by recovering tumor cells from tumor-bearing animals and measuring NCAM levels by flow cytometry. These observations show that overexpression of NCAM 140 decreases the long-range spread of CNS-1 glioma along basal lamina pathways but enhances local infiltration of neuropil.

The human leukemic T-cell line, TALL-104, is cytotoxic to human malignant brain tumors and traffics through brain tissue: implications for local adoptive immunotherapy.

Preclinical studies with the human MHC nonrestricted cytotoxic T-cell leukemic line, TALL-104, were performed in anticipation of its use in cellular immunotherapy trials for primary malignant brain tumors. In this study, we have: (a) quantitated the in vitro brain tumor cell lysis; (b) measured the cytokine secretion upon coincubation of TALL-104 cells with brain tumor cells; (c) investigated the effect of dexamethasone on brain tumor cell cytolysis by TALL-104 cells; (d) explored the effects of lethal irradiation and cryopreservation on TALL-104 cell viability and lytic efficacy; and (e) estimated the damage TALL-104 cells induce to murine normal and tumor brain cells and their trafficking patterns in both normal and tumor-bearing rat brain upon intracranial infusion. In vitro coincubation of TALL-104 cells with human brain tumor cells, explants, and cell lines resulted in significant lysis of them, but normal brain cells were spared. Lysis of tumor at 4 h was unaffected by dexamethasone or lethal irradiation. Secretion of tumor necrosis factor-alpha, tumor necrosis factor-beta, IFN-gamma, or granulocyte/macrophage-colony stimulating factor upon TALL-104 cell coincubation with brain tumor cells variably occurred without always correlating with lysis. In vivo experiments using irradiated TALL-104 cells, placed at multiple times into normal cannulated rat brain, produced focal sterile abscesses at the instillation site but no widespread allergic encephalitic reaction. Cells morphologically consistent with TALL-104 cells specifically trafficked from the site of instillation through the neuropil, occasionally into the contralateral brain, and egressed at perivascular and leptomeningeal spaces. In vivo experiments with cannulated rats bearing 9L gliosarcoma showed a preferential localization of the TALL-104 cells in tumor compared with normal brain. Taken together, these data support the concept that TALL-104 cells can be used as a novel nontoxic and efficacious paradigm for cellular immunotherapy trials in human primary malignant brain tumors.

Characterization of a glycoprotein fusogen isolated from Sendai virus.

After isolation from Sendai virus, the glycoproteins HN and F retained their ability to induce hemagglutination and both heterologous and homologous cell-cell fusion. Both methods for demonstrating cell fusion indicated that the isolated HN and F glycoproteins compared favorably with whole Sendai virus as a fusogen. Conditions affecting the degree of fusion were examined and optimized. Whole virus and isolated glycoprotein preparations were characterized by electron microscopy and by SDS-polyacrylamide gel electrophoresis. Lipid analysis of the glycoprotein preparations by thin layer chromatography and gas chromatography/mass spectrometry indicated that they were partially lipid-depleted during the isolation protocol and the ratio of cholesterol to phospholipid was higher than in the whole virus. A complete fatty acid analysis was performed on lipid extracts from whole virus and from glycoprotein preparations. Detergent was removed from the glycoproteins by dialysis and by incubation with Amberlite XAD-2 resin. The detergent content of the glycoprotein preparations was monitored by gas chromatography and with [3H]Triton X-100. Both methods showed that virtually all (greater than or equal to 99.8%) of the originally added detergent was removed. Electron microscopy of the negatively-stained HN and F preparations showed primarily spherical particles 120 +/- 20 A in diameter (range 80-250 A). Since no organization reminiscent of envelopes could be demonstrated, we conclude that the fusogenic activity of Sendai virus resides in the glycoproteins per se rather than in bilayer integrated lipid-protein complexes.

Microinjection of arginase into enzyme-deficient cells with the isolated glycoproteins of Sendai virus as fusogen.

A method of introducing enzymes into the cytoplasm of fibroblasts in culture is described. Erythrocytes obtained from normal and arginase-deficient individuals were loaded with arginase in vitro and fused to arginase-deficient mouse and human fibroblasts. Erythrocyte ghost-fibroblast fusion was quantified by a 14C-radioactive assay for arginase in solubilized fibroblasts. Fusion was successfully induced by Sendai virus and also by the isolated glycoproteins of Sendai virus. After fusion the arginase activity associated with the Fibroblasts was 700--1500 U of arginase/mg of cell protein; this enzyme activity was 5- to 10-times higher than that normally found in the fibroblasts. The enrichment in arginase activity indicated that between four and ten ghosts had fused per fibroblast. The use of isolated viral proteins to mediate the transfer of enzymes into cells in vivo might alleviate clinical complications inherent in the use of whole virions. The enzyme replacement technique described in this report for a hyperargininemic model cell system should be applicable to the group of inborn errors of metabolism characterized by deficiency of an enzyme normally localized in the cytoplasmic compartment of cells.

Chemo-immunotherapy and chemo-adoptive immunotherapy of cancer.

The chemo-immunotherapy (CIT) and chemo-adoptive immunotherapy (CAIT) regimens tested in the past decade are summarized. From them we have learned a great deal about the interactions between various chemotherapeutic agents, immune modulating agents and effector cells. The most commonly reported result in multi-modality experiments with CAIT has been a synergistic enhancement in antitumor activity. Clinical trials usually demonstrated improvement in patient quality of life, an extension of survival time, and occasional complete regression of tumor. In many animal models, this enhancement often meant the complete regression and apparent cure of tumor in the animal. One mechanism by which this synergistic enhancement takes place appears to be a suppression of tumor-associated suppressor T cell activity by the chemotherapeutic agents, thereby inducing enhanced cytolytic activity against tumor by the adoptively transferred, activated effector cells. In CAIT the most commonly used drug has been cyclophosphamide. In CIT a wide variety of chemotherapy agents have been used but none of the clinical trials made use of cyclophosphamide. Thus, direct comparisons are not possible. Suggestive of the intricate regulatory processes involved, many CIT studies indicate a synergy only when specific doses of chemotherapy and immunotherapy agents are given, and in a specific sequence. CIT has become less toxic, is being handled on a cost-effective outpatient basis, while maintaining similar objective response rates to earlier inpatient treatments. In the future, CAIT and CIT will probably have an increasing role in the management of patients with specific cancers.

Purified herpes simplex thymidine kinase retroviral particles. II. Influence of clinical parameters and bystander killing mechanisms.

High-titer, purified herpes simplex virus thymidine kinase (HSV-TK) retroviral particles, followed with intraperitoneal ganciclovir (GCV) were tested in Fischer rats bearing 1-week established 9L gliosarcomas. 9L cells were infused intracranially through a cannula on day 0, given intracranial infusions of HSV-TK retroviral particles on days 7-12, and given 5 or 10 daily doses of intraperitoneal GCV starting at day 14. Tumor volumetric studies performed on rat brains at day 26 after tumor infusion revealed significant differences in experimental groups receiving HSV-TK retroviral particles plus 10-day GCV or the 100% transduced 9L-TK group receiving GCV versus control groups treated with either buffer, HSV-TK vector, or either 5- or 10-day regimens of GCV alone. The duration of GCV administration and the volume of tumor burden influenced efficacy. Adjuvant dexamethasone did not significantly affect efficacy. Experiments in which 9L rechallenge of animals treated with 9L-TK/GCV or 9L tumors treated with HSV-TK vector/GCV indicated that a host immune response was involved in rejecting the rechallenge tumor. Outcome was dependent upon the site and size of the rechallenge inoculum. In vitro, bystander effects were significant but were especially marked when cell-to-cell contact was maintained. Cumulatively, the data indicate that both the bystander effect and the host immune response are responsible for the efficacy observed in the suicide gene therapy paradigm using purified HSV-TK retroviral particles and GCV to treat smaller tumor burden in rats with 9L gliosarcoma.

Purified herpes simplex thymidine kinase Retrovector particles. I. In vitro characterization, in situ transduction efficiency, and histopathological analyses of gene therapy-treated brain tumors.

Replication-defective, highly purified retroviral vectors (Retrovector), at titers of 10(8) colony forming units/mL, were prepared that conferred either beta-galactosidase or herpes simplex thymidine kinase (HSV-TK) activity. 9L gliosarcoma cells, transduced efficiently in vitro, were highly sensitive to ganciclovir (GCV). The mean frequency of in situ transduction, measured by flow cytometry of single-cell tumor suspensions isolated from rat brains, was 3.2 +/- 0.6%; similar assessments were made by staining of beta-galactosidase or by immunohistochemistry with anti-HSV-TK. In vitro HSV-TK-transduced and G418-selected 9L-TK gliosarcoma tumors treated with GCV were eradicated in approximately 53% of the animals (10/19) at day 26, however, 89% (17/19) histologically showed < 1% tumor volume. Histologic evaluation at day 26 of animals with established 9L tumors treated with intralesional injection of HSV-TK vector followed by GCV treatment showed that 29% (4/14) had no tumor; 50% (7/14) had < 1% tumor volume. Regression of tumors proceeded over the time since the complete rate was increased at day 60. Neither HSV-TK vector particles nor GCV alone altered the histological profile of 9L tumors, but substantial numbers of CD4+ and CD8+ lymphocytes infiltrated the tumors of animals treated with both. In cured animals, the former tumor bed contained cell debris, immune cells, and fibroblasts and was without damage to adjacent brain. The efficacy of suicide gene therapy for rat gliosarcoma using highly purified virion vectors approaches that of packaging cell lines.

Migration of activated lymphocytes when adoptively transferred into cannulated rat brain.

Migration of fluorescent DNA-labeled or 111Indium-labeled activated lymphocytes was studied in normal rat brain bearing surgically implanted cannulas. The migration of allogeneic cytotoxic T lymphocytes (CTL), derived from the DA rat (DA anti Fischer CTL), and of syngeneic concanavalin A (ConA)-activated lymphocytes (Fischer Con A blasts), was determined in Fischer rats between 2 h and 7 days post instillation into parietal brain. Whole body nuclear imaging indicated that the majority of the radiolabeled lymphocytes, either syngeneic or allogeneic, were present in the brain at 2 and 18 h. Autoradiography of brain slices demonstrated that label was located throughout the brain and in both hemispheres at all time points. By direct tissue radioassay, approximately 60% of the injected dose was present between 2 and 18 h; this decreased to 18% by day 7. By fluorescence microscopy, large numbers of lymphocytes were visible up to 3-4 days. The lymphocytes traveled from the instillation site into both cerebral hemispheres primarily following white matter tracts. Preferential localization of fluorescently labeled lymphocytes was seen in the corpus callosum, internal and external capsules, anterior commissures, lateral olfactory tracts, white matter connections in the caudate and putamen, mammillothalamic and optic tracts. Overall, gray matter contained fewer cells although perivascular spaces within it had high concentrations of cells, indicating these spaces may act as points of egress.

Alveolar soft part sarcoma: an unusually long interval between presentation and brain metastasis.

While alveolar soft part sarcoma is an uncommon soft tissue tumor known for late metastases to lung, bone, and brain, and interval of 33 years between primary presentation and development of brain metastasis has not been described. We document a patient with a removal of an alveolar soft part sarcoma from the pectoralis major muscle at the age of 10 years, a lung metastasis at the age of 31 years, and brain and renal masses at the age of 43 years. The patient received surgical resections each time, but never radiotherapy or chemotherapy. He is currently alive and well. Immunohistochemistry, karyotypic analysis, flow cytometry, and gene expression were analyzed on primary tumor and established cell cultures in the hopes of further elucidating the histogenesis of this unusual neoplasm.

Suicide gene therapy with Herpes simplex virus thymidine kinase and ganciclovir is enhanced with connexins to improve gap junctions and bystander effects.

Connexins are proteins that form gap junctions between cells in various mammalian tissues. Because of their role in intercellular communication, connexins are important in the bystander cell death seen in Herpes simplex virus-thymidine kinase (HSV-TK) gene therapy for brain tumors. A selective review of connexin transduction/transfection studies with particular emphasis to central nervous system tumor cells is presented. In addition, specific references to studies with cell types that demonstrate low gap junction intercellular communication are presented. Data are included with the HT-29 colorectal tumor cell line to support the concept that enhancing gap junction protein expression in otherwise low gap junction communicating HT-29 cells increases bystander cell death and reduces tumor burden beyond what might be expected from HSV-TK and ganciclovir (GCV) treatment alone. Maximum in vitro bystander cell death was always produced when GCV treated co-cultures of TK-transduced and non-TK-transduced HT-29 cell lines were also transduced with connexin-43. When connexin was present in only one group of cells in the co-culture, there was more bystander cell death observed with connexin transduced into the non-TK-transduced cells, rather than the TK-transduced cells. The data presented reinforces conclusions made from earlier findings from cell line mixing experiments in which the non-TK-transduced cell population determined the level of bystander cell death (Burrows et al., 2002).

A 9L gliosarcoma transplantation model for studying adoptive immunotherapy into the brains of conscious rats.

A rat model for brain tumor immunotherapy is described that closely mimics the type of treatment that could be administered to humans. It involves surgical implantation of a permanent cannula in the brain, through which tumor cells and various effector cells and/or cytokines can be injected. The advantage of this system over more conventional animal surgical procedures is that conscious animals can be treated multiple times while avoiding morbidity and mortality associated with reoperative procedures. Using this system to study adoptive immunotherapy for brain tumors, we provide evidence that the 9L gliosarcoma tumor from the Fischer rat strain can be reduced or destroyed in situ following adoptive immunotherapy with specifically activated cytotoxic T lymphocytes.

Establishment and characterization of a human glioblastoma multiforme cell line.

Cell lines provide a useful system for further understanding the biology of glioblastoma multiforme. In this study, a new glioblastoma multiforme cell line, GATAGM-96 (Gulhane Askeri Tip Akademisi-Gliblastoma Multiforme-96), was established from a tumor specimen removed from an 80-year-old male patient who underwent surgery for intracranial tumor. Morphologic examination, immunocytochemical staining, growth kinetics, and karyotypic characteristics of this cell line were studied. The cytoskeleton was positive for neuron-specific enolase, vimentin, and neurofilament, and it was negative for glial fibrillary acidic protein, S-100 protein, p53 protein, epidermal growth factor, and transforming growth factor alpha. Growth kinetic studies demonstrated an approximate population doubling time of 38 to 42 h and a colony forming efficiency of 83.3%. The karyotype of the cells demonstrated it as hyperdiploid, with a large subpopulation of polyploid cells. There were numerous structural and numerical chromosome aberrations; most of them were present as clonal events. The phenotypic and chromosomal features detailed on the GATAGM-96 cell line should make it a useful addition to the cell lines currently available for in vitro and in vivo studies of glioblastoma multiforme.

Strategies using the immune system for therapy of brain tumors.

Phase II immunotherapy and gene therapy studies should be pursued because of encouraging results in many phase I studies. Future testing in this field may consider modifications of some of the above-mentioned combined strategies. For instance, in the immunization and adoptive transfer studies performed by Holladay et al and by Plautz et al, the systemic adoptive transfer could be altered to intratumoral placements of effector cells. This permutation may be more efficacious because local adoptive immunotherapy approaches involve placement of effector cells where they are needed. Additionally, new avenues of gene therapy are being explored that may offer added beneficial effects for immunization, local or systemic adoptive immunotherapy, or combined chemotherapy and adoptive immunotherapy of tumors. With new genetic tools, such as microarray analyses, SEREX, and creation of cDNA libraries from tumor cells, significant progress in the treatment of neoplasms in the immunologically privileged brain should be forthcoming.

Long-term follow-up of patients with recurrent malignant gliomas treated with adjuvant adoptive immunotherapy.

Between August 1986 and October 1987, the Denver Brain Tumor Research Group conducted a clinical trial using autologous human recombinant interleukin-2 (rIL-2)-activated lymphocytes to treat 20 patients with recurrent high-grade gliomas. The trial involved surgical resection and/or decompression followed by intracavitary implantation of lymphokine-activated killer (LAK) cells and autologous stimulated lymphocytes (ASL) along with rIL-2 in a plasma clot. One month later, stimulated lymphocytes and rIL-2 were infused through a Rickham reservoir attached to a catheter directed into the tumor bed. The LAK cells were rIL-2-activated peripheral blood lymphocytes cultured for 4 days; the ASL were lectin- and rIL-2-activated peripheral blood lymphocytes cultured for 10 days. Of the 20 patients treated, 11 were evaluated as a group (mean age, 44 years, range, 15-61 years; mean Karnofsky rating, 69, range, 50-100; mean Decadron dose at entry, 14 mg/d, range, 0-32). The average number of lymphocytes implanted was 7.6 x 10(9) (range, 1.9-27.5 x 10(9], together with 1 to 4 x 10(6) U of rIL-2. To date, 10 of the 11 patients died, all from recurrent tumor growth. The median overall survival time was 63 weeks (range, 36-201; mean, 86). The median survival time after immunotherapy was 18 weeks (range, 11-151; mean, 39). No significant difference in survival after immunotherapy was found between those patients who had received previous chemotherapy and those who had not. The use of steroids or prior chemotherapy did not influence the in vitro generation of ASL or LAK cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Thirty year experience with predictive lumbar sympathectomy. Method for selection of patients.

An office or bedside procedure to predict benefit from or contraindications to lumbar sympathectomy has been presented. Significant benefit and no instance of paradoxic gangrene occurred in an experience of more than 30 years with the test. This personal series of over 70 patients illustrates significant benefits, including healing of gangrenous digits, successful ray (transmetatarsal) amputation, and relief of ischemic rest pain in selected patients, even in the absence of femoral (groin) pulses. A criterion of an increase of 2 degrees or more in skin temperature of the ipsilateral great toe after lumbar sympathetic block at the approximate levels of the second, third, and fourth lumbar vertebrae has proved to be an adequate parameter or indication of subsequent benefit from surgical lumbar sympathectomy. A favorable response is predictable by the method described.

Receptor expression, cytogenetic, and molecular analysis of six continuous human glioma cell lines.

Six human glioma cell lines were established from tissues obtained from five patients diagnosed with Kernohan grade IV glioblastoma multiforme and one from a patient with a grade II astrocytoma. One line was from a recurrent patient who had received prior therapy; the other lines were derived from patients at initial diagnosis and/or before cytoreductive therapies other than surgery were given. Considerable variability in phenotypic, karyotypic, and cell surface marker expression was displayed between the six human glioma cell lines. The karyotypes ranged from apparently normal (grade II astrocytoma) to those with complex rearrangements. Trisomy of chromosome 7 was the most common abnormality. The extensive cytogenetic and molecular characterization of these lines may facilitate their utilization in cellular and molecular biologic studies.

Cytotoxicity against autologous, allogeneic, and xenogeneic tumor targets by human recombinant interleukin-2-activated lymphocytes from healthy dogs and dogs with lung tumors.

Before dogs with lung tumors were treated by adoptive immunotherapy, the ability of canine blood lymphocytes (PBL) from the peripheral circulation to differentiate in vitro in the presence of human recombinant interleukin-2 (rIL-2) and become tumoricidal was investigated. The PBL from healthy dogs (n = 6) and dogs with lung tumors (n = 5) were grown in culture medium alone, in the presence of rIL-2 to generate lymphokine-activated killer (LAK) cells, or with phytohemagglutinin (PHA) and rIL-2 to generate autologous-stimulated lymphocytes (ASL). After 4 days, cytotoxicity by the ASL, LAK, and PBL was determined in a 4-hour 51chromium-release assay. Target cells in the assay were short-term cultured enzyme digests of autologous (self), allogeneic (genetically different) primary tumors, and Raji, the xenogeneic human lymphoma cell line. The PBL cultured without rIL-2 were not cytotoxic against any tumor. However, when a dog's PBL were activated in vitro, they killed the dog's own tumor, ASL more effectively than LAK cells. Pulmonary adenocarcinomas and an osteosarcoma metastasis to lung were among the autologous tumors assayed. Against an allogeneic canine osteosarcoma, ASL generated from healthy dogs were significantly more cytolytic than LAK from healthy dogs, or than ASL generated from tumor-bearing dogs. Cytotoxicity was greater against allogeneic tumor than against Raji. Lectin-dependent cellular cytotoxicity, tested by including PHA in the assay medium with lymphocytes and Raji cells, by ASL and LAK was greater than cytotoxicity of Raji without PHA. Because ASL were more cytolytic than LAK against all targets in vitro, they may be more beneficial than LAK for immunotherapy of canine tumors.

The role of toxic oxygen metabolites in interleukin-2-induced vascular leak syndrome.

We demonstrated that IL-2 causes injury by a blood mediator not by a direct action on endothelial cells and that toxic oxygen metabolites may play a role.