High-efficiency transduction of freshly isolated human tumor cells using adenoviral interleukin-2 vectors.

Tumor cells genetically modified to express immunostimulatory molecules can produce high levels of antitumor immunity in rodent models. Although a number of clinical trials are currently in progress to assess the value of the approach in human disease, almost all require ex vivo transduction of cultured tumor cells with retroviral vectors. This process is not feasible for many human malignancies, hampering clinical evaluation of the approach. We have used an E1a,1b/E3 deletion mutant of adenovirus containing either the lacZ or the human interleukin-2 (IL-2) gene to transduce human neuroblastoma cells. This vector transduces fresh neuroblastoma cells and neuroblastoma cell lines with an efficiency of 80-90%, compared to an efficiency of 0-14% obtained with retroviral vectors. Cells transduced with the IL-2 adenovector produce up to 12,000 pg of IL-2/10(6) cells/24 hr. IL-2 adenovector-transduced neuroblasts are immunostimulatory; when they are cultured with patient lymphocytes, they increase the proportion of DR+ T cells and generate major histocompatibility complex (MHC) unrestricted cytotoxic effector cells active against parental (nontransduced) tumor cells. We conclude that IL-2 adenovector can be used to transduce freshly isolated human tumor cells efficiently, which will then produce immunomodulatory quantities of the cytokine. The use of adenoviral rather than retroviral vectors facilitates preparation of human tumor "vaccines" and these vectors are now being used in our clinical study of neuroblastoma patients.

Induction of therapeutic antitumor antiangiogenesis by intratumoral injection of genetically engineered endostatin-producing Semliki Forest virus.

Antiangiogenic therapy using Semliki Forest virus (SFV) carrying Endostatin gene for malignant brain tumor was investigated to improve the therapeutic efficacy. The efficiency of SFV-mediated gene delivery was first evaluated for B 16 cells and compared with the efficiency in cells of endothelial origin (HMVECs). HMVECs are more susceptible to SFV infection than B 16 cells. For the in vivo treatment model, phosphate-buffered saline, SFV-LacZ, retrovirus vector GCsap-Endostatin, and SFV-Endostatin were injected to mice bearing B 16 brain tumors. A very significant inhibition of tumor growth was observed in the group that had been treated with SFV-Endostatin. A marked reduction of intratumoral vascularization was seen in the tumor sections from the SFV-Endostatin group compared with tumor sections from the SFV-LacZ or GCsap-Endostatin groups. Moreover, at day 7 after intravenous administration of SFV-Endostatin, the serum level of endostatin was augmented more than 3-fold compared to that after intravenous administration of GCsap-Endostatin. The results indicated that treatment with SFV-Endostatin inhibited the angiogenesis with established tumors. Gene therapy with Endostatin delivered via SFV may be a candidate for the development of new therapy for brain tumors.

Enhancement of antitumor immune response in glioma models in mice by genetically modified dendritic cells pulsed with Semliki forest virus-mediated complementary DNA.

OBJECT: The aim of this study was to further investigate dendritic cell (DC)-based immunotherapy for malignant glioma to improve its therapeutic efficacy. METHODS: Dendritic cells were isolated from the bone marrow and pulsed with phosphate-buffered saline, tumor RNA, tumor lysate, Semliki Forest virus (SFV)-LacZ, SFV-mediated B16 complementary (c)DNA, or SFV-mediated 203 glioma cDNA, respectively, to treat mice bearing tumors of the 203 glioma cell line. The results indicated that pre-immunization with DCs pulsed with the same type of cDNA as in the tumor by a self-replicating RNA vector (that is, SFV) protected mice from tumor challenge, and that therapeutic immunization prolonged the survival of mice with established tumors. The SFV induced apoptosis in DCs and their death facilitated the uptake of apoptotic cells by other DCs, thus providing a potential mechanism for enhanced immunogenicity. CONCLUSIONS: Therapy with DCs that have been pulsed with SFV-mediated tumor cDNA may be an excellent procedure for the development of new cancer vaccines.

Ionizing radiation induces up-regulation of functional beta1-integrin in human lung tumour cell lines in vitro.

PURPOSE: Cell-matrix interactions are in part mediated through the beta1-integrin pathway regulating cell survival, proliferation, adhesion and migration. This study was performed to elucidate alterations of expression of the beta1-integrin and its co-localized protein kinase, integrin-linked kinase (ILK), after exposure to ionizing radiation in two lung carcinoma cell lines in the presence or absence of different beta1-integrin-dependent matrix proteins. MATERIALS AND METHODS: Exponentially growing A549 and SKMES1 cells grown on fibronectin, laminin, BSA or plastic were exposed to 2 Gy or 6 Gy. Besides colony formation assays (0.5-8 Gy) and immediate plating experiments, flow cytometry (for beta1-integrin) and immunoblotting (for beta1-integrin and ILK) were carried out to analyze the protein expression. The localization of both proteins plus filamentous (f-) actin was further examined by immunofluorescence staining and laser confocal scanning microscopy. Functionality of the beta1 receptor subunit after irradiation was investigated in adhesion assays. RESULTS: A549 and SKMES1 cells grown on fibronectin or laminin demonstrated a significant increase in cell survival after irradiation compared to cells grown on BSA or plastic. Immediate plating of cells after irradiation on fibronectin did not show an improved survival. Flow cytometric and Western blot data showed a dose- and matrix-dependent induction of beta1-integrin and ILK expression after irradiation within 48 h. Adhesion to fibronectin or laminin compared to BSA or plastic was increased by 10-fold after irradiation, demonstrating these specific cell surface receptors to be functional. The staining of beta1-integrin and ILK in A549 cells confirmed the radiation-induced up-regulation of both proteins. Additionally, beta1-integrin and ILK co-localized with accumulated actin fibers at the cytoplasmic face of the cell membrane at confined areas. CONCLUSIONS: Ionizing radiation strongly induced the expression of functional beta1-integrin and ILK in the two lung cancer cell lines, A549 and SKMES1, dependent on different matrices used. Additionally, the subcellular localization of both proteins was altered by irradiation, and the individual cellular radiosensitivity was reduced in the presence of an extracellular matrix. On the one hand, this may result in aggravated therapeutic approaches and on the other hand, cells could adhere more strongly in their environment by the increase in functional surface receptor density preventing metastasis. Concerning intravascular located tumour cells, beta1-integrin up-regulation might enable these cells to adhere to the endothelium, which represents a prerequisite for metastatic disease. Identification of such mechanisms will provide considerable insights into the understanding of tumorigenicity and metastatic phenotypes, possibly leading to new, optimized radiochemotherapeutic regimens.

CRABP I expression and the mediation of the sensitivity of human tumour cells to retinoic acid and irradiation.

PURPOSE: To examine the role cytoplasmic retinoic acid binding protein type 1 (CRABP I) and retinoic acid receptor beta 2 (RAR-beta 2) in mediating radiosensitization of human tumour cells in vitro by retinoic acid. MATERIALS AND METHODS: Human squamous cell carcinoma cell lines of different types were treated with retinoic acid followed by irradiation. Radiation response under drug treatment was detected by colony-formation assay. mRNA and protein expression levels of CRABP I, RAR-beta and cyclin D1 were investigated under different treatment conditions by room temperature polymerase chain reaction and Western blotting. The retinoic acid-sensitive cell line HTB35 was transfected for inducible CRABP I overexpression to test the role of this protein in modulating the sensitivity to retinoic acid and radiation as well as in regulating RAR-beta 2 and cyclin D1 expression. RESULTS: The basal CRABP I level clearly correlated with the clonogenic survival of tumour cells and normal fibroblasts after treatment with retinoic acid and ionizing irradiation (IR). Cells expressing high basal CRABP I were more resistant to combined retinoic acid radiation treatment than cells with low basal expression. Overexpression of CRABP I in retinoic acid-sensitive HTB35 cells induced a retinoic acid-insensitive phenotype resistant to combined treatment with retinoic acid and radiation. This effect was independent of RAR-beta 2 expression. CRABP I overexpression resulted in stimulated cyclin D1 expression indicating the dependency of this cell cycle control protein on retinoic acid metabolism. CONCLUSION: CRABP I plays an important role not only in mediating the retinoid effects, but also in modulating the radiation sensitivity of tumour cells after combined retinoic acid radiation treatment.

Fibronectin and laminin increase resistance to ionizing radiation and the cytotoxic drug Ukrain in human tumour and normal cells in vitro.

PURPOSE: Cell-extracellular matrix (ECM) interactions are thought to mediate drug and radiation resistance. Dependence of cell survival, beta1-integrin expression and cell cycling on the ECM proteins and beta1-integrin ligands fibronectin (FN) and laminin (LA) were examined in malignant and normal cells exposed to the cytotoxic drug Ukrain plus/minus irradiation. MATERIALS AND METHODS: Human A549 lung cancer and MDAMB231 (MDA231) breast cancer cells and normal fibroblasts (HSF1) grown on FN, LA, bovine serum albumin (BSA) or polystyrene were treated with Ukrain (1 microg ml(-1), 24 h) plus/minus irradiation (2-8 Gy) and the effects studied using colony formation assays, flow cytometry (beta1-integrin, DNA analysis) and adhesion assays. RESULTS: FN and LA reduced the cytotoxic effect of single Ukrain treatment compared with polystyrene and BSA. FN and LA also abolished Ukrain-dependent radiosensitization in A549 cells and decreased the radiosensitivity of MDA231 and HSF1 cells. Single Ukrain exposure on polystyrene significantly reduced beta1-integrin expression and promoted G2-phase accumulation of A549 cells. In contrast, Ukrain-treated MDA231 and HSF1 cells showed elevated beta1-integrin expression and no Ukrain-specific cell cycle effect. Under Ukrain-radiation exposure, irradiation, FN or LA abolished Ukrain-mediated reduction of beta1-integrin expression and G2-phase accumulation in A549 cells, whereas in MDA231 cells and fibroblasts beta1-integrin expression and cell cycle distribution were stabilized. Cell adhesion to FN or LA was significantly impaired (A549) or improved (MDA231, HSF1) upon Ukrain treatment. CONCLUSIONS: The data corroborate the findings of other groups that cell adhesion-mediated resistance to either single or combined drug and radiation exposure is tightly correlated to specific ECM proteins. By demonstrating a strong modulatory impact of FN and LA on the radiosensitivity-modifying activity of the drug Ukrain, the set findings are also highly important for the assessment of drug and radiation effects within in vitro cytotoxicity studies. The data give the first mechanistic insights into specific FN- and LA-modulated cellular resistance mechanisms as well as into the important role for beta1-integrins using the unique cytotoxic and radiosensitivity-modifying drug Ukrain.

Induction of a therapeutic antitumor immunological response by intratumoral injection of genetically engineered Semliki Forest virus to produce interleukin-12.

OBJECT: The authors investigated immunogene therapy for malignant glioma to determine whether its therapeutic efficacy could be improved. METHODS: Four groups of 203-glioma-bearing mice were treated with injections of phosphate-buffered saline, Semliki Forest virus (SFV)-LacZ, retrovirus vector DFG-interleukin (IL)-12, and SFV-IL12, respectively. The results indicated that therapeutic immunization with SFV-IL12 prolonged the survival of mice with established tumors. Semliki Forest virus induces apoptotic death to glioma cells, which facilitates the uptake of apoptotic cells by dendritic cells, providing a potential mechanism for enhanced immunogenicity. CONCLUSIONS: Immunogene therapy with IL-12 via SFV may be an excellent candidate for the development of new cancer vaccines.

Measurement of polyclonal immunoglobulin synthesis using the reverse plaque technique.

This unit describes the reverse hemolytic plaque assay, an effective method for measuring the number of immunoglobulin (Ig)-secreting cells present in a cell population at any particular time. Cell populations that can be assayed using the technique include peripheral blood mononuclear cells or cells from tissues such as the tonsils. The basic protocol is divided into three stages. First, protein A-sensitized sheep red blood cells (SRBC), guinea pig complement, and anti-Ig antibody are prepared. Test samples are then combined and incubated with the SRBC, complement, and antibody in appropriate chambers. Finally, the resulting plaques are scored. A support protocol describes the preparation of plaquing chambers.

Glucocorticosteroids stimulate polyclonal immunoglobulin production by cord blood mononuclear cells.

Glucocorticosteroids (GCS) are in vitro polyclonal activators of immunoglobulin (Ig) production for human mononuclear cells (MC) from virtually all adult donors. However, GCS treatment of cord blood MC resulted in Ig production in only 12/41 samples. This GCS effect is T cell and monocyte dependent and is mediated in part by a soluble T cell replacing factor. The inconsistent response of cord MC could be due to either cellular or soluble factor differences from adults. In 11/12 samples tested, irradiated cord T cells did help adult B cells, but less than did irradiated allogeneic adult T cells. T cell suppression in cord samples is unlikely inasmuch as higher cord T cell numbers and proportions increased the number of responding cord samples. Cord monocytes function adequately, because monocytes supported GCS responses when cord non-T cells had sufficient T cell help. The T cell replacing factor was found in supernatants of unstimulated cord as well as in adult MC cultures, but was less than 50% as potent. Cord B cells did not develop GCS-induced Ig production with such supernatants, suggesting that cord B cells may require a higher concentration or more prolonged exposure to T cell help. With a 2:1 ratio of irradiated adult T cells to cord non-T cells, only 25% of cord samples responded to GCS (compared to greater than 95% of adult samples), but with a ratio of 4:1, 75% responded. IgM was the predominant isotype secreted by GCS-stimulated cord cells, but 6/14 samples also produced IgG and 8/14 produced IgA. Thus, the functional immaturity of both cord T and B cells exists for GCS-induced Ig production, but with appropriate conditions GCS can activate most samples of cord B cells to synthesize Ig.

Activation of suppressor T cells during Epstein-Barr-virus-induced infectious mononucleosis.

Infectious mononucleosis is caused by the Epstein-Barr virus (EBV), an unusual human pathogen because it preferentially infects B lymphocytes and consequently activates them to produce immunoglobulins. When cultures of lymphocytes from patients with infectious mononucleosis were stimulated with polyclonal activators, unseparated cells failed to produce immunoglobulins, whereas purified B cells responded normally. Cocultures demonstrated profound suppressor T-cell activity in blood from patients with infectious mononucleosis. Early in this disease, circulating immunoglobulin-secreting cells were elevated, but during the second week their number was strikingly depressed. These data indicate that during infectious mononucleosis, EBV causes polyclonal activation of B cells, reflected by hypergammaglobulinemia and increased circulating immunoglobulin-secreting cells. Next, suppressor T cells become activated and inhibit further B-cell activation. Thus, activation of suppressor T cells in infectious mononucleosis provides a unique additional mechanism of host defense because these T cells inhibit the activation and proliferation of an important target of the causative virus.

Hodgkin's disease occurring in a patient with the Wiskott-Aldrich syndrome.

Hodgkin's disease, nodular sclerosing, developed in a 16-year-old man with the Wiskott-Aldrich syndrome. Two brothers and two nephews had documented Wiskott-Aldrich syndrome and had died of infectious complications in childhood. While the patient reported here had lifelong thrombocytopenia and recurrent upper respiratory infections, he had no severe infection prior to the development of Hodgkin's disease. Skin test sensitization with dinitrochlorobenzene was unsuccessful. No antibodies were found after immunization with pneumococcal polysaccharides. Platelet aggregation studies were abnormal in the patient, his mother, and one of his nephews. A complete response of short duration occurred after treatment with nitrogen mustard, vincristine, procarbazine, and prednisone. On recurrence, he proved unresponsive to further chemotherapy or radiation therapy. Infection with four different fungi was found at autopsy. This patient is the third recorded case of Hodgkin's disease associated with the Wiskott-Aldrich syndrome.

Deficient DR antigen expression on human cord blood monocytes: reversal with lymphokines.

Expression of DR antigen on cord blood (neonatal) human monocytes using complement-mediated cytotoxicity with anti-DR alloantisera and fluorescent-activated cell sorting (FACS) utilizing a battery of anti-DR mouse monoclonal antibodies was assessed. Forty preparations of neonatal cord blood monocytes were purified by adherence and elution from plastic petri dishes: lymphocyte contamination was less than 5% as indicated by esterase and peroxidase stains and cell sizing. By cytotoxicity tests 22 +/- 5.5% (SD) of neonatal monocytes expressed DR compared to its expression on 78.6 +/- 3.1% of adult monocytes. By FACS analysis, the frequency of DR expression on neonatal monocytes was 19-33% compared to 71-82% for adult monocytes. Incubation of neonatal monocytes with concanavalin A (Con A) or phytohemagglutinin (PHA)-stimulated human peripheral blood mononuclear cell culture supernatants (lymphokine) or recombinant interferon-alpha (IFN-alpha) increased the expression of DR antigens in a dose- and time-dependent manner. A Con A-supplemented culture supernatant of unstimulated peripheral blood mononuclear cells had no effect on DR expression. Neonatal monocytes that were pretreated with anti-DR and complement in order to remove DR-positive cells were induced to express DR antigen after 2 days in culture with lymphokine. Thus DR-negative neonatal monocytes can be induced to express DR antigen. These results suggest a correctable maturational deficiency of neonatal monocytes. The inducibility of DR antigen expression by lymphokines and recombinant IFN-alpha suggests that they play an important role in the regulation of immune responses.

Naturally occurring antibodies to phosphocholine as a potential index of antibody responsiveness to polysaccharides.

Naturally occurring antibodies reactive with the phosphocholine (PC) determinant of pneumococcal teichoic acids may be useful for evaluating the potential of patients to make antibodies to polysaccharides. Antibodies to PC are present in most adults under the age of 60 years, are absent in very young children, and are present at low levels in Wiscott-Aldrich patients and in IgG2-deficient adults. These last three groups respond very poorly to polysaccharide antigens. Antibodies to PC are also found at low levels in the elderly, a group that has previously been shown to have low levels of antibody to blood groups A and B carbohydrates. The levels of antibody to PC over time were constant in most individuals and, in adults, seemed to show slightly less variation than did titers of antibody to blood group B. Our findings suggest that titers of antibody to PC may be superior to titers of antibody to blood group A or B for monitoring responsiveness to carbohydrate antigens.

Natural killer cell function and interferon generation in patients with primary immunodeficiencies.

Patients with primary immunodeficiency disorders were evaluated for three aspects of natural defense: natural killer (NK) cells which lyse HSV-infected fibroblasts [NK(HSV-FS)], NK cells which lyse K562 tumor targets [NK(K562)], and interferon-alpha generation. In addition, capacity to make interferon upon challenge with other commonly used inducers was also evaluated. Most patients with severe combined immunodeficiency disease (SCID) and deficits of both T- and B-cell function demonstrated normal NK function with one or both targets. Six of eight SCID patients generated interferon-alpha at or below the lower limit of normal while only two made clearly normal levels. Six of 10 patients with Wiskott-Aldrich syndrome (WAS) had normal NK(K562) and five of 10 generated normal levels of interferon-alpha but all had severely deficient NK(HSV-FS). Patients with Bruton's agammaglobulinemia demonstrated normal NK and interferon generation, as did patients with common variable immunodeficiency, even when subdivided into patients with T-cell proliferative deficiencies and those with only hypogammaglobulinemia. Natural defense parameters may help categorize patients with SCID and WAS and help define these heterogeneous diseases.

Localization of the gene for the Wiskott-Aldrich syndrome between two flanking markers, TIMP and DXS255, on Xp11.22-Xp11.3.

The Wiskott-Aldrich syndrome (WAS) is an X-linked recessive genetic disease in which the basic molecular defect is unknown. We previously located the WAS gene between two DNA markers, DXS7 (Xp11.3) and DXS14 (Xp11), and mapped it to the proximal short arm of the human X chromosome (Kwan et al., 1988, Genomics 3:39-43). In this study, further mapping was performed on 17 WAS families with two additional RFLP markers, TIMP and DXS255. Our data suggest that DXS255 is closer to the WAS locus than any other markers that have been previously described, with a multipoint maximum lod score of Z = 8.59 at 1.2 cM distal to DXS255 and thus further refine the position of the WAS gene on the short arm of the X chromosome. Possible locations for the WAS gene are entirely confined between TIMP (Xp11.3) and DXS255 (Xp11.22). Use of these markers thus represents a major improvement in genetic prediction in WAS families.

Genetic mapping of the Wiskott-Aldrich syndrome with two highly-linked polymorphic DNA markers.

The Wiskott-Aldrich syndrome (WAS) is an X-linked recessive genetic disease in which the molecular defect is unknown. In 15 families with WAS, seven restriction fragment length polymorphic loci from the X chromosome were used to map the disease locus. Of the eight intervals studied, the likelihood of the WAS gene lying between DXS7 (Xp11.3) and DXS14 (Xp11) was at least 128 times higher than that for any other interval. The most likely gene order is DXS84-OTC-DXS7-WAS-DXS14-DXS1-PGK-DXYS1. Close genetic linkage to DXS7 and DXS14 permits accurate prenatal diagnosis and carrier detection with greater than 98% confidence in fully informative WAS families.

The development of gene therapy for the treatment of cancer.

OBJECTIVE: The authors sought to develop new treatments for patients with cancer based on the genetic modification of immune lymphocytes and tumor cells designed to increase the host immune reaction against growing cancers. METHODS: Retroviral-mediated gene transduction was used to introduce genes into tumor-infiltrating lymphocytes (TIL), and these genetically altered TIL were administered to patients with cancer. Genes coding for cytokines were introduced into tumor cells, and these cells were used to immunize patients against their autologous cancers. RESULTS: In initial studies, the gene for neomycin phosphotransferase was introduced into the TIL of ten patients with advanced cancer to study the survival and distribution of TIL in humans. These studies showed that retroviral gene transduction is a safe and practical method for adding genes to human cells and led to clinical trials in which the gene for tumor necrosis factor (TNF) was inserted into TIL in an effort to increase their therapeutic effectiveness. Phase I trials are currently underway using TIL that secrete up to 100 times the normal level of TNF. More recently, animal experiments have revealed that transduction of tumor cells with cytokine genes can enhance tumor immunogenicity and, thus, increase the recognition of the tumor as foreign by the host. Clinical trials based on these observations have begun in which patients are immunized against their own autologous tumors that were transduced with the genes for TNF or interleukin-2. CONCLUSIONS: Attempts at gene therapy for cancer are underway and have opened new possibilities for the development of cancer treatments.