Activation of Raf by ionizing radiation.

The critical pathways through which ionizing radiation induces malignant transformation and cell death are not well defined. Raf-1, a cytoplasmic serine-threonine protein kinase, mediates the transmission of mitogenic signals initiated at the cell membrane to the nucleus, resulting in the activation of transcription factors that regulate cell growth and proliferation. Moreover, Raf-1 overexpression and activation increases the survival response of mammalian cells to the toxic effects of ionizing radiation by an as-yet unknown mechanism (refs 3, 4 and V.

Physical and functional association of Fc mu receptor on human natural killer cells with the zeta- and Fc epsilon RI gamma-chains and with src family protein tyrosine kinases.

We recently reported that Fc mu R on NK cells is a signal transducing protein that stimulates a rapid increase in the level of cytoplasmic free calcium upon binding of IgM. This study was designed to examine signal transduction via the Fc mu R on NK cells and to characterize intracellular second messengers activated by IgM. Immunoprecipitation of IgM-bound Fc mu R by IgM-specific Ab coimmunoprecipitated the zeta- and Fc epsilon RI gamma-chains.

Expression and activity of signaling molecules in T lymphocytes obtained from patients with metastatic melanoma before and after interleukin 2 therapy.

Recent studies have demonstrated altered expression and function of signaling molecules in T and natural killer cells in patients with cancer. The impairment of immune cell functions in advanced cancer may result from defects in signal transduction. We studied purified T cells obtained from peripheral blood or tumor-involved lymph nodes (LNs) of 45 patients with advanced metastatic melanoma for the presence of abnormalities in expression or activity of various signaling molecules.

Nonsecretory apoptotic killing by human NK cells.

We defined a novel constitutive mechanism of cell-mediated cytotoxicity, utilized by nonactivated human peripheral blood NK cells against a variety of tumor cell targets resistant to secretory (i.e., perforin/granzyme-mediated) NK cell killing.

Increased expression of interferon-gamma in a rat model of chronic intestinal allograft rejection.

Chronic rejection remains a major cause of late graft dysfunction. Although much research has focused on acute rejection, little is known about the mechanisms of chronic rejection. Our group has recently reported evidence of significant intestinal smooth muscle hypertrophy and hyperplasia associated with abnormal contractile and electrical activities in a rat model of chronic intestinal rejection.

The role of apoptosis in antibody-dependent cell-mediated cytotoxicity against monolayers of human squamous cell carcinoma of the head and neck targets.

Antibody-dependent cellular cytotoxicity (ADCC) against squamous cell carcinoma of the head and neck (SCCHN) targets in the presence of human/mouse chimeric monoclonal antibodies (cMAbs), SF-25 and 323/A3, is mediated by natural killer (NK) cells. In 4-hr 51Cr-release assays with SSCHN targets in suspension, ADCC was always significantly better (P < 0.01) than that measured in parallel with the same target cells in monolayers.

Antitumor effects of cytolytic T lymphocytes (CTL) and natural killer (NK) cells in head and neck cancer.

Immune effector cells were generated in vitro and compared for activity against human head and neck cancer (HNC). A subset of human IL2-activated natural killer (A-NK) cells was able to eliminate tumor cells in vitro by necrosis, apoptosis or cytokine-mediated effects and to induce regression of established tumors in vivo, in a xenograft nude mouse model of HNC. Cytolytic T lymphocytes (CTL) with specificity for a human oral carcinoma were as effective as A-NK cells in mediating killing of autologous tumor cells or entering spheroids.

A phase I trial of a synthetic mucin peptide vaccine. Induction of specific immune reactivity in patients with adenocarcinoma.

We tested a 105 amino acid synthetic mucin MUC-1 peptide that has 5 repeated immunodominant epitopes to evaluate toxicity and detect mucin-specific immune responses in patients with adenocarcinoma. We also studied the enhancement of these responses by vaccinating patients with the synthetic mucin peptide admixed with BCG. Mucins are glycoproteins present on the luminal surface of ductal epithelial cells and on tumors derived from them.

Divergent effects of Fc gamma RIIIA ligands on the functional activities of human natural killer cells in vitro.

The Fc gamma receptor (R)IIIA (CD 16) plays an important role in regulating the cytotoxic and non-cytotoxic functions of human natural killer (NK) cells. Some anti-CD 16 monoclonal antibodies (mAb) have been shown to stimulate NK activity, while human monomeric (m) IgG induces dose-dependent inhibition of NK activity. To explore further these interactions mediated via Fc gamma RIIIA, purified NK cells were cultured for 2-3 days in the presence of mIgG, 3G8 mAb, interleukin-2 (IL-2) or a combination of mIgG or 3G8 with IL-2.

Effect of influenza A virus infection on natural and adaptive cellular immunity.

Influenza A virus (FLU) is an important pathogen in humans. Although many features of the antiviral immune response have been elucidated in murine and human models of disease, little is known about the role of NK cells, which provide natural, innate immunity. The effects of experimental intranasal FLU (H1N1) inoculation on NK cells and other immune parameters were studied in 18 healthy, adult volunteers during the acute and convalescent phases of infection.

Protective effects of interferon-gamma on squamous-cell carcinoma of head and neck targets in antibody-dependent cellular cytotoxicity mediated by human natural killer cells.

An in vitro model of antibody-dependent cellular cytotoxicity (ADCC) was established, using squamous-cell carcinoma of the head and neck (SCCHN) targets,human/mouse chimeric monoclonal antibodies (cMAbs) SF-25 and 323/A3 and human peripheral blood mononuclear cells (PBMC). We previously showed that natural killer (NK) cells are the main effector population mediating ADCC in the presence of the cMAbs. ADCC was significantly inhibited by the overnight pre-treatment of SCCHN targets with exogenous interferon-gamma (IFN-gamma).

Generation of activated natural killer (A-NK) cells in patients with chronic myelogenous leukaemia and their role in the in vitro disappearance of BCR/abl-positive targets.

Activated natural killer (A-NK) cells, a subset of CD56(dim)CD3- lymphocytes, are obtained from PBMC of normal donors by adherence to plastic and culture in the presence of IL2. In this study we tested the feasibility of generating A-NK cells in patients with Ph+ chronic myeloid leukaemia (CML). Cultures obtained from patients with early chronic phase (ECP; n=7) contained a mean (+/-SD) of83 +/- 7% of CD3- cells, and those from patients with advanced chronic phase (ACP; n=7) contained 27+/-33% CD56+CD3- cells.

Antitumor Functions of Natural Killer Cells and Control of Metastases.

Natural killer (NK) cells have been recognized as effector cells responsible for the elimination of blood-borne metastases. Newer evidence suggests that NK cells play an important role in the control of solid tissue metastases. NK cells have the ability to kill a broad variety of fresh or cultured "NK-resistant" tumor cell targets by mechanisms that are not dependent on perforin or granzyme secretion.

Measurements of Natural Killer (NK) Activity and NK-Cell Quantification.

Natural killer (NK) cells are a subset of lymphocytes with a distinct morphology and the ability to kill certain target cells via one or more cytolytic mechanisms. Measurements of in vitro NK activity from heparinized whole blood, or lymphocytes isolated from human peripheral blood or tissue, may be performed by a variety of techniques. Procedures using standard 51Cr-release assays as well as nonradioactive labeling materials are described in detail in this article, and advantages and disadvantages of these methods are discussed.

Elimination of established liver metastases by human interleukin 2-activated natural killer cells after locoregional or systemic adoptive transfer.

An in vivo model of liver metastasis induced by human gastric carcinoma was established in nude mice and used for locoregional or systemic immunotherapy with a subset of human A-natural killer (NK) cells defined previously. A single intrasplenic (i.s.

Successful culture and selection of cytokine gene-modified human dermal fibroblasts for the biologic therapy of patients with cancer.

Human autologous dermal fibroblasts have been cultured, transduced with the interleukin-4 (IL-4) gene and used as a vaccine together with irradiated autologous tumor cells in patients with cancer participating in a phase I/II clinical trial at the University of Pittsburgh Cancer Institute. In support of this clinical trial, methods have been devised to facilitate isolation of fibroblasts from freshly harvested skin specimens, to enhance their outgrowth in large-scale cultures, and to assay cytokine (IL-4) production following transduction with the cytokine gene +/- irradiation. Fibroblasts were isolated from skin specimens by enzymatic digestion, grown in primary cultures, and transduced with a retroviral vector containing the gene for human IL-4 and the NeoR gene as a selectable marker.

Divergent phosphotyrosine signaling via Fc gamma RIIIA on human NK cells.

Previous studies have indicated that interaction of Fc gamma RIIIA on natural killer (NK) cells with various immunoglobulin ligands or monoclonal antibodies (mAbs) can have either stimulatory or inhibitory effects on cytotoxic activity, but the basis for such divergent functional effects has been unclear. We report here that stimulation of NK cells via Fc gamma RIIIA by monoclonal anti-human CD16 (3G8), monomeric IgG (mIgG), or dimeric IgG (dIgG), used either alone or cross-linked by secondary Ab (goat anti-mouse IgG or goat anti-human IgG), resulted in different phosphotyrosine protein patterns. These results suggest that distinct substrates are involved in signaling pathways activated via various agonists of the same triggering surface molecule.

Alterations in expression and function of signal-transducing proteins in tumor-associated T and natural killer cells in patients with ovarian carcinoma.

Tumor-associated lymphocytes (TALs) freshly isolated from patients with cancer usually manifest reduced proliferative and cytolytic functions. To determine whether alterations in signal transduction contribute to functional impairments seen in TALs, we purified populations of T and natural killer (NK) cells by negative selection from ascites of seven patients with ovarian carcinoma. The average purity was 84 +/- 5% for CD3(+) TALs and 77 +/- 10% for CD3(-)CD56(+)CD16(+) TALs.

Growth stimulation of human head and neck squamous cell carcinoma cell lines by interleukin 4.

Interleukin 4 (IL-4) has been reported recently to inhibit growth of acute lymphoblastic lymphoma, non-Hodgkin's lymphoma, melanoma, sarcoma, breast, gastric, colon, and renal tumor cell lines, and treatment of murine tumors with IL-4 gene-transduced cells has been therapeutically successful. Therefore, we sought to determine the effect of IL-4 on the growth of human squamous cell carcinoma of the head and neck (SCCHN) cell lines. Growth of SCCHN cell lines incubated in the presence of various concentrations of IL-4 was measured in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric assays and by cell counts.

Constitutive production of IL-2 by human carcinoma cells, expression of IL-2 receptor, and tumor cell growth.

Human carcinomas spontaneously express abundant IL-2R beta but little IL-2R alpha on the cell surface, contain mRNA for IL-2R beta- and IL-2R alpha-chains, and may be inhibited in growth by exogenous IL-2. To study the relationship between IL-2R expression and growth inhibition by IL-2, carcinoma cells were transduced with IL-2R alpha and IL-2R gamma cDNAs or IL-2R beta antisense cDNA. Transfectants with the IL-2R alpha gene expressed high levels of the alpha- and beta-receptor chains and showed increased binding of [125I]IL-2.

The role of natural killer cells in immune surveillance of cancer.

In the past year, a subset of natural killer cells designated 'A-NK cells' has been characterized. These immune cells appear to be able to enter solid tissues, migrate to sites of metastasis and eliminate malignant tissue cells, but spare normal tissue cells. They appear to be ideal surveillance cells, readily capable of upregulating antitumor functions in response to local activation signals.

Cytokines, immunoglobulins, and bacterial pathogens in middle ear effusions.

Objective: To elucidate the role of cytokines, immunoglobulins, and bacterial pathogens in the middle ear effusions (MEEs) of children with otitis media (OM).

Design: Paired MEEs and serum samples collected from consecutive patients were assayed for immunoglobulins. Middle ear effusions were cultured for bacterial pathogens and assayed for interleukin-1 beta, interleukin-6, tumor necrosis factor alpha, and interferon gamma.

The role of natural killer cells in the treatment of chronic myeloid leukemia.

The success of chemotherapy in patients with leukemia whose marrow appears to be replaced by leukemia cells must be due to the persistence of normal stem cells. In this normal population are the progenitors of the cells of the immune system. Natural killer (NK) cells originate in the bone marrow.