Ex vivo generation of genetically modified dendritic cells for immunotherapy: implications of lymphocyte contamination.

Genetically modified dendritic cell (DC) vaccines expressing tumor-associated antigens are currently used for cancer immunotherapy. Peripheral blood (PB) monocyte precursors are a relatively convenient source of DCs for use in clinical studies, but are often contaminated by lymphocytes. The current study was conducted to examine the impact of T-lymphocyte contamination on genetically modified DC product. PB monocyte-derived DCs were efficiently transduced (75-95%) with an HIV-1-based self-inactivating lentiviral vector encoding a model antigen, the enhanced green fluorescent protein (eGFP). The lymphocyte-free DC culture transduced with Lenti-eGFP showed stable expression of eGFP without measurable decline in viability. In contrast, the eGFP-positive DCs disappeared rapidly in transduced DC cultures containing lymphocyte contaminants, concurrent with detectable activation and expansion of T-lymphocytes. Upon antigen recall, these T cells elicited major histocompatability complex-restricted antigen-specific cytotoxicity against eGFP-positive autologous DCs and mitogen-stimulated T lymphoblasts, mainly through the perforin-mediated pathway. In summary, this study demonstrate that the relative purity of DC cultures could determine the persistence of gene-modified DC, which may affect the induction of effective immune responses by DC vaccination strategies.

Immune modulation in cancer patients after adoptive transfer of anti-CD3/anti-CD28-costimulated T cells-phase I clinical trial.

Anti-CD3/anti-CD28 monoclonal antibody-coactivated T cells (COACTs) proliferate, secrete tumoricidal cytokines, and mediate non-major histocompatibility complex (MHC)-restricted cytotoxicity. This phase I study was done to determine the safety, maximum tolerated dose, technical limits of expansion, and modulation of immune functions in cancer patients given COACTs. Coactivated T cells were produced by stimulating peripheral blood mononuclear cells (PBMCs) with OKT3 anti-CD3 and 9.3 (anti-CD28)-coated beads in the presence of 100 IU interleukin (IL)-2 per milliliter for 14 days. The beads were removed after 4 days of culture. Ten courses of COACTs were given to eight patients with renal cell (1), ovarian (2), breast (1), and colorectal (4) carcinomas; two patients received two courses of COACTs. Patients were given up to 10 x 10 9 COACTs twice a week for 3 weeks without dose-limiting toxicities. Patients at the first and second dose levels received a mean total of 17.6 and 42.4 x 10 9 COACTs, respectively. After 14 days of culture, the COACTs contained a mean of 57.5% CD4+ cells and 42.5% CD8+ cells, exhibited non-MHC-restricted cytotoxicity, and produced significant amounts of interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, and granulocyte macrophage colony-stimulating factor (GM-CSF). Infusions were safe and induced measurable serum levels of IFNgamma, TNFalpha, and IL-4 in two patients. Peripheral blood mononuclear cells from patients who received COACTs secreted higher amounts of IFNgamma and GM-CSF on in vitro anti-CD3/anti-CD28 restimulation than PBMCs obtained before immunotherapy. The detection of cytokines in patient sera and enhanced in vitro production of cytokines by anti-CD3/anti-CD28-stimulated patient PBMCs after COACT infusions suggest that COACTs were modulating immune responses in cancer patients.

Predicting slips and falls considering required and available friction.

This study investigated the relationship among measurements of friction, the biomechanics of gait, and actual slip and fall events. The goal was to develop a method for estimating the probability of slips and falls based on measurements of available friction and required friction. Five subjects wearing safety harnesses walked down a ramp at various angles with either a tile or carpeted surface under dry, wet or soapy conditions. Ramp angles of 0 degree, 10 degrees and 20 degrees were used to vary the shear and normal foot force requirements. The dynamic coefficient of friction (DCOF) of shoe, floor surface and contaminant interfaces was measured. Required friction was assessed by examining the foot forces during walking trials when no slips occurred. Slips with recoveries and slips resulting in falls were recorded and categorized using a force plate and high-speed video camera. These data were then incorporated into a logistic regression to model the probability of a slip or fall event occurring based on the difference between the COF required by the foot forces generated and the measured DCOF. The results showed that the number of slip and fall events increased as the difference between the required COF and the measured DCOF increased. The logistic regression model fit the data well, resulting in an estimate of the probability of a slip or fall event based on the difference between the measured and required friction. This type of model could be used in the future to evaluate slip resistance measurement devices under various environments and assist in the design of safer work environments.

Autoimmune Addison's disease after treatment with interleukin-2 and tumor-infiltrating lymphocytes.

Autoimmune thyroiditis with hypothyroidism is a well-known complication of immunotherapy with interleukin-2 (IL-2) with or without lymphokine-activated killer (LAK) cells. To date, however, no cases of IL-2/LAK-induced autoimmune adrenalitis with adrenal insufficiency have been reported. We describe a patient who developed primary adrenal insufficiency following IL-2/tumor-infiltrating lymphocytes (TIL) immunotherapy for renal cell carcinoma. A 64-year-old male with renal cell carcinoma metastatic to bone, skin, lung, and the central nervous system presented for IL-2/TIL treatment. Nine months earlier, he had undergone a right nephrectomy and adrenalectomy. He had already received two courses of IL-2 and one course of IL-4 following surgery. Dynamic studies of adrenal function performed prior to IL-2/TIL immunotherapy demonstrated intact cortisol and aldosterone responses to ACTH as well as negative adrenal antibodies. One week after IL-2/TIL therapy, the patient developed a nonanion gap metabolic acidosis, hypotension and hypoglycemia. Adrenocortical function was re-evaluated demonstrating blunted cortisol and aldosterone responses to ACTH with an elevated plasma ACTH confirming the presence of primary adrenal insufficiency. Adrenal antibodies were now positive. Hydrocortisone and fludrocortisone were given with a good clinical response. We suggest immunotherapy with IL-2/TIL may cause adrenal insufficiency by activating autoimmune adrenalitis.

Molecular cytogenetic mapping of the human melanoma antigen (MAGE) gene family to chromosome region Xq27-qter: implications for MAGE immunotherapy.

We have defined the chromosomal location of the human MAGE (melanoma antigen) gene family by polymerase chain reaction amplification of several segments of the MAGE genes from somatic cell hybrids containing well-defined groups of human chromosomes and hybrids containing human derivative chromosomes. The data show that the three known MAGE family members (MAGEs -1, -2, & -3) are syntenic and map to the human X chromosome region q27-qter. Because males and females are hemizygous for most X-linked genes, the frequency of antigen-loss variants for the MAGE system is expected to be greater in comparison to antigens encoded by somatic chromosomes. In this regard, we believe that patients enrolled in MAGE-specific immunotherapy trials should be carefully monitored for the presence of MAGE antigen-loss variants.