Arsenic exposure is associated with decreased DNA repair in vitro and in individuals exposed to drinking water arsenic.

The mechanism(s) by which arsenic exposure contributes to human cancer risk is unknown ; however, several indirect cocarcinogenesis mechanisms have been proposed. Many studies support the role of As in altering one or more DNA repair processes. In the present study we used individual-level exposure data and biologic samples to investigate the effects of As exposure on nucleotide excision repair in two study populations, focusing on the excision repair cross-complement 1 (ERCC1) component. We measured drinking water, urinary, or toenail As levels and obtained cryopreserved lymphocytes of a subset of individuals enrolled in epidemiologic studies in New Hampshire (USA) and Sonora (Mexico). Additionally, in corroborative laboratory studies, we examined the effects of As on DNA repair in a cultured human cell model. Arsenic exposure was associated with decreased expression of ERCC1 in isolated lymphocytes at the mRNA and protein levels. In addition, lymphocytes from As-exposed individuals showed higher levels of DNA damage, as measured by a comet assay, both at baseline and after a 2-acetoxyacetylaminofluorene (2-AAAF) challenge. In support of the in vivo data, As exposure decreased ERCC1 mRNA expression and enhanced levels of DNA damage after a 2-AAAF challenge in cell culture. These data provide further evidence to support the ability of As to inhibit the DNA repair machinery, which is likely to enhance the genotoxicity and mutagenicity of other directly genotoxic compounds, as part of a cocarcinogenic mechanism of action.

Advantages of hydrophobic culture bags over flasks for the generation of monocyte-derived dendritic cells for clinical applications.

Dendritic cells (DC), potent antigen presenting cells capable of activating both naïve and primed T cells, are currently being pursued clinically in the development of cancer vaccines. Variations in the literature regarding DC source, culture conditions, maturation state, dose, and route of immunization make comparisons of clinical trial data difficult. In order to define and optimize the culture conditions for DC generation, we have performed a careful comparison of two culture methods, as well as different methods of DC maturation. Our studies demonstrate that high viability DC can be produced and matured in gas permeable hydrophobic culture bags. These cells express surface molecules characteristic of DC and have superior yield, viability, and function to cells cultured in plastic tissue culture flasks. These results suggest that hydrophobic culture bags are ideal for the preparation of clinical DC vaccines, as DC can be generated, antigen-loaded, and matured in a closed system, a scheme we have found to be superior to previously described flask culture methods.

Multiparameter precursor analysis of T-cell responses to antigen.

Triggering of the T-cell receptor by cognate antigen induces a variety of cellular events leading to cell proliferation and differentiation. While the plasticity and diversity of T-cell responses have been recognized for a long time, few quantitative studies have been conducted to measure what proportion of specific T cells will enter a given differentiation program after antigen stimulation. In the present study, we analyzed human T cells cultured with influenza-peptide-loaded dendritic cells. We compared three individual methods for assaying the frequency of antigen-specific T cells: ELISPOT, tetramer-binding, and proliferation. The three methods yielded similar but not identical results. In order to study these differences at the single cell level, we developed a multiparameter flow cytometric method, which allows simultaneous analysis of antigen-specific tetramer binding, T-cell proliferation, and cytokine production. Based on these data, we used flow precursor frequency analysis to calculate the proportion of eight different precursor subsets in the original, resting population. We conclude that approximately half of the cells that bound specific tetramers actually proliferated and synthesized IFNgamma in response to antigen. In addition, similar numbers of cells that did not bind tetramer proliferated (but did not synthesize IFNgamma). The method allows for an estimate of the precursor frequency of each functional subset within the initial population. It could be applied to additional markers of function and differentiation, combining all parameters into a description of the complex response potential of a T-cell pool.

The pharmacokinetics of the bispecific antibody MDX-H210 when combined with interferon gamma-1b in a multiple-dose phase I study in patients with advanced cancer.

INTRODUCTION: MDX-H210 is a Fab'xFab' bispecific antibody (BsAb) constructed chemically by crosslinking Fab' mAb 520C9 (anti-HER-2/neu) and humanized Fab' mAbH22 (anti-CD64). STUDY DESIGN AND OBJECTIVES: This was a phase I dose-escalation study of intravenous MDX-H210 (1-70 mg/m(2)) combined with subcutaneous IFN-gamma, 50 microg/m(2) given 24 h before the BsAb, both drugs being given three times a week for 3 weeks. The major objectives of the study were to define the safety, tolerability and pharmacokinetics of MDX-H210 when given with IFN-gamma on this schedule. RESULTS: The study group comprised 23 patients (19 female, 4 male; median age 51.5 years, range 25-72 years) with advanced HER-2/neu-positive cancers (19 breast, 3 prostate and 1 lung). Inspection of the log plasma MDX-H210 concentrations-time data for both days 1 and 17 of treatment revealed monoexponential decay in the majority of patients with adequate concentration-time data points. The MDX-H210 T(1/2) ranged from 2.9 to 21.9 h. The MDX-H210 C(max) on day 1 (means+/-SD) increased from 0.30+/-0.22 microg/ml at the 1-mg/m(2) dose tier to 86.91+/-6.46 microg/ml at 70 mg/m(2). Equivalent day-17 values were 0.27+/-0.30 microg/ml increasing to 147.85+/-40.23 microg/ml. The MDX-H210 T(max) occurred at or after the end of the infusion for all treatments. The mean MDX-H210 total body clearance (Cl) was in the range 0.01-0.34 ml/min per kg and the mean MDX-H210 apparent volume of distribution at steady-state (Vd(ss)) in the range 20-170 ml/kg, compatible with distribution primarily limited to the intravascular space. MDX-H210 T(1/2) increased with dose (ANOVA P=0.001) and Cl decreased with dose (ANOVA P=0.006). There were no significant changes in MDX-H210 C(max), AUC, Cl or Vd(ss) between day 1 and day 17. CONCLUSIONS: MDX-H210 pharmacokinetics appeared saturable over the dose range 1-70 mg/m(2), and there was no significant change in MDXH210 pharmacokinetics over the course of the study, or evidence of excessive accumulation of MDX-H210 on this multiple dosing schedule. When MDX-H210 was combined with IFN-gamma, the estimated MDX-H210 pharmacokinetic parameters were similar to the published data for single-agent MDX-H210.

Use of cell-tracking dyes to determine proliferation precursor frequencies of antigen-specific T cells.

The T-cell receptor provides T cells with specificity for antigens of particular molecular structure (the "epitope"); the T-cell pool in an individual responds to the presence of many different antigenic epitopes, but any particular T cell will respond preferentially to one defined epitope. After stimulation of a T cell by the binding of its receptor to its cognate antigen in the context of a major histocompatibility complex (MHC) molecule on an antigen-presenting cell, the T cell will begin to proliferate and synthesize cytokines. Tetramer binding and the enzyme-linked immunospot (ELISPOT) method have been used to determine what proportion of cells in the T-cell pool can bind to a defined antigenic peptide or will secrete cytokines in response to a particular antigenic stimulation. The method described here uses tracking dyes to determine what proportion of T cells will proliferate in response to stimulation. As a flow cytometric "single-cell" method, it can be combined with tetramer and cytokine staining to determine the precursor frequencies of cells in the T-cell pool able to bind tetramer, to synthesize cytokines, and to proliferate in response to antigen.

Immunological effects of granulocyte-macrophage colony-stimulating factor and autologous tumor vaccine in patients with renal cell carcinoma.

PURPOSE: Biological therapy for renal cell carcinoma (RCC) uses agents that mobilize immune effector cells which are able to recognize and destroy cancer. We evaluated the effects of weekly then monthly autologous tumor vaccine combined with daily granulocyte macrophage-colony stimulating factor (GM-CSF) in patients with RCC as a method of stimulating antigen presenting cells. MATERIALS AND METHODS: Eligible patients with pathological stage II to IV RCC were entered into this pilot study. Autologous tumor vaccine (0.5 to 1 x 107 irradiated tumor cells) admixed with 250 microg GM-CSF per vaccine was given subcutaneously weekly for 4 weeks and then monthly for 4 months. GM-CSF (125 microg/m2) was given subcutaneously for 13 days after vaccine injection 1 and injections 4 to 8. Treatment related tumor specific CD4 and CD8 positive T cell precursors were assessed. RESULTS: A total of 22 patients were entered into this study. Patients were stratified by bulk of disease (group 1, 9 patients with micrometastatic disease, and group 2, 13 patients with macrometastatic disease). In general treatment was well tolerated. Of 9 patients in group 1 7 remained disease-free after nephrectomy. In group 2, 6 patients had stable (46.2%) and 7 patients had progressive disease (53.8%). Statistically significant treatment related increases in CD4 (p = 0.028) and CD8 (p = 0.018) positive tumor specific T cell precursors were observed for the entire group of patients. Changes in CD4 and CD8 positive precursors correlated significantly with each other (p = 0.0001). This correlation was seen in the 2 patient subpopulations as well (group 1 p = 0.003, group 2 p = 0.013). Patients with minimal disease, and with changes in CD4 and CD8 positive tumor specific T cell precursors greater than the median appeared to have an improved time to progression as well as a survival benefit. CONCLUSIONS: GM-CSF and autologous vaccine can be given safely in combination to patients with renal cell cancer. We observed treatment related changes in tumor specific circulating lymphocyte populations.