Chad T. Robinson

Sodium tungstate (Na2WO4) exposure increases apoptosis in human peripheral blood lymphocytes.

The potential for adverse health effects of using tungsten and its alloys in military munitions are an important concern to both civilians and the US military. The toxicological implications of exposure to tungsten, its alloys, and the soluble tungstate (Na2WO4) are currently under investigation. To examine tungstate toxicity, a series of experiments to determine its in vitro effects on cells of the immune system were performed. We identified alterations in isolated human peripheral blood lymphocytes (PBL) treated in vitro with sodium tungstate (0.01, 0.1, 1.0, and 10 mM). Analyses of apoptosis with annexin V and propidium iodide revealed a dose- and time-dependent increase in the quantity of cells in early apoptosis after tungstate exposure. Reductions in the number of cells entering into the cell cycle were also noted. Exposure of PBL to tungstate (1 mM) and Concanavalin A (ConA) for 72 h reduced the number of cells in S and G2/M phases of the cell cycle. There were alterations in the numbers of cells in G0/G1, S, and G2/M phases of the cell cycle in long-term THP-1 (acute leukemic monocytes) cultures treated with tungstate (0.01, 0.1, 1.0, and 10 mM). Gel electrophoresis, silver staining, and LC-MS/MS showed the cytoplasmic presence of histone H1b and H1d after 72 h of tungstate exposure. The addition of tungstate to cultures resulted in significant reductions in the quantity of interleukin-10 (IL-10), tumor necrosis factor-α (TNF-α), and IL-6 produced by stimulated [CD3/CD28, ConA, or lipopolysaccharide (LPS)] and tungstate-treated lymphocytes. Taken together, these data indicate that tungstate increases apoptosis of PBL, alters cell cycle progression, reduces cytokine production, and therefore warrants further investigation.

Concerns over interspecies transcriptional comparisons in mice and humans after trauma

We have read with interest the study by Seok et al. (1) describing transcriptional responses of the immune systems of humans and mice. The authors perform Affymetrix GeneChip-based microarray assays on blood samples collected from blunt trauma, burn, and endotoxemia patients and mouse models of these pathologies. They report little correlation between human and murine genomic responses. Furthermore, the authors assert that mouse models of human disease are of questionable value due to the low biological similarity they observed.

Circulating microparticles do not all share biophysical light scatter properties with immune complexes when analyzed by flow cytometry

To the editor: Recent discussions have been published on the flow cytometric analysis of cell-derived microparticles (MPs) and the confounding effects of insoluble immune complexes (ICs) on the interpretation of data,[1][1][⇓][2]–[3][3] with Gyorgy et al stating that MPs and ICs share

Oral tungstate (Na2WO4) exposure reduces adaptive immune responses in mice after challenge

Abstract Tungstate has been identified as a ground water contaminant at military firing ranges and can be absorbed by ingestion. In this study, C57BL6 mice were exposed to sodium tungstate (Na2WO4·2H2O) (0, 2, 62.5, 125, and 200 mg/kg/day) in their drinking water for an initial 28-day screen and in a one-generation (one-gen) model. Twenty-four hours prior to euthanasia, mice were intraperitoneally injected with Staphylococcal enterotoxin B (SEB) (20 μg/mouse) or saline as controls. After euthanasia, splenocytes and blood were collected and stained with lymphocyte and/or myeloid immunophenotyping panels and analyzed by flow cytometry. In the 28-day and one-gen exposure, statistically significant reductions were observed in the quantities of activated cytotoxic T-cells (TCTL; CD3+CD8+CD71+) and helper T-cells (TH; CD3+CD4+CD71+) from spleens of SEB-treated mice. In the 28-day exposures, CD71+ TCTL cells were 12.87 ± 2.05% (SE) in the 0 tungstate (control) group compared to 4.44 ± 1.42% in the 200 mg/kg/day (p < 0.001) group. TH cells were 4.85 ± 1.23% in controls and 2.76 ± 0.51% in the 200 mg/kg/day (p < 0.003) group. In the one-gen exposures, TCTL cells were 7.98 ± 0.49% and 6.33 ± 0.49% for P and F1 mice after 0 mg/kg/day tungstate vs 1.58 ± 0.23% and 2.52 ± 0.25% after 200 mg/kg/day of tungstate (p < 0.001). Similarly, TH cells were reduced to 6.21 ± 0.39% and 7.20 ± 0.76%, respectively, for the 0 mg/kg/day P and F1 mice, and 2.28 ± 0.41% and 2.85 ± 0.53%, respectively, for the 200 mg/kg/day tungstate P and F1 groups (p < 0.001). In delayed-type hypersensitivity Type IV experiments, tungstate exposure prior to primary and secondary antigen challenge significantly reduced footpad swelling at 20 and 200 mg/kg/day. These data indicate that exposure to tungstate can result in immune suppression that may, in turn, reduce host defense against pathogens.

Protocol Standardization Reveals MV Correlation to Healthy Donor BMI

Microvesicles (MVs) are cell-derived vesicles which are of interest in a clinical setting, as they may be predictive of early signs of disease and/or of treatment progression. However, there are growing concerns about using conventional flow cytometry (cFMC) for the detection and quantification of microvesicles. These concerns range from error-sources in collection through to the physical limitations of detection. Here we present a standardized method for collection and analysis which shows that the MV numbers detected by cFCM correlate to donor Body Mass Index (BMI). Although unlikely to be comprehensive, we also demonstrate how cFCM is a useful and valid tool in the analysis of MVs.

The Effects of (-)-Epigallocatechin-3-Galate on Wound Closure and Infections in Mice

In this study, we investigated the effects of (-)-epigallocatechin-3-gallate (EGCG) on wound healing both in vitro and in vivo with and without infection. EGCG has antimicrobial properties and could be useful as a topical agent to prevent and/or treat wound infections. Normal fibroblasts were isolated from the dermis of C57BL/6 mice and cultured with 0, 0.001 to 0.400 mg/ml of EGCG. In vitro assays demonstrated that migration, proliferation, and apop- tosis were inhibited at EGCG concentrations of 0.100-0.400 mg/ml. Expression of α-smooth muscle actin (α-SMA) was also reduced. In vivo experiments measured closure/contraction of full-thickness dorsal wounds that were treated with 0, 0.3, 3.0, and 30.0 mg/ml of EGCG every 24 hours. Macrophages (F4/80), neutrophils (Ly-6G) and myofibroblasts (α-SMA) were assessed at 48 and 168 hours. By 168 hours there was a significant reduction in presence with the 30 mg/ml dose vs. 0.3 and 3.0 mg/ml (P<0.009, P<0.006, respectively). The percentage of wound closure at one week in EGCG treated wounds was 87.87% (0.03 mg), 85.23% (0.3 mg) and 40.06% (30 mg/ml) compared to controls. Reduced quantities of α-SMA myofibroblasts were observed in the 3 mg EGCG treatment group compared to controls at 168 hours. We previously demonstrated that EGCG has antimicrobial properties (MIC 50 ~0.3 mg/ml). This data suggests that EGCG could potentially be applied to the wound surface as an antimicrobial without negatively influencing healing. To this end we applied EGCG (10 mg/ml) to a model of an infected traumatic wound. EGCG treatment significantly reduced bacterial load after one and two dose regimens.