Dye exclusion tests are used to determine the number of live and dead cells. These assays are based on the principle that intact plasma membranes in live cells exclude specific dyes, whereas dead cells do not. Although widely used, the trypan blue (TB) exclusion assay has limitations. The dye can be incorporated by live cells after a short exposure time, and personal reliability, related to the expertise of the analyst, can affect the results. We propose an alternative assay for evaluating cell viability that combines the TB exclusion test and the high sensitivity of the flow cytometry technique. Previous studies have demonstrated the ability of TB to emit fluorescence when complexed with proteins. According to our results, TB/bovine serum albumin and TB/cytoplasmic protein complexes emit fluorescence at 660 nm, which is detectable by flow cytometry using a 650-nm low-pass band filter. TB at 0.002% (w/v) was defined as the optimum concentration for distinguishing unstained living cells from fluorescent dead cells, and fluorescence emission was stable for 30 min after cell treatment. Although previous studies have shown that TB promotes green fluorescence quenching, TB at 0.002% did not interfere with green fluorescence in human live T-cells stained with anti-CD3/fluorescein isothiocyanate (FITC) monoclonal antibody. We observed a high correlation between the percentage of propidium iodide+CD3/FITC⁺ and TB+CD3/FITC⁺ cells, as well as similar double-stained cell profiles in flow cytometry dot-plot graphs. Taken together, the results indicate that a TB exclusion assay by flow cytometry can be employed as an alternative tool for quick and reliable cell viability analysis.

Many studies have demonstrated that monocyte-derived macrophages display critical activities in immunity to parasites. The ability of these cells to process and present antigens, produce cytokines, and provide costimulatory signals demonstrates their pivotal role in initiating immune responses. Although potential modulatory function has been attributed to monocytes from patients with Chagas' disease, a systematic phenotypic and functional analysis of these cells has not been performed. In this work, we analyzed the ex vivo expression of important surface molecules (CD11b and HLA-DR) and immunoregulatory cytokines (interleukin-10 [IL-10], IL-12 and tumor necrosis factor alpha [TNF-α]) in CD14⁺and CD14⁻monocytes from Chagas' disease patients with polar clinical forms of the disease: indeterminate or severe cardiac. We also evaluated the influence of in vitro infection withT. cruziin the expression of such molecules. We observed that monocytes from indeterminate-disease patients display lower levels of HLA-DR than those from noninfected individuals both ex vivo and after in vitro infection withT. cruzi.Although ex vivo expression of CD11b was similar among the groups, in vitro infection led to decreased expression of this molecule by monocytes from Chagas' disease patients but not from noninfected individuals. Analysis of the expression of immunoregulatory cytokines showed that while monocytes from indeterminate-disease patients are committed to IL-10 expression, a higher percentage of monocytes from cardiac-disease patients express TNF-α after exposure to live parasites. These results suggest that monocytes from indeterminate-disease patients display modulatory characteristics related to low HLA-DR and high IL-10 expression whereas monocytes from cardiac-disease, patients may be committed to induction of inflammatory responses related to high TNF-α expression.

Objectives: The study objectives were to evaluate the effects of adding whole-body vibration to squat training on functional performance and self-report of disease in elderly individuals with knee osteoarthritis (OA). Design: This was a prospective, randomized trial in which selected variables were evaluated at three periods: 3 weeks prior to the training, immediately prior, and after the end of the training. Subjects: Twenty-three (23) elderly subjects were evaluated using four functional performance tests: Berg Balance Scale (BBS), Timed Get Up and Go Test (TGUG), Chair Stand Test (CST), and 6-Minute Walk Test (6MWT), and a self-report of the status of disease (WOMAC). Interventions: The intervention lasted for 12 weeks, 3 times per week. The participants were randomized into two groups: (1) squat training with whole-body vibration, and (2) squat training without vibration. Results: Although there was no statistical difference in functional performance and self-report of disease status between the groups, performance in all the functional tests and in all the domains of WOMAC improved in the vibration group compared to their initial status. In the exercise group, performance improved only two tests (BBS and 6MWT), and there was a reduction in self-reported pain (WOMAC) compared to their initial status. Conclusions: Although the addition of whole-body vibration to squat training failed to result in a significant improvement in functional performance and self-reported status of knee osteoarthritis in the elderly, the intragroup results suggest that whole-body vibration may represent a feasible and effective way of improving the functionality and self-perception of disease status in older adults with knee OA.

Background: Autoimmunity is increasingly recognized as a key contributing factor in heart muscle diseases. The functional features of cardiac autoimmunity in humans remain undefined because of the challenge of studying immune responses in situ. We previously described a subset of c-mesenchymal epithelial transition factor (c-Met)–expressing (c-Met ⁺ ) memory T lymphocytes that preferentially migrate to cardiac tissue in mice and humans. Methods: In-depth phenotyping of peripheral blood T cells, including c-Met ⁺ T cells, was undertaken in groups of patients with inflammatory and noninflammatory cardiomyopathies, patients with noncardiac autoimmunity, and healthy controls. Validation studies were carried out using human cardiac tissue and in an experimental model of cardiac inflammation. Results: We show that c-Met ⁺ T cells are selectively increased in the circulation and in the myocardium of patients with inflammatory cardiomyopathies. The phenotype and function of c-Met ⁺ T cells are distinct from those of c-Met–negative (c-Met ⁻ ) T cells, including preferential proliferation to cardiac myosin and coproduction of multiple cytokines (interleukin-4, interleukin-17, and interleukin-22). Furthermore, circulating c-Met ⁺ T cell subpopulations in different heart muscle diseases identify distinct and overlapping mechanisms of heart inflammation. In experimental autoimmune myocarditis, elevations in autoantigen-specific c-Met ⁺ T cells in peripheral blood mark the loss of immune tolerance to the heart. Disease development can be halted by pharmacologic c-Met inhibition, indicating a causative role for c-Met ⁺ T cells. Conclusions: Our study demonstrates that the detection of circulating c-Met ⁺ T cells may have use in the diagnosis and monitoring of adaptive cardiac inflammation and definition of new targets for therapeutic intervention when cardiac autoimmunity causes or contributes to progressive cardiac injury.

We evaluated the effect of different water immersion (WI) temperatures on post-exercise cardiac parasympathetic reactivation. Eight young, physically active men participated in four experimental conditions composed of resting (REST), exercise session (resistance and endurance exercises), post-exercise recovery strategies, including 15 min of WI at 15°C (CWI), 28°C (TWI), 38°C (HWI) or control (CTRL, seated at room temperature), followed by passive resting. The following indices were assessed before and during WI, 30 min post-WI and 4 hours post-exercise: mean R-R (mR-R), the natural logarithm (ln) of the square root of the mean of the sum of the squares of differences between adjacent normal R–R (ln rMSSD) and the ln of instantaneous beat-to-beat variability (ln SD1). The results showed that during WI mRR was reduced for CTRL, TWI and HWI versus REST, and ln rMSSD and ln SD1 were reduced for TWI and HWI versus REST. During post-WI, mRR, ln rMSSD and ln SD1 were reduced for HWI versus REST, and mRR values for CWI were higher versus CTRL. Four hours post exercise, mRR was reduced for HWI versus REST, although no difference was observed among conditions. We conclude that CWI accelerates, while HWI blunts post-exercise parasympathetic reactivation, but these recovery strategies are short-lasting and not evident 4 hours after the exercise session.