Inhibitory effect of the Pseudobrickellia brasiliensis (Spreng) R.M. King & H. Rob. aqueous extract on human lymphocyte proliferation and IFN-γ and TNF-α production in vitro

Pseudobrickellia brasiliensis (Asteraceae) is a plant commonly known as arnica-do-campo and belongs to the native flora of the Brazilian Cerrado. The alcoholic extract of the plant has been used as an anti-inflammatory agent in folk medicine, but the biological mechanism of action has not been elucidated. The present study evaluated the composition of P. brasiliensis aqueous extract and its effects on pro-inflammatory cytokine production and lymphocyte proliferation. The extracts were prepared by sequential maceration of P. brasiliensis leaves in ethanol, ethyl acetate, and water. Extract cytotoxicity was evaluated by trypan blue exclusion assay, and apoptosis and necrosis were measured by staining with annexin V-FITC and propidium iodide. The ethanolic (ETA) and acetate (ACE) extracts showed cytotoxic effects. The aqueous extract (AQU) was not cytotoxic. Peripheral blood mononuclear cells stimulated with phorbol myristate acetate and ionomycin and treated with AQU (100 μg/mL) showed reduced interferon (IFN)-γ and tumor necrosis factor (TNF)-α expression. AQU also inhibited lymphocyte proliferative response after nonspecific stimulation with phytohemagglutinin. The aqueous extract was analyzed by liquid chromatography coupled with photodiode array detection and mass spectrometry. Quinic acid and its derivatives 5-caffeoylquinic acid and 3,5-dicaffeoylquinic acid, as well as the flavonoids luteolin and luteolin dihexoside, were detected. All these compounds are known to exhibit anti-inflammatory activity. Taken together, these findings demonstrate that P. brasiliensis aqueous extract can inhibit the pro-inflammatory cytokine production and proliferative response of lymphocytes. These effects may be related to the presence of chemical substances with anti-inflammatory actions previously reported in scientific literature.

Aerobic training modulates T cell activation in elderly women with knee osteoarthritis

Osteoarthritis of the knee (kOA) is a disease that mainly affects the elderly and can lead to major physical and functional limitations. However, the specific effects of walking, particularly on the immune system, are unknown. Therefore, this study aimed to analyze the effect of 12 weeks of walking (3×/week) on the leukocyte profile and quality of life (QL) of elderly women with kOA. Sixteen women (age: 67±4 years, body mass index: 28.07±4.16 kg/m2) participated in a walking program. The variables were assessed before and after 12 weeks of training with a progressively longer duration (30–55 min) and higher intensity (72–82% of HRmax determined using a graded incremental treadmill test). The QL was assessed using the Medical Outcomes Study 36-Item Short Form Health Survey (SF-36), and blood samples were collected for analysis with a cell counter and the San Fac flow cytometer. Walking training resulted in a 47% enhancement of the self-reported QL (P<0.05) and a 21% increase in the VO2max (P<0.0001) in elderly women with kOA. Furthermore, there was a reduction in CD4+ cells (pre=46.59±7%, post=44.58±9%, P=0.0189) and a higher fluorescence intensity for CD18+CD4+ (pre=45.30±10, post=64.27±33, P=0.0256) and CD18+CD8+ (pre=64.2±27, post=85.02±35, P=0.0130). In conclusion, the walking program stimulated leukocyte production, which may be related to the immunomodulatory effect of exercise. Walking also led to improvements in the QL and physical performance in elderly women with kOA.

Trypan blue exclusion assay by flow cytometry

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.

Whole-body vibration decreases the proliferativeb response of TCD4+ cells in elderly individuals with knee osteoarthritis

The aim of this study was to investigate the effect of adding whole-body vibration (WBV; frequency = 35 to 40 Hz; amplitude = 4 mm) to squat training on the T-cell proliferative response of elderly patients with osteoarthritis (OA) of the knee. This study was a randomized controlled trial in which the selected variables were assessed before and after 12 weeks of training. Twenty-six subjects (72 ± 5 years of age) were divided into three groups: 1) squat training with WBV (WBV, N = 8); 2) squat training without WBV (N = 10), and 3) a control group (N = 8). Women who were ≥60 years of age and had been diagnosed with OA in at least one knee were eligible. The intervention consisted of 12 uninterrupted weeks of squatting exercise training performed 3 times/week. Peripheral blood mononuclear cells were obtained from peripheral blood collected before and after training. The proliferation of TCD4+ and TCD8+ cells was evaluated by flow cytometry measuring the carboxyfluorescein succinimidyl ester fluorescence decay before and after the intervention (∆). The proliferative response of TCD4+ cells (P = 0.02, effect size = 1.0) showed a significant decrease (23%) in the WBV group compared to the control group, while there was no difference between groups regarding the proliferative response of TCD8+ cells (P = 0.12, effect size = 2.23). The data suggest that the addition of WBV to squat exercise training might modulate T-cell-mediated immunity, minimizing or slowing disease progression in elderly patients with OA of the knee.