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.

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.

IL-10 produced by CD4+ and CD8+ T cells emerge as a putative immunoregulatory mechanism to counterbalance the monocyte-derived TNF-alpha and guarantee asymptomatic clinical status during chronic HTLV-I infection.

Although it is believed widely that distinct patterns of the host immune response are associated with the outcome of chronic human T cell lymphotropic virus type 1 (HTLV-I) infection toward asymptomatic or symptomatic neurodegenerative myelopathy (HAM/TSP), the exact mechanism underlying these immunological events still remains unknown. In this study, we have evaluated the cytokine pattern [interleukin (IL)-12, interferon (IFN)-gamma, tumour necrosis factor (TNF)-alpha, IL-4 and IL-10] of innate and adaptive immunity cells present at the peripheral blood from non-infected (NI) and HTLV-I infected individuals [asymptomatic (AS), oligosymptomatic (OL) and HAM/TSP-HT], following in vitro short-term incubation in the absence/presence of phorbol myristate acetate (PMA) pan-leucocyte stimulation. In the absence of PMA stimulation, our data demonstrate that despite the overall immunological profile of AS mimicry that observed for NI, the high frequency of IL-12(+) neutrophils and TNF-alpha(+) monocytes are also a hallmark of this group of individuals. However, the outstanding positive correlation between the high frequency of TNF-alpha(+) monocytes and high levels CD4(+) IL-10(+) and CD8(+) IL-10(+) T cells suggests the establishment of immunoregulatory mechanisms that guarantee their asymptomatic clinical status. On the other hand, OL and HT did not present any association between the high frequency and TNF-alpha(+) neutrophils and monocytes and this immunoregulatory profile at their adaptive immunity cells. Upon PMA-index analysis, high levels of type 1 CD4(+) T cells, as well as higher IFN-gamma/IL-10 and TNF-alpha/IL-10 ratios, were observed in HT, and re-emphasize the role of Th1-cytokines from CD4(+) cells to HTLV-I immunity and disease. Moreover, increasing frequency of CD8(+) IFN-gamma(+) and CD8(+) TNF-alpha(+) cells were observed in the HT, which corroborates the marked inflammatory profile underlying this pathological condition and the role of CD8(+) T cells in the pathogenesis of HAM/TSP.

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.

Establishing phenotypic features associated with morbidity in human T-cell lymphotropic virus type 1 infection.

The human T-cell lymphotropic virus type 1 (HTLV-1) is the causative agent of HTLV-1-associated myelopathy/tropical spastic paraparesis (HT). Although it is widely believed that virus infection and host immune response are involved in the pathogenic mechanisms, the role of the immune system in the development and/or maintenance of HT remains unknown. We performed an analysis of the peripheral blood leukocyte phenotype for two different subcohorts of HTLV-1-infected individuals to verify the existence of similar immunological alterations, possible laboratory markers for HT. The leukocyte population balance, the activation status of the T lymphocytes, and the cellular migratory potential of T lymphocytes, monocytes, and neutrophils were evaluated in the peripheral blood of HTLV-1-infected individuals classified as asymptomatic individuals, oligosymptomatic individuals, and individuals with HT. Data analysis demonstrated that a decreased percentage of B cells, resulting in an increased T cell/B cell ratio and an increase in the CD8+ HLA-DR+ T lymphocytes, exclusively in the HT group could be identified in both subcohorts, suggesting its possible use as a potential immunological marker for HT for use in the laboratory. Moreover, analysis of likelihood ratios showed that if an HTLV-1-infected individual demonstrated B-cell percentages lower than 7.0%, a T cell/B cell ratio higher than 11, or a percentage of CD8+ HLA-DR+ T lymphocytes higher than 70.0%, this individual would have, respectively, a 12-, 13-, or 22-times-greater chance of belonging to the HT group. Based on these data, we propose that the T cell/B cell ratios and percentages of circulating B cells and activated CD8+ T lymphocytes in HTLV-1-infected patients are important immunological indicators which could help clinicians monitor HTLV-1 infection and differentiate the HT group from the asymptomatic and oligosymptomatic groups.

Phenotypic study of peripheral blood leucocytes in HTLV-I-infected individuals from Minas Gerais, Brazil.

The human T-cell lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) associated with the HTLV-I is a well-defined clinical-pathological entity in which the virus and host immune responses contribute to the pathological mechanism. In this study, flow cytometric analysis of whole peripheral blood leucocytes (PBL) was performed to evaluate the immunological status of HTLV-I-infected individuals in an effort to better understand the role of the immune system in the development of HAM/TSP. We have evaluated three groups of infected patients including asymptomatic (AS = 18), ambulatory/oligosymptomatic (AM = 14) and hospitalized HAM/TSP individuals (HO = 42). Noninfected healthy blood donors were used for the control group (NI = 32). Our results demonstrated that the HO group presents an increased percentage of circulating T cells and a decreased percentage of B and natural killer (NK) cells, leading to the highest T/B-cell ratio in comparison with the other groups. Interestingly, while an increased percentage of activated CD4+HLA-DR+ T lymphocytes was observed in both AM and HO, only HO presented higher percentage of activated CD8+HLA-DR+ in combination with the highest CD18 surface expression. This was true for all cell populations analysed, including T lymphocytes, monocytes and neutrophils. Moreover, the HO group was distinguished by a dramatic decrease in the percentage of CD8+CD28+ lymphocytes. Taken together, these findings demonstrate a potent cellular immune activation response involving primarily CD8+ T cells that is concomitant with disease progression in HAM/TSP. We also show that an upregulation of CD18 expression, a hallmark for increased cell migratory potential, might play a critical role in the development/maintenance of HAM/TSP.