Photobiomodulation therapy on expression of HSP70 protein and tissue repair in experimental acute Achilles tendinitis

Lasers in Medical Science - The aim of the present study was to investigate the effects of photobiomodulation (PBM) therapy on the expression of heat shock protein 70 (HSP70) and tissue repair in...     

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.     

Infrared photobiomodulation (PBM) therapy improves glucose metabolism and intracellular insulin pathway in adipose tissue of high-fat fed mice

Obesity represents a continuously growing global epidemic and is associated with the development of type 2 diabetes mellitus. The etiology of type 2 diabetes is related to the resistance of insulin-sensitive tissues to its action leading to impaired blood glucose regulation. Photobiomodulation (PBM) therapy might be a non-pharmacological, non-invasive strategy to improve insulin resistance. It has been reported that PBM therapy in combination with physical exercise reduces insulin resistance. Therefore, the aim of this study was to investigate the effects of PBM therapy on insulin resistance in obese mice. Male Swiss albino mice received low-fat control diet (n?=?16, LFC) or high-fat diet (n?=?18, HFD) for 12 weeks. From 9th to 12th week, the mice received PBM therapy (LASER) or Sham (light off) treatment and were allocated into four groups: LFC Sham (n?=?8), LFC PBM (n?=?8), HFD Sham (n?=?9), and HFD PBM (n?=?9). The PBM therapy was applied in five locations: to the left and right quadriceps muscle, upper limbs and center of the abdomen, during 40 s at each point, once a day, 5 days a week, for 4 weeks (780 nm, 250 mW/cm², 10 J/cm², 0.4 J per site; 2 J total dose per day). Insulin signaling pathway was evaluated in the epididymal adipose tissue. PBM therapy improved glucose tolerance and phosphorylation of Akt (Ser473) and reversed the HFD-induced reduction of GLUT4 content and phosphorylation of AS160 (Ser588). Also, PBM therapy reversed the increased area of epididymal and mesenteric adipocytes. The results showed that chronic PBM therapy improved parameters related to obesity and insulin resistance in HFD-induced obesity in mice.     

Swim training does not protect mice from skeletal muscle oxidative damage following a maximum exercise test

We investigated whether swim training protects skeletal muscle from oxidative damage in response to a maximum progressive exercise. First, we investigated the effect of swim training on the activities of the antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), in the gastrocnemius muscle of C57Bl/6 mice, 48 h after the last training session. Mice swam for 90 min, twice a day, for 5 weeks at 31°C (± 1°C). The activities of SOD and CAT were increased in trained mice (P < 0.05) compared to untrained group. However, no effect of training was observed in the activity of GPx. In a second experiment, trained and untrained mice were submitted to a maximum progressive swim test. Compared to control mice (untrained, not acutely exercised), malondialdehyde (MDA) levels were increased in the skeletal muscle of both trained and untrained mice after maximum swim. The activity of GPx was increased in the skeletal muscle of both trained and untrained mice, while SOD activity was increased only in trained mice after maximum swimming. CAT activity was increased only in the untrained compared to the control group. Although the trained mice showed increased activity of citrate synthase in skeletal muscle, swim performance was not different compared to untrained mice. Our results show an imbalance in the activities of SOD, CAT and GPx in response to swim training, which could account for the oxidative damage observed in the skeletal muscle of trained mice in response to maximum swim, resulting in the absence of improved exercise performance.     

Leukocytosis,muscle damage and increased lymphocyte proliferative response after an adventure sprint race

The effect of an adventure sprint race (ASR) on T-cell proliferation, leukocyte count and muscle damage was evaluated. Seven young male runners completed an ASR in the region of Serra do Espinhaço, Brazil. The race induced a strong leukocytosis (6.22±2.04×10³ cells/mm³ before vs 14.81±3.53×10³ cells/mm³ after the race), marked by a significant increase of neutrophils and monocytes (P<0.05), but not total lymphocytes, CD3⁺CD4⁺ or CD3⁺CD8⁺ cells. However, the T-cell proliferative response to mitogenic stimulation was increased (P=0.025) after the race, which contradicted our hypothesis that ASR, as a high-demand competition, would inhibit T-cell proliferation. A positive correlation (P=0.03, r=0.79) was observed between the proliferative response of lymphocytes after the race and the time to complete the race, suggesting that the proliferative response was dependent on exercise intensity. Muscle damage was evident after the race by increased serum levels of aspartate amino transferase (24.99±8.30 vs 50.61±15.76 U/L, P=0.003). The results suggest that humoral factors and substances released by damaged muscle may be responsible for lymphocyte activation, which may be involved in muscle recovery and repair.     

IL-33 in obesity: where do we go from here?

Inflammation Research - IL-33 is a cytokine that belongs to the IL-1 family and is classically associated with type 2-like immune responses. In the adipose tissue, IL-33 is related to the beiging...     

Monocytes from patients with indeterminate and cardiac forms of Chagas' disease display distinct phenotypic and functional characteristics associated with morbidity

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-alpha]) 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 with T. cruzi in 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 with T. 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-alpha 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-alpha expression.     

Histopathological outcome of Leishmania major-infected BALB/c mice is improved by oral treatment with N-acetyl-l-cysteine

Leishmania major infected BALB/c mice were treated with N-acetyl-l-cysteine (NAC), a glutathione precursor, to evaluate the role of in vivo glutathione on lesion pathology and cytokine profiles following infection. Mice were maintained on NAC-containing water 2 days before infection for a total of 14 weeks. The BALB/c response to L. major infection was improved by oral administration of NAC, at the level of histopathological outcome, lesion progression and cytokine profile. A significantly improved histopathological outcome of the footpad lesion, characterized by a mixed inflammatory infiltrate organized in a focal pattern with little tissue destruction and a reduced parasite load, was observed in NAC-treated BALB/c mice. Histopathological modulation was accompanied by a modified cytokine pattern from popliteal lymph node cells, demonstrated by a sustained higher frequency of interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha)-producing cells. This work points to an important role for glutathione in the modulation of effector responses in BALB/c mice.     

Effect of aerobic training on plasma cytokines and soluble receptors in elderly women with knee osteoarthritis, in response to acute exercise

The aim of this study was to evaluate levels of interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α), and soluble forms of the TNF-α receptor (sTNFR1 and sTNFR2) from plasma taken from the peripheral blood of elderly individuals presenting with osteoarthritis (OA) of the knee. These patients underwent aerobic treatment through the use of physical exercises. The study consisted of a longitudinal analysis of older individuals presenting clinical and radiographic diagnosis of knee OA that were submitted to 12 weeks of aerobic treatment. The individuals were evaluated during acute exercise or after chronic exercise. During acute exercise (walking slowly on the mat), blood samples of the patients were collected before, immediately after, and 30 min following the end of training. After chronic exercise (aerobic walking training, three times/week for 12 weeks), patient blood samples were obtained for comparison. Additionally, clinical and functional assessments (WOMAC test and 6-min walk) were performed at the end of all physical exercises. Plasma concentrations of cytokines and soluble receptors were measured by ELISA. Aerobic training increased the plasma concentration of sTNR1; however, it decreased the plasma concentration of sTNFR2, when compared with levels of resting patients. Acute exercise differentially affects the levels of sTNFR1 dependent on when the samples were taken, before and after aerobic training. However, the levels of sTNFR2 were not affected by training. For the population studied, we observed differences in the levels of sTNFR1 and sTNFR2 following acute and chronic exercise. Other additional factors, like the level of inactivity of the individual and the type of physical exercise that patients are exposed to, need to be considered as well. The variation in the levels of soluble receptors correlated with functional improvement; however, the inflammatory osteoarthritis markers (IL-6 and TNF-α) were unaffected by the walking exercises.