Effects of Conjugated Linoleic Acid Associated With Endurance Exercise on Muscle Fibres and Peroxisome Proliferator-Activated Receptor γ Coactivator 1 α Isoforms.

Conjugated linoleic acid (CLA) has been reported to improve muscle hypertrophy, steroidogenesis, physical activity, and endurance capacity in mice, although the molecular mechanisms of its actions are not completely understood. The aim of the present study was to identify whether CLA alters the expression of any of the peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α) isoforms, and to evaluate the possible existence of fibre-type-specific hypertrophy in the gastrocnemius and plantaris muscles. Mice were randomly assigned to one of four groups: placebo sedentary, CLA sedentary, placebo trained, or CLA trained. The CLA groups were gavaged with 35 µl per day of Tonalin® FFA 80 food supplement containing CLA throughout the 6-week experimental period, whereas the placebo groups were gavaged with 35 µl sunflower oil each day. Each administered dose of CLA corresponded to approximately 0.7 g/kg or 0.5%, of the dietary daily intake. Trained groups ran 5 days per week on a Rota-Rod for 6 weeks at increasing speeds and durations. Mice were sacrificed by cervical dislocation and hind limb posterior muscle groups were dissected and used for histological and molecular analyses. Endurance training stimulated mitochondrial biogenesis by PGC1α isoforms (tot, α1, α2, and α3) but CLA supplementation did not stimulate PGC1α isoforms or mitochondrial biogenesis in trained or sedentary mice. In the plantaris muscle, CLA supplementation induced a fibre-type-specific hypertrophy of type IIx muscle fibres, which was associated with increased capillary density and was different from the fibre-type-specific hypertrophy induced by endurance exercise (of types I and IIb muscle fibres). J. Cell. Physiol. 232: 1086-1094, 2017. © 2016 Wiley Periodicals, Inc.

Heat shock protein 60 levels in tissue and circulating exosomes in human large bowel cancer before and after ablative surgery.

BACKGROUND: Heat shock protein 60 (Hsp60) is a chaperonin involved in tumorigenesis, but its participation in tumor development and progression is not well understood and its value as a tumor biomarker has not been fully elucidated. In the current study, the authors presented evidence supporting the theory that Hsp60 has potential as a biomarker as well as a therapeutic target in patients with large bowel cancer. METHODS: The authors studied a population of 97 subjects, including patients and controls. Immunomorphology, Western blot analysis, and quantitative real-time polymerase chain reaction were performed on tissue specimens. Exosomes were isolated from blood and characterized by electron microscopy, biochemical tests, and Western blot analysis. RESULTS: Hsp60 was found to be increased in cancerous tissue, in which it was localized in the tumor cell plasma membrane, and in the interstitium associated with cells of the immune system, in which it was associated with exosomes liberated by tumor cells and, as such, circulated in the blood. An interesting finding was that these parameters returned to normal shortly after tumor removal. CONCLUSIONS: The data from the current study suggested that Hsp60 is a good candidate for theranostics applied to patients with large bowel carcinoma and encourage similar research among patients with other tumors in which Hsp60 has been implicated.

The involvement of MMP-2 and MMP-9 in heart exercise-related angiogenesis.

BACKGROUND: Little is known about the involvement of matrix metalloproteinases (MMPs) in cardiac vascular remodelling induced by exercise. Our aim was to evaluate and localize MMP-2 and MMP-9's activities in relation to capillary proliferation in mouse hearts trained for 15, 30 and 45 days. METHODS: Sixty-three mice were randomly assigned to 7 groups: four control sedentary groups (C0, C15, C30 and C45) and three groups trained by an endurance protocol (T15, T30 and T45). MMP-2 and MMP-9 were examined with zymography and immunostaining analyses. Capillary proliferation was evaluated counting the number of CD31-positive cells. RESULTS: Different activity patterns of the latent form of both MMPs were found. Pro-MMP-9 increased after 15 days of training; whereas pro-MMP-2 gradually decreased after 30 and 45 days of training below the control groups. The latter was inversely correlated with capillary growth. MMP-9 was mainly localized in myocardiocytes and less evident in capillaries. Conversely, MMP-2 was more intense in capillary endothelial cells and slightly in myocardiocytes. CONCLUSIONS: A different spatiotemporal modulation of pro-MMP-2 and pro-MMP-9 activities has been detected in the myocardium during angiogenesis related to the aerobic training. These results can be useful to draw up training protocols for improving the performance of healthy and diseased human hearts.

Heat stroke risk for open-water swimmers during long-distance events.

Open-water swimming is a rapidly growing sport discipline worldwide, and clinical problems associated with long-distance swimming are now better recognized and managed more effectively. The most prevalent medical risk associated with an open-water swimming event is hypothermia; therefore, the Federation Internationale De Natation (FINA) has instituted 2 rules to reduce this occurrence related to the minimum water temperature and the time taken to complete the race. Another medical risk that is relevant to open-water swimmers is heat stroke, a condition that can easily go unnoticed. The purpose of this review is to shed light on this physiological phenomenon by examining the physiological response of swimmers during long-distance events, to define a maximum water temperature limit for competitions. We conclude that competing in water temperatures exceeding 33°C should be avoided.

Effect of conjugated linoleic acid on testosterone levels in vitro and in vivo after an acute bout of resistance exercise.

The purposes of the present study were to investigate the effect of conjugated linoleic acid (CLA) supplementation on testosterone levels in vitro on a cell line derived from Leydig cells (R2C) and in vivo in the blood of physically active subjects before and after a resistance exercise bout. In vitro R2C cells were treated with different CLA concentrations (0-30 µM) for 24 and 48 hours. After treatment, supernatant media were tested to determine testosterone secretion. The CLA increased the testosterone secretion only after 48 hours. In vivo, 10 resistance-trained male subjects, in a double-blind placebo-controlled and crossover study design were randomized for 3 weeks of either 6 g·d⁻¹ CLA or placebo. Blood was drawn pre and post each resistance exercise bout to determine the total testosterone and sex hormone-binding globulin (SHBG) levels. No significant differences were observed for total testosterone or SHBG pre and post each resistance exercise bout; although after the resistance exercise bouts, total testosterone increased moderately (effect size = moderate), whereas after CLA supplementation, there was a large increase in total testosterone (effect size = large). CLA supplementation induced an increase in testosterone levels in Leydig cells in vitro after 48 hours but not in vivo before and after a resistance exercise bout. These findings suggest that CLA supplementation may promote testosterone synthesis through a molecular pathway that should be investigated in the future, although this effect did not have an anabolic relevance in our in vivo model.

Physiactisome: A New Nanovesicle Drug Containing Heat Shock Protein 60 for Treating Muscle Wasting and Cachexia.

Currently, no commercially available drugs have the ability to reverse cachexia or counteract muscle wasting and the loss of lean mass. Here, we report the methodology used to develop Physiactisome-a conditioned medium released by heat shock protein 60 (Hsp60)-overexpressing C2C12 cell lines enriched with small and large extracellular vesicles. We also present evidence supporting its use in the treatment of cachexia. Briefly, we obtain a nanovesicle-based secretion by genetically modifying C2C12 cell lines with an Hsp60-overexpressing plasmid. The secretion is used to treat naïve C2C12 cell lines. Physiactisome activates the expression of PGC-1α isoform 1, which is directly involved in mitochondrial biogenesis and muscle atrophy suppression, in naïve C2C12 cell lines. Proteomic analyses show Hsp60 localisation inside isolated nanovesicles and the localisation of several apocrine and merocrine molecules, with potential benefits for severe forms of muscle atrophy. Considering that Physiactisome can be easily obtained following tissue biopsy and can be applied to autologous muscle stem cells, we propose a potential nanovesicle-based anti-cachexia drug that could mimic the beneficial effects of exercise. Thus, Physiactisome may improve patient survival and quality of life. Furthermore, the method used to add Hsp60 into nanovesicles can be used to deliver other drugs or active proteins to vesicles.

Oxidative stress induces myeloperoxidase expression in endocardial endothelial cells from patients with chronic heart failure.

Increased oxidative stress has been implicated in the pathogenesis of a number of cardiovascular diseases. Recent findings suggest that myeloperoxidase (MPO) may play a key role in the initiation and maintenance of chronic heart failure (CHF) by contributing to the depletion of the intracellular reservoir of nitric oxide (NO). NO consumption through MPO activity may lead to protein chlorination or nitration, leading to tissue damage. Primary cultures of human endocardial endothelial cells (EEC) obtained at heart transplantation of patients with CHF and human umbilical vein endothelial cells (HUVEC) were subjected to oxidative stress by incubation with hydrogen peroxide at non lethal (60 microM) dose for different exposure times (3 and 6 h). Treated and control cells were tested by immunohistochemistry and RT-PCR for MPO and 3-chlorotyrosine expression. Both endothelial cell types expressed myeloperoxidase following oxidative stress, with higher levels in EEC. Moreover, 3-chlorotyrosine accumulation in treated cells alone indicated the presence of MPO-derived hypochlorous acid. Immunohistochemistry on sections from post-infarcted heart confirmed in vivo the endothelial positivity to MPO, 3-chlorotyrosine and, to a minor extent, nitrotyrosine. Immunohistochemical observations were confirmed by detection of MPO mRNA in both stimulated EEC and HUVEC cells. This study demonstrates for the first time that EEC can express MPO after oxidative stress, both in vitro and in vivo, followed by accumulation of 3-chlorotyrosine, an end product of oxidative stress. Deregulation of endothelial functions may contribute to the development of a number of cardiovascular diseases, including CHF. The results also highlight the notion that endothelium is not only a target but also a key player in oxidative-driven cardiovascular stress.

Isolation and characterization of Oct-4+/HLA-G+ mesenchymal stem cells from human umbilical cord matrix: differentiation potential and detection of new markers.

The presence of multipotent cells in several adult and embryo-related tissues opened new paths for their use in regenerative medicine. Extraembryonic tissues such as umbilical cord are considered a promising source of stem cells, potentially useful in therapy. The characterization of cells from the umbilical cord matrix (Wharton's Jelly) and amniotic membrane revealed the presence of a population of mesenchymal-like cells, sharing a set of core-markers expressed by "mesenchymal stem cells". Several reports enlightened the differentiation capabilities of these cells, even if at times the lack of an extensive characterization of surface markers and immune co-stimulators expression revealed hidden pitfalls when in vivo transplantation was performed. The present work describes a novel isolation protocol for obtaining mesenchymal stem cells from the umbilical cord matrix. These cells are clonogenic, retain long telomeres, can undergo several population doublings in vitro, and can be differentiated in mature mesenchymal tissues as bone and adipose. We describe for the first time that these cells, besides expressing all of the core-markers for mesenchymal stem cells, feature also the expression, at both protein and mRNA level, of tolerogenic molecules and markers of all the three main lineages, potentially important for both their differentiative potential as well as immunological features.

Cigarette smoke exposure inhibits extracellular MMP-2 (gelatinase A) activity in human lung fibroblasts.

BACKGROUND: Exposure to cigarette smoke is considered a major risk factor for the development of lung diseases, since its causative role has been assessed in the induction and maintenance of an inflamed state in the airways. Lung fibroblasts can contribute to these processes, due to their ability to produce proinflammatory chemotactic molecules and extracellular matrix remodelling proteinases. Among proteolytic enzymes, gelatinases A and B have been studied for their role in tissue breakdown and mobilisation of matrix-derived signalling molecules. Multiple reports linked gelatinase deregulation and overexpression to the development of inflammatory chronic lung diseases such as COPD. METHODS: In this study we aimed to determine variations in the gelatinolytic pattern of human lung fibroblasts (HFL-1 cell line) exposed to cigarette smoke extract (CSE). Gelatinolytic activity levels were determined by using gelatin zymography for the in-gel detection of the enzymes (proenzyme and activated forms), and the subsequent semi-quantitative densitometric evaluation of lytic bands. Expression of gelatinases was evaluated also by RT-PCR, zymography of the cell lysates and by western blotting. RESULTS: CSE exposure at the doses used (1-10%) did not exert any significant cytotoxic effects on fibroblasts. Zymographic analysis showed that CSE exposure resulted in a linear decrease of the activity of gelatinase A. Control experiments allowed excluding a direct inhibitory effect of CSE on gelatinases. Zymography of cell lysates confirmed the expression of MMP-2 in all conditions. Semi-quantitative evaluation of mRNA expression allowed assessing a reduced transcription of the enzyme, as well as an increase in the expression of TIMP-2. Statistical analyses showed that the decrease of MMP-2 activity in conditioned media reached the statistical significance (p = 0.0031 for 24 h and p = 0.0012 for 48 h), while correlation analysis showed that this result was independent from CSE cytotoxicity (p = 0.7833 for both exposures). CONCLUSION: Present work describes for the first time that, apart well characterized proinflammatory responses, human lung fibroblasts may react to CSE with a significant reduction of extracellular MMP-2 lytic activity. Therefore, fibroblasts may actively participate to the alteration of the proteolysis/antiproteolysis balance, which reflects the defective repair of the extracellular matrix. Such event should provide a further contribution to the maintenance of the inflamed state in the lungs.

Comparative analysis of the structure of temporomandibular joint in human and rabbit.

In order to increase knowledge on the morphology and structure of the articular disc of the TMJ for a better understanding of the functional role of the same, it proceeded with an investigation on histological samples in the block of 'TMJ and periarticular tissues of adult rabbits and human fetuses at different stage of development.

Skeletal muscle Heat shock protein 60 increases after endurance training and induces peroxisome proliferator-activated receptor gamma coactivator 1 α1 expression.

Heat shock protein 60 (Hsp60) is a chaperone localizing in skeletal muscle mitochondria, whose role is poorly understood. In the present study, the levels of Hsp60 in fibres of the entire posterior group of hindlimb muscles (gastrocnemius, soleus, and plantaris) were evaluated in mice after completing a 6-week endurance training program. The correlation between Hsp60 levels and the expression of four isoforms of peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC1α) were investigated only in soleus. Short-term overexpression of hsp60, achieved by in vitro plasmid transfection, was then performed to determine whether this chaperone could have a role in the activation of the expression levels of PGC1α isoforms. The levels of Hsp60 protein were fibre-type specific in the posterior muscles and endurance training increased its content in type I muscle fibers. Concomitantly with the increased levels of Hsp60 released in the blood stream of trained mice, mitochondrial copy number and the expression of three isoforms of PGC1α increased. Overexpressing hsp60 in cultured myoblasts induced only the expression of PGC1 1α, suggesting a correlation between Hsp60 overexpression and PGC1 1 α activation.

Nutrition, oxidative stress and intestinal dysbiosis: Influence of diet on gut microbiota in inflammatory bowel diseases.

BACKGROUND: Microbiota refers to the population of microorganisms (bacteria, viruses and fungi) that inhabit the entire gastrointestinal tract, more particularly the colon whose role is to maintain the integrity of the intestinal mucosa and control the proliferation of pathogenic bacteria. Alteration in the composition of the gut microbiota is called dysbiosis. Dysbiosis redisposes to inflammatory bowel diseases such as ulcerative colitis, Crohn disease and indeterminate colitis. METHODS: The purpose of this literature review is to elucidate the influence of diet on the composition of the gastrointestinal microbiota in the healthy gut and the role of diet in the development of dysbiosis. CONCLUSION: The "Western diet", in particular a low - fiber high fat/high carbohydrate diet is one factor that can lead to severe dysbiosis. In contrast, "mediterranean" and vegetarian diets that includes abundant fruits, vegetables, olive oil and oily fish are known for their anti-inflammatory effects and could prevent dysbiosis and subsequent inflammatory bowel disease.

Quantitative patterns of Hsps in tubular adenoma compared with normal and tumor tissues reveal the value of Hsp10 and Hsp60 in early diagnosis of large bowel cancer.

Large bowel carcinogenesis involves accumulation of genetic alterations leading to transformation of normal mucosa into dysplasia and, lastly, adenocarcinoma. It is pertinent to elucidate the molecular changes occurring in the pre-neoplastic lesions to facilitate early diagnosis and treatment. Heat shock proteins (Hsps), many of which are molecular chaperones, are implicated in carcinogenesis, and their variations with tumor progression encourage their study as biomarkers. There are many reports on Hsps and cancer but none to our knowledge on their systematic quantification in pre-neoplastic lesions of the large bowel. We performed immunohistochemical determinations of Hsp10, Hsp60, Hsp70, and Hsp90 in biopsies of large bowel tubular adenomas with moderate grade of dysplasia and compared to normal mucosa and adenocarcinoma with a moderate grade of differentiation (G2). A significant elevation of Hsp10 and Hsp60 only, i.e., in the absence of elevation of Hsp70 or Hsp90, in both epithelium and lamina propria was found in tubular adenoma by comparison with normal mucosa. In contrast, adenocarcinoma was characterized by the highest levels of Hsp10 and Hsp60 in epithelium and lamina propria, accompanied by the highest levels of Hsp70 only in epithelium and of Hsp90 only in lamina propria, by comparison with normal and tubular adenoma counterparts. Hsp10 and Hsp60 are promising biomarkers for early diagnosis of tubular adenoma and for its differentiation from more advanced malignant lesions. Hsp10 and Hsp60 may be implicated in carcinogenesis from its very early steps and, thus, are potentially convenient targets for therapy.

Alcoholic Liver Disease: A Mouse Model Reveals Protection by Lactobacillus fermentum.

OBJECTIVES: Alcoholism is one of the most devastating diseases with high incidence, but knowledge of its pathology and treatment is still plagued with gaps mostly because of the inherent limitations of research with patients. We developed an animal model for studying liver histopathology, Hsp (heat-shock protein)-chaperones involvement, and response to treatment. METHODS: The system was standardized using mice to which ethanol was orally administered alone or in combination with Lactobacillus fermentum following a precise schedule over time and applying, at predetermined intervals, a battery of techniques (histology, immunohistochemistry, western blotting, real-time PCR, immunoprecipitation, 3-nitrotyrosine labeling) to assess liver pathology (e.g., steatosis, fibrosis), and Hsp60 and iNOS (inducible form of nitric oxide synthase) gene expression and protein levels, and post-translational modifications. RESULTS: Typical ethanol-induced liver pathology occurred and the effect of the probiotic could be reliably monitored. Steatosis score, iNOS levels, and nitrated proteins (e.g., Hsp60) decreased after probiotic intake. CONCLUSIONS: We describe a mouse model useful for studying liver disease induced by chronic ethanol intake and for testing pertinent therapeutic agents, e.g., probiotics. We tested L. fermentum, which reduced considerably ethanol-induced tissue damage and deleterious post-translational modifications of the chaperone Hsp60. The model is available to test other agents and probiotics with therapeutic potential in alcoholic liver disease.

The expression of HSP60 and HSP10 in large bowel carcinomas with lymph node metastase.

BACKGROUND: The involvement of Heat Shock Proteins (HSP) in cancer development and progression is a widely debated topic. The objective of the present study was to evaluate the presence and expression of HSP60 and HSP10 in a series of large bowel carcinomas and locoregional lymph nodes with and without metastases. METHODS: 82 Astler and Coller's stage C2 colorectal cancers, of which 48 well-differentiated and 34 poorly-differentiated, were selected along with 661 lymph nodes, including 372 with metastases and 289 with reactive hyperplasia only, from the same tumours. Primitive tumours and both metastatic and reactive lymph nodes were studied; specifically, three different compartments of the lymph nodes, secondary follicle, paracortex and medullary sinus, were also analysed. An immunohistochemical research for HSP60 and HSP10 was performed and the semiquantitative results were analysed by statistical analysis to determine the correlation between HSPs expression and 1) tumour grading; 2) degree of inflammation; 3) number of lymph nodes involved; 4) lymph node compartment hyperplasia. Moreover, western blotting was performed on a smaller group of samples to confirm the immunohistochemical results. RESULTS: Our data show that the expression of HSP60, in both primary tumour and lymph node metastasis, is correlated with the tumoral grade, while the HSP10 expression is not. Nevertheless, the levels of HSP10 are commonly higher than the levels of HSP60. In addition, statistical analyses do not show any correlation between the degree of inflammation and the immunopositivity for both HSP60 and HSP10. Moreover, we find a significant correlation between the presence of lymph node metastases and the positivity for both HSP60 and HSP10. In particular, metastatic lymph nodes show a higher percentage of cells positive for both HSP60 and HSP10 in the secondary follicles, and for HSP10 in the medullary sinuses, when compared with hyperplastic lymph nodes. CONCLUSION: HSP60 and HSP10 may have diagnostic and prognostic significance in the management of this tumour and their overexpression in tumoral cells may be functionally related to tumoral progression. We hypothesise that their expression in follicular and medullary cells of lymph nodes may be induced by formation of metastases. Further studies based on these observations could lead to a better understanding of the HSPs involvement in colorectal cancer progression, as well as other neoplasms.

Senescence-associated HSP60 expression in normal human skin fibroblasts.

Normal mammalian fibroblasts cultured in vitro undergo a limited number of divisions before entering a senescent phase in which they can be maintained for long periods but cannot be induced to divide. Senescent cells become unresponsive to growth-promoting signals and exhibit senescent cell morphology with flattened and enlarged cell shape. Several chaperones have a direct effect on cellular senescence. HSP60 has been largely studied in our laboratories and it has been associated with uncontrolled cell proliferation in tumor cells. Since senescence is firmly regulated during cell cycle progression, we wanted to investigate HSP60 protein level during cellular senescence. Our data show that HSP60 increases during the initial stage of senescence and that it is localized in cellular compartments, resembling mitochondria. An increase in HSP60 protein amount is associated with a cell cycle slow-down and it may have a role in cell cycle progression.

Cigarette smoke causes caspase-independent apoptosis of bronchial epithelial cells from asthmatic donors.

BACKGROUND: Epidemiologic studies have demonstrated important links between air pollution and asthma. Amongst these pollutants, environmental cigarette smoke is a risk factor both for asthma pathogenesis and exacerbation. As the barrier to the inhaled environment, the bronchial epithelium is a key structure that is exposed to cigarette smoke. OBJECTIVES: Since primary bronchial epithelial cells (PBECs) from asthmatic donors are more susceptible to oxidant-induced apoptosis, we hypothesized that they would be susceptible to cigarette smoke-induced cell death. METHODS: PBECs from normal and asthmatic donors were exposed to cigarette smoke extract (CSE); cell survival and apoptosis were assessed by fluorescence-activated cell sorting, and protective effects of antioxidants evaluated. The mechanism of cell death was evaluated using caspase inhibitors and immunofluorescent staining for apoptosis-inducing factor (AIF). RESULTS: Exposure of PBEC cultures to CSE resulted in a dose-dependent increase in cell death. At 20% CSE, PBECs from asthmatic donors exhibited significantly more apoptosis than cells from non-asthmatic controls. Reduced glutathione (GSH), but not ascorbic acid (AA), protected against CSE-induced apoptosis. To investigate mechanisms of CSE-induced apoptosis, caspase-3 or -9 inhibitors were tested, but these failed to prevent apoptosis; in contrast, CSE promoted nuclear translocation of AIF from the mitochondria. GSH reduced the number of nuclear-AIF positive cells whereas AA was ineffective. CONCLUSION: Our results show that PBECs from asthmatic donors are more susceptible to CSE-induced apoptosis. This response involves AIF, which has been implicated in DNA damage and ROS-mediated cell-death. Epithelial susceptibility to CSE may contribute to the impact of environmental tobacco smoke in asthma.

CD1a and antitumour immune response.

Primary immune response is based on the capacity of local professional antigen-presenting cells (whose prototype is represented by dendritic cells, DCs) to take up and present antigens to selected clones of T cells, but also to non-specific effector cells such as macrophages or natural killer cells. The four CD1 proteins, all of which share a limited homology to class I MHC proteins, are differently expressed in various cell types, of both mesenchymal and, as recently described, epithelial lineage. Regarding the role of CD1 molecules in the anti-tumour response, it has been reported that CD1+ dendritic cells are involved in the first steps of the primary immune response in a number of malignancies. Moreover, the presence of a high number of DCs in the tumoral or peritumoral area, as well as in the draining lymph nodes, has been shown to correlate with a better prognosis. A recent report on the presence of CD1a in metaplastic epithelial cells of Barrett esophagus introduced new questions about CD1a expression patterns. Moreover, the strong correlation between the lack of CD1a+ cells and the malignant evolution of the lesion may indicate a possible role of non-professional APCs in mediating and/or potentiating immune responses to tumours.