High blood levels of IL-6 nicely correlate with animal survival in trained C26 bearing mice

Exercise is a beneficial adjunct therapy to maintain or enhance quality of life in cancer patients. Recently, few studies demonstrated a correlation between high concentrations of IL-6 and a poor survival. This depends on the equilibrium between the concentrations of IL-6 and sIL-6R. Exercise induces a beneficial increase in circulating IL-6 (1). Fresh fragments of solid C26 tumor were inoculated in healthy 3 months-old mice (n=230, M=115 and F=115). The experimental procedure were 12 weeks long. During the first 6 weeks, mice were randomly assigned to one of the experimental conditions: sedentary (SED) or progressive training (TRP). After the first 6 weeks, all mice were inoculated with a fresh fragment of tumor. All trained adult mice after the tumor inoculation were randomly assigned to a different training program: low intensity training (TRL), moderate intensity training (TRM) and high intensity training (TRH). Mice run 5 days per week on a Rota-Rod following one of the specific training program (TRP ,TRL, TRM and TRH) (2). After tumor inoculation the mice were daily weighted and tumor size monitored until death. Moreover, 8 mice for each group were sacrificed when cachexia occurred (>9% body weight loss), and blood samples were stored for CBA Enhanced flex set flow-cytometric assays (IL-6 and TNF-alpha). The TRM and TRH training protocol performed by trained adult male mice extend the median survival compared to the sedentary adult mice and trained female mice. Interesting the beneficial effect of exercise seemed to be mediated extending the survival days. Significant high blood levels of IL-6 were recorded among the male trained mice (TRM and TRH) groups in comparison with sedentary adult mice and trained female mice (TRM and TRH). The results suggest that endurance exercise as adjuvant therapy is gender and physical training level specific. This effect seems to be mediated by IL-6 blood levels

Endurance training induces apoptosis in the tumor mass in the C26-bearing mouse model

Cachexia, sarcopenia and anorexia are characterised by muscle wasting. This condition is a weakening, shrinking, and loss of muscle caused by a disease or lack of use. The loss of muscle causes a decrease in strength and inability to move compromising the quality of life. Recently we demonstrated that the skeletal muscle of endurance trained Balb/c mice release IL-6 and Hsp60 (inside exosomes) in the blood stream. We studied the expression of Hsp60 in the muscles of trained and untrained C26-bearing mice, to understand if Hsp60 was over-expression may improve muscle performance and reduce cachexia. Four different interleukins have been also studied in cachectic mice, to understand which was their effect on Hsp60 expression both in the tumor mass and the trained muscle. In the present study we demonstrated that: 1) IL-6 is released by the trained muscle; 2) IL-6 is release also by the tumor mass, 3) in animals inoculated with the C26 tumor and trained after inoculation, IL-6 is synthesized mainly by the skeletal muscle and the tumor mass undergo apoptosis

Conjugated linoleic acid (CLA) stimulates mitochondrial biogenesis by PGC-1alpha in trained mice

Conjugated linoleic acid (CLA) has been reported to improve muscle hypertrophy, steroidogenesis, physical activity and endurance capacity in mice (1,2), however the mode of action is not completely understood. The aims of the present study were to identify the pathway stimulated by CLA supplementation on mitochondrial biogenesis, one of the most important adaptive response in skeletal muscle after endurance exercise. Mice were randomly assigned to one of four groups (n = 8 per group): placebo sedentary (PLA-SED); CLA sedentary (CLA-SED); placebo trained (PLA-TR); or CLA trained (CLA-TR). The CLA groups were gavaged with 35 μL per day (corresponding to the 0.5% of food ingested, approximately 4 g) Tonalin® FFA 80 food supplement containing CLA throughout the 6 week experimental period, while the placebo groups were gavaged with 35 μL per day sunflower oil. Trained groups performed progressive running on the rotarod for 6 weeks at increasing speed and duration (3). Preliminary findings may suggest that CLA supplementation potentiate mitochondrial biogenesis in trained skeletal muscle via PGC-1 alpha, although further studies need to be conducted to delineate the signaling cascade

Hsp60 and interleukins expression in the skeletal muscle and its implications in exercise and cachexia

Heat shock protein 60 (Hsp60) is a chaperon localizing in skeletal muscle mitochondria, whose role is poorly understood. This chaperone has been found also in other cellular localizations. In the three years we studied the levels of Hsp60 in fibres of the entire posterior group of hindlimb muscles (gastrocnemius, soleus, and plantaris) in mice after completing a 6-week endurance training program. In this evaluation we correlated Hsp60 levels with the expression of four isoforms of the peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α). Moreover, the short-term overexpression of hsp60, achieved by in vitro plasmid transfection was performed to determine whether this chaperon could have a role in the activation of the expression levels of PGC-1α 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 PGC-1α increased. Overexpressing hsp60 in cultured myoblasts induced only the expression of PGC-1 α1, suggesting a correlation between Hsp60 overexpression and PGC-1 α1 activation. We are now studying the expression of Hsp60 in the muscles of trained and untrained C26-bearing mice, to understand if Hsp60 over expression may improve muscle performance and reduce cachexia. Four different interleukins have been also studied in cachectic mice, to understand which can be the effect of them on Hsp60 expression both in the tumor mass and the trained muscle.This work was funded by PRIN2009 - Prof. G. Zummo and PRIN2012 - Prof. Farina F

Skeletal muscle heat shock protein 60 increases after endurance training in mice and induces peroxisome proliferation-activated receptor-γ coactivator-1 α1 expression

Heat shock protein (Hsp60) is a mitochondrial chaperonin whose unconventional cellular localizations and functions are discovered day by day. In the present study, the levels of Hsp60 in fibres of the soleus muscle and its correlation to the expression of four isoforms of peroxisome proliferation-activated receptor-γ (PPAR-γ) coactivator-1α (PGC1α) were investigated in 72 young (7-weeks old) healthy male mice (BALB/c AnNHsd) at baseline and after completing a 6-week endurance training program. The mice were assigned to one of the two experimental groups: SED (sedentary) or TR (trained). Short-term overexpression of hsp60, achieved by in vitro plasmid transfection, was then performed to determine whether this chaperonin could have a role in the activation of the expression levels of PGC-1α isoforms. The levels of Hsp60 protein were fibre-type specific in the posterior muscles at baseline, 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 PGC-1α increased. Overexpressing hsp60 in cultured myoblasts induced only the expression of PGC-1 α1, letting us suppose a direct correlation between Hsp60 overexpression and PGC-1 α1 activation. Overall, these results suggest that during endurance training Hsp60 is upregulated and activates the mitochondrial biogenesis pathway, probably as a response to the oxidative stress induced by exercise. This study reveals a molecular response of skeletal muscle to a mechanical stress induced by training which involves the molecular chaperonin Hsp60 and the transcriptional co-activator PGC-1 α1. The role of these proteins in aerobic adaptation and pathological conditions as cancer cachexia warrants further investigations

HSP90 and eNOS partially co-localize and change cellular localization in relation to different ECM components in 2D and 3D cultures of adult rat cardiomyocytes.

Background information. Cultivation techniques promoting three-dimensional organization of mammalian cells are of increasing interest, since they confer key functionalities of the native ECM (extracellular matrix) with a power for regenerative medicine applications. Since ECM compliance influences a number of cell functions, Matrigel-based gels have become attractive tools, because of the ease with which their mechanical properties can be controlled. In the present study, we took advantage of the chemical and mechanical tunability of commonly used cell culture substrates, and co-cultures to evaluate, on both two- and three-dimensional cultivated adult rat cardiomyocytes, the impact of ECM chemistry and mechanics on the cellular localization of two interacting signalling proteins: HSP90 (heat-shock protein of 90 kDa) and eNOS (endothelial nitric oxide synthase). Results. Freshly isolated rat cardiomyocytes were cultured on fibronectin, Matrigel gel or laminin, or in co-culture with cardiac fibroblasts, and tested for both integrity and viability. As validation criteria, integrity of both plasma membrane and mitochondria was evaluated by transmission electron microscopy. Cell sensitivity to microenvironmental stimuli was monitored by immunofluorescence and confocal microscopy. We found that HSP90 and eNOS expression and localization are affected by changes in ECM composition. Elaboration of the images revealed, on Matrigel-cultured cardiomyocytes, areas of high co-localization between HSP90 and eNOS and co-localization coefficients, which indicated the highest correlation with respect to the other substrates. Conclusions. Our three-dimensional adult cardiomyocyte cultures are suitable for both analysing cell–ECM interactions at electron and confocal microscopy levels and monitoring micro-environment impact on cardiomyocyte phenotype

Clinical anatomic, immunomorphologic and molecular anatomic data suggest interplay of thyroidal molecules, autoantibodies and Hsp60 in Hashimoto’s disease

Hsp60 is, typically, a mitochondrial protein, but it also occurs in the cytosol, vesicles, and plasma membrane, and in the intercellular space and biological fluids, e.g., blood. Changes in the levels and distribution of Hsp60 are linked to several pathologies, including cancer and chronic inflammatory and autoimmune disorders. What is the histopathological pattern of Hsp60 in the thyroid of Hashimoto’s patients? Are there indications of a pathogenic role of Hsp60 that may make Hashimoto’s thyroiditis a chaperonopathy? Experiments reported here provide information regarding those questions. We found by various immunomorphological techniques increased levels of Hsp60 in the thyroid from HT patients, localized to thyrocytes of small and degenerated follicles and to oncocytes (Hurtle cells). Immunofluorescence showed the chaperonin both inside the cells and also in the plasma membrane, especially in oncocytes. We also found that Hsp60 levels in the blood of HT patients were increased compared to controls and correlated with those of autoantibodies against two distinctive thyroidal proteins, thyroglobulin (TG) and thyroid peroxidase (TPO) (r=0.379, p=0.0103; r=0.484, p=0.0008; respectively). Molecular analysis of these two proteins in comparison with Hsp60 demonstrated various regions of high structural similarity shared by them, which could very well be immunologically crossreactive epitopes. Thus, it is likely that the three proteins potentiate each other as immunogens to elicit autoantibodies and, as antigens, to cause antigen-antibody reactions at those sites in which Hsp60 is exposed, for example the surface of oncocytes. This would lead to inflammation and oncocyte lysis with destruction of thyroidal tissue. The cytometric bead assay revealed that recombinant Hsp60 did not induce increment of cytokine production by peripheral blood mononuclear cells from HT patients. Consequently, we propose that Hsp60 is implicated in the pathogenesis of Hashimoto’s thyroiditis as autoantigen, via a participation of autoantibodies that also recognize TG and TPO, whereas participation of inflammatory cytokines induced by the chaperonin is unlikely. Supported by IEMEST (FC and AJLM)

Clinical anatomic, immunomorphologic and molecular anatomic data suggest interplay of thyroidal molecules, autoantibodies and Hsp60 in Hashimoto’s disease

Hsp60 is, typically, a mitochondrial protein, but it also occurs in the cytosol, vesicles, and plasma membrane, and in the intercellular space and biological fluids, e.g., blood. Changes in the levels and distribution of Hsp60 are linked to several pathologies, including cancer and chronic inflammatory and autoimmune disorders. What is the histopathological pattern of Hsp60 in the thyroid of Hashimoto’s patients? Are there indications of a pathogenic role of Hsp60 that may make Hashimoto’s thyroiditis a chaperonopathy? Experiments reported here provide information regarding those questions. We found by various immunomorphological techniques increased levels of Hsp60 in the thyroid from HT patients, localized to thyrocytes of small and degenerated follicles and to oncocytes (Hurtle cells). Immunofluorescence showed the chaperonin both inside the cells and also in the plasma membrane, especially in oncocytes. We also found that Hsp60 levels in the blood of HT patients were increased compared to controls and correlated with those of autoantibodies against two distinctive thyroidal proteins, thyroglobulin (TG) and thyroid peroxidase (TPO) (r=0.379, p=0.0103; r=0.484, p=0.0008; respectively). Molecular analysis of these two proteins in comparison with Hsp60 demonstrated various regions of high structural similarity shared by them, which could very well be immunologically crossreactive epitopes. Thus, it is likely that the three proteins potentiate each other as immunogens to elicit autoantibodies and, as antigens, to cause antigen-antibody reactions at those sites in which Hsp60 is exposed, for example the surface of oncocytes. This would lead to inflammation and oncocyte lysis with destruction of thyroidal tissue. The cytometric bead assay revealed that recombinant Hsp60 did not induce increment of cytokine production by peripheral blood mononuclear cells from HT patients. Consequently, we propose that Hsp60 is implicated in the pathogenesis of Hashimoto’s thyroiditis as autoantigen, via a participation of autoantibodies that also recognize TG and TPO, whereas participation of inflammatory cytokines induced by the chaperonin is unlikely. Supported by IEMEST (FC and AJLM)

Role of different endurance training program on cancer cachexia: pointing particular attention to the gender and age differences

Evidence from recent publications indicates that repeated exercise may enhance the quality of life of cancer patients (Maddocks et al., 2012). Regular physical activity may attenuate the adverse effects of cancer therapy, prevent or reverse cachexia and improve survival, although not all the patients are able or willing to undertake programs currently being offered. The aims of this study were to analyze: i) the effects of a progressive endurance exercise (progressive Training, pTR) on survival and cachexia in sedentary (SED) mice inoculated (I) with a fresh fragment of solid C26 tumor [SED-I-pTR; SED-I-SED]; ii) the effect of different protocols of endurance exercise (Trained for 30 min, TR30; Trained for 60 min, TR60; Trained for 120 min, TR120) on survival and cachexia in trained mice inoculated (I) with a fresh fragment of solid C26 tumor [pTR-I-TR30’; pTR-I-TR60’; pTR-I-TR120’]. All the conditions were tested to evaluate the gender (male and female) and age differences (young, 7-weeks old; adult, 3-months old; old, 15-months old). Mice were trained on a rota-rod for 6 weeks (5 times per week). Male sedentary mice (SED-I-SED) showed a higher median survival than sedentary female mice (for each age group); moreover adult mice survive more than sedentary young and old mice (for both gender groups). The endurance training improved the survival of mice in which the tumor was more aggressive (young and old), especially in female mice. Moreover, in the female old mice the progressive training exercise conducted before the inoculation seems to prolong survival. The data suggest that the endurance exercise as adjuvant therapy in cachexia needs to be gender and age specific

Fibromyalgia: Could hyperbaric oxygen therapy make the difference? Our experience

Key Clinical Message Fibromyalgia is a rare disease, difficult to diagnose and to treat. We think that hyperbaric oxygen therapy could improve its signs and symptoms although more evidences have to be accumulated