Key pointsr Weaning from mechanical ventilation (MV) of long-term intensive care unit (ICU) patients is delayed by impaired respiratory muscle function; however, the mechanisms that cause the impairment are not fully understood.r A novel experimental rat ICU model was used for time-resolved analyses (6 h to 2 weeks) of the effects of MV on diaphragm muscle fibre structure and function, and on gene and protein expression.r A prompt and progressive decline of diaphragm muscle fibre function, preceding atrophy, occurred with MV, and at the end of 2 weeks residual diaphragm muscle fibre function was <15% of control levels.r Cellular and subcellular analyses indicated that oxidative stress-triggered protein modifications had significantly diminished diaphragm muscle fibre function.r The novel finding that activation of proteolytic pathways and regulation of contractile protein synthesis were different in diaphragm and limb muscles has direct implications for the design of muscle-specific intervention strategies.Abstract Controlled mechanical ventilation (CMV) plays a key role in triggering the impaired diaphragm muscle function and the concomitant delayed weaning from the respirator in critically ill intensive care unit (ICU) patients. To date, experimental and clinical studies have primarily focused on early effects on the diaphragm by CMV, or at specific time points. To improve our understanding of the mechanisms underlying the impaired diaphragm muscle function in response to mechanical ventilation, we have performed time-resolved analyses between 6 h and 14 days using an experimental rat ICU model allowing detailed studies of the diaphragm in response to long-term CMV. A rapid and early decline in maximum muscle fibre force and preceding muscle fibre atrophy was observed in the diaphragm in response to CMV, resulting in an 85% reduction in residual diaphragm fibre function after 9-14 days of CMV. A modest loss of contractile proteins was observed and linked to an early activation of the ubiquitin proteasome pathway, myosin:actin ratios were not affected and the transcriptional regulation of myosin isoforms did not show any dramatic changes during the observation period. Furthermore, small angle X-ray diffraction analyses demonstrate that myosin can bind to actin in an ATP-dependent manner even after 9-14 days of exposure to CMV. Thus, quantitative changes in muscle fibre size and contractile proteins are not the dominating factors underlying the dramatic decline in diaphragm muscle function in response to CMV, in contrast to earlier observations in limb muscles. The observed early loss of subsarcolemmal neuronal nitric oxide synthase activity, onset of oxidative stress, intracellular lipid accumulation and post-translational protein modifications strongly argue for significant qualitative changes in contractile proteins causing the severely impaired residual function in diaphragm fibres after long-term mechanical ventilation. For the first time, the present study demonstrates novel changes in the diaphragm struct...

BackgroundAcute quadriplegic myopathy (AQM) or critical illness myopathy (CIM) is frequently observed in intensive care unit (ICU) patients. To elucidate duration-dependent effects of the ICU intervention on molecular and functional networks that control the muscle wasting and weakness associated with AQM, a gene expression profile was analyzed at time points varying from 6 hours to 14 days in a unique experimental rat model mimicking ICU conditions, i.e., post-synaptically paralyzed, mechanically ventilated and extensively monitored animals.ResultsDuring the observation period, 1583 genes were significantly up- or down-regulated by factors of two or greater. A significant temporal gene expression pattern was constructed at short (6 h-4 days), intermediate (5-8 days) and long (9-14 days) durations. A striking early and maintained up-regulation (6 h-14d) of muscle atrogenes (muscle ring-finger 1/tripartite motif-containing 63 and F-box protein 32/atrogin-1) was observed, followed by an up-regulation of the proteolytic systems at intermediate and long durations (5-14d). Oxidative stress response genes and genes that take part in amino acid catabolism, cell cycle arrest, apoptosis, muscle development, and protein synthesis together with myogenic factors were significantly up-regulated from 5 to 14 days. At 9-14 d, genes involved in immune response and the caspase cascade were up-regulated. At 5-14d, genes related to contractile (myosin heavy chain and myosin binding protein C), regulatory (troponin, tropomyosin), developmental, caveolin-3, extracellular matrix, glycolysis/gluconeogenesis, cytoskeleton/sarcomere regulation and mitochondrial proteins were down-regulated. An activation of genes related to muscle growth and new muscle fiber formation (increase of myogenic factors and JunB and down-regulation of myostatin) and up-regulation of genes that code protein synthesis and translation factors were found from 5 to 14 days.ConclusionsNovel temporal patterns of gene expression have been uncovered, suggesting a unique, coordinated and highly complex mechanism underlying the muscle wasting associated with AQM in ICU patients and providing new target genes and avenues for intervention studies.

Mast cells have been implicated in malignant processes, mainly through clinical correlative studies and by experiments performed using animals lacking mast cells due to defective c-kit signaling. However, mast cell-deficient mouse models based on c-kit defects have recently been questioned for their relevance. Here we addressed the effect of mast cells in a tumor setting by using transgenic Mcpt5-Cre+ R-DTA+ mice, in which the deficiency of mast cells is independent of c-kit defects. Melanoma cells (B16.F10) were administered either subcutaneously or intravenously into Mcpt5-Cre+ R-DTA+ mice or Mcpt5-Cre− R-DTA+ littermate controls, followed by the assessment of formed tumors. In the subcutaneous model, mast cells were abundant in the tumor stroma of control mice but were absent in Mcpt5-Cre+ R-DTA+ mice. However, the absence of mast cells did not affect tumor size. In contrast, after intravenous administration of B16.F10 cells, melanoma colonization of the lungs was markedly reduced in Mcpt5-Cre+ R-DTA+ vs. Mcpt5-Cre− R-DTA+ animals. Decreased melanoma colonization of the lungs in Mcpt5-Cre+ R-DTA+ animals was accompanied by increased inflammatory cell recruitment into the bronchoalveolar lavage fluid, suggesting that mast cells suppress inflammation in this setting. Further, qPCR analysis revealed significant alterations in the expression of Twist and E-cadherin in lungs of Mcpt5-Cre+ R-DTA+ vs. control Mcpt5-Cre− R-DTA+ animals, suggesting an impact of mast cells on epithelial-mesenchymal transition. In conclusion, this study reveals that mast cells promote melanoma colonization of the lung.

Mast cell secretory granules are densely packed with various bioactive mediators including proteases of chymase, tryptase and CPA3 type. Previous studies have indicated that mast cells can affect the outcome of melanoma but the contribution of the mast cell granule proteases to such effects has not been clear. Here we addressed this issue by assessing mice lacking either the chymase Mcpt4, the tryptase Mcpt6 or carboxypeptidase A3 (Cpa3), as well as mice simultaneously lacking all three proteases, in a model of melanoma dissemination from blood to the lung. Although mice with individual deficiency in the respective proteases did not differ significantly from wildtype mice in the extent of melanoma colonization, mice with multiple protease deficiency (Mcpt4/Mcpt6/Cpa3-deficient) exhibited a higher extent of melanoma colonization in lungs as compared to wildtype animals. This was supported by higher expression of melanoma-specific genes in lungs of Mcpt4/Mcpt6/CPA3-deficient vs. wildtype mice. Cytokine profiling showed that the levels of CXCL16, a chemokine with effects on T cell populations and NKT cells, were significantly lower in lungs of Mcpt4/Mcpt6/Cpa3-deficient animals vs. controls, suggesting that multiple mast cell protease deficiency might affect T cell or NKT cell populations. In line with this, we found that the Mcpt4/Mcpt6/Cpa3-deficiency was associated with a reduction in cells expressing CD1d, a MHC class 1-like molecule that is crucial for presenting antigen to invariant NKT (iNKT) cells. Together, these findings indicate a protective role of mast cell-specific proteases in melanoma dissemination, and suggest that this effect involves a CXCL16/CD1d/NKT cell axis.

Mastitis is an inflammatory condition of the mammary tissue and represents a major problem for the dairy industry worldwide. The present study was undertaken to study how experimentally induced acute bovine mastitis affects inflammatory parameters and changes in the metabolome. To this end, we induced experimental mastitis in nine cows by intramammary infusion of 100 µg purified Escherichia coli lipopolysaccharide (LPS) followed by kinetic assessments of cytokine responses (by enzyme-linked immunosorbent assay), changes in the metabolome (assessed by nuclear magnetic resonance), clinical parameters (heat, local pain perception, redness, swelling, rectal temperature, clot formation, and color changes in the milk), and milk somatic cell counts, at several time points post LPS infusion. Intramammary LPS infusion induced clinical signs of mastitis, which started from 2 h post infusion and had returned to normal levels within 24–72 h. Milk changes were seen with a delay compared with the clinical signs and persisted for a longer time. In parallel, induction of IL-6 and TNF-α were seen in milk, and there was also a transient elevation of plasma IL-6 whereas plasma TNF-α was not significantly elevated. In addition, a robust increase in CCL2 was seen in the milk of LPS-infused cows, whereas G-CSF, CXCL1, and histamine in milk were unaffected. By using a metabolomics approach, a transient increase of plasma lactose was seen in LPS-induced cows. In plasma, significant reductions in ketone bodies (3-hydroxybutyrate and acetoacetate) and decreased levels of short-chain fatty acids, known to be major products released from the gut microbiota, were observed after LPS infusion; a profound reduction of plasma citrate was also seen. Intramammary LPS infusion also caused major changes in the milk metabolome, although with a delay in comparison with plasma, including a reduction of lactose. We conclude that the LPS-induced acute mastitis rapidly affects the plasma metabolome and cytokine induction with similar kinetics as the development of the clinical signs, whereas the corresponding effects in milk occurred with a delay.

Here we addressed the potential impact of chymase, a mast-cell restricted protease, on mouse bone phenotype. We show that female mice lacking the chymase Mcpt4 acquired a persistent expansion of diaphyseal bone in comparison with wild type controls, reaching a 15% larger diaphyseal cross sectional area at 12 months of age. Mcpt4-/- mice also showed increased levels of a bone anabolic serum marker and higher periosteal bone formation rate. However, they were not protected from experimental osteoporosis, suggesting that chymase regulates normal bone homeostasis rather than the course of osteoporosis. Further, the absence of Mcpt4 resulted in age-dependent upregulation of numerous genes important for bone formation but no effects on osteoclast activity. In spite of the latter, Mcpt4-/- bones had increased cortical porosity and reduced endocortical mineralization. Mast cells were found periosteally and, notably, bone-proximal mast cells in Mcpt4-/- mice were degranulated to a larger extent than in wild type mice. Hence, chymase regulates degranulation of bone mast cells, which could affect the release of mast cell-derived factors influencing bone remodelling. Together, these findings reveal a functional impact of mast cell chymase on bone. Further studies exploring the possibility of using chymase inhibitors as a strategy to increase bone volume may be warranted.

Current chemotherapy for treatment of pediatric acute leukemia, although generally successful, is still a matter of concern due to treatment resistance, relapses and life-long side effects for a subset of patients. Inhibition of dynamin, a GTPase involved in clathrin-mediated endocytosis and regulation of the cell cycle, has been proposed as a potential anti-cancer regimen, but the effects of dynamin inhibition on leukemia cells has not been extensively addressed. Here we adopted single cell and whole-population analysis by flow cytometry and live imaging, to assess the effect of dynamin inhibition (Dynasore, Dyngo-4a, MitMAB) on pediatric acute leukemia cell lines (CCRF-CEM and THP-1), human bone marrow biopsies from patients diagnosed with acute lymphoblastic leukemia (ALL), as well as in a model of lymphoma (EL4)-induced tumor growth in mice. All inhibitors suppressed proliferation and induced pronounced caspase-dependent apoptotic cell death in CCRF-CEM and THP-1 cell lines. However, the inhibitors showed no effect on bone marrow biopsies, and did not prevent EL4-induced tumor formation in mice. We conclude that dynamin inhibition affects highly proliferating human leukemia cells. These findings form a basis for evaluation of the potential, and constraints, of employing dynamin inhibition in treatment strategies against leukemia and other malignancies.

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