Increased glycolysis and HIF-1α activity are characteristics of cells under hypoxic or inflammatory conditions. Besides, in normal O
environments, elevated rates of glycolysis support critical ...cellular mechanisms such as cell survival. The purpose of this study was to analyze the contribution of HIF-1α to the energy metabolism and survival of human synovial fibroblasts (SF) under normoxic conditions. HIF-1α was silenced using lentiviral vectors or small-interfering RNA (siRNA) duplexes. Expression analysis by qRT-PCR and western blot of known HIF-1α target genes in hypoxia demonstrated the presence of functional HIF-1α in normoxic SF and confirmed the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a HIF-1α target even in normoxia. HIF-1α silencing induced apoptotic cell death in cultured SF and, similarly, treatment with glycolytic, but not with OXPHOS inhibitors, induced SF death. Finally, in vivo HIF-1α targeting by siRNA showed a significant reduction in the viability of human SF engrafted into a murine air pouch. Our results demonstrate that SF are highly dependent on glycolytic metabolism and that HIF-1α plays a regulatory role in glycolysis even under aerobic conditions. Local targeting of HIF-1α provides a feasible strategy to reduce SF hyperplasia in chronic arthritic diseases.
The functional relevance of synovial ectopic lymphoid neogenesis (ELN) in rheumatoid arthritis (RA) remains unknown. As ELN correlates with the degree of tissue inflammation, we investigated whether ...ELN was associated with specific cytokine profiles.
Synovial ELN was determined by immunohistology and long CD21 isoform (CD21L) expression. Cytokine expression was determined by multiplex enzyme-linked immunosorbent assay (ELISA) and quantitative polymerase chain reaction (PCR) as well as immunohistology in synovial fluid (SF) (n = 44) and tissue (ST) (n = 108), respectively. Production of ELN-associated chemokines by fibroblast-like synoviocytes (FLS) was studied in vitro.
Screening analysis of SF by multiplex ELISA showed higher protein levels of interleukin (IL)-23 (p = 0.018) and IL-17F (p = 0.028) in ELN+ versus ELN- samples. Other cytokines, including IL-17A, IL-6, and tumor necrosis factor (TNF)-α, were not different. The association between IL-23 and ELN was not biased by disease activity or other clinical features and was confirmed by higher IL-23 mRNA expression in ELN+ versus ELN- ST samples (p = 0.030), a correlation between IL-23 and CD21L expression in the same samples (r = 0.70 p < 0.0001), and a similar correlation in two independent ST sample sets (r = 0.778 p < 0.0001 and r = 0.817 p = 0.011). IL-23 p19 staining was neither restricted nor enhanced in close proximity of ectopic lymphoid follicles, and neither IL-23 nor IL-17A stimulation induced expression of the ELN-associated CC chemokine ligand, CCL21 and CXC chemokine ligand CXCL13, by FLS. Downstream of IL-23, CD21L expression was significantly associated with IL-17F, IL-21, and IL-22, but not IL-17A in two independent ST sample sets.
Synovial ELN in RA is strongly associated with activation of the IL-23 pathway but not with IL-17A.
The early stages of the COVID-19 pandemic presented the characteristics of a traumatic event that could trigger post-traumatic stress disorder. Emergency Medical Services workers are already a ...high-risk group due to their professional development. The research project aimed to analyse the impact of the COVID-19 pandemic on EMS professionals in terms of their mental health. For this purpose, we present a descriptive crosssectional study with survey methodology. A total of 317 EMS workers (doctors, nurses, and emergency medical technicians) were recruited voluntarily. Psychological distress, post-traumatic stress disorder, and insomnia were assessed. The instruments were the General Health Questionnaire-12 (GHQ-12), the Davidson Trauma Scale (DTS-8), and the Athens Insomnia Scale (AIS-8). We found that 36% of respondents had psychological distress, 30.9% potentially had PTSD, and 60.9% experienced insomnia. Years of work experience were found to be positively correlated, albeit with low effect, with the PTSD score (r = 0.133). Finally, it can be stated that the COVID-19 pandemic has been a traumatic event for EMS workers. The number of professionals presenting psychological distress, possible PTSD, or insomnia increased dramatically during the early phases of the pandemic. This study highlights the need for mental health disorder prevention programmes for EMS workers in the face of a pandemic.
High-risk hematological malignancies are a privileged setting for infection by opportunistic microbes, with invasive mycosis being one of the most serious complications. Recently, genetic background ...has emerged as an unanticipated risk factor. For this reason, polymorphisms for genes encoding archetypal receptors involved in the opsonic and nonopsonic clearance of microbes, pentraxin-3 (PTX3) and Dectin-1, respectively, were studied and correlated with the risk of infection. Fungal, bacterial, and viral infections were registered for a group of 198 patients with high-risk hematological malignancies. Polymorphisms for the pentraxin-3 gene (
) showed a significant association with the risk of fungal infection by
spp. and, especially, by
spp. This link remained even for patients undergoing antifungal prophylaxis, thus demonstrating the clinical relevance of PTX3 in the defense against fungi.
polymorphisms did not show any definite correlation with the risk of invasive mycosis, nor did they influence the expression of Dectin-1 isoforms generated by alternative splicing. The
mRNA expression level was significantly lower in samples from healthy volunteers who showed these polymorphisms, although no differences were observed in the extents of induction elicited by bacterial lipopolysaccharide and heat-killed
, thus suggesting that the expression of PTX3 at the start of infection may influence the clinical outcome.
mRNA expression can be a good biomarker to establish proper antifungal prophylaxis in immunodepressed patients.
Objective
Acquired tracheal stenosis (ATS) is an unusual disease often secondary to prolonged mechanical trauma. Acquired tracheal stenosis pathogenesis involves inflammation and subsequent fibrosis ...with narrowing of the tracheal lumen. Transforming growth factor‐β1 (TGF‐β) represents a pivotal factor in most fibrotic processes, and therefore a potential target in this context. The aim of this study is to analyze the role of TGF‐β as a target for anti‐fibrotic interventions in tracheal stenosis.
Methods
Human stenotic tracheobronchial tissues from patients with benign airway stenosis and normal controls from pneumonectomy specimens were analyzed. Tracheal stenosis was induced in adult NZ rabbits by a circumferential thermal injury to the mucosa during open surgery and re‐anastomosis. Rabbits were treated postoperatively with a peritracheal collagen sponge containing a TGF‐β peptide antagonist (p17) or vehicle. Fibrosis was determined by Masson's trichrome staining, and smooth muscle cell α‐actin+ (α‐SMA+ Confirm accuracy.) myofibroblasts, connective tissue growth factor (CTGF), and p‐Smad2/3 expression by immunohistochemistry.
Results
Human and rabbit stenotic tissues showed extensive submucosal fibrosis, characterized by significantly increased α‐SMA+ myofibroblasts and CTGF expression. In human stenotic lesions, increased p‐Smad2/3+ nuclei were also observed. p17 treatment significantly reduced the fibrotic thickness, as well as the density of α‐SMA+ myofibroblasts and CTGF+ cells in rabbit stenotic lesions, but failed to improve the luminal area.
Conclusion
ATS is characterized by a TGF‐β dependent fibrotic process, but reduction of the fibrotic component by TGF‐β1 antagonist therapy was not sufficient to improve tracheal narrowing, suggesting that fibrosis may not be the main contributor to luminal stenosis.
Level of Evidence
NA. Laryngoscope, 127:561–567, 2017
Thalidomide is an immunomodulatory drug (IMiD) with proven therapeutic action in several autoimmune/inflammatory diseases; however, its inherent high toxicity has led to the development of more ...powerful and safer thalidomide analogs, including lenalidomide and pomalidomide. These are new generation IMiDs that exhibit direct antitumor activity as well as anti-inflammatory/immunomodulatory properties, and are FDA-approved for the treatment of several hematological malignances. Here we investigated the potential therapeutic effects of lenalidomide and pomalidomide in several experimental murine models of autoimmune/inflammatory diseases: 2,4,6-trinitrobenzene sulfonic acid- and dextran sulfate sodium-induced inflammatory bowel disease and type II collagen-induced arthritis. Lenalidomide displayed a strong therapeutic effect in all these models of autoimmune/inflammatory diseases, while the effect of pomalidomide was less pronounced. In vitro experiments confirmed the immunosuppressive effect of both IMiDs on the proliferative response of stimulated human lymphocytes and on the balance of secreted cytokines toward an anti-inflammatory profile. We conclude that lenalidomide may offer a therapeutic opportunity against autoimmune/inflammatory diseases.
Graft-versus-host disease (GvHD) remains the major obstacle to successful allogeneic hematopoietic stem cell transplantation, despite of the immunosuppressive regimens administered to control T cell ...alloreactivity. PI3K/AKT/mTOR pathway is crucial in T cell activation and function and, therefore, represents an attractive therapeutic target to prevent GvHD development. Recently, numerous PI3K inhibitors have been developed for cancer therapy. However, few studies have explored their immunosuppressive effect.
The effects of a selective PI3K inhibitor (BKM120) and a dual PI3K/mTOR inhibitor (BEZ235) on human T cell proliferation, expression of activation-related molecules, and phosphorylation of PI3K/AKT/mTOR pathway proteins were analyzed. Besides, the ability of BEZ235 to prevent GvHD development in mice was evaluated.
Simultaneous inhibition of PI3K and mTOR was efficient at lower concentrations than PI3K specific targeting. Importantly, BEZ235 prevented naïve T cell activation and induced tolerance of alloreactive T cells, while maintaining an adequate response against cytomegalovirus, more efficiently than BKM120. Finally, BEZ235 treatment significantly improved the survival and decreased the GvHD development in mice.
These results support the use of PI3K inhibitors to control T cell responses and show the potential utility of the dual PI3K/mTOR inhibitor BEZ235 in GvHD prophylaxis.
Activation of oval cells (OCs) has been related to hepatocyte injury during chronic liver diseases including non-alcoholic fatty liver disease (NAFLD). However, OCs plasticity can be affected under ...pathological environments. We previously found protection against hepatocyte cell death by inhibiting protein tyrosine phosphatase 1B (PTP1B). Herein, we investigated the molecular and cellular processes involved in the lipotoxic susceptibility in OCs expressing or not PTP1B. Palmitic acid (PA) induced apoptotic cell death in wild-type (Ptpn1
) OCs in parallel to oxidative stress and impaired autophagy. This lipotoxic effect was attenuated in OCs lacking Ptpn1 that showed upregulated antioxidant defences, increased unfolded protein response (UPR) signaling, higher endoplasmic reticulum (ER) content and elevated stearoyl CoA desaturase (Scd1) expression and activity. These effects in Ptpn1
OCs concurred with an active autophagy, higher mitochondrial efficiency and a molecular signature of starvation, favoring lipid droplet (LD) formation and dynamics. Autophagy blockade in Ptpn1
OCs reduced Scd1 expression, mitochondrial fitness, LD formation and restored lipoapoptosis, an effect also recapitulated by Scd1 silencing. PTP1B immunostaining was detected in OCs from mouse liver and, importantly, LDs were found in OCs from Ptpn1
mice with NAFLD. In conclusion, we demonstrated that Ptpn1 deficiency restrains lipoapoptosis in OCs through a metabolic rewiring towards a "starvation-like" fate, favoring autophagy, mitochondrial fitness and LD formation. Dynamic LD-lysosomal interations likely ensure lipid recycling and, overall, these adaptations protect against lipotoxicity. The identification of LDs in OCs from Ptpn1
mice with NAFLD opens therapeutic perspectives to ensure OC viability and plasticity under lipotoxic liver damage.
Fatty acid metabolism in the hypothalamus has an important role in food intake, but its specific role in AgRP neurons is poorly understood. Here, we examined whether carnitinea palmitoyltransferase ...1A (CPT1A), a key enzyme in mitochondrial fatty acid oxidation, affects energy balance.
To obtain Cpt1aKO mice and their control littermates, Cpt1a
mice were crossed with tamoxifen-inducible AgRP
mice. Food intake and body weight were analyzed weekly in both males and females. At 12 weeks of age, metabolic flexibility was determined by ghrelin-induced food intake and fasting-refeeding satiety tests. Energy expenditure was analyzed by calorimetric system and thermogenic activity of brown adipose tissue. To study fluid balance the analysis of urine and water intake volumes; osmolality of urine and plasma; as well as serum levels of angiotensin and components of RAAS (renin-angiotensin-aldosterone system) were measured. At the central level, changes in AgRP neurons were determined by: (1) analyzing specific AgRP gene expression in RiboTag-Cpt1aKO mice obtained by crossing Cpt1aKO mice with RiboTag mice; (2) measuring presynaptic terminal formation in the AgRP neurons with the injection of the AAV1-EF1a-DIO-synaptophysin-GFP in the arcuate nucleus of the hypothalamus; (3) analyzing AgRP neuronal viability and spine formations by the injection AAV9-EF1a-DIO-mCherry in the arcuate nucleus of the hypothalamus; (4) analyzing in situ the specific AgRP mitochondria in the ZsGreen-Cpt1aKO obtained by breeding ZsGreen mice with Cpt1aKO mice. Two-way ANOVA analyses were performed to determine the contributions of the effect of lack of CPT1A in AgRP neurons in the sex.
Changes in food intake were just seen in male Cpt1aKO mice while only female Cpt1aKO mice increased energy expenditure. The lack of Cpt1a in the AgRP neurons enhanced brown adipose tissue activity, mainly in females, and induced a substantial reduction in fat deposits and body weight. Strikingly, both male and female Cpt1aKO mice showed polydipsia and polyuria, with more reduced serum vasopressin levels in females and without osmolality alterations, indicating a direct involvement of Cpt1a in AgRP neurons in fluid balance. AgRP neurons from Cpt1aKO mice showed a sex-dependent gene expression pattern, reduced mitochondria and decreased presynaptic innervation to the paraventricular nucleus, without neuronal viability alterations.
Our results highlight that fatty acid metabolism and CPT1A in AgRP neurons show marked sex differences and play a relevant role in the neuronal processes necessary for the maintenance of whole-body fluid and energy balance.
Recently, it was proposed that the thiophene ring is capable of promoting mitochondrial accumulation when linked to fluorescent markers. As a noncharged group, thiophene presents several advantages ...from a synthetic point of view, making it easier to incorporate such a side moiety into different molecules. Herein, we confirm the general applicability of the thiophene group as a mitochondrial carrier for drugs and fluorescent markers based on a new concept of nonprotonable, noncharged transporter. We implemented this concept in a medicinal chemistry application by developing an antitumor, metabolic chimeric drug based on the pyruvate dehydrogenase kinase (PDHK) inhibitor dichloroacetate (DCA). The promising features of the thiophene moiety as a noncharged carrier for targeting mitochondria may represent a starting point for the design of new metabolism-targeting drugs.
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