The INSPIRE Bio-Resource Research Platform for Healthy Aging and Geroscience: Focus on the Human Translational Research Cohort (The INSPIRE-T Cohort).

BACKGROUND: The Geroscience field focuses on the core biological mechanisms of aging, which are involved in the onset of age-related diseases, as well as declines in intrinsic capacity (IC) (body functions) leading to dependency. A better understanding on how to measure the true age of an individual or biological aging is an essential step that may lead to the definition of putative markers capable of predicting healthy aging. OBJECTIVES: The main objective of the INStitute for Prevention healthy agIng and medicine Rejuvenative (INSPIRE) Platform initiative is to build a program for Geroscience and healthy aging research going from animal models to humans and the health care system. The specific aim of the INSPIRE human translational cohort (INSPIRE-T cohort) is to gather clinical, digital and imaging data, and perform relevant and extensive biobanking to allow basic and translational research on humans. METHODS: The INSPIRE-T cohort consists in a population study comprising 1000 individuals in Toulouse and surrounding areas (France) of different ages (20 years or over - no upper limit for age) and functional capacity levels (from robustness to frailty, and even dependency) with follow-up over 10 years. Diversified data are collected annually in research facilities or at home according to standardized procedures. Between two annual visits, IC domains are monitored every 4-month by using the ICOPE Monitor app developed in collaboration with WHO. Once IC decline is confirmed, participants will have a clinical assessment and blood sampling to investigate markers of aging at the time IC declines are detected. Biospecimens include blood, urine, saliva, and dental plaque that are collected from all subjects at baseline and then, annually. Nasopharyngeal swabs and cutaneous surface samples are collected in a large subgroup of subjects every two years. Feces, hair bulb and skin biopsy are collected optionally at the baseline visit and will be performed again during the longitudinal follow up. EXPECTED RESULTS: Recruitment started on October 2019 and is expected to last for two years. Bio-resources collected and explored in the INSPIRE-T cohort will be available for academic and industry partners aiming to identify robust (set of) markers of aging, age-related diseases and IC evolution that could be pharmacologically or non-pharmacologically targetable. The INSPIRE-T will also aim to develop an integrative approach to explore the use of innovative technologies and a new, function and person-centered health care pathway that will promote a healthy aging.

Protease-activated receptor 2 contributes to Toxoplasma gondii-mediated gut inflammation.

Toxoplasma gondii is a widespread intracellular parasite, which naturally enters the organism via the oral route and crosses the intestinal barrier to disseminate. In addition to neuronal and ocular pathologies, this pathogen also causes gut inflammation in a number of animals. This infection-triggered inflammation has been extensively studied in the C57BL/6 mice, highlighting the importance of the immune cells and their mediators in the development of gut pathology. However, despite their importance in inflammation, the role of protease-activated receptors (PAR) was never reported in the context of T.gondii-mediated small intestine inflammation. Using genetically modified mice, we show that PAR2 plays a pathogenic role in the development of gut inflammatory lesions. We find that PAR2 controls the innate inflammatory mediators IL-6, KC/CXCL1, PGE2 as well as neutrophil infiltration in T. gondii-triggered gut damage. These results bring new knowledge on the mechanisms operating in the gut in response to T. gondii infection.

F16357, a novel protease-activated receptor 1 antagonist, improves urodynamic parameters in a rat model of interstitial cystitis.

BACKGROUND AND PURPOSE: The aims of the present study were to characterize the role of PAR1 in rat bladder under inflammatory conditions and determine whether a selective PAR1 antagonist, F16357, can prevent the pathophysiological symptoms of cyclophosphamide-induced interstitial cystitis (IC). EXPERIMENTAL APPROACH: Immunohistochemistry, contractile activity in isolated bladder and urodynamics were determined before and after cyclophosphamide treatment. F16357 was administered intravesically during the acute phase of inflammation, and effects on PAR1 and PAR1-related bladder contraction evaluated 24 h after cyclophosphamide injection. Urodynamics and associated voided volumes were recorded 7 and 24 h after cyclophosphamide. KEY RESULTS: In control conditions, PAR1 was present only in some umbrella cells. Cyclophosphamide disrupted the urothelium and expression of PAR1 by all remaining urothelial cells. After F16357 treatment, urothelial damage was absent and PAR1 immunoreactivity similar to control tissues. Thrombin and TFLLR-NH2 induced bladder contractions. These were increased in inflammatory conditions and antagonized by F16357 in a concentration-dependent manner. In telemetric experiments, furosemide increased urine production and voiding frequency for 60 min, 7 h after cyclophosphamide injection. Intravesical administration of F16357 blocked these changes with a return to a physiological profile; 24 h after cyclophosphamide, the volume of micturition was still lower with no increase in number of micturitions. F16357 30 µM reduced the number of micturitions and improved bladder capacity, but did not affect diuresis. Under similar experimental conditions, lidocaine 2% induced comparable effects. CONCLUSIONS AND IMPLICATIONS: PAR1 is expressed in rat bladder, overactivated in inflammatory conditions and involved in bladder function and sensation. F16357 could represent an interesting candidate for IC treatment.

A novel orally administered trimebutine compound (GIC-1001) is anti-nociceptive and features peripheral opioid agonistic activity and Hydrogen Sulphide-releasing capacity in mice.

BACKGROUND: Trimebutine maleate, a noncompetitive spasmolytic agent with some affinity for peripheral µ- and κ-opioid receptors has been evaluated as a treatment in a limited number of patients undergoing sedation-free full colonoscopy. The efficiency of such treatment was comparable to sedation-based colonoscopies to relieve from pain and discomfort. METHODS: A new and improved trimebutine salt capable of releasing in vivo hydrogen sulphide (H2S), a gaseous mediator known to reduce nociception, has been developed. This drug salt (GIC-1001) is composed of trimebutine bearing a H2S-releasing counterion (3-thiocarbamoylbenzoate, 3TCB), the latter having the ability to release H2S. GIC-1001 has been tested here in a mouse model of colorectal distension. RESULTS: In mice, while orally given trimebutine (the maleate salt, non-H2 S-releaser) only slightly reduced the nociceptive response to increasing pressures of colorectal distension, oral administration of GIC-1001 (the H2S-releaser) was able to significantly reduce nociceptive response to all noxious stimuli, in a dose-dependent manner. This effect of GIC-1001 was significantly better than the effects of its parent compound trimebutine administered at equimolar doses. CONCLUSIONS: Taken together, these results demonstrated increased antinociceptive properties for GIC-1001 compared to trimebutine, suggesting that this compound would be a better option to relieve from visceral pain and discomfort induced by lumenal distension.

Sex differences in the GSK3ß-mediated survival of adherent leukemic progenitors.

Therapeutic resistance of acute myeloid leukemia stem cells, enriched in the CD34(+)38(-)123(+) progenitor population, is supported by extrinsic factors such as the bone marrow niche. Here, we report that when adherent onto fibronectin or osteoblast components, CD34(+)38(-)123(+) progenitors survive through an integrin-dependent activation of glycogen synthase kinase 3ß (GSK3ß) by serine 9-dephosphorylation. Strikingly, GSK3ß-mediated survival was restricted to leukemic progenitors from female patients. GSK3ß inhibition restored sensitivity to etoposide, and impaired the clonogenic capacities of adherent leukemic progenitors from female patients. In leukemic progenitors from female but not male patients, the scaffolding protein RACK1, activated downstream of α(5)ß(1)-integrin engagement, was specifically upregulated and controlled GSK3ß activation through the phosphatase protein phosphatase 2A (PP2A). In a mirrored manner, survival of adherent progenitors (CD34(+)38(-)) from male but not female healthy donors was partially dependent on this pathway. We conclude that the GSK3ß-dependent survival pathway might be sex-specific in normal immature population and flip-flopped upon leukemogenesis. Taken together, our results strengthen GSK3ß as a promising target for leukemic stem cell therapy and reveal gender differences as a new parameter in anti-leukemia therapy.

Protease-activated receptor-2 (PAR(2)) in human periodontitis.

No evidence for the role of protease-activated receptor-2 (PAR(2)) in human periodontal disease has been demonstrated so far. Thus, we sought to investigate the expression of PAR(2) mRNA in chronic periodontitis, and to examine whether its expression is related to the presence of PAR(2) potential activators. Microbiological and gingival crevicular fluid samples were collected from individuals with chronic periodontitis and control individuals, and the presence of neutrophil serine proteinase 3 (P3) and Porphyromonas gingivalis was evaluated. PAR(2) mRNA expression was higher (p < 0.001) in those with chronic periodontitis compared with control individuals, and it was statistically decreased (p = 0.0006) after periodontal treatment. Furthermore, those with chronic periodontitis presented higher (p < 0.05) levels of IL-1alpha, IL-6, IL-8, and TNF-alpha, total proteolytic activity, P. gingivalis prevalence, and P3mRNA expression compared with control individuals. We conclude that PAR(2) mRNA expression and its potential activators are elevated in human chronic periodontitis, therefore suggesting that PAR(2) may play a role in periodontal inflammation.

Combined challenge of mice with Citrobacter rodentium and ionizing radiation promotes bacterial translocation.

PURPOSE: Both enteric infection and exposure to ionizing radiation are associated with increased intestinal permeability. However, the combined effect of irradiation and enteric infection has not been described. We combined infection of mice with the enteric pathogen, Citrobacter rodentium, with exposure to ionizing radiation and assessed the impact on colonic epithelial ion transport, permeability and bacterial translocation. MATERIALS AND METHODS: Mice were infected with C. rodentium and then received whole-body exposure to 5 Gray gamma-radiation 7 days later. Three days post-irradiation, mice were euthanized and colons removed. Control groups included sham-infected mice that were irradiated and mice that were infected, but not irradiated. RESULTS: Macroscopic damage score and colonic wall thickness were increased by C. rodentium infection, but these parameters were not exacerbated by irradiation. Infection caused an increase in myeloperoxidase activity that was reduced by irradiation. Irradiation reduced the secretory response to electrical field stimulation, forskolin and carbachol; these changes were not altered by infection with C. rodentium. None of the treatments caused an increase in permeability to 51Cr-ethylenediaminetetraacetic acid (EDTA). However, combined infection and irradiation synergistically increased bacterial translocation to mesenteric lymph nodes, liver, spleen and blood. CONCLUSIONS: Although the combination of irradiation and infection did not exacerbate the individual effects of these challenges on ion secretion and mucosal permeability to 51Cr-EDTA, it dramatically increased susceptibility to bacterial translocation and bacteremia. These results have important implications for patients who develop an enteric infection during the course of abdominopelvic radiotherapy.

Role of protease-activated receptor-2 in inflammation, and its possible implications as a putative mediator of periodontitis

Proteinase-activated receptor-2 (PAR2) belongs to a novel subfamily of G-protein-coupled receptors with seven-transmembrane domains. This receptor is widely distributed throughout the body and seems to be importantly involved in inflammatory processes. PAR2 can be activated by serine proteases such as trypsin, mast cell tryptase, and bacterial proteases, such as gingipain produced by Porphyromonas gingivalis. This review describes the current stage of knowledge of the possible mechanisms that link PAR2 activation with periodontal disease, and proposes future therapeutic strategies to modulate the host response in the treatment of periodontitis.

Proteinase-activated receptor-2 and hyperalgesia: A novel pain pathway.

Using a combined pharmacological and gene-deletion approach, we have delineated a novel mechanism of neurokinin-1 (NK-1) receptor-dependent hyperalgesia induced by proteinase-activated receptor-2 (PAR2), a G-protein-coupled receptor expressed on nociceptive primary afferent neurons. Injections into the paw of sub-inflammatory doses of PAR2 agonists in rats and mice induced a prolonged thermal and mechanical hyperalgesia and elevated spinal Fos protein expression. This hyperalgesia was markedly diminished or absent in mice lacking the NK-1 receptor, preprotachykinin-A or PAR2 genes, or in rats treated with a centrally acting cyclooxygenase inhibitor or treated by spinal cord injection of NK-1 antagonists. Here we identify a previously unrecognized nociceptive pathway with important therapeutic implications, and our results point to a direct role for proteinases and their receptors in pain transmission.

Protease-activated receptor-1 stimulates Ca(2+)-dependent Cl(-) secretion in human intestinal epithelial cells.

The thrombin receptor, protease-activated receptor-1 (PAR-1), has wide tissue distribution and is involved in many physiological functions. Because thrombin is in the intestinal lumen and mucosa during inflammation, we sought to determine PAR-1 expression and function in human intestinal epithelial cells. RT-PCR showed PAR-1 mRNA expression in SCBN cells, a nontransformed duodenal epithelial cell line. Confluent SCBN monolayers mounted in Ussing chambers responded to PAR-1 activation with a Cl(-)-dependent increase in short-circuit current. The secretory effect was blocked by BaCl2 and the Ca(2+)-ATPase inhibitor thapsigargin, but not by the L-type Ca(2+) channel blocker verapamil or DIDS, the nonselective inhibitor of Ca(2+)-dependent Cl(-) transport. Responses to thrombin and PAR-1-activating peptides exhibited auto- and crossdesensitization. Fura 2-loaded SCBN cells had increased fluorescence after PAR-1 activation, indicating increased intracellular Ca(2+). RT-PCR showed that SCBN cells expressed mRNA for the cystic fibrosis transmembrane conductance regulator (CFTR) and hypotonicity-activated Cl(-) channel-2 but not for the Ca(2+)-dependent Cl(-) channel-1. PAR-1 activation failed to increase intracellular cAMP, suggesting that the CFTR channel is not involved in the Cl(-) secretory response. Our data demonstrate that PAR-1 is expressed on human intestinal epithelial cells and regulates a novel Ca(2+)-dependent Cl(-) secretory pathway. This may be of clinical significance in inflammatory intestinal diseases with elevated thrombin levels.

Proteinase-activated receptor-2-activating peptides induce leukocyte rolling, adhesion, and extravasation in vivo.

Proteinase-activated receptor 2 (PAR2) has been suggested to play a role in inflammatory reactions. Because leukocyte-endothelial cell interactions are critical events during inflammatory reactions, and because PAR2 is expressed both on endothelium and leukocytes, we have examined the effects of PAR2-activating peptides (PAR2-APs) on leukocyte rolling and adhesion in mesenteric venules and on leukocyte recruitment into the peritoneal cavity. Using intravital microscopy, leukocyte rolling, flux, and adhesion in rat mesenteric postcapillary venules were quantified. Topical addition of PAR2-APs (10 microM) for 1 min to the superfused venule induced a significant increase in leukocyte rolling and adherence. The increase in leukocyte adherence was not affected by pretreatment with a mast cell stabilizer (sodium cromoglycate) nor by prior degranulation of mast cells with compound 48/80. Nonetheless, both leukocyte rolling and adhesion were completely inhibited by pretreatment with a platelet-activating factor receptor antagonist (WEB 2086). Intraperitoneal injections of a selective PAR2-AP (SLIGRL-NH2) caused a significant increase in leukocyte migration into the peritoneal cavity. The effect of SLIGRL-NH2 on peritoneal leukocyte infiltration was completely inhibited by WEB 2086. These data suggest that PAR2 activation could contribute to several early events in the inflammatory reaction, including leukocyte rolling, adherence, and recruitment, by a mechanism dependent on platelet-activating factor release.

Inflammatory neutrophils secrete annexin 1 during experimentally induced colitis in rats.

Immunoblotting and immunohistochemical analysis were performed to identify the cells expressing and secreting annexin 1 during experimental rat colitis induced by trinitrobenzene sulfonic acid. Annexin 1 expression was increased during the inflammation. Likewise, annexin 1 secretion was induced in inflamed colons at one, three, six, and nine days after trinitrobenzene sulfonic acid treatment but was not detected in colons from controls and rats at 12 days. Immunohistochemistry showed that the rise in annexin 1 expression resulted from the infiltration of two types of leukocytes highly positive for annexin 1: neutrophils (the most abundant) and macrophages. At day 1 after treatment, neutrophils of the inflammatory site, in mucosa and submucosa, are the only cells expressing annexin 1. Immunoblotting showed that they secreted annexin 1 whereas neutrophils from blood or tunica muscularis did not. This indicates that, during this colitis, annexin 1 can be secreted by neutrophils located in the inflammatory site.

Characterization of the inflammatory response to proteinase-activated receptor-2 (PAR2)-activating peptides in the rat paw.

In the present study, we have observed the development of an inflammatory reaction in the rat hindpaw, following the injection of specific agonists of PAR2 (two PAR2 activating peptides). This inflammation was characterized by oedema and granulocyte infiltration. Two selective PAR2 activating peptides, SLGRL-NH2 and trans-cinnamoyl-LIGRLO-NH2 induced significant oedema in the rat hindpaw from 1-6 h following subplantar injection. Six hours after the PAR2-activating peptide injection, the paw tissues showed a complete disruption of tissue architecture along with an inflammatory cell infiltrate. In the inflamed paw, PAR2-immunoreactivity was expressed on endothelial cells as well as on the infiltrating inflammatory cells. The oedema induced by the injection of the two PAR2 activating peptides was slightly reduced in rats pre-treated with compound 48/80, but was not modified by pre-treatment of rats with cromolyn, a mast cell stabilizer. Pre-treatment of rats with a cyclo-oxygenase inhibitor (indomethacin) or a nitric oxide synthase inhibitor (L-N(omega)-nitro-L-arginine methyl ester) had no effect on the oedema induced by the PAR2-activating peptides. These results demonstrate that the administration of PAR2-activating peptides into the rat paw induced an acute inflammatory response characterized by a persistent oedema (at least 6 h) and granulocyte infiltration. The PAR2-induced inflammatory response occurred through a mechanism largely independent of mast cell activation, and of the production of prostanoids and nitric oxide.