Human Osteoblast-Conditioned Media Can Influence Staphylococcus aureus Biofilm Formation.

Osteoblasts are bone-forming and highly active cells participating in bone homeostasis. In the case of osteomyelitis and more specifically prosthetic joint infections (PJI) for which Staphylococcus aureus (S. aureus) is mainly involved, the interaction between osteoblasts and S. aureus results in impaired bone homeostasis. If, so far, most of the studies of osteoblasts and S. aureus interactions were focused on osteoblast response following direct interactions with co-culture and/or internalization models, less is known about the effect of osteoblast factors on S. aureus biofilm formation. In the present study, we investigated the effect of human osteoblast culture supernatant on methicillin sensitive S. aureus (MSSA) SH1000 and methicillin resistant S. aureus (MRSA) USA300. Firstly, Saos-2 cell line was incubated with either medium containing TNF-α to mimic the inflammatory periprosthetic environment or with regular medium. Biofilm biomass was slightly increased for both strains in the presence of culture supernatant collected from Saos-2 cells, stimulated or not with TNF-α. In such conditions, SH1000 was able to develop microcolonies, suggesting a rearrangement in biofilm organization. However, the biofilm matrix and regulation of genes dedicated to biofilm formation were not substantially changed. Secondly, culture supernatant obtained from primary osteoblast culture induced varied response from SH1000 strain depending on the different donors tested, whereas USA300 was only slightly affected. This suggested that the sensitivity to bone cell secretions is strain dependent. Our results have shown the impact of osteoblast secretions on bacteria and further identification of involved factors will help to manage PJI.

Inhibition of Recruitment and Activation of Neutrophils by Pyridazinone-Scaffold-Based Compounds.

In inflammatory diseases, polymorphonuclear neutrophils (PMNs) are known to produce elevated levels of pro-inflammatory cytokines and proteases. To limit ensuing exacerbated cell responses and tissue damage, novel therapeutic agents are sought. 4aa and 4ba, two pyridazinone-scaffold-based phosphodiesterase-IV inhibitors are compared in vitro to zardaverine for their ability to: (1) modulate production of pro-inflammatory mediators, reactive oxygen species (ROS), and phagocytosis; (2) modulate degranulation by PMNs after transepithelial lung migration. Compound 4ba and zardaverine were tested in vivo for their ability to limit tissue recruitment of PMNs in a murine air pouch model. In vitro treatment of lipopolysaccharide-stimulated PMNs with compounds 4aa and 4ba inhibited the release of interleukin-8, tumor necrosis factor-α, and matrix metalloproteinase-9. PMNs phagocytic ability, but not ROS production, was reduced following treatment. Using a lung inflammation model, we proved that PMNs transmigration led to reduced expression of the CD16 phagocytic receptor, which was significantly blunted after treatment with compound 4ba or zardaverine. Using the murine air pouch model, LPS-induced PMNs recruitment was significantly decreased upon addition of compound 4ba or zardaverine. Our data suggest that new pyridazinone derivatives have therapeutic potential in inflammatory diseases by limiting tissue recruitment and activation of PMNs.

Overexpression of RANKL in osteoblasts: a possible mechanism of susceptibility to bone disease in cystic fibrosis.

Bone fragility and loss are a significant cause of morbidity in patients with cystic fibrosis (CF), and the lack of effective therapeutic options means that treatment is more often palliative rather than curative. A deeper understanding of the pathogenesis of CF-related bone disease (CFBD) is necessary to develop new therapies. Defective CF transmembrane conductance regulator (CFTR) protein and chronic inflammation in bone are important components of the CFBD development. The receptor activator of nuclear factor kappa-B ligand (RANKL) and osteoprotegerin (OPG) drive the regulation of bone turnover. To investigate their roles in CFBD, we evaluated the involvement of defective CFTR in their production level in CF primary human osteoblasts with and without inflammatory stimulation, in the presence or not of pharmacological correctors of the CFTR. No major difference in cell ultrastructure was noted between cultured CF and non-CF osteoblasts, but a delayed bone matrix mineralization was observed in CF osteoblasts. Strikingly, resting CF osteoblasts exhibited strong production of RANKL protein, which was highly localized at the cell membrane and was enhanced in TNF (TNF-α) or IL-17-stimulated conditions. Under TNF stimulation, a defective response in OPG production was observed in CF osteoblasts in contrast to the elevated OPG production of non-CF osteoblasts, leading to an elevated RANKL-to-OPG protein ratio in CF osteoblasts. Pharmacological inhibition of CFTR chloride channel conductance in non-CF osteoblasts replicated both the decreased OPG production and the enhanced RANKL-to-OPG ratio. Interestingly, using CFTR correctors such as C18, we significantly reduced the production of RANKL by CF osteoblasts, in both resting and TNF-stimulated conditions. In conclusion, the overexpression of RANKL and high membranous RANKL localization in osteoblasts are related to defective CFTR, and may worsen bone resorption, leading to bone loss in patients with CF. Targeting osteoblasts with CFTR correctors may represent an effective strategy to treat CFBD. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

An increase in Wharton's jelly membrane osteocompatibility by a genipin-cross-link.

Wharton's Jelly (WJ) has attracted significant interest in the field of tissue healing thanks to its biological properties, including antibacterial activity and immunomodulation. However, due to the fast degradation and poor mechanical behavior in biological environment, its application in bone regeneration is compromised. Here, we proposed to use genipin as an efficient cross-linking agent to significantly improve the elasticity and the enzymatical stability of the WJ matrix. The degree of cross-linking, linear elastic moduli, and collagenase resistance varied over a wide range depending on genipin concentration. Furthermore, our results highlighted that an increase in genipin concentration led to a decreased surface wettability, therefore impairing cell attachment and proliferation. The genipin cross-linking prevented rapid in vitro and in vivo degradation, but led to an adverse host reaction and calcification. When implanted in the parietal bone defect, a limited parietal bone regeneration to the dura was observed. We conclude that genipin-cross-linked WJ is a versatile medical device however, a careful selection is required with regards to the genipin concentration.

Cytocompatibility of titanium and poly(etheretherketone) surfaces after O2 non-thermal plasma sterilization.

The sterilization of medical devices is paramount to achieve an acceptable level of sterility assurance and to prevent hospital-acquired infections. However, some medical devices cannot be sterilized by usual processes such as autoclave (AC) and gamma-ray irradiation (GI). A new non-thermal plasma (NTP) process using sealed bag that preserves the sterile state of the devices could be used as an alternative sterilization method. The aim of the study was to assess the cytocompatibility of titanium and poly(etheretherketone) (PEEK) surfaces after O2-NTP sterilization compared to GI and AC. MG-63 osteoblast-like cells were seeded on titanium (TA6V) and PEEK disks sterilized by AC, GI and O2-NTP. The cells' viability and proliferation, determined by WST-1 and DNA quantification respectively, were enhanced whatever the material types from 3 to 10 days. When seeded on titanium, MG-63 cells showed a higher viability and proliferation after GI and O2-NTP treatment compared to AC treatment. When cultured on PEEK, MG-63 cells showed a higher viability after O2-NTP treatment. No difference of proliferation was observed whatever the sterilization processes. The cell colonization of the materials' surface was confirmed by scanning electron microscopy. Lactate dehydrogenase (LDH) assay revealed no cytotoxicity. Thus, O2-NTP led to similar cell responses to AC and GI and could be a cost-effective alternative process to the usual sterilization methods for fragile medical devices.

Tissue factor pathway inhibitor: a new link among arterial stiffness, pulse pressure, and coagulation in postmenopausal women.

OBJECTIVE: To investigate in women older than 60 whether aortic stiffness or pulse pressure (PP) is associated with selected procoagulant or anticoagulant factors and to examine whether pulsatile stretch influences these factors in human vascular smooth muscle cells (VSMCs) in vitro. METHODS AND RESULTS: Aortic pulse wave velocity (PWV) and carotid PP were studied in 123 apparently healthy postmenopausal women. PWV, PP, von Willebrand factor, and free tissue factor pathway inhibitor (TFPI), but not mean arterial pressure, increased with age. Free TFPI and PWV were positively correlated, even after adjustment for age and PP and other confounding parameters. In vitro, 5% or 10% pulsatile stretch (at 1 Hz) enhanced TFPI synthesis and secretion by VSMCs in a time-independent manner (1 to 48 hours) without changes in protein level of smooth muscle myosin heavy chain. Application of 5% static stretch had no effect. CONCLUSIONS: In postmenopausal women, free TFPI increases as vascular wall function deteriorates and PP increases. These findings are supported by the increase in TFPI synthesized by VSMCs in response to cyclic stress in vitro. They suggest that VSMCs require pulsatility to interfere with the coagulation process and highlight the relevance of plasma free TFPI levels to cardiovascular diseases.

[Home delivered meals: impasse or solution?]. / Portage des repas à domicile: impasse ou solution?

Undernutrition is common in elderly people living at home. A loss of autonomy can lead them to use home help services, such as home delivered meals. However this solution is not always effective and having meals brought to the home seems to aggravate their isolation, as the results of a survey of twenty people showed.

Health-related quality of life in French adolescents and adults: norms for the DUKE Health Profile.

BACKGROUND: The continual monitoring of population health-related quality of life (HRQoL) with validated instruments helps public health agencies assess, protect, and promote population health. This study aimed to determine norms for the French adolescent and adult general population for the Duke Health Profile (DUKE) questionnaire in a large representative community sample. METHODS: We randomly selected 17,733 French people aged 12 to 75 years old in 2 steps, by households and individuals, from the National Health Barometer 2005, a periodic population study by the French National Institute for Prevention and Health Education. Quality of life and other data were collected by computer-assisted telephone interview. RESULTS: Normative data for the French population were analyzed by age, gender and self-reported chronic disease. Globally, function scores (best HRQoL=100) for physical, mental, social, and general health, as well as perceived health and self-esteem, were 72.3 (SEM 0.2), 74.6 (0.2), 66.8 (0.1), 71.3 (0.1), 71.3 (0.3), 76.5 (0.1), respectively. Dysfunction scores (worst HRQoL=100) for anxiety, depression, pain and disability domains were 30.9 (0.1), 27.6 (0.2), 34.3 (0.3), 3.1 (0.1), respectively. CONCLUSION: The French norms for adolescents and adults for the DUKE could be used as a reference for other studies assessing HRQoL, for specific illnesses, in France and for international comparisons.

Osteoclastogenesis and sphingosine-1-phosphate secretion from human osteoclast precursor monocytes are modulated by the cystic fibrosis transmembrane conductance regulator.

Osteopenia and increased fracture rates are well-recognized in patients with cystic fibrosis (CF) disease. In CF pathology, F508del is the most common CFTR mutation, with more than 85% of patients carrying it on at least one allele. The underlying molecular defect in CFTR caused by the F508del-CFTR mutation in osteoclastogenesis, i.e., on the generation and bone-resorption activity of osteoclasts (OCs) from peripheral blood-derived monocytes (PBMCs) remained unexplored. We therefore investigated whether the F508del mutation could affect the osteoclastogenic capacity of PBMCs collected from 15 adult patients bearing the F508del-CFTR mutation, to modulate their bone-resorptive abilities and the level of sphingosine-1-phosphate (S1P) produced by OCs, a key factor in the bone mineral density and formation. In the present study, a severe, defective differentiation of CF-F508del PBMCs to CF-F508del OCs without any significant difference in nuclei number per OC was found compared to non-CF healthy PBMCs from 13 subjects after 7-14-days culture periods. We observed a reduced number of formed non-CF healthy OCs in the presence of a selective inhibitor of CFTR chloride conductance (CFTR-Inh172). Our data regarding OCs resorptive capabilites revealed that a loss of CFTR chloride activity in OCs led to a marked reduction in their trench-resorption mode. A 7-fold increase of the S1P release by CF-F508del OCs was found compared to non-CF healthy OCs after a 21-days culture period. We hypothesize that defective maturation of F508del-OCs precursor monocytes associated with high S1P production in the bone environment might contribute to low bone mineral density observed in the CF population.

Pyridazinone Derivatives Limit Osteosarcoma-Cells Growth In Vitro and In Vivo.

Osteosarcoma is a rare primary bone cancer that mostly affects children and young adults. Current therapeutic approaches consist of combining surgery and chemotherapy but remain unfortunately insufficient to avoid relapse and metastases. Progress in terms of patient survival has remained the same for 30 years. In this study, novel pyridazinone derivatives have been evaluated as potential anti-osteosarcoma therapeutics because of their anti-type 4 phosphodiesterase activity, which modulates the survival of several other cancer cells. By using five-four human and one murine osteosarcoma-cell lines, we demonstrated differential cytotoxic effects of four pyridazinone scaffold-based compounds (mitochondrial activity and DNA quantification). Proapoptotic (annexin V positive cells and caspase-3 activity), anti-proliferative (EdU integration) and anti-migratory effects (scratch test assay) were also observed. Owing to their cytotoxic activity in in vitro conditions and their ability to limit tumor growth in a murine orthotopic osteosarcoma model, our data suggest that these pyridazinone derivatives might be hit-candidates to develop new therapeutic strategies against osteosarcoma.

The Nexus Between Telomere Length and Lymphocyte Count in Seniors Hospitalized With COVID-19.

Profound T-cell lymphopenia is the hallmark of severe coronavirus disease 2019 (COVID-19). T-cell proliferation is telomere length (TL) dependent and telomeres shorten with age. Older COVID-19 patients, we hypothesize, are, therefore, at a higher risk of having TL-dependent lymphopenia. We measured TL by the novel Telomere Shortest Length Assay (TeSLA), and by Southern blotting (SB) of the terminal restriction fragments in peripheral blood mononuclear cells of 17 COVID-19 and 21 non-COVID-19 patients, aged 87 ± 8 (mean ± SD) and 87 ± 9 years, respectively. TeSLA tallies and measures single telomeres, including short telomeres undetected by SB. Such telomeres are relevant to TL-mediated biological processes, including cell viability and senescence. TeSLA yields 2 key metrics: the proportions of telomeres with different lengths (expressed in %) and their mean (TeSLA mTL), (expressed in kb). Lymphocyte count (109/L) was 0.91 ± 0.42 in COVID-19 patients and 1.50 ± 0.50 in non-COVID-19 patients (p < .001). In COVID-19 patients, but not in non-COVID-19 patients, lymphocyte count was inversely correlated with the proportion of telomeres shorter than 2 kb (p = .005) and positively correlated with TeSLA mTL (p = .03). Lymphocyte count was not significantly correlated with SB mTL in either COVID-19 or non-COVID-19 patients. We propose that compromised TL-dependent T-cell proliferative response, driven by short telomere in the TL distribution, contributes to COVID-19 lymphopenia among old adults. We infer that infection with SARS-CoV-2 uncovers the limits of the TL reserves of older persons. Clinical Trials Registration Number: NCT04325646.

CFTR-deficient pigs display alterations of bone microarchitecture and composition at birth.

BACKGROUND: The lack of cystic fibrosis transmembrane conductance regulator (CFTR) function causes cystic fibrosis (CF), predisposing to severe lung disease, reduced growth and osteopenia. Both reduced bone content and strength are increasingly recognized in infants with CF before the onset of significant lung disease, suggesting a developmental origin and a possible role in bone disease pathogenesis. The role of CFTR in bone metabolism is unclear and studies on humans are not feasible. Deletion of CFTR in pigs (CFTR -/- pigs) displays at birth severe malformations similar to humans in the intestine, respiratory tract, pancreas, liver, and male reproductive tract. METHODS: We compared bone parameters of CFTR -/- male and female pigs with those of their wild-type (WT) littermates at birth. Morphological and microstructural properties of femoral cortical and trabecular bone were evaluated using micro-computed tomography (µCT), and their chemical compositions were examined using Raman microspectroscopy. RESULTS: The integrity of the CFTR -/- bone was altered due to changes in its microstructure and chemical composition in both sexes. Low cortical thickness and high cortical porosity were found in CFTR -/- pigs compared to sex-matched WT littermates. Moreover, an increased chemical composition heterogeneity associated with higher carbonate/phosphate ratio and higher mineral crystallinity was found in CFTR -/- trabecular bone, but not in CFTR -/- cortical bone. CONCLUSIONS: The loss of CFTR directly alters the bone composition and metabolism of newborn pigs. Based on these findings, we speculate that bone defects in patients with CF could be a primary, rather than a secondary consequence of inflammation and infection.

A Mechanism for Severity of Disease in Older Patients with COVID-19: The Nexus between Telomere Length and Lymphopenia.

BACKGROUND: Lymphopenia due to a plummeting T-cell count is a major feature of severe COVID-19. T-cell proliferation is telomere length (TL)-dependent and TL shortens with age. Older persons are disproportionally affected by severe COVID-19, and we hypothesized that those with short TL have less capacity to mount an adequate T-cell proliferative response to SARS-CoV-2. This hypothesis predicts that among older patients with COVID-19, shorter telomeres of peripheral blood mononuclear cells (PBMCs) will be associated with a lower lymphocyte count. METHODS: Our sample comprised 17 COVID-19 and 21 non-COVID-19 patients, aged 87(8) (mean(SD)) and 87 (9) years, respectively. We measured TL by the Telomere Shortest Length Assay, a novel method that measures and tallies the short telomeres directly relevant to telomere-mediated biological processes. The primary analysis quantified TL as the proportion of telomeres shorter than 2 kilobases. For comparison, we also quantified TL by Southern blotting, which measures the mean length of telomeres. RESULTS: Lymphocyte count (109/L) was 0.91 (0.42) in COVID-19 patients and 1.50(0.50) in non-COVID-19 patients (P < 0.001). In COVID-19 patients, but not in non-COVID-19 patients, lymphocyte count was inversely correlated with the proportion of telomeres shorter than 2 kilobases (P = 0.005) and positively correlated with the mean of telomeres measured by TeSLA (P = 0.03). Lymphocyte counts showed no statistically significant correlations with Southern blotting results in COVID-19 or non-COVID-19 patients. CONCLUSIONS: These results support the hypothesis that a compromised TL-dependent T-cell proliferative response contributes to lymphopenia and the resulting disproportionate severity of COVID-19 among old adults. We infer that infection with SARS-CoV-2 uncovers the limits of the TL reserves of older persons.

Mechanisms of antiphospholipid-induced thrombosis: effects on the protein C system.

An acquired resistance to activated protein C (APC) has been demonstrated in patients with antiphospholipid antibodies (aPL). Recent studies report interactions between beta2 glycoprotein I (beta2GPI) and prothrombin-binding antibodies and the protein C system. Some aPL in patients recognize one or more conformational epitopes shared by beta2GPI and catalytic domains of APC. Both beta2GPI and anti-prothrombin antibodies are associated with APC resistance. Several clinical studies have focused on an association between aPL and APC resistance, determined by classic activated partial thromboplastin time-based tests. It has been shown in most studies that APC resistance was associated with lupus anticoagulants. APC resistance is also associated with thrombosis, especially venous thromboembolism. Several recent studies have reported a novel integrated approach of coagulation using calibrated automated thrombography. This technique allows an approach of APC sensitivity without interference with lupus anticoagulants. Clinical associations between APC resistance and thromboembolic events have been demonstrated.