Hepatic miR-149-5p upregulation fosters steatosis, inflammation and fibrosis development in mice and in human liver organoids.

Background & Aims: The incidence of metabolic dysfunction-associated steatotic liver disease (MASLD) is increasing worldwide. Alterations of hepatic microRNA (miRNA) expression/activity significantly contribute to the development and progression of MASLD. Genetic polymorphisms of miR-149 are associated with an increased susceptibility to MASLD development in humans. Aberrant expression of miR-149 was also associated with metabolic alterations in several organs, but the impact of hepatic miR-149-5p deregulation in MASLD remains poorly characterized. Methods: MiR-149-5p was downregulated in the livers of mice by in vivo transduction with hepatotropic adeno-associated virus 8 harboring short-hairpin RNAs (shRNAs) specific for miR-149-5p (shmiR149) or scrambled shRNAs (shCTL). MASLD was then induced with a methionine/choline-deficient (MCD, n = 7 per group) diet or a fructose/palmitate/cholesterol-enriched (FPC, n = 8-12 per group, per protocol) diet. The impact of miR-149-5p modulation on MASLD development was assessed in vivo and in vitro using multi-lineage 3D human liver organoids (HLOs) and Huh7 cells. Results: MiR-149-5p expression was strongly upregulated in mouse livers from different models of MASLD (2-4-fold increase in ob/ob, db/db mice, high-fat and FPC-fed mice). In vivo downregulation of miR-149-5p led to an amelioration of diet-induced hepatic steatosis, inflammation/fibrosis, and to increased whole-body fatty acid consumption. In HLOs, miR-149-5p overexpression promoted lipid accumulation, inflammation and fibrosis. In vitro analyses of human Huh7 cells overexpressing miR-149-5p indicated that glycolysis and intracellular lipid accumulation was promoted, while mitochondrial respiration was impaired. Translatomic analyses highlighted deregulation of multiple potential miR-149-5p targets in hepatocytes involved in MASLD development. Conclusions: MiR-149-5p upregulation contributes to MASLD development by affecting multiple metabolic/inflammatory/fibrotic pathways in hepatocytes. Our results further demonstrate that HLOs are a relevant 3D in vitro model to investigate hepatic steatosis and inflammation/fibrosis development. Impact and implications: Our research shows compelling evidence that miR-149-5p plays a pivotal role in the development and progression of MASLD. By employing in vivo and innovative in vitro models using multi-lineage human liver organoids, we demonstrate that miR-149-5p upregulation significantly impacts hepatocyte energy metabolism, exacerbating hepatic steatosis and inflammation/fibrosis by modulating a wide network of target genes. These findings not only shed light on the intricate miR-149-5p-dependent molecular mechanisms underlying MASLD, but also underscore the importance of human liver organoids as valuable 3D in vitro models for studying the disease's pathogenesis.

CCR5 deficiency: Decreased neuronal resilience to oxidative stress and increased risk of vascular dementia.

INTRODUCTION: As the chemokine receptor5 (CCR5) may play a role in ischemia, we studied the links between CCR5 deficiency, the sensitivity of neurons to oxidative stress, and the development of dementia. METHODS: Logistic regression models with CCR5/apolipoprotein E (ApoE) polymorphisms were applied on a sample of 205 cognitively normal individuals and 189 dementia patients from Geneva. The impact of oxidative stress on Ccr5 expression and cell death was assessed in mice neurons. RESULTS: CCR5-Δ32 allele synergized with ApoEε4 as risk factor for dementia and specifically for dementia with a vascular component. We confirmed these results in an independent cohort from Italy (157 cognitively normal and 620 dementia). Carriers of the ApoEε4/CCR5-Δ32 genotype aged ≥80 years have an 11-fold greater risk of vascular-and-mixed dementia. Oxidative stress-induced cell death in Ccr5-/- mice neurons. DISCUSSION: We propose the vulnerability of CCR5-deficient neurons in response to oxidative stress as possible mechanisms contributing to dementia.

NOX family NADPH oxidases in mammals: Evolutionary conservation and isoform-defining sequences.

NADPH oxidases are superoxide-producing enzymes that play a role in host defense, biosynthetic pathways, as well as cellular signaling. Humans have 7 NOX isoforms (NOX1-5, DUOX1,2), while mice and rats lack NOX5 and therefore have only 6 NOX isoforms. Whether all human NOX isoforms or their subunits (CYBA, NCF1, 2, 4, NOXO1, NOXA1, DUOXA1, 2) are present and conserved in other mammalian species is unknown. In this study, we have analyzed the conservation of the NOX family during mammalian evolution using an in-silico approach. Complete genomic sequences of 164 mammalian species were available. The possible absence of genes coding for NOX isoforms was investigated using the NCBI orthologs database followed by manual curation. Conservation of a given NOX isoform during mammalian evolution was evaluated by multiple alignment and identification of highly conserved sequences. There was no convincing evidence for the absence of NOX2, 3, 4, and DUOX1, 2 in all the available mammalian genome. However, NOX5 was absent in 27 of 31 rodent, in 2 of 3 lagomorph and in 2 out of 18 bat species. NOX1 was absent in all sequenced Afrotheria and Monotremata species, as well as in 3 of 18 bat species. NOXA1 was absent in all Afrotheria and in 3 out of 4 Eulipotyphla species. We also investigated amino acid sequence conservation among given NOX isoforms. Highly conserved sequences were observed for most isoforms except for NOX5. Interestingly, the highly conserved region of NOX2 sequence was relatively small (11 amino acids), as compared to NOX1, 3, 4. The highly conserved domains are different from one NOX isoform to the other, raising the possibility of distinct evolutionary conserved functional domains. Our results shed a new light on the essentiality of different NOX isoforms. We also identified isoform-defining sequences, i.e., hitherto undescribed conserved domains within specific NOX isoforms.

Wnt-Independent SARS-CoV-2 Infection in Pulmonary Epithelial Cells.

The Wnt signaling pathway within host cells regulates infections by several pathogenic bacteria and viruses. Recent studies suggested that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection depends on ß-catenin and can be inhibited by the antileprotic drug clofazimine. Since clofazimine has been identified by us as a specific inhibitor of Wnt/ß-catenin signaling, these works could indicate a potential role of the Wnt pathway in SARS-CoV-2 infection. Here, we show that the Wnt pathway is active in pulmonary epithelial cells. However, we find that in multiple assays, SARS-CoV-2 infection is insensitive to Wnt inhibitors, including clofazimine, acting at different levels within the pathway. Our findings assert that endogenous Wnt signaling in the lung is unlikely required or involved in the SARS-CoV-2 infection and that pharmacological inhibition of this pathway with clofazimine or other compounds is not a universal way to develop treatments against the SARS-CoV-2 infection. IMPORTANCE The development of inhibitors of the SARS-CoV-2 infection remains a need of utmost importance. The Wnt signaling pathway in host cells is often implicated in infections by bacteria and viruses. In this work, we show that, despite previous indications, pharmacological modulation of the Wnt pathway does not represent a promising strategy to control SARS-CoV-2 infection in lung epithelia.

Cancer Spheroids and Organoids as Novel Tools for Research and Therapy: State of the Art and Challenges to Guide Precision Medicine.

Spheroids and organoids are important novel players in medical and life science research. They are gradually replacing two-dimensional (2D) cell cultures. Indeed, three-dimensional (3D) cultures are closer to the in vivo reality and open promising perspectives for academic research, drug screening, and personalized medicine. A large variety of cells and tissues, including tumor cells, can be the starting material for the generation of 3D cultures, including primary tissues, stem cells, or cell lines. A panoply of methods has been developed to generate 3D structures, including spontaneous or forced cell aggregation, air-liquid interface conditions, low cell attachment supports, magnetic levitation, and scaffold-based technologies. The choice of the most appropriate method depends on (i) the origin of the tissue, (ii) the presence or absence of a disease, and (iii) the intended application. This review summarizes methods and approaches for the generation of cancer spheroids and organoids, including their advantages and limitations. We also highlight some of the challenges and unresolved issues in the field of cancer spheroids and organoids, and discuss possible therapeutic applications.

Adipose-Derived Stromal Cells within a Gelatin Matrix Acquire Enhanced Regenerative and Angiogenic Properties: A Pre-Clinical Study for Application to Chronic Wounds.

This study evaluates the influence of a gelatin sponge on adipose-derived stromal cells (ASC). Transcriptomic data revealed that, compared to ASC in a monolayer, a cross-linked porcine gelatin sponge strongly influences the transcriptome of ASC. Wound healing genes were massively regulated, notably with the inflammatory and angiogenic factors. Proteomics on conditioned media showed that gelatin also acted as a concentrator and reservoir of the regenerative ASC secretome. This secretome promoted fibroblast survival and epithelialization, and significantly increased the migration and tubular assembly of endothelial cells within fibronectin. ASC in gelatin on a chick chorioallantoic membrane were more connected to vessels than an empty sponge, confirming an increased angiogenesis in vivo. No tumor formation was observed in immunodeficient nude mice to which an ASC gelatin sponge was transplanted subcutaneously. Finally, ASC in a gelatin sponge prepared from outbred rats accelerated closure and re-vascularization of ischemic wounds in the footpads of rats. In conclusion, we provide here preclinical evidence that a cross-linked porcine gelatin sponge is an optimal carrier to concentrate and increase the regenerative activity of ASC, notably angiogenic. This formulation of ASC represents an optimal, convenient and clinically compliant option for the delivery of ASC on ischemic wounds.

NADPH oxidase 4 deficiency attenuates experimental osteoarthritis in mice.

OBJECTIVE: Low-grade inflammation plays a pivotal role in osteoarthritis (OA) through exposure to reactive oxygen species (ROS). In chondrocytes, NADPH oxidase 4 (NOX4) is one of the major ROS producers. In this study, we evaluated the role of NOX4 on joint homoeostasis after destabilisation of the medial meniscus (DMM) in mice. METHODS: Experimental OA was simulated on cartilage explants using interleukin-1ß (IL-1ß) and induced by DMM in wild-type (WT) and NOX4 knockout (NOX4-/-) mice. We evaluated NOX4 expression, inflammation, cartilage metabolism and oxidative stress by immunohistochemistry. Bone phenotype was also determined by micro-CT and histomorphometry. RESULTS: Whole body NOX4 deletion attenuated experimental OA in mice, with a significant reduction of the OARSI score at 8 weeks. DMM increased total subchondral bone plate (SB.Th), epiphysial trabecular thicknesses (Tb.Th) and bone volume fraction (BV/TV) in both NOX4-/- and wild-type (WT) mice. Interestingly, DDM decreased total connectivity density (Conn.Dens) and increased medial BV/TV and Tb.Th only in WT mice. Ex vivo, NOX4 deficiency increased aggrecan (AGG) expression and decreased matrix metalloproteinase 13 (MMP13) and collagen type I (COL1) expression. IL-1ß increased NOX4 and 8-hydroxy-2'-deoxyguanosine (8-OHdG) expression in WT cartilage explants but not in NOX4-/-. In vivo, absence of NOX4 increased anabolism and decreased catabolism after DMM. Finally, NOX4 deletion decreased synovitis score, 8-OHdG and F4/80 staining following DMM. CONCLUSION: NOX4 deficiency restores cartilage homoeostasis, inhibits oxidative stress, inflammation and delays OA progression after DMM in mice. These findings suggest that NOX4 represent a potential target to counteract for OA treatment.

Discovery and validation of new Hv1 proton channel inhibitors with onco-therapeutic potential.

The voltage-gated hydrogen channel Hv1 encoded in humans by the HVCN1 gene is a highly selective proton channel that allows large fluxes of protons across biological membranes. Hv1 form functional dimers of four transmembrane spanning proteins resembling the voltage sensing domain of potassium channels. Each subunit is highly selective for protons and is controlled by changes in the transmembrane voltage and pH gradient. Hv1 is most expressed in phagocytic cells where it sustains NADPH oxidase-dependent bactericidal function and was reported to facilitate antibody production by B cells and to promote the maturation and motility of spermatocytes. Hv1 contributes to neuroinflammation following brain damage and favors cancer progression possibly by extruding protons generated during aerobic glycolysis of cancer cells. Lack of specific Hv1 inhibitors has hampered translation of this knowledge to treat immune, fertility, or malignancy diseases. In this study, we show that the genetic deletion of Hv1 delays tumor development in a mouse model of granulocytic sarcoma and report the discovery and characterization of two novel bioavailable inhibitors of Hv1 channels that we validate by orthogonal assays and electrophysiological recordings.

Articular Cartilage Repair After Implantation of Hyaline Cartilage Beads Engineered From Adult Dedifferentiated Chondrocytes: Cartibeads Preclinical Efficacy Study in a Large Animal Model.

BACKGROUND: Chondrocyte-based cell therapy to repair cartilage has been used for >25 years despite current limitations. This work presents a new treatment option for cartilage lesions. HYPOTHESIS: High-quality hyaline cartilage microtissues called Cartibeads are capable of treating focal chondral lesions once implanted in the defect, by complete fusion of Cartibeads among themselves and their integration with the surrounding native cartilage and subchondral bone. STUDY DESIGN: Controlled laboratory study. METHODS: Cartibeads were first produced from human donors and characterized using histology (safranin O staining of glycosaminoglycan [GAG] and immunohistochemistry of collagen I and II) and GAG dosage. Cartibeads from 6 Göttingen minipigs were engineered and implanted in an autologous condition in the knee (4 or 5 lesions per knee). One group was followed up for 3 months and the other for 6 months. Feasibility and efficacy were measured using histological analysis and macroscopic and microscopic scores. RESULTS: Cartibeads revealed hyaline features with strong staining of GAG and collagen II. High GAG content was obtained: 24.6-µg/mg tissue (wet weight), 15.52-µg/mg tissue (dry weight), and 35 ± 3-µg GAG/bead (mean ± SD). Histological analysis of Göttingen minipigs showed good integration of Cartibeads grafts at 3 and 6 months after implantation. The Bern Score of the histological assay comparing grafted versus empty lesions was significant at 3 months (grafted, n = 10; nongrafted, n = 4; score, 3.3 and 5.3, respectively) and 6 months (grafted, n = 11; nongrafted, n = 3; score, 1.6 and 5.1). CONCLUSION: We developed an innovative 3-step method allowing, for the first time, the use of fully dedifferentiated adult chondrocytes with a high number of cell passage (owing to the extensive amplification in culture). Cartibeads engineered from chondrocytes hold potential as an advanced therapy medicinal product for treating cartilage lesions with established efficacy. CLINICAL RELEVANCE: This successful preclinical study, combined with standardized manufacturing of Cartibeads according to good manufacturing practice guidelines, led to the approval of first-in-human clinical trial by the ethics committee and local medical authority. The generated data highlighted a promising therapy to treat cartilage lesions from a small amount of starting biopsy specimen. With our innovative cell amplification technology, very large lesions can be treated, and older active patients can benefit from it.

NADPH oxidase 4 is dispensable for skin myofibroblast differentiation and wound healing.

Differentiation of fibroblasts to myofibroblasts is governed by the transforming growth factor beta (TGF-ß) through a mechanism involving redox signaling and generation of reactive oxygen species (ROS). Myofibroblasts synthesize proteins of the extracellular matrix (ECM) and display a contractile phenotype. Myofibroblasts are predominant contributors of wound healing and several pathological states, including fibrotic diseases and cancer. Inhibition of the ROS-generating enzyme NADPH oxidase 4 (NOX4) has been proposed to mitigate fibroblast to myofibroblast differentiation and to offer a therapeutic option for the treatment of fibrotic diseases. In this study, we addressed the role of NOX4 in physiological wound healing and in TGF-ß-induced myofibroblast differentiation. We explored the phenotypic changes induced by TGF-ß in primary skin fibroblasts isolated from Nox4-deficient mice by immunofluorescence, Western blotting and RNA sequencing. Mice deficient for Cyba, the gene coding for p22phox, a key subunit of NOX4 were used for confirmatory experiments as well as human primary skin fibroblasts. In vivo, the wound healing was similar in wild-type and Nox4-deficient mice. In vitro, despite a strong upregulation following TGF-ß treatment, Nox4 did not influence skin myofibroblast differentiation although a putative NOX4 inhibitor GKT137831 and a flavoprotein inhibitor diphenylene iodonium mitigated this mechanism. Transcriptomic analysis revealed upregulation of the mitochondrial protein Ucp2 and the stress-response protein Hddc3 in Nox4-deficient fibroblasts, which had however no impact on fibroblast bioenergetics. Altogether, we provide extensive evidence that NOX4 is dispensable for wound healing and skin fibroblast to myofibroblast differentiation, and suggest that another H2O2-generating flavoprotein drives this mechanism.

Adipose-Derived Stromal Cells for Chronic Wounds: Scientific Evidence and Roadmap Toward Clinical Practice.

Chronic wounds, ie, non-healing ulcers, have a prevalence of ~1% in the general population. Chronic wounds strongly affect the quality of life and generate considerable medical costs. A fraction of chronic wounds will heal within months of appropriate treatment; however, a significant fraction of patients will develop therapy-refractory chronic wounds, leading to chronic pain, infection, and amputation. Given the paucity of therapeutic options for refractory wounds, cell therapy and in particular the use of adipose-derived stromal cells (ASC) has emerged as a promising concept. ASC can be used as autologous or allogeneic cells. They can be delivered in suspension or in 3D cultures within scaffolds. ASC can be used without further processing (stromal vascular fraction of the adipose tissue) or can be expanded in vitro. ASC-derived non-cellular components, such as conditioned media or exosomes, have also been investigated. Many in vitro and preclinical studies in animals have demonstrated the ASC efficacy on wounds. ASC efficiency appears to occurs mainly through their regenerative secretome. Hitherto, the majority of clinical trials focused mainly on safety issues. However more recently, a small number of randomized, well-controlled trials provided first convincing evidences for a clinical efficacy of ASC-based chronic wound therapies in humans. This brief review summarizes the current knowledge on the mechanism of action, delivery and efficacy of ASC in chronic wound therapy. It also discusses the scientific and pharmaceutical challenges to be solved before ASC-based wound therapy enters clinical reality.

Genetic knockout of NTRK2 by CRISPR/Cas9 decreases neurogenesis and favors glial progenitors during differentiation of neural progenitor stem cells.

The tropomyosin receptor kinase B (TrkB) is encoded by the NTRK2 gene. It belongs to the family of transmembrane tyrosine kinases, which have key roles in the development and maintenance of the nervous system. Brain-derived neurotrophic factor (BDNF) and the neurotrophins NT3 and NT4/5 have high affinity for TrkB. Dysregulation of TrkB is associated to a large spectrum of diseases including neurodegeneration, psychiatric diseases and some cancers. The function of TrkB and its role in neural development have mainly been decrypted using transgenic mouse models, pharmacological modulators and human neuronal cell lines overexpressing NTRK2. In this study, we identified high expression and robust activity of TrkB in ReNcell VM, an immortalized human neural progenitor stem cell line and generated NTRK2-deficient (NTRK2-/-) ReNcell VM using the CRISPR/Cas9 gene editing technology. Global transcriptomic analysis revealed major changes in expression of specific genes responsible for neurogenesis, neuronal development and glial differentiation. In particular, key neurogenic transcription factors were massively down-regulated in NTRK2-/- cells, while early glial progenitor markers were enriched in NTRK2-/- cells compared to NTRK2+/+. This indicates a previously undescribed inhibitory role of TrkB on glial differentiation in addition to its well-described pro-neurogenesis role. Altogether, we have generated for the first time a human neural cell line with a loss-of-function mutation of NTRK2, which represents a reproducible and readily available cell culture system to study the role of TrkB during human neural differentiation, analyze the role of TrkB isoforms as well as validate TrkB antibodies and pharmacological agents targeting the TrkB pathway.

Hyaline Cartilage Microtissues Engineered from Adult Dedifferentiated Chondrocytes: Safety and Role of WNT Signaling.

The repair of damaged articular cartilage is an unmet medical need. Chondrocyte-based cell therapy has been used to repair cartilage for over 20 years despite current limitations. Chondrocyte dedifferentiation upon expansion in monolayer is well known and is the main obstacle to their use as cell source for cartilage repair. Consequently, current approaches often lead to fibrocartilage, which is biomechanically different from hyaline cartilage and not effective as a long-lasting treatment. Here, we describe an innovative 3-step method to engineer hyaline-like cartilage microtissues, named Cartibeads, from high passage dedifferentiated chondrocytes. We show that WNT5A/5B/7B genes were highly expressed in dedifferentiated chondrocytes and that a decrease of the WNT signaling pathway was instrumental for full re-differentiation of chondrocytes, enabling production of hyaline matrix instead of fibrocartilage matrix. Cartibeads showed hyaline-like characteristics based on GAG quantity and type II collagen expression independently of donor age and cartilage quality. In vivo, Cartibeads were not tumorigenic when transplanted into SCID mice. This simple 3-step method allowed a standardized production of hyaline-like cartilage microtissues from a small cartilage sample, making Cartibeads a promising candidate for the treatment of cartilage lesions.

A Cellular Assay for Spike/ACE2 Fusion: Quantification of Fusion-Inhibitory Antibodies after COVID-19 and Vaccination.

Not all antibodies against SARS-CoV-2 inhibit viral entry, and hence, infection. Neutralizing antibodies are more likely to reflect real immunity; however, certain tests investigate protein/protein interaction rather than the fusion event. Viral and pseudoviral entry assays detect functionally active antibodies but are limited by biosafety and standardization issues. We have developed a Spike/ACE2-dependent fusion assay, based on a split luciferase. Hela cells stably transduced with Spike and a large fragment of luciferase were co-cultured with Hela cells transduced with ACE2 and the complementary small fragment of luciferase. Cell fusion occurred rapidly allowing the measurement of luminescence. Light emission was abolished in the absence of Spike and reduced in the presence of proteases. Sera from COVID-19-negative, non-vaccinated individuals or from patients at the moment of first symptoms did not lead to a significant reduction of fusion. Sera from COVID-19-positive patients as well as from vaccinated individuals reduced the fusion. This assay was more correlated to pseudotyped-based entry assay rather than serology or competitive ELISA. In conclusion, we report a new method measuring fusion-inhibitory antibodies in serum, combining the advantage of a complete Spike/ACE2 interaction active on entry with a high degree of standardization, easily allowing automation in a standard bio-safety environment.

Transposon-Directed Insertion-Site Sequencing Reveals Glycolysis Gene gpmA as Part of the H2O2 Defense Mechanisms in Escherichia coli.

Hydrogen peroxide (H2O2) is a common effector of defense mechanisms against pathogenic infections. However, bacterial factors involved in H2O2 tolerance remain unclear. Here we used transposon-directed insertion-site sequencing (TraDIS), a technique allowing the screening of the whole genome, to identify genes implicated in H2O2 tolerance in Escherichia coli. Our TraDIS analysis identified 10 mutants with fitness defect upon H2O2 exposure, among which previously H2O2-associated genes (oxyR, dps, dksA, rpoS, hfq and polA) and other genes with no known association with H2O2 tolerance in E. coli (corA, rbsR, nhaA and gpmA). This is the first description of the impact of gpmA, a gene involved in glycolysis, on the susceptibility of E. coli to H2O2. Indeed, confirmatory experiments showed that the deletion of gpmA led to a specific hypersensitivity to H2O2 comparable to the deletion of the major H2O2 scavenger gene katG. This hypersensitivity was not due to an alteration of catalase function and was independent of the carbon source or the presence of oxygen. Transcription of gpmA was upregulated under H2O2 exposure, highlighting its role under oxidative stress. In summary, our TraDIS approach identified gpmA as a member of the oxidative stress defense mechanism in E. coli.

Development and in vivo validation of small interfering RNAs targeting NOX3 to prevent sensorineural hearing loss.

The reactive oxygen species (ROS)-generating enzyme NOX3 has recently been implicated in the pathophysiology of several acquired forms of sensorineural hearing loss, including cisplatin-, noise- and age-related hearing loss. NOX3 is highly and specifically expressed in the inner ear and therefore represents an attractive target for specific intervention aiming at otoprotection. Despite the strong rationale to inhibit NOX3, there is currently no specific pharmacological inhibitor available. Molecular therapy may represent a powerful alternative. In this study, we developed and tested a collection of small interfering (si) RNA constructs to establish a proof of concept of NOX3 inhibition through local delivery in the mouse inner ear. The inhibitory potential of 10 different siRNA constructs was first assessed in three different cells lines expressing the NOX3 complex. Efficacy of the most promising siRNA construct to knock-down NOX3 was then further assessed in vivo, comparing middle ear delivery and direct intracochlear delivery through the posterior semi-circular canal. While hearing was completely preserved through the intervention, a significant downregulation of NOX3 expression in the mouse inner ear and particularly in the spiral ganglion area at clinically relevant levels (>60%) was observed 48 h after treatment. In contrast to successful intracochlear delivery, middle ear administration of siRNA failed to significantly inhibit Nox3 mRNA expression. In conclusion, intracochlear delivery of NOX3-siRNAs induces a robust temporal NOX3 downregulation, which could be of relevance to prevent predictable acute insults such as cisplatin chemotherapy-mediated ototoxicity and other forms of acquired hearing loss, including post-prevention of noise-induced hearing loss immediately after trauma. Successful translation of our concept into an eventual clinical use in humans will depend on the development of atraumatic and efficient delivery routes into the cochlea without a risk to induce hearing loss through the intervention.

Decreased Levels of SARS-CoV-2 Fusion-Inhibitory Antibodies in the Serum of Aged COVID-19 Patients.

Background: The SARS-CoV-2 pandemic was particularly devastating for elderly people, and the underlying mechanisms of the disease are still poorly understood. In this study, we investigated fusion inhibitory antibodies (fiAbs) in elderly and younger COVID-19 patients and analyzed predictive factors for their occurrence. Methods: Data and samples were collected in two cohorts of hospitalized patients. A fusion assay of SARS-CoV-2 spike-expressing cells with ACE2-expressing cells was used to quantify fiAbs in the serum of patients. Results: A total of 108 patients (52 elderly (mean age 85 ± 7 years); 56 young (mean age 52 ± 10 years)) were studied. The concentrations of fiAbs were lower in geriatric patients, as evidenced at high serum dilutions (1/512). The association between fiAbs and anti-Spike Ig levels was weak (correlation coefficient < 0.3), but statistically significant. Variables associated with fusion were the delay between the onset of symptoms and testing (HR = −2.69; p < 0.001), clinical frailty scale (HR = 4.71; p = 0.035), and WHO severity score (HR = −6.01, p = 0.048). Conclusions: Elderly patients had lower fiAbs levels after COVID-19 infection. The decreased fiAbs levels were associated with frailty.

Periodontitis and peri-implantitis in elderly people experiencing institutional and hospital confinement.

An increasing number of elderly people retain their natural teeth into old age and further, the prevalence of endosseous implants for supporting oral prosthesis is ever increasing. These teeth and implants now present a considerable challenge in terms of maintenance, especially when patients become dependent on care. Periodontal and peri-implant diseases are more prevalent in elderly than in younger age cohorts. There are distinct differences related to the inflammatory response between periodontal and peri-implant tissues, both in young and old age. The age-related reasons for the increase in periodontal infections may be related to poor oral hygiene because of a loss of dexterity or vision, but also to immunosenescence. This term describes the aging of the immune system and the decline of its effectiveness with age. Low-grade infections, like chronic periodontitis, may cause low-grade inflammation and subsequently increase the likelihood of developing chronic diseases. In return, treatment of periodontitis may improve general health, as demonstrated for diabetes. A second mechanism illustrating how poor oral health translates into systemic disease is the risk of developing aspiration pneumonia. The treatment options in old age should be evaluated with regard to the issues of general health and maintenance. Systematic periodontal maintenance therapy, as performed in younger age cohorts, may be difficult to implement in elderly people experiencing institutional or hospital confinement because of logistics, barriers related to patients and caregivers, or cost. The scale of periodontal disease in old age represents a public health issue.

Alpha-1 Antitrypsin Reduces Disease Progression in a Mouse Model of Charcot-Marie-Tooth Type 1A: A Role for Decreased Inflammation and ADAM-17 Inhibition.

Charcot-Marie-Tooth disease type 1 (CMT1A) is a hereditary peripheral neuropathy for which there is no available therapy. Alpha-1 antitrypsin (AAT) is an abundant serine protease inhibitor with anti-inflammatory and immunomodulating properties. Here, we tested whether treatment with human AAT (hAAT) would have a therapeutic effect on CMT1A in a PMP22 transgenic mouse model. Our results show that hAAT significantly improved compound muscle action potential and histopathological features and decreased circulating IL-6 in CMT1A mice. We also investigated some of the possible underlying mechanisms in vitro. We confirmed that hAAT inhibits ADAM-17, a protease that has been implicated in blocking myelination. Furthermore, both hAAT and recombinant human AAT (rhAAT) were able to attenuate the activation of a macrophage/microglia cell line, markedly decreasing the activation of the MHC class II promoter and the expression of pro-inflammatory genes such as IL-1ß and the endoplasmic reticulum (ER) stress marker ATF3. Taken together, our results demonstrate for the first time that hAAT is able to reduce the progression of CMT1A, possibly by dampening inflammation and by regulating ADAM-17. Given the already well-established safety profile of hAAT, specifically in AAT deficiency disease (AATD), we suggest that the findings of our study should be promptly investigated in CMT1A patients.

Transcriptomic Analysis of E. coli after Exposure to a Sublethal Concentration of Hydrogen Peroxide Revealed a Coordinated Up-Regulation of the Cysteine Biosynthesis Pathway.

Hydrogen peroxide (H2O2) is a key defense component of host-microbe interaction. However, H2O2 concentrations generated by immune cells or epithelia are usually insufficient for bacterial killing and rather modulate bacterial responses. Here, we investigated the impact of sublethal H2O2 concentration on gene expression of E. coli BW25113 after 10 and 60 min of exposure. RNA-seq analysis revealed that approximately 12% of bacterial genes were strongly dysregulated 10 min following exposure to 2.5 mM H2O2. H2O2 exposure led to the activation of a specific antioxidant response and a general stress response. The latter was characterized by a transient down-regulation of genes involved in general metabolism, such as nucleic acid biosynthesis and translation, with a striking and coordinated down-regulation of genes involved in ribosome formation, and a sustained up-regulation of the SOS response. We confirmed the rapid transient and specific response mediated by the transcription factor OxyR leading to up-regulation of antioxidant systems, including the catalase-encoding gene (katG), that rapidly degrade extracellular H2O2 and promote bacterial survival. We documented a strong and transient up-regulation of genes involved in sulfur metabolism and cysteine biosynthesis, which are under the control of the transcription factor CysB. This strong specific transcriptional response to H2O2 exposure had no apparent impact on bacterial survival, but possibly replenishes the stores of oxidized cysteine and glutathione. In summary, our results demonstrate that different stress response mechanisms are activated by H2O2 exposure and highlight the cysteine synthesis as an antioxidant response in E. coli.