GEF-H1 controls microtubule-dependent sensing of nucleic acids for antiviral host defenses.

Detailed understanding of the signaling intermediates that confer the sensing of intracellular viral nucleic acids for induction of type I interferons is critical for strategies to curtail viral mechanisms that impede innate immune defenses. Here we show that the activation of the microtubule-associated guanine nucleotide exchange factor GEF-H1, encoded by Arhgef2, is essential for sensing of foreign RNA by RIG-I-like receptors. Activation of GEF-H1 controls RIG-I-dependent and Mda5-dependent phosphorylation of IRF3 and induction of IFN-ß expression in macrophages. Generation of Arhgef2(-/-) mice revealed a pronounced signaling defect that prevented antiviral host responses to encephalomyocarditis virus and influenza A virus. Microtubule networks sequester GEF-H1 that upon activation is released to enable antiviral signaling by intracellular nucleic acid detection pathways.

Norrie disease protein is essential for cochlear hair cell maturation.

Mutations in the gene for Norrie disease protein (Ndp) cause syndromic deafness and blindness. We show here that cochlear function in an Ndp knockout mouse deteriorated with age: At P3-P4, hair cells (HCs) showed progressive loss of Pou4f3 and Gfi1, key transcription factors for HC maturation, and Myo7a, a specialized myosin required for normal function of HC stereocilia. Loss of expression of these genes correlated to increasing HC loss and profound hearing loss by 2 mo. We show that overexpression of the Ndp gene in neonatal supporting cells or, remarkably, up-regulation of canonical Wnt signaling in HCs rescued HCs and cochlear function. We conclude that Ndp secreted from supporting cells orchestrates a transcriptional network for the maintenance and survival of HCs and that increasing the level of ß-catenin, the intracellular effector of Wnt signaling, is sufficient to replace the functional requirement for Ndp in the cochlea.

A CNN Sound Classification Mechanism Using Data Augmentation.

Sound classification has been widely used in many fields. Unlike traditional signal-processing methods, using deep learning technology for sound classification is one of the most feasible and effective methods. However, limited by the quality of the training dataset, such as cost and resource constraints, data imbalance, and data annotation issues, the classification performance is affected. Therefore, we propose a sound classification mechanism based on convolutional neural networks and use the sound feature extraction method of Mel-Frequency Cepstral Coefficients (MFCCs) to convert sound signals into spectrograms. Spectrograms are suitable as input for CNN models. To provide the function of data augmentation, we can increase the number of spectrograms by setting the number of triangular bandpass filters. The experimental results show that there are 50 semantic categories in the ESC-50 dataset, the types are complex, and the amount of data is insufficient, resulting in a classification accuracy of only 63%. When using the proposed data augmentation method (K = 5), the accuracy is effectively increased to 97%. Furthermore, in the UrbanSound8K dataset, the amount of data is sufficient, so the classification accuracy can reach 90%, and the classification accuracy can be slightly increased to 92% via data augmentation. However, when only 50% of the training dataset is used, along with data augmentation, the establishment of the training model can be accelerated, and the classification accuracy can reach 91%.

Circulatory antigen processing by mucosal dendritic cells controls CD8(+) T cell activation.

Circulatory antigens transit through the small intestine via the fenestrated capillaries in the lamina propria prior to entering into the draining lymphatics. But whether or how this process controls mucosal immune responses remains unknown. Here we demonstrate that dendritic cells (DCs) of the lamina propria can sample and process both circulatory and luminal antigens. Surprisingly, antigen cross-presentation by resident CX3CR1(+) DCs induced differentiation of precursor cells into CD8(+) T cells that expressed interleukin-10 (IL-10), IL-13, and IL-9 and could migrate into adjacent compartments. We conclude that lamina propria CX3CR1(+) DCs facilitate the surveillance of circulatory antigens and act as a conduit for the processing of self- and intestinally absorbed antigens, leading to the induction of CD8(+) T cells, that partake in the control of T cell activation during mucosal immune responses.

Transcriptional response to Wnt activation regulates the regenerative capacity of the mammalian cochlea.

Lack of sensory hair cell (HC) regeneration in mammalian adults is a major contributor to hearing loss. In contrast, the neonatal mouse cochlea retains a transient capacity for regeneration, and forced Wnt activation in neonatal stages promotes supporting cell (SC) proliferation and induction of ectopic HCs. We currently know little about the temporal pattern and underlying mechanism of this age-dependent regenerative response. Using an in vitro model, we show that Wnt activation promotes SC proliferation following birth, but prior to postnatal day (P) 5. This age-dependent decline in proliferation occurs despite evidence that the Wnt pathway is postnatally active and can be further enhanced by Wnt stimulators. Using an in vivo mouse model and RNA sequencing, we show that proliferation in the early neonatal cochlea is correlated with a unique transcriptional response that diminishes with age. Furthermore, we find that augmenting Wnt signaling through the neonatal stages extends the window for HC induction in response to Notch signaling inhibition. Our results suggest that the downstream transcriptional response to Wnt activation, in part, underlies the regenerative capacity of the mammalian cochlea.

Skin nerve pathology: Biomarkers of premanifest and manifest amyloid neuropathy.

OBJECTIVE: Small-fiber sensory and autonomic symptoms are early presentations of familial amyloid polyneuropathy (FAP) with transthyretin (TTR) mutations. This study aimed to explore the potential of skin nerve pathologies as early and disease-progression biomarkers and their relationship with skin amyloid deposits. METHODS: Skin biopsies were performed in patients and carriers to measure intraepidermal nerve fiber (IENF) density, sweat gland innervation index of structural protein gene product 9.5 (SGII[PGP9.5]) and peptidergic vasoactive intestinal peptide (SGII[VIP]), and cutaneous amyloid index. These skin pathologies were analyzed with clinical disability assessed by FAP stage score (stage 0-4) and compared to neurophysiological and psychophysical tests. RESULTS: There were 70 TTR-mutant subjects (22 carriers and 48 patients), and 66 cases were TTR-A97S. Skin nerve pathologies were distinct according to stage. In carriers, both skin denervation and peptidergic sudomotor denervation were evident: (1) IENF density was gradually reduced from stage 0 through 4, and (2) SGII(VIP) was markedly reduced from stage 1 to 2. In contrast, SGII(PGP9.5) was similar between carriers and controls, but it declined in patients from stage 2. Skin amyloids were absent in carriers and became detectable from stage 1. Cutaneous amyloid index was correlated with SGII(PGP9.5) and stage in a multivariate mixed-effect model. When all tests were compared, only IENF density, SGII(PGP9.5), and cutaneous amyloid index were correlated with stage, and IENF density had the highest abnormal rate in carriers. INTERPRETATION: Biomarkers of sensory and sudomotor innervation exhibited a stage-dependent progression pattern, with sensory nerve degeneration as the early skin nerve pathology. Ann Neurol 2019;85:560-573.

Temporal expression patterns of distinct cytokines and M1/M2 macrophage polarization regulate rheumatoid arthritis progression.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation of synovial joints and often associated with chronic pain. Chronic joint inflammation is attributed to severe proliferation of synoviocytes and resident macrophages and infiltration of immune cells. These cells secrete pro-inflammatory cytokines such as tumor necrosis factor α (TNF-α), interleukin 6 (IL-6) and IL-17 to overcome actions of anti-inflammatory cytokines, thereby maintaining chronic inflammation and pain. The imbalance between pro-inflammatory cytokines (produced by M1 macrophages) and anti-inflammatory cytokines (produced by M2 macrophages) is a feature of RA progression, but the switch time of M1/M2 polarization and which receptor regulates the switch remain unsolved. Here we used an established RA mouse model to demonstrate that TNF-α expression was responsible for the initial acute stage of inflammation and pain (1-4 weeks), IL-17 expression the transition stage (4-12 weeks), and IL-6 expression the later maintenance stage (> 12 weeks). The switch time of M1/M2 polarization occurred at 4-8 weeks. We also identified a potential compound, anthra[2,1-c][1,2,5] thiadiazole-6,11-dione (NSC745885), that specifically inhibited T-cell death-associated gene 8 (TDAG8) function and expression. NSC745885 decreased joint inflammation and destruction and attenuated pain by reducing cytokine production and regulating the M1/M2 polarization switch. TDAG8 may participate in regulating the M1/M2 polarization and temporal expression of distinct cytokines to control RA progression.

Microtubule-Mediated NLRP3 Inflammasome Activation Is Independent of Microtubule-Associated Innate Immune Factor GEF-H1 in Murine Macrophages.

Inflammasomes are intracellular multiple protein complexes that mount innate immune responses to tissue damage and invading pathogens. Their excessive activation is crucial in the development and pathogenesis of inflammatory disorders. Microtubules have been reported to provide the platform for mediating the assembly and activation of NLRP3 inflammasome. Recently, we have identified the microtubule-associated immune molecule guanine nucleotide exchange factor-H1 (GEF-H1) that is crucial in coupling microtubule dynamics to the initiation of microtubule-mediated immune responses. However, whether GEF-H1 also controls the activation of other immune receptors that require microtubules is still undefined. Here we employed GEF-H1-deficient mouse bone marrow-derived macrophages (BMDMs) to interrogate the impact of GEF-H1 on the activation of NLRP3 inflammasome. NLRP3 but not NLRC4 or AIM2 inflammasome-mediated IL-1ß production was dependent on dynamic microtubule network in wild-type (WT) BMDMs. However, GEF-H1 deficiency did not affect NLRP3-driven IL-1ß maturation and secretion in macrophages. Moreover, α-tubulin acetylation and mitochondria aggregations were comparable between WT and GEF-H1-deficient BMDMs in response to NLRP3 inducers. Further, GEF-H1 was not required for NLRP3-mediated immune defense against Salmonella typhimurium infection. Collectively, these findings suggest that the microtubule-associated immune modulator GEF-H1 is dispensable for microtubule-mediated NLRP3 activation and host defense in mouse macrophages.

Helminthostachys zeylanica alleviates hepatic steatosis and insulin resistance in diet-induced obese mice.

BACKGROUND: Obesity and its associated health conditions, type 2 diabetes mellitus (T2DM) and nonalcoholic fatty liver disease (NAFLD), are worldwide health problems. It has been shown that insulin resistance is associated with increased hepatic lipid and causes hepatic steatosis through a myriad of mechanisms, including inflammatory signaling. METHODS: Helminthostachys zeylanica (HZ) is used widely as a common herbal medicine to relieve fever symptoms and inflammatory diseases in Asia. In the present study, we evaluated whether HZ has therapeutic effects on obesity, NAFLD and insulin resistance. The protective effects of HZ extract were examined using free fatty acid-induced steatosis in human HuS-E/2 cells and a high-fat diet-induced NAFLD in mice. RESULTS: The major components of the HZ extract are ugonins J and K, confirmed by HPLC. Incubation of human hepatocytes, HuS-E/2 cells, with palmitate markedly increased lipid accumulation and treatment with the HZ extract significantly decreased lipid deposition and facilitated AMPK and ACC activation. After 12 weeks of a high-fat diet with HZ extract treatment, the HFD mice were protected from hyperlipidemia and hyperglycemia. HZ extract prevented body weight gain, adipose tissue expansion and adipocyte hypertrophy in the HFD mice. In addition, fat accumulation was reduced in mice livers. Moreover, the insulin sensitivity-associated index, which evaluates insulin function, was also significantly restored. CONCLUSIONS: These results suggest that HZ has a promising pharmacological effect on high-fat diet-induced obesity, hepatic steatosis and insulin resistance, which may have the potential for clinical application.

Diphtheria Toxin-Induced Cell Death Triggers Wnt-Dependent Hair Cell Regeneration in Neonatal Mice.

UNLABELLED: Cochlear hair cells (HCs), the sensory cells that respond to sound, do not regenerate after damage in adult mammals, and their loss is a major cause of deafness. Here we show that HC regeneration in newborn mouse ears occurred spontaneously when the original cells were ablated by treatment with diphtheria toxin (DT) in ears that had been engineered to overexpress the DT receptor, but was not detectable when HCs were ablated in vivo by the aminoglycoside antibiotic neomycin. A variety of Wnts (Wnt1, Wnt2, Wnt2b, Wnt4, Wnt5a, Wnt7b, Wnt9a, Wnt9b, and Wnt11) and Wnt pathway component Krm2 were upregulated after DT damage. Nuclear ß-catenin was upregulated in HCs and supporting cells of the DT-damaged cochlea. Pharmacological inhibition of Wnt decreased spontaneous regeneration, confirming a role of Wnt signaling in HC regeneration. Inhibition of Notch signaling further potentiated supporting cell proliferation and HC differentiation that occurred spontaneously. The absence of new HCs in the neomycin ears was correlated to less robust Wnt pathway activation, but the ears subjected to neomycin treatment nonetheless showed increased cell division and HC differentiation after subsequent forced upregulation of ß-catenin. These studies suggest, first, that Wnt signaling plays a key role in regeneration, and, second, that the outcome of a regenerative response to damage in the newborn cochlea is determined by reaching a threshold level of Wnt signaling rather than its complete absence or presence. SIGNIFICANCE STATEMENT: Sensory HCs of the inner ear do not regenerate in the adult, and their loss is a major cause of deafness. We found that HCs regenerated spontaneously in the newborn mouse after diphtheria toxin (DT)-induced, but not neomycin-induced, HC death. Regeneration depended on activation of Wnt signaling, and regeneration in DT-treated ears correlated to a higher level of Wnt activation than occurred in nonregenerating neomycin-treated ears. This is significant because insufficient regeneration caused by a failure to reach a threshold level of signaling, if true in the adult, has the potential to be exploited for development of clinical approaches for the treatment of deafness caused by HC loss.

Mitochondrial immobilization mediated by syntaphilin facilitates survival of demyelinated axons.

Axonal degeneration is a primary cause of permanent neurological disability in individuals with the CNS demyelinating disease multiple sclerosis. Dysfunction of axonal mitochondria and imbalanced energy demand and supply are implicated in degeneration of chronically demyelinated axons. The purpose of this study was to define the roles of mitochondrial volume and distribution in axonal degeneration following acute CNS demyelination. We show that the axonal mitochondrial volume increase following acute demyelination of WT CNS axons does not occur in demyelinated axons deficient in syntaphilin, an axonal molecule that immobilizes stationary mitochondria to microtubules. These findings were supported by time-lapse imaging of WT and syntaphilin-deficient axons in vitro. When demyelinated, axons deficient in syntaphilin degenerate at a significantly greater rate than WT axons, and this degeneration can be rescued by reducing axonal electrical activity with the Na(+) channel blocker flecainide. These results support the concept that syntaphilin-mediated immobilization of mitochondria to microtubules is required for the volume increase of axonal mitochondria following acute demyelination and protects against axonal degeneration in the CNS.

Sudomotor innervation in transthyretin amyloid neuropathy: Pathology and functional correlates.

OBJECTIVE: Autonomic neuropathy is a major component of familial amyloid polyneuropathy (FAP) due to mutated transthyretin, with sudomotor failure as a common manifestation. This study aimed to investigate the pathology and clinical significance of sudomotor denervation. METHODS: Skin biopsies were performed on the distal leg of FAP patients with a follow-up duration of 3.8 ± 1.6 years. Sudomotor innervation was stained with 2 markers: protein gene product 9.5 (PGP 9.5), a general neuronal marker, and vasoactive intestinal peptide (VIP), a sudomotor nerve functional marker, followed by quantitation according to sweat gland innervation index (SGII) for PGP 9.5 (SGIIPGP 9.5) and VIP (SGIIVIP). RESULTS: There were 28 patients (25 men) with Ala97Ser transthyretin and late onset (59.9 ± 6.0 years) disabling neuropathy. Autonomic symptoms were present in 22 patients (78.6%) at the time of skin biopsy. The SGIIPGP 9.5 and SGIIVIP of FAP patients were significantly lower than those of age- and gender-matched controls. The reduction of SGIIVIP was more severe than that of SGIIPGP 9.5 (p = 0.002). Patients with orthostatic hypotension or absent sympathetic skin response at palms were associated with lower SGIIPGP 9.5 (p = 0.019 and 0.002, respectively). SGIIPGP 9.5 was negatively correlated with the disability grade at the time of skin biopsy (p = 0.004), and was positively correlated with the interval from the time of skin biopsy to the time of wheelchair usage (p = 0.029). INTERPRETATION: This study documented the pathological evidence of sudomotor denervation in FAP. SGIIPGP 9.5 was functionally correlated with autonomic symptoms, autonomic tests, ambulation status, and progression of disability.

Effects of Chain-Chain Associations on Hybridization in DNA Brushes.

Hybridization of solution nucleic acids to DNA brushes is widely encountered in diagnostic and materials science applications. Typically, brush chain lengths of ten or more nucleotides are used to provide the needed sequence specificity and binding affinity. At these lengths, coincidental occurrence of complementary regions is expected to lead to associations between the nominally single-stranded brush chains due to intra- or interchain base pairing. This report investigates how these associations impact the brushes' hybridization activity toward complementary "target" sequences. Brushes were prepared from 20-mer chains with four-nucleotide-long "adhesive regions" through which neighboring chains could interact. The affinity and position of the adhesive region along the chain backbone were varied. DNA brushes were exposed to complementary solution targets, and the corresponding melting transitions were measured to estimate free energies of the brush-target hybridization. These results revealed that higher affinity adhesive regions more extensively suppressed brush hybridization relative to hybridization in solution. Associations near the middle of the chains were found to be more penalizing than those at the immobilized or the free end of the chains. Provided that the brush chains were close enough to associate, changes in brush density did not exert a significant effect on hybridization thermodynamics within the investigated coverage window. Comparison of the DNA brush results with those from commercial Affymetrix single-nucleotide-polymorphism (SNP) microarrays revealed agreement in the impact of chain associations on hybridization.

Mitochondrial fission augments capsaicin-induced axonal degeneration.

Capsaicin, an agonist of transient receptor potential vanilloid receptor 1, induces axonal degeneration of peripheral sensory nerves and is commonly used to treat painful sensory neuropathies. In this study, we investigated the role of mitochondrial dynamics in capsaicin-induced axonal degeneration. In capsaicin-treated rodent sensory axons, axonal swellings, decreased mitochondrial stationary site length and reduced mitochondrial transport preceded axonal degeneration. Increased axoplasmic Ca(2+) mediated the alterations in mitochondrial length and transport. While sustaining mitochondrial transport did not reduce axonal swellings in capsaicin-treated axons, preventing mitochondrial fission by overexpression of mutant dynamin-related protein 1 increased mitochondrial length, retained mitochondrial membrane potentials and reduced axonal loss upon capsaicin treatment. These results establish that mitochondrial stationary site size significantly affects axonal integrity and suggest that inhibition of Ca(2+)-dependent mitochondrial fission facilitates mitochondrial function and axonal survival following activation of axonal cationic channels.

HSP27 Modulates Neuropathic Pain by Inhibiting P2X3 Degradation.

The role of heat shock protein 27 (HSP27), a chaperone, in neuropathic pain after nerve injury has not been systematically surveyed despite its neuroprotective and regeneration-promoting effects. In this study, we found that HSP27 expression in sensory neurons of the dorsal root ganglia (DRG) mediated nerve injury-induced neuropathic pain. Neuropathic pain behaviors were alleviated by silencing HSP27 in the DRG of a rat spinal nerve ligation (SNL) model. Local injection of an HSP27-overexpression construct into the DRG of naïve rats elicited neuropathic pain behaviors. HSP27 interacted with a purinergic receptor, P2X3, and their expression patterns corroborated the induction and reversal of neuropathic pain according to two lines of evidence: colocalization immunohistochemically and immunoprecipitation biochemically. In a cell model cotransfected with HSP27 and P2X3, the degradation rate of P2X3 was reduced in the presence of HSP27. Such an alteration was mediated by reducing P2X3 ubiquitination in SNL rats and was reversed after silencing HSP27 in the DRGs of SNL rats. In summary, the interaction of HSP27 with P2X3 provides a new mechanism of injury-induced neuropathic pain that could serve as an alternative therapeutic target.

Candida tropicalis-derived vitamin B3 exerts protective effects against intestinal inflammation by promoting IL-17A/IL-22-dependent epithelial barrier function.

Candida tropicalis-a prevalent gut commensal fungus in healthy individuals - contributes to intestinal health and disease. However, how commensal C. tropicalis influences intestinal homeostasis and barrier function is poorly understood. Here, we demonstrated that the reference strain of C. tropicalis (MYA-3404) ameliorated intestinal inflammation in murine models of chemically induced colitis and bacterial infection. Intestinal colonization of C. tropicalis robustly upregulated the expression of IL-17A and IL-22 to increase barrier function and promote proliferation of intestinal epithelial cells in the mouse colon. Metabolomics analysis of fecal samples from mice colonized with C. tropicalis revealed alterations in vitamin B3 metabolism, promoting conversion of nicotinamide to nicotinic acid. Although nicotinamide worsened colitis, treatment with nicotinic acid alleviated disease symptoms and enhanced epithelial proliferation and Th17 cell differentiation. Oral gavage of C. tropicalis mitigated nicotinamide-induced intestinal dysfunction in experimental colitis. Blockade of nicotinic acid production with nicotinamidase inhibitors lowered the protective effects against colitis in mice treated with C. tropicalis. Notably, a clinical C. tropicalis strain isolated from patients with candidemia lacked the protective effects against murine colitis observed with the reference strain. Together, our results highlight a novel role for C. tropicalis in resolving intestinal inflammation through the modulation of vitamin B3 metabolism.

• Protection against colitis conferred by intestinal colonization of Candida tropicalis depends on metabolic activity and strain• C. tropicalis MYA-3404 supplementation promotes intestinal epithelial barrier function through IL-17A and IL-22 expressed by Th17 cells, γδ T cells, and ILC3• MYA-3404 strain uses its enzymatic activity to modulate vitamin B3 metabolism for protective benefits.

Histones of Neutrophil Extracellular Traps Directly Disrupt the Permeability and Integrity of the Intestinal Epithelial Barrier.

BACKGROUND: Increased neutrophil extracellular trap (NET) formation and abundant NET-associated proteins are frequently found in the inflamed colon of patients with inflammatory bowel disease. Peptidyl arginine deiminase 4 (PAD4) activation is essential for the generation of NET and NET-mediated pathogenesis. However, the role of PAD4-dependent NET formation in murine inflammatory bowel disease models and the molecular mechanisms responsible for the altered gut barrier function are unknown. METHODS: Wild-type and Pad4 knockout (Pad4-/-) mice were administrated 3% dextran sulfate sodium (DSS) in their drinking water. Caco-2 monolayers were used to test the effect of NETs on intestinal barrier function and cytotoxicity. Histones were intrarectally administrated to wild-type mice to determine their effects on intestinal barrier function and cytotoxicity in vivo. RESULTS: PAD4 deficiency reduced the severity of DSS-induced colitis with decreased intestinal NET formation and enhanced gut barrier function and integrity in mice. NETs disrupted the barrier function in intestinal epithelial Caco-2 monolayers through their protein, rather than DNA, components. Pretreatment of NETs with histone inhibitors abrogated the effects on epithelial permeability. Consistent with these observations, adding purified histone proteins to Caco-2 monolayers significantly damaged epithelial barrier function, which was associated with the abnormal distribution and integrity of tight junctions as well as with increased cell death. Furthermore, intrarectal administration of histones damaged the intestinal barrier integrity and induced cytotoxicity in the mouse colon epithelium. CONCLUSIONS: PAD4-mediated NET formation has a detrimental role in acute colitis. NET-associated histones directly inhibit intestinal barrier function, resulting in cytotoxicity in vitro and in vivo.

Peptidyl arginine deiminase 4­dependent neutrophil extracellular trap formation is detrimental to intestinal barrier function in acute colitis. Neutrophil extracellular trap­associated histones altered the integrity of tight junction and adherens junction proteins as well as induced intestinal epithelial cell death that resulted in increased gut epithelium permeability.

Optimisation of anti-interleukin-6 therapy: Precision medicine through mathematical modelling.

Background: Dysregulated interleukin (IL)-6 production can be characterised by the levels present, the kinetics of its rise and its inappropriate location. Rapid, excessive IL-6 production can exacerbate tissue damage in vital organs. In this situation, therapy with an anti-IL-6 or anti-IL-6 receptor (IL-6R) monoclonal antibody, if inappropriately dosed, may be insufficient to fully block IL-6 signalling and normalise the immune response. Methods: We analysed inhibition of C-reactive protein (CRP) - a biomarker for IL-6 activity - in patients with COVID-19 or idiopathic multicentric Castleman disease (iMCD) treated with tocilizumab (anti-IL-6R) or siltuximab (anti-IL-6), respectively. We used mathematical modelling to analyse how to optimise anti-IL-6 or anti-IL-6R blockade for the high levels of IL-6 observed in these diseases. Results: IL-6 signalling was insufficiently inhibited in patients with COVID-19 or iMCD treated with standard doses of anti-IL-6 therapy. Patients whose disease worsened throughout therapy had only partial inhibition of CRP production. Our model demonstrated that, in a scenario representative of iMCD with persistent high IL-6 production not controlled by a single dose of anti-IL-6 therapy, repeated administration more effectively inhibited IL-6 activity. In a situation with rapid, high, dysregulated IL-6 production, such as severe COVID-19 or a cytokine storm, repeated daily administration of an anti-IL-6/anti-IL-6R agent, or alternating daily doses of anti-IL-6 and anti-IL-6R therapies, could neutralise IL-6 activity. Conclusion: In clinical practice, IL-6 inhibition should be individualised based on pathophysiology to achieve full blockade of CRP production. Funding: EUSA Pharma funded medical writing assistance and provided access to the phase II clinical data of siltuximab for analysis.

Exploitation of a rod-shaped, acid-labile curcumin-loaded polymeric nanogel system in the treatment of systemic inflammation.

Curcumin is proven to have potent anti-inflammatory activity, but its low water solubility and rapid degradation in physiological conditions limit its clinical use, particularly in intravenous drug delivery. In this study, we fabricated rod-shaped, acid-labile nanogels, using high biosafe and biocompatible polymers, for intravenous application in systemic inflammation treatment. The constituent polymers of the nanogels were prepared via the conjugation of vitamin B6 derivatives, including pyridoxal and pyridoxamine, onto poly(glutamate) with ester bonds. The aldehyde groups of the pyridoxal and amine groups of the pyridoxamine on the polymers enable crosslinking using a Schiff base during the solvent evaporation procedure for the preparation of the rod-shaped nanogels. Our study is the first to introduce this linkage, which is generated from two vitamin B6 derivatives into a nanogel system. It is also the first to fabricate a rod-shaped nanogel system via simple solvent evaporation. Under acidic conditions, such as those encountered in the endosomes and lysosomes within inflammatory macrophage cells spread in the whole body, imine bonds are cleaved and release payloads. The nanogel polymers were successfully synthesized and characterized, and the formation and disappearance of the Schiff base under neutral and acidic conditions were also confirmed using Fourier transform infrared spectroscopy. Following curcumin encapsulation, the long, rod-shaped nanogels were able to rapidly internalize into macrophage cells in static or adhere to cells under the flows, release their payloads in the acid milieus, and, thus, mitigate curcumin degradation. Consequently, curcumin-loaded, rod-shaped nanogels displayed exceptional anti-inflammatory activity both in vitro and in vivo, by efficiently inhibiting pro-inflammatory mediator secretion. These results demonstrate the feasibility of our acid-labile, rod-shaped nanogels for the treatment of systemic inflammation.

Delayed intervention with a novel SGLT2 inhibitor NGI001 suppresses diet-induced metabolic dysfunction and non-alcoholic fatty liver disease in mice.

BACKGROUND AND PURPOSE: Non-alcoholic fatty liver disease (NAFLD), including non-alcoholic steatohepatitis, is closely related to metabolic diseases such as obesity and diabetes. Despite an accumulating number of studies, no pharmacotherapy that targets NAFLD has received general approval for clinical use. EXPERIMENTAL APPROACH: Inhibition of the sodium-glucose cotransporter 2 (SGLT2) is a promising approach to treat diabetes, obesity, and associated metabolic disorders. In this study, we investigated the effect of a novel SGLT2 inhibitor, NGI001, on NAFLD and obesity-associated metabolic symptoms in high-fat diet (HFD)-induced obese mice. KEY RESULTS: Delayed intervention with NGI001 protected against body weight gain, hyperglycaemia, hyperlipidaemia, and hyperinsulinaemia, compared with HFD alone. Adipocyte hypertrophy was prevented by administering NGI001. NGI001 inhibited impaired glucose metabolism and regulated the secretion of adipokines associated with insulin resistance. In addition, NGI001 supplementation suppressed hepatic lipid accumulation and inflammation but had little effect on kidney function. In-depth investigations showed that NGI001 ameliorated fat deposition and increased AMPK phosphorylation, resulting in phosphorylation of its major downstream target, acetyl-CoA carboxylase, in human hepatocyte HuS-E/2 cells. This cascade ultimately led to the down-regulation of downstream fatty acid synthesis-related molecules and the up-regulation of downstream ß oxidation-associated molecules. Surprisingly, NGI001 decreased gene and protein expression of SGLT1 and SGLT2 and glucose uptake in oleic acid-treated HuS-E/2 cells. CONCLUSION AND IMPLICATIONS: Our findings suggest the novel SGLT2 inhibitor, NGI001 has therapeutic potential to attenuate or delay the onset of diet-induced metabolic diseases and NAFLD.