Appropriate glycemic management protects the germline but not the uterine environment in hyperglycemia.

Emerging evidence indicates that parental diseases can impact the health of subsequent generations through epigenetic inheritance. Recently, it was shown that maternal diabetes alters the metaphase II oocyte transcriptome, causing metabolic dysfunction in offspring. However, type 1 diabetes (T1D) mouse models frequently utilized in previous studies may be subject to several confounding factors due to severe hyperglycemia. This limits clinical translatability given improvements in glycemic control for T1D subjects. Here, we optimize a T1D mouse model to investigate the effects of appropriately managed maternal glycemic levels on oocytes and intrauterine development. We show that diabetic mice with appropriate glycemic control exhibit better long-term health, including maintenance of the oocyte transcriptome and chromatin accessibility. We further show that human oocytes undergoing in vitro maturation challenged with mildly increased levels of glucose, reflecting appropriate glycemic management, also retain their transcriptome. However, fetal growth and placental function are affected in mice despite appropriate glycemic control, suggesting the uterine environment rather than the germline as a pathological factor in developmental programming in appropriately managed diabetes.

Risk factors for Charcot foot development in individuals with diabetes mellitus.

AIMS/HYPOTHESIS: Charcot foot is a complication of diabetes mellitus that has potentially disastrous consequences. Although it was first described in 1868 and found to be associated with diabetes in 1936, there is still uncertainty about the risk factors affecting the development of the condition. Here, we aim to identify risk factors for Charcot foot in a nationwide cohort study. METHODS: A retrospective register-based cohort study was performed for the period 2001-2016, using nationwide registries. Individuals with diabetes and Charcot foot were identified and matched by diabetes type and with similar diabetes duration with individuals with diabetes but not Charcot foot. Logistic regression analyses were used to identify risk factors. RESULTS: A total of 3397 participants with diabetes mellitus and Charcot foot and 27,662 control participants with diabetes but without Charcot foot were included. HbA1c, duration of diabetes, micro- and macroalbuminuria, retinopathy and atherosclerosis (general and peripheral) were identified as risk factors for Charcot foot in participants with type 1 diabetes and participants with type 2 diabetes. CONCLUSIONS/INTERPRETATION: In the most extensive study on Charcot foot to date, we identified distinctive and common risk factors associated with the development of Charcot foot in individuals with type 1 diabetes and type 2 diabetes.

Identification of early risk factors for anti-citrullinated-protein-antibody positive rheumatoid arthritis-a prospective cohort study.

OBJECTIVE: Individuals positive for anti-cyclic-peptide-antibodies (anti-CCP) and musculoskeletal complaints (MSK-C) are at risk for developing rheumatoid arthritis (RA). In this study we aimed to investigate factors involved in arthritis progression. METHODS: Anti-CCP2-positive individuals with MSK-C referred to a rheumatologist were recruited. Individuals lacked arthritis at clinical and ultrasound examination and were followed for ≥three years or until clinical arthritis diagnosis. Blood samples from inclusion were analyzed for; nine anti-citrullinated-protein-antibody (ACPA) reactivities (citrullinated α-1-enolase, fibrinogen, filaggrin, histone, vimentin and tenascin peptides); 92 inflammation-associated proteins; and HLA-shared epitope alleles. Cox regression was applied to the data to identify independent predictors in a model. RESULTS: 267 individuals were included with median follow up of 49 months (IQR: 22-60). 101 (38%) developed arthritis after median 14 months (IQR: 6-27). The analysis identified that presence of at least one ACPA reactivity (HR 8.0, 95% CI 2.9-22), ultrasound detected tenosynovitis (HR 3.4, 95% CI 2.0-6.0), IL6 levels (HR 1.5, 95% CI 1.2-1.8) and IL15-Rα levels (HR 0.6, 95% CI 0.4-0.9) are significant independent predictors for arthritis progression in a prediction model (Harrell's C 0.76 [SE 0.02], AUC 0.82 [95% CI 0.76-0.89], cross-validated AUC 0.70 [95% CI 0.56-0.85]). CONCLUSION: We propose a high-Risk-RA phase characterized by presence of ACPA reactivity, tenosynovitis, IL6, and IL15-Rα and suggest that these factors need to be further investigated for their biological effects and clinical values, to identify individuals at particular low risk and high risk for arthritis progression.

The 5:2 Diet Affects Markers of Insulin Secretion and Sensitivity in Subjects with and without Type 2 Diabetes-A Non-Randomized Controlled Trial.

This non-randomized controlled trial aimed to compare the effect of the 5:2 diet on insulin levels as a primary outcome and markers of insulin secretion (connecting peptide (C-peptide) and insulin-like growth factor binding protein-1 (IGFBP-1)) and sensitivity (Homeostatic Model Assessment for Insulin Resistance (HOMA-IR)), as well as body composition as secondary outcomes in overweight/obese individuals with and without type 2 diabetes (T2D). Ninety-seven participants (62% women), 35 with T2D and 62 BMI- and waist-matched controls without T2D, followed the 5:2 diet (two days per week of fasting) for six months with a 12-month follow-up. At six months, there was no loss to follow-up in the T2D group, whereas four controls discontinued this study. Overall, 82% attended the 12-month follow-up. After the intervention, insulin levels decreased in the control group and glucose decreased in the T2D group, while C-peptide, HOMA-IR, waist circumference, BMI, trunk, and total fat% decreased in both groups. Furthermore, low IGFBP-1, indicating hyperinsulinemia, improved in the T2D group. The changes in fasting glucose and waist measurement were significantly more improved in the T2D group than in the controls. Persistent positive effects were observed at the 12-month follow-up. The 5:2 diet for six months was feasible and efficient to reduce markers of insulin secretion and resistance and therefore holds promise as management of overweight/obesity in subjects with and without T2D.

Triggering of a Dll4-Notch1 loop impairs wound healing in diabetes.

Diabetic foot ulcerations (DFUs) represent a major medical, social, and economic problem. Therapeutic options are restricted due to a poor understanding of the pathogenic mechanisms. The Notch pathway plays a pivotal role in cell differentiation, proliferation, and angiogenesis, processes that are profoundly disturbed in diabetic wounds. Notch signaling is activated upon interactions between membrane-bound Notch receptors (Notch 1-4) and ligands (Jagged 1-2 and Delta-like 1, 3, 4), resulting in cell-context-dependent outputs. Here, we report that Notch1 signaling is activated by hyperglycemia in diabetic skin and specifically impairs wound healing in diabetes. Local inhibition of Notch1 signaling in experimental wounds markedly improves healing exclusively in diabetic, but not in nondiabetic, animals. Mechanistically, high glucose levels activate a specific positive Delta-like 4 (Dll4)-Notch1 feedback loop. Using loss-of-function genetic approaches, we demonstrate that Notch1 inactivation in keratinocytes is sufficient to cancel the repressive effects of the Dll4-Notch1 loop on wound healing in diabetes, thus making Notch1 signaling an attractive locally therapeutic target for the treatment of DFUs.

Hypoxia and hypoxia-inducible factors in diabetes and its complications.

Hypoxia-inducible factors (HIFs) are the key regulators of oxygen homeostasis in response to hypoxia. In diabetes, multiple tissues are hypoxic but adaptive responses to hypoxia are impaired due to insufficient activation of HIF signalling, which results from inhibition of HIF-1α stability and function due to hyperglycaemia and elevated fatty acid levels. In this review, we will summarise and discuss current findings about the regulation of HIF signalling in diabetes and the pathogenic roles of hypoxia and dysregulated HIF signalling in the development of diabetes and its complications. The therapeutic potential of targeting HIF signalling for the prevention and treatment of diabetes and related complications is also discussed.

Anticitrullinated protein antibodies facilitate migration of synovial tissue-derived fibroblasts.

OBJECTIVES: Rheumatoid arthritis (RA)-specific anti-citrullinated protein/peptide antibodies (ACPAs) might contribute to bone loss and arthralgia before the onset of joint inflammation. We aimed to dissect additional mechanisms by which ACPAs might contribute to development of joint pathology. METHODS: Fibroblast-like synoviocytes (FLS) were isolated from the synovial membrane of patients with RA. The FLS cultures were stimulated with polyclonal ACPAs (anti-CCP-2 antibodies) purified from the peripheral blood of patients with RA or with monoclonal ACPAs derived from single synovial fluid B cells. We analysed how ACPAs modulate FLS by measuring cell adhesion and mobility as well as cytokine production. Expression of protein arginine deiminase (PAD) enzymes and protein citrullination were analysed by immunofluorescence, and signal transduction was studied using immunoblotting. RESULTS: Challenge of FLS by starvation-induced stress or by exposure to the chemokine interleukin-8 was essential to sensitise the cells to ACPAs. These challenges led to an increased PAD expression and protein citrullination and an ACPA-mediated induction of FLS migration through a mechanism involving phosphoinositide 3-kinase activation. Inhibition of the PAD enzymes or competition with soluble citrullinated proteins or peptides completely abolished the ACPA-induced FLS migration. Different monoclonal ACPAs triggered distinct cellular effects in either fibroblasts or osteoclasts, suggesting unique roles for individual ACPA clones in disease pathogenesis. CONCLUSION: We propose that transient synovial insults in the presence of a certain pre-existing ACPA repertoire might result in an ACPA-mediated increase of FLS migration.

A Notch-independent mechanism contributes to the induction of Hes1 gene expression in response to hypoxia in P19 cells.

Hes1 is a Notch target gene that plays a major role during embryonic development. Previous studies have shown that HIF-1α can interact with the Notch intracellular domain and enhance Notch target gene expression. In this study, we have identified a Notch-independent mechanism that regulates the responsiveness of the Hes1 gene to hypoxia. Using P19 cells we show that silencing the Notch DNA binding partner CSL does not prevent hypoxia-dependent upregulation of Hes1 expression. In contrast to CSL, knockdown of HIF-1α or Arnt expression prevents Hes1 induction in hypoxia. Deletion analysis of the Hes1 promoter identified a minimal region near the transcription start site that is still responsive to hypoxia. In addition, we show that mutating the GA-binding protein (GABP) motif significantly reduced Hes1 promoter-responsiveness to hypoxia or to HIF-1 overexpression whereas mutation of the hypoxia-responsive element (HRE) present in this region had no effect. Chromatin immunoprecipitation assays demonstrated that HIF-1α binds to the proximal region of the Hes1 promoter in a Notch-independent manner. Using the same experimental approach, the presence of GABPα and GABPß1 was also observed in the same region of the promoter. Loss- and gain-of-function studies demonstrated that Hes1 gene expression is upregulated by hypoxia in a GABP-dependent manner. Finally, co-immunoprecipitation assays demonstrated that HIF-1α but not HIF-2α is able to interact with either GABPα or GABPß1. These results suggest a Notch-independent mechanism where HIF-1 and GABP contribute to the upregulation of Hes1 gene expression in response to hypoxia.

Identification of a novel chemokine-dependent molecular mechanism underlying rheumatoid arthritis-associated autoantibody-mediated bone loss.

OBJECTIVES: Rheumatoid arthritis (RA)-specific anti-citrullinated protein/peptide antibodies (ACPAs) appear before disease onset and are associated with bone destruction. We aimed to dissect the role of ACPAs in osteoclast (OC) activation and to identify key cellular mediators in this process. METHODS: Polyclonal ACPA were isolated from the synovial fluid (SF) and peripheral blood of patients with RA. Monoclonal ACPAs were isolated from single SF B-cells of patients with RA. OCs were developed from blood cell precursors with or without ACPAs. We analysed expression of citrullinated targets and peptidylarginine deiminases (PAD) enzymes by immunohistochemistry and cell supernatants by cytometric bead array. The effect of an anti-interleukin (IL)-8 neutralising antibody and a pan-PAD inhibitor was tested in the OC cultures. Monoclonal ACPAs were injected into mice and bone structure was analysed by micro-CT before and after CXCR1/2 blocking with reparixin. RESULTS: Protein citrullination by PADs is essential for OC differentiation. Polyclonal ACPAs enhance OC differentiation through a PAD-dependent IL-8-mediated autocrine loop that is completely abolished by IL-8 neutralisation. Some, but not all, human monoclonal ACPAs derived from single SF B-cells of patients with RA and exhibiting distinct epitope specificities promote OC differentiation in cell cultures. Transfer of the monoclonal ACPAs into mice induced bone loss that was completely reversed by the IL-8 antagonist reparixin. CONCLUSIONS: We provide novel insights into the key role of citrullination and PAD enzymes during OC differentiation and ACPA-induced OC activation. Our findings suggest that IL8-dependent OC activation may constitute an early event in the initiation of the joint specific inflammation in ACPA-positive RA.

Disturbed hypoxic responses as a pathogenic mechanism of diabetic foot ulcers.

Diabetic foot ulceration (DFU) is a chronic complication of diabetes that is characterized by impaired wound healing in the lower extremities. DFU remains a major clinical challenge because of poor understanding of its pathogenic mechanisms. Impaired wound healing in diabetes is characterized by decreased angiogenesis, reduced bone marrow-derived endothelial progenitor cell (EPC) recruitment, and decreased fibroblast and keratinocyte proliferation and migration. Recently, increasing evidence has suggested that increased hypoxic conditions and impaired cellular responses to hypoxia are essential pathogenic factors of delayed wound healing in DFU. Hypoxia-inducible factor-1 (HIF-1, a heterodimer of HIF-1α and HIF-1ß) is a master regulator of oxygen homeostasis that mediates the adaptive cellular responses to hypoxia by regulating the expression of genes involved in angiogenesis, metabolic changes, proliferation, migration, and cell survival. However, HIF-1 signalling is inhibited in diabetes as a result of hyperglycaemia-induced HIF-1α destabilization and functional repression. Increasing HIF-1α expression and activity using various approaches promotes angiogenesis, EPC recruitment, and granulation, thereby improving wound healing in experimental diabetes. The mechanisms underlying HIF-1α regulation in diabetes and the therapeutic strategies targeting HIF-1 signalling for the treatment of diabetic wounds are discussed in this review. Further investigations of the pathways involved in HIF-1α regulation in diabetes are required to advance our understanding of the mechanisms underlying impaired wound healing in diabetes and to provide a foundation for developing novel therapeutic approaches to treat DFU.

Coenzyme Q10 prevents peripheral neuropathy and attenuates neuron loss in the db-/db- mouse, a type 2 diabetes model.

Diabetic peripheral neuropathy (DPN) is the most common complication in both type 1 and type 2 diabetes. Here we studied some phenotypic features of a well-established animal model of type 2 diabetes, the leptin receptor-deficient db(-)/db(-) mouse, and also the effect of long-term (6 mo) treatment with coenzyme Q10 (CoQ10), an endogenous antioxidant. Diabetic mice at 8 mo of age exhibited loss of sensation, hypoalgesia (an increase in mechanical threshold), and decreases in mechanical hyperalgesia, cold allodynia, and sciatic nerve conduction velocity. All these changes were virtually completely absent after the 6-mo, daily CoQ10 treatment in db(-)/db(-) mice when started at 7 wk of age. There was a 33% neuronal loss in the lumbar 5 dorsal root ganglia (DRGs) of the db(-)/db(-) mouse versus controls at 8 mo of age, which was significantly attenuated by CoQ10. There was no difference in neuron number in 5/6-wk-old mice between diabetic and control mice. We observed a strong down-regulation of phospholipase C (PLC) ß3 in the DRGs of diabetic mice at 8 mo of age, a key molecule in pain signaling, and this effect was also blocked by the 6-mo CoQ10 treatment. Many of the phenotypic, neurochemical regulations encountered in lumbar DRGs in standard models of peripheral nerve injury were not observed in diabetic mice at 8 mo of age. These results suggest that reactive oxygen species and reduced PLCß3 expression may contribute to the sensory deficits in the late-stage diabetic db(-)/db(-) mouse, and that early long-term administration of the antioxidant CoQ10 may represent a promising therapeutic strategy for type 2 diabetes neuropathy.

Hyperbaric oxygen therapy activates hypoxia-inducible factor 1 (HIF-1), which contributes to improved wound healing in diabetic mice.

Hyperbaric oxygen (HBO) therapy has been used as an adjunctive therapy for diabetic foot ulcers, although its mechanism of action is not completely understood. Recently, it has been shown that HBO mobilizes the endothelial progenitor cells (EPCs) from bone marrow that eventually will aggregate in the wound. However, the gathering of the EPCs in diabetic wounds is impaired because of the decreased levels of local stromal-derived factor-1α (SDF-1α). Therefore, we investigated the influence of HBO on hypoxia-inducible factor 1 (HIF-1), which is a central regulator of SDF-1α and is down-regulated in diabetic wounds. The effects of HBO on HIF-1α function were studied in human dermal fibroblasts, SKRC7 cells, and HIF-1α knock-out and wild-type mouse embryonic fibroblasts using appropriate techniques (Western blot, quantitative polymerase chain reaction, and luciferase hypoxia-responsive element reporter assay). Cellular proliferation was assessed using H(3) -thymidine incorporation assay. The effect of HIF in combination with HBOT was tested by inoculating stable HIF-1α-expressing adenovirus (Adv-HIF) into experimental wounds in db/db mice exposed to HBO. HBO activates HIF-1α at several levels by increasing both HIF-1α stability (by a non-canonical mechanism) and activity (as shown both by induction of relevant target genes and by a specific reporter assay). HIF-1α induction has important biological relevance because the induction of fibroblast proliferation in HBO disappears when HIF-1α is knocked down. Moreover, the local transfer of stable HIF-1α-expressing adenovirus (Adv-HIF) into experimental wounds in diabetic (db/db mice) animals has an additive effect on HBO-mediated improvements in wound healing. In conclusion, HBO stabilizes and activates HIF-1, which contributes to increased cellular proliferation. In diabetic animals, the local transfer of active HIF further improves the effects of HBO on wound healing.

Hyperoxia inhibits glucose-induced insulin secretion and mitochondrial metabolism in rat pancreatic islets.

Isolated pancreatic islets containing the insulin-producing beta cells are devoid of circulation. They may therefore experience hypoxia with possible negative effects on beta cell function and survival. We investigated (1) whether hyperoxia in vitro would be beneficial by counteracting putative effects of lost circulation and, further, (2) whether previous hyperoxia would attenuate the impact of subsequently induced severe hypoxia. Islets from Sprague-Dawley rats were exposed to 95% O2 for 18 h. This hyperoxic exposure diminished glucose-induced insulin secretion by 47% and inhibited oxygen consumption by 39-41%. Mitochondrial complexes I-III were decreased by 29-37%. Negative effects on insulin secretion and complexes III and IV waned after a 22 h period of normoxia following hyperoxia whereas complexes I and II were still diminished, ROS production was increased and rates of apoptosis tended to be increased (P=0.07). The effects of previous hyperoxia on susceptibility to damage by subsequent hypoxia were tested after 5.5h of 0.8% O2. Previous hyperoxia did not affect hypoxia-induced enhancement of HIF-1 alpha but modestly and significantly attenuated hypoxia-induced decreases in insulin contents. We conclude that hyperoxia exerts largely negative effects on beta cells, effects which are functional and possibly also toxic. A paradoxical positive finding (attenuation of hypoxia-induced effects) could be secondary to a protective effect of the hyperoxia-induced reduction of oxidative metabolism.

A protein-rich meal provides beneficial glycemic and hormonal responses as compared to meals enriched in carbohydrate, fat or fiber, in individuals with or without type-2 diabetes.

Introduction: Diet stands as a pivotal modifiable risk factor influencing weight gain and the onset of type-2 diabetes (T2D). This study delves into the variation in glucose and regulatory pancreatic hormone levels subsequent to the consumption of meals with differing macronutrient compositions. Methods: The cohort comprised 20 individuals diagnosed with T2D and 21 without diabetes. Participants underwent a cross-over design, consuming four isocaloric meals (600 kcal) enriched in carbohydrate, fiber, fat and protein. Plasma glucose, insulin and glucagon levels were measured at -30, and -5 min, followed by subsequent measurements every 30 min for 240 min post meal intake. Quantification of alterations in the postprandial state was accomplished through the incremental area under the curve (iAUC) and the incremental peak height for the insulin:glucagon ratio (IGR) and plasma glucose levels. The meal demonstrating the lowest responses across these variables was deemed the optimal meal. Results: Meals rich in protein and fat, and consequently low in carbohydrate, exhibited reduced incremental peak and iAUC for both glucose and the IGR in comparison to the other meals. While the protein-enriched meal neared optimal standards, it proved less efficient for individuals without T2D and possessing a low BMI, as well as in those with T2D and poor glycemic control. Conclusion: Our findings endorse the adoption of protein-enriched, low-carbohydrate meals to curtail the meal-induced anabolic hormonal response while averting excessive fluctuations in glucose levels.

PIONEER REAL Sweden: A Multicentre, Prospective, Real-World Observational Study of Oral Semaglutide Use in Adults with Type 2 Diabetes in Swedish Clinical Practice.

INTRODUCTION: The study was designed to assess outcomes with once-daily oral semaglutide in adults with type 2 diabetes (T2D) naïve to injectable glucose-lowering agents, in Swedish clinical practice. METHODS: In this non-interventional, multicentre study, participants initiated oral semaglutide and were followed for 34-44 weeks. The primary endpoint was glycated haemoglobin (HbA1c) change from baseline to end of study (EOS). Secondary endpoints included body weight (BW) change from baseline to EOS, proportion of participants achieving HbA1c < 7%, and proportion achieving both a HbA1c reduction ≥ 1% and BW reduction of ≥ 3% or ≥ 5%, at EOS. Participants completed Diabetes Treatment Satisfaction Questionnaires (DTSQ status/change) and a dosing conditions questionnaire. RESULTS: A total of 187 participants (mean age 62.5 years) initiated oral semaglutide. Baseline mean HbA1c and BW were 7.8% (n = 177) and 96.9 kg (n = 165), respectively. Estimated mean changes in HbA1c and BW were - 0.88%-points (95% confidence interval [CI] - 1.01 to - 0.75; P < 0.0001) and - 4.72% (95% CI - 5.58 to - 3.86; P < 0.0001), respectively. At EOS, 64.6% of participants had HbA1c < 7%, and 22.9% achieved HbA1c reduction of ≥ 1% and BW reduction of ≥ 5%. DTSQ status and change scores improved by 1.44 (P = 0.0260) and 12.3 points (P < 0.0001), respectively. Oral semaglutide was easy or very easy to consume for 86.4% of participants. Most common adverse events (AEs) were gastrointestinal disorders; nine participants (4.8%) had serious AEs; one (0.5%) experienced severe hypoglycaemia. CONCLUSION: In this real-world study population, we observed significant reductions in HbA1c and BW in people living with T2D when prescribed semaglutide tablets as part of routine clinical practice in Sweden, with improved treatment satisfaction among participants and no new safety concerns. TRIAL REGISTRATION: NCT04601753.

Development of a three-dimensional scoring model for the assessment of continuous glucose monitoring data in type 1 diabetes.

INTRODUCTION: Despite the improvements in diabetes management by continuous glucose monitoring (CGM) it is difficult to capture the complexity of CGM data in one metric. We aimed to develop a clinically relevant multidimensional scoring model with the capacity to identify the most alarming CGM episodes and/or patients from a large cohort. RESEARCH DESIGN AND METHODS: Retrospective CGM data from 2017 to 2020 available in electronic medical records were collected from n=613 individuals with type 1 diabetes (total 82 114 days). A scoring model was developed based on three metrics; glycemic variability percentage, low blood glucose index and high blood glucose index. Values for each dimension were normalized to a numeric score between 0-100. To identify the most representative score for an extended time period, multiple ways to combine the mean score of each dimension were evaluated. Correlations of the scoring model with CGM metrics were computed. The scoring model was compared with interpretations of a clinical expert board (CEB). RESULTS: The dimension of hypoglycemia must be weighted to be representative, whereas the other two can be represented by their overall mean. The scoring model correlated well with established CGM metrics. Applying a score of ≥80 as the cut-off for identifying time periods with a 'true' target fulfillment (ie, reaching all targets for CGM metrics) resulted in an accuracy of 93.4% and a specificity of 97.1%. The accuracy of the scoring model when compared with the CEB was high for identifying the most alarming CGM curves within each dimension of glucose control (overall 86.5%). CONCLUSIONS: Our scoring model captures the complexity of CGM data and can identify both the most alarming dimension of glycemia and the individuals in most urgent need of assistance. This could become a valuable tool for population management at diabetes clinics to enable healthcare providers to stratify care to the patients in greatest need of clinical attention.

Rare coding variants in NOX4 link high ROS levels to psoriatic arthritis mutilans.

Psoriatic arthritis mutilans (PAM) is the rarest and most severe form of psoriatic arthritis, characterized by erosions of the small joints and osteolysis leading to joint disruption. Despite its severity, the underlying mechanisms are unknown, and no susceptibility genes have hitherto been identified. We aimed to investigate the genetic basis of PAM by performing massive parallel sequencing in sixty-one patients from the PAM Nordic cohort. We found rare variants in the NADPH oxidase 4 (NOX4) in four patients. In silico predictions show that the identified variants are potentially damaging. NOXs are the only enzymes producing reactive oxygen species (ROS). NOX4 is specifically involved in the differentiation of osteoclasts, the cells implicated in bone resorption. Functional follow-up studies using cell culture, zebrafish models, and measurement of ROS in patients uncovered that these NOX4 variants increase ROS levels both in vitro and in vivo. We propose NOX4 as the first candidate susceptibility gene for PAM. Our study links high levels of ROS caused by NOX4 variants to the development of PAM, offering a potential therapeutic target.

Five sessions of hyperbaric oxygen for critically ill patients with COVID-19-induced ARDS: A randomised, open label, phase II trial.

BACKGROUND: Few treatment options exist for patients with COVID-19-induced acute respiratory distress syndrome (ARDS). Data on the benefits and harms of hyperbaric oxygen treatment (HBOT) for this condition is limited. OBJECTIVE: To evaluate benefits and harms of HBOT in patients with COVID-19 induced ARDS. METHODS: In this open-label trial conducted at three hospitals in Sweden and Germany, patients with moderate to severe ARDS and at least two risk factors for unfavourable outcome, were randomly assigned (1:1) to medical oxygen 100 %, 2·4 Atmospheres absolute (ATA), 80 min (HBOT) adjuvant to best practice or to best practice alone (Control). Randomisation was stratified by sex and site. The primary endpoint was ICU admission by Day 30. RESULTS: Between June 4, 2020, and Dec 1, 2021, 34 subjects were randomised to HBOT (N = 18) or Control (N = 16). The trial was prematurely terminated for futility. There was no statistically significant difference in ICU admission, 5 (50 %) in Control vs 13 (72 %) in HBOT. OR 2·54 [95 % CI 0·62-10·39], p = 0·19. HARMS: 102 adverse events (AEs) were recorded. 16 (94 %) subjects in the HBOT group and 14 (93 %) in the control group had at least one AE. Three serious adverse events (SAEs), were at least, possibly related to HBOT. All deaths were unlikely related to HBOT. CONCLUSIONS: HBOT did not reduce ICU admission or mortality in patients with COVID-19-induced ARDS. The trial cannot conclude definitive benefits or harms. Treating COVID-19-induced ARDS with HBOT is feasible with a favourable harms profile.

Influence of liver-X-receptor on tissue cholesterol, coenzyme Q and dolichol content.

The organ content of the mevalonate pathway lipids was investigated in liver-X-receptor (LXR) α, ß and double knock-out mice. An extensive or moderate increase of total cholesterol in the double KO mice was found in all organs elicited by the increase of the esterified form. In LXRα and double KO mice, coenzyme Q (CoQ) was decreased in liver and increased in spleen, thymus and lung, while dolichol was increased in all organs investigated. This effect was confirmed using LXR- agonist GW 3965. Analysis of CoQ distribution in organelles showed that the modifications are present in all cellular compartments and that the increase of the lipid in mitochondria was the result of a net increase of CoQ without changing the number of mitochondria. It appears that LXR influences not only cellular cholesterol homeostasis but also the metabolism of CoQ and dolichol, in an indirect manner.