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PURPOSE: Immune-related thyroid adverse events (irTAEs) occur frequently following immune checkpoint inhibitor (ICI) therapy. The purpose of this study is to provide knowledge about the incidence, clinical timeline characteristics, associated factors of irTAEs, and potential impact on treatment efficacy in patients with melanoma receiving adjuvant ICI therapy. METHODS: A national multicenter retrospective cohort study of patients with resected stage III/IV melanoma treated with adjuvant PD-1 inhibitors between November 2018 and December 2020. Data were extracted from the Danish Metastatic Melanoma Database. The irTAEs were defined as two consecutive abnormal TSH values and subdivided into transient or persistent. RESULTS: Of 454 patients, 99 developed an irTAE (21.8%), of these were 46 transient (46.5%) and 53 persistent (53.5%). Median time to transient and persistent irTAE was 55 and 44 days, respectively (p = 0.57). A hyperthyroid phase followed by hypothyroidism was seen in 73.6% of persistent irTAEs, whereas 87% of transient irTAEs developed an isolated hypo- or hyperthyroid phase. Multiple variable analysis demonstrated an association between irTAE and female sex (HR 2.45; 95% CI 1.63-3.70; p < 0.001), but no association with recurrence-free survival (HR 0.86; 95% CI 0.50-1.48; p = 0.587) or overall survival (HR 1.05; 95% CI 0.52-2.12, p = 0.891). CONCLUSIONS: IrTAE is a common side effect to PD-1 inhibitors primarily occurring within the first 3 months, with a high risk of persistency. Female sex is a strong predictive factor. IrTAE was not associated with improved clinical outcome.

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We aimed to understand the impact of the interplay between bile salts and cyclodextrins on the dissolution-permeation of poorly soluble drug compounds with a moderate-strong binding constant to cyclodextrin. Phase diagrams were prepared on the chosen model compound albendazole in phosphate buffer, fasted state simulated intestinal fluid (FaSSIF), and a modified fed state simulated intestinal fluid (FeSSIFmod) with (2-hydroxypropyl)-beta-cyclodextrin (HP-ß-CD) concentrations of up to 10 % (m/m). Then we investigated the dissolution/permeation interplay of albendazole dissolved/suspended in the different media through a biomimetic barrier on a 96-well in vitro model. The apparent solubility of albendazole was enhanced by HP-ß-CD and FaSSIF/FeSSIFmod separately. However, when albendazole was dissolved in HP-ß-CD and biomimetic media together, the solubility was significantly lower than the predicted additive solubility from the solubilizing effects. It is postulated that this is due to the sodium taurocholate from the biomimetic media displacing albendazole from the hydrophobic cavity of HP-ß-CD. In the permeation experiments, the highest permeation was observed at cyclodextrin concentrations able to solubilize close to the total dose of albendazole without a major surplus of solubilization capacity. Furthermore, an over-proportional permeation enhancement was observed when both, cyclodextrin and biomimetic media were present. These results indicate that the interplay between bile salts and cyclodextrins can enhance the free (molecularly dissolved) fraction of drug in solution to a greater extent than could be obtained with one of the solubilizing components alone. In conclusion, at carefully selected cyclodextrin-concentrations in combination with biomimetic media, obviously, a transient supersaturation is induced, which is made responsible for the observed major permeation enhancement.

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Remote ischemic conditioning (RIC) is a procedure that can attenuate ischemic-reperfusion injury by conducting brief cycles of ischemia and reperfusion in the arm or leg. Extracellular vesicles (EVs) circulating in the bloodstream can release their content into recipient cells to confer protective function on ischemia-reperfusion injured (IRI) organs. Skeletal muscle cells are potential candidates to release EVs as a protective signal during RIC. In this study, we used C2C12 cells as a model system and performed cyclic hypoxia-reoxygenation (HR) to mimic RIC. EVs were collected and subjected to small RNA profiling and proteomics. HR induced a distinct shift in the miRNA profile and protein content in EVs. HR EV treatment restored cell viability, dampened inflammation, and enhanced tube formation in in vitro assays. In vivo, HR EVs showed increased accumulation in the ischemic brain compared to EVs secreted from normoxic culture (N EVs) in a mouse undergoing transient middle cerebral artery occlusion (tMCAO). We conclude that HR conditioning changes the miRNA and protein profile in EVs released by C2C12 cells and enhances the protective signal in the EVs to recipient cells in vitro.

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BACKGROUND: Anterior cruciate ligament (ACL) rupture is a serious injury with a high prevalence worldwide, and subsequent ACL reconstructions (ACLR) appear to be most commonly performed using hamstring-derived (semitendinosus tendon) autografts. Recovery of maximal muscle strength to ≥90% of the healthy contralateral limb is considered an important criterion for safe return to sports. However, the speed of developing muscular force (ie, the rate of force development [RFD]) is also important for the performance of many types of activities in sports and daily living, yet RFD of the knee extensor and flexor muscles has apparently never been examined in patients who undergo ACLR with hamstring autograft (HA). PURPOSE: To examine potential deficits in RFD, maximal muscle strength (ie, maximal voluntary isometric contraction [MVIC]), and functional capacity of ACLR-HA limbs in comparison with the healthy contralateral leg and matched healthy controls 3 to 9 months after surgery. STUDY DESIGN: Cross-sectional study; Level of evidence: 3. METHODS: A total of 23 young patients who had undergone ACLR-HA 3 to 9 months earlier were matched by age to 14 healthy controls; both groups underwent neuromuscular screening. Knee extensor and flexor MVIC and RFD, as well as functional capacity (single-leg hop for distance [SLHD] test, timed single-leg sit-to-stand [STS] test), were assessed on both limbs. Furthermore, patient-reported knee function (Knee injury and Osteoarthritis Outcome Score) was assessed. RESULTS: Knee extensor and flexor MVIC and RFD were markedly compromised in ACLR-HA limbs compared with healthy contralateral limbs (MVIC for extensor and flexor, 13% and 26%, respectively; RFD, 14%-17% and 32%-39%) and controls (MVIC, 16% and 31%; RFD, 14%-19% and 30%-41%) (P < .05-.001). Further, ACLR-HA limbs showed reduced functional capacity (reduced SLHD and STS performance) compared with contralateral limbs (SLHD, 11%; STS, 14%) and controls (SLHD, 20%; STS, 31%) (P < .01-.001). Strength (MVIC) and functional (SLHD) parameters were positively related to the duration of time after surgery (P < .05), although this relationship was not observed for RFD and STS. CONCLUSION: Knee extensor and flexor RFD and maximal strength, as well as functional single-leg performance, remained substantially reduced in ACLR-HA limbs compared with noninjured contralateral limbs and healthy controls 3 to 9 months after reconstructive surgery.

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INTRODUCTION: Intervening effectively to prevent students' harmful use of alcohol remains a challenge. Harmful alcohol use has been noted as the most dominant public health problem facing universities today. This study sought to investigate the diversity in staff and student perceptions of the contribution alcohol makes to student life in a Danish university setting. Increasing understanding of staff and students' perceptions of how alcohol fits into student life is required to amend future public health intervention for this population. MATERIALS AND METHODS: This Q methodology study included 38 staff members and 105 students from Aarhus University, Denmark. Participants used online Q sorting software, to rank 40 statements about the contribution alcohol makes to the university student experience from strongly agree to disagree. To support the interpretation of the factors, self-reported alcohol consumption and demographic data were collected. In addition qualitative data was collected on the participant's reasons for the ranking of the items they most strongly agreed or disagreed with. RESULTS AND DISCUSSION: Using principal components analysis, five statistically independent viewpoints for students and four for staff were identified. The findings provide evidence to inform approaches to prevent harmful alcohol use. Some viewpoints suggest a need for tailored secondary and tertiary prevention and intervention that focusses on individuals and/or sub-groups who are at risk of consuming alcohol at harmful levels. Other viewpoints suggest the need for primary universal prevention to support the maintenance of healthy norms which can prevent harmful alcohol behaviour. Public health campaigns need to ensure that interventions targeting harmful alcohol use at universities challenge problematic perceptions and attitudes while also bolstering exposure to positive norms.

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Ubiquitination is a post-translational modification (PTM) that is essential for balancing numerous physiological processes. To enable delineation of protein ubiquitination at a site-specific level, we generated an antibody, denoted UbiSite, recognizing the C-terminal 13 amino acids of ubiquitin, which remain attached to modified peptides after proteolytic digestion with the endoproteinase LysC. Notably, UbiSite is specific to ubiquitin. Furthermore, besides ubiquitination on lysine residues, protein N-terminal ubiquitination is readily detected as well. By combining UbiSite enrichment with sequential LysC and trypsin digestion and high-accuracy MS, we identified over 63,000 unique ubiquitination sites on 9,200 proteins in two human cell lines. In addition to uncovering widespread involvement of this PTM in all cellular aspects, the analyses reveal an inverse association between protein N-terminal ubiquitination and acetylation, as well as a complete lack of correlation between changes in protein abundance and alterations in ubiquitination sites upon proteasome inhibition.

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BACKGROUND: The protective effect of farm upbringing on allergic rhinitis is well known, but how upbringing in other environments influences the development of allergic rhinitis is scarcely investigated. The aim of this study was to investigate the association between place of upbringing and pet keeping in childhood and allergic rhinitis and nasal symptoms in adulthood. METHODS: The population-based Respiratory Health in Northern Europe study includes subjects from Denmark, Norway, Sweden, Iceland, and Estonia born in 1945-1973. This paper analyses 13,376 participants of the third study wave. Six categories of place of upbringing were defined: farm with livestock, farm without livestock, village in rural area, small town, city suburb, and inner city. Pets in the home at birth and during childhood were recorded. Data were analysed using adjusted logistic regression models. RESULTS: Livestock farm upbringing predicted less adult allergic rhinitis [odds ratio (OR) 0.68, 0.54-0.85] and nasal symptoms (OR 0.82, 0.68-0.99) than city upbringing, and an urban-rural gradient with decreasing risk per level of urbanisation was observed (OR 0.92, 0.88-0.94). Pets in the home at birth (OR 0.78, 0.68-0.88) and during childhood (OR 0.83, 0.74-0.93) were associated with less subsequent allergic rhinitis. Pet keeping did not explain the protective effect of place of upbringing. CONCLUSION: Risk of allergic rhinitis and nasal symptoms in adulthood was inversely associated with the level of urbanisation during upbringing. Pets at birth decreased the risk further, but did not explain the urban-rural gradient. Persistent beneficial effects of microbial diversity in early life might be an explanation for the findings.

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BACKGROUND AND PURPOSE: Two of the most utilized outcome measures to assess knee joint range of motion (ROM) and intra-articular effusion are goniometry and circumference, respectively. Neither goniometry nor circumference of the knee joint have been examined for both intra-tester and inter-tester in patients with total knee arthroplasty (TKA). The purpose of this study was to determine the intra-tester and inter-tester reliability of active and passive knee joint ROM and circumference in patients with TKA when administered by physiotherapists (testers) with different clinical experience. METHOD: The design was an intra-tester, inter-tester and intra-day reliability study. Nineteen outpatients (10 females) having received a TKA were examined by an inexperienced and an experienced physiotherapist. Following a standardized protocol, active and passive knee joint ROM and circumference measurements were obtained using a universal goniometer and a tape measure, respectively. To establish reliability, intraclass correlation coefficients (ICC(2,1)) and smallest real difference (SRD) were calculated. RESULTS: The knee joint ROM and circumference measurements were generally reliable (ICC > 0.8) within and between physiotherapists (except passive knee extension). Changes in knee joint ROM of more than 6.6 degrees and 10 degrees (except active knee flexion) and knee joint circumference of more than 1.0 cm and 1.63 cm represent a real clinical improvement (SRD) or deterioration for a single individual within and between physiotherapists, respectively. Generally, the experienced tester recorded larger knee joint ROM and lower circumference values than that of the inexperienced tester. CONCLUSIONS: In clinical practice, we suggest that repeated knee goniometric and circumferential measurements should be recorded by the same physiotherapist in individual patients with TKA. Tester experience appears not to influence the degree of reliability.

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BACKGROUND: The Belgian shepherd Groenendael and Tervueren is believed to be at higher risk of developing epilepsy than dogs of the common population. This epidemiological study was designed to estimate the prevalence of epilepsy in the Danish population of Groenendael and Tervueren born between 1995 and 2004. Furthermore, it was the intention to describe the clinical manifestation (seizure types and phenomenology) of epilepsy and to identify risk factors for euthanasia once the dog was diagnosed as having epilepsy. METHODS: All owners of Groenendael and Tervueren dogs born between January 1995 and December 2004 and registered in the Danish Kennel Club (1,248 dogs) were contacted and asked to answer a mailed questionnaire concerning epilepsy. Positive responders were subsequently validated in a follow-up interview conducted by telephone using a standardized questionnaire. Owners were questioned about age at first seizure, seizure frequency, seizure duration, a detailed description of seizure phenomenology, post-ictal signs and if a veterinarian had diagnosed the dog with epilepsy. RESULTS: Prevalence of epilepsy was estimated at 9.5%. Mean age of epilepsy debut was 3.3 years (range 0.5-8.0 years). There was an almost equal number of Groenendael (25) and Tervueren (24). The distribution of females and males was 31 and 18 respectively. Twenty-five per cent experienced focal seizures, 53% experienced focal seizures with secondary generalization and 18% experienced primary generalized seizures. In four percent seizures were unclassifiable. The most commonly reported focal seizure phenomenology included ataxia, crawling, swaying, fearful behavior, salivation, excessive attention seeking and disorientation. In 16% of the cases, epilepsy led to euthanasia. Intact dogs with epilepsy had a significantly increased risk of being euthanized because of epilepsy compared to neutered dogs with epilepsy. In 22% of the cases the owners reported that anxiety/hyperactivity/stress could act as a seizure provoking factor. CONCLUSION: A high prevalence of epilepsy appears to be present in the Danish Groenendael and Tervueren population. The relatively late debut age of epilepsy in this breed contributes greatly to the increased prevalence of epileptic individuals, because dogs developing epilepsy late in life are used for breeding unintended.

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Apolipoprotein A-V (apoA-V) is present in low amounts in plasma and has been found to modulate triacylglycerol levels in humans and in animal models. ApoA-V displays affinity for members of the low density lipoprotein receptor (LDL-R) gene family, known as the classical lipoprotein receptors, including LRP1 and SorLA/LR11. In addition to LDL-A binding repeats, the mosaic receptor SorLA/LR11 also possesses a Vps10p domain. Here we show that apoA-V also binds to sortilin, a receptor from the Vsp10p domain gene family that lacks LDL-A repeats. Binding of apoA-V to sortilin was competed by neurotensin, a ligand that binds specifically to the Vps10p domain. To investigate the biological fate of receptor-bound apoA-V, binding experiments were conducted with cultured human embryonic kidney cells transfected with either SorLA/LR11 or sortilin. Compared with nontransfected cells, apoA-V binding to SorLA/LR11- and sortilin-expressing cells was markedly enhanced. Internalization experiments, live imaging studies, and fluorescence resonance energy transfer analyses demonstrated that labeled apoA-V was rapidly internalized, co-localized with receptors in early endosomes, and followed the receptors through endosomes to the trans-Golgi network. The observed decrease of fluorescence signal intensity as a function of time during live imaging experiments suggested ligand uncoupling in endosomes with subsequent delivery to lysosomes for degradation. This interpretation was supported by experiments with (125)I-labeled apoA-V, demonstrating clear differences in degradation between transfected and nontransfected cells. We conclude that apoA-V binds to receptors possessing LDL-A repeats and Vsp10p domains and that apoA-V is internalized into cells via these receptors. This could be a mechanism by which apoA-V modulates lipoprotein metabolism in vivo.

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Environmental fracturing offers assistance to remediation efforts at contaminated, low-permeability sites via creation of active fracture networks, and hence, reduction of mass transport limitations set by diffusion in low-permeability matrices. A pilot study of pneumatic fracturing, focusing on direct documentation of fracture propagation patterns and spacing, was performed at a typical basal clay till site. The study applied a novel package of documentation methods, including injection of five tracers with different characteristics (bromide, uvitex, fluorescein, rhodamine WT, and brilliant blue), subsequent tracer-filled fracture documentation via direct and indirect methods, and geological characterization of the fractured site. The direct documentation methods consisted of Geoprobe coring, augering, and excavation. A mass balance and conceptual model have been established for the distribution of the injected tracers in the subsurface. They reveal that tracer was distributed within 2 m of the fracturing well, mainly in existing fractures above the redox boundary (2 to 4 m.b.s.; 5 to 10 cm spacing). Spacing of observed tracer-filled fractures was large (>1 m) at greater depths. The number of fractures induced/activated could possibly be increased via adjustments to the fracturing equipment design.

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