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Anti-SSA-autoantibodies are common in patients with rheumatologic disease, especially Sjögren's syndrome, systemic lupus erythematosus and rheumatoid arthritis. They consist of both autoantibodies towards Ro60 and Ro52, the latter also known as TRIM21. TRIM21 is an intracellular protein consisting of four domains; PRY/SPRY, Coiled-Coil, B-box and RING. The aim of this study was to establish an indirect ELISA detecting autoantibodies towards both the full-length TRIM21 protein and its four domains. We expressed the five constructs, created, and validated indirect ELISA protocols for each target using plasma from anti-SSA positive patients and healthy controls. Our findings were validated to the clinically used standards. We measured significantly higher levels of autoantibodies towards our full-length TRIM21, and the PRY/SPRY, Coiled-Coil and RING domains in patients compared to healthy controls. No significant difference in the level of autoantibodies were detected against the B-box domain. Our setups had a signal to noise ratio in the range of 30 to 184, and an OD between 2 and 3. Readings did not decline using NaCl of 500 mM as wash, affirming the high binding affinity of the autoantibodies measured. Our protocols allow us to further study the different autoantibodies of anti-SSA positive patients. This creates the possibility to stratify our patients into subgroups regarding autoantibody profile and specific pheno- or endotype.

This review aims to make clinicians aware of the newly described syndrome, VEXAS. VEXAS should become an obvious differential diagnosis in cases of unexplained inflammation, anemia, and rheumatological and/or hematological manifestations. Patients with VEXAS are typically male aged > 60, with inflammation, and macrocytic anaemia. On suspicion of cancer or infections patients have frequently been exposed to extensive diagnostic procedures and hospital admissions. In this review, we summarise the current knowledge of VEXAS regarding pathogenesis, symptoms, diagnosis, and treatment.

Background: Vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic (VEXAS) is an autoinflammatory condition with overlapping features of rheumatology and haematology caused by somatic mutations in the UBA1 gene. Patients present with highly variable symptoms and their path towards diagnosis are often complicated and characterised by extensive examinations. It is, therefore, pivotal that clinicians become familiar with the clinical presentation of VEXAS to advance identification of patients with the disease., Objectives: We aimed to (1) characterise patients diagnosed with VEXAS in a tertiary rheumatology referral centre, (2) identify common rheumatological biomarkers that may distinguish VEXAS from other rheumatic diseases and (3) suggest which clinical findings should motivate genetic testing for VEXAS., Methods: Patients were identified and diagnosed at the department of Rheumatology, Aarhus University Hospital (AUH), Denmark. Blood samples were examined for VEXAS-associated UBA1 variants by Sanger sequencing at the department of Clinical Immunology, AUH. Clinical and biochemical data were retrieved from the hospital electronic patient chart., Results: Eleven male patients with clinical suspicion of VEXAS underwent sequencing. Five of these carried known VEXAS-associated variants. Median age at diagnosis was 84 (75-87) years. All patients had significantly elevated inflammatory markers with a median C-reactive protein (CRP) of 297 (196-386) mg/L and macrocytic anaemia. None of the patients presented common biomarkers for autoimmunity., Conclusion: Danish patients with VEXAS syndrome are men with persistent inflammation, constitutional symptoms and heterogeneous clinical presentations. Shared features for all patients in this study were highly elevated inflammatory markers, macrocytic anaemia and negative autoimmune biomarkers., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Serological assays for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are needed to support clinical diagnosis and epidemiological investigations. Recently, assays for large-scale detection of total antibodies (Ab), immunoglobulin G (IgG), and IgM against SARS-CoV-2 antigens have been developed, but there are limited data on the diagnostic accuracy of these assays. This study was a Danish national collaboration and evaluated 15 commercial and one in-house anti-SARS-CoV-2 assays in 16 laboratories. Sensitivity was evaluated using 150 samples from individuals with asymptomatic, mild, or moderate COVID-19, nonhospitalized or hospitalized, confirmed by nucleic acid amplification tests (NAAT); samples were collected 13 to 73 days either from symptom onset or from positive NAAT (patients without symptoms). Specificity and cross-reactivity were evaluated in samples collected prior to the SARS-CoV-2 epidemic from >586 blood donors and patients with autoimmune diseases, cytomegalovirus or Epstein-Barr virus infections, and acute viral infections. A specificity of ≥99% was achieved by all total-Ab and IgG assays except one, DiaSorin Liaison XL IgG (97.2%). Sensitivities in descending order were Wantai ELISA total Ab (96.7%), CUH-NOVO in-house ELISA total Ab (96.0%), Ortho Vitros total Ab (95.3%), YHLO iFlash IgG (94.0%), Ortho Vitros IgG (93.3%), Siemens Atellica total Ab (93.2%), Roche Elecsys total Ab (92.7%), Abbott Architect IgG (90.0%), Abbott Alinity IgG (median 88.0%), DiaSorin Liaison XL IgG (median 84.6%), Siemens Vista total Ab (81.0%), Euroimmun/ELISA IgG (78.0%), and Snibe Maglumi IgG (median 78.0%). However, confidence intervals overlapped for several assays. The IgM results were variable, with the Wantai IgM ELISA showing the highest sensitivity (82.7%) and specificity (99%). The rate of seropositivity increased with time from symptom onset and symptom severity., (Copyright © 2021 American Society for Microbiology.)

A natural and frequently occurring replication problem is generated by the action of topoisomerase I (Top1). Trapping of Top1 in a cleavage complex on the DNA generates a protein-linked DNA nick (PDN), which upon DNA replication can be transformed into a one-ended double-strand break (DSB). Break-induced replication (BIR) has been recognized as a critical repair mechanism of one-ended DSBs. Here, we have investigated resection at a one-ended DSB formed exclusively during replication due to Top1-mimicking damage. We show that resection is minimal, and only when strand invasion is abolished is extensive resection detected. When DNA synthesis is slowed by hydroxyurea treatment, extended resection is not observed, which suggests that strand invasion and/or heteroduplex formation restrains resection. Our results demonstrate that the BIR pathway acting during S phase is tailored to prevent hazardous effects of naturally and frequently occurring DNA breaks such as Top1-generated PDNs., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Chronic hepatitis B (CHB) infection increases the risk of developing severe liver disease including cirrhosis and hepatocellular carcinoma (HCC). As microRNAs may modulate host - virus interactions, we here investigated if hepatitis B virus (HBV) infection modulate microRNA expression using an in vitro HepG2 cell model system with inducible HBV replication. We found that HBV replication was associated with upregulation of miR-192-5p, miR-194-5p and miR-215-5p, of which miR-192-5p and miR-215-5p have identical seed sequences. Bioinformatics analyses revealed a significant enrichment of potential target genes involved in apoptosis signaling of all three microRNAs. In line with this, transfection with a mimic of miR-192-5p suppressed the protein level of pro-apoptotic BIM and reduced endoplasmic reticulum (ER) stress-induced apoptosis in HepG2 cells. In contrast, transfection with a mimic of miR-194-5p downregulated the anti-apoptotic proteins SODD and cFLIP, and sensitized HepG2 cells to both ER stress- and cytokine-induced apoptosis. In conclusion, our study suggests that HBV upregulates the expression of miR-192-5p and miR-194-5p in the host cell. These microRNAs target important apoptosis-regulatory proteins, and may thus contribute to the development of HBV-related liver disease., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Hepatitis B virus (HBV) infection is a major global health burden as chronic hepatitis B (CHB) is associated with the development of liver diseases including hepatocellular carcinoma (HCC). To gain insight into the mechanisms causing HBV-related HCC, we investigated the effects of HBV replication on global host cell gene expression using human HepG2 liver cells. By microarray analysis, we identified 54 differentially expressed genes in HBV-replicating HepG2 cells. One of the differentially-expressed genes was insulin-like growth factor binding protein 1 (IGFBP1) which was downregulated in HBV-replicating cells. Consistent with the gene expression data, IGFBP1 was suppressed at both the cellular and secreted protein levels in the presence of HBV replication. Transient transfection experiments with an inducible plasmid encoding the HBV X protein (HBx) revealed that HBx alone was sufficient to modulate IGFBP1 expression. Small interference RNA (siRNA)-mediated loss of function studies revealed that knockdown of IGFBP1 reduced apoptosis induced by either thapsigargin (TG) or staurosporine (STS). Treatment of cells with recombinant insulin-like growth factor 1 (IGF-1) decreased both TG- or STS-induced apoptosis. Interestingly, addition of recombinant IGFBP1 reversed the anti-apoptotic effect of IGF-1 on TG-induced, but not STS-induced, apoptosis. In conclusion, our results suggest an anti-apoptotic autocrine function of HBV-mediated downregulation of IGFBP1 in HepG2 cells. Such an effect may contribute to the development of HBV-mediated HCC by increasing pro-survival and anti-apoptotic IGF-1 effects., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: MicroRNAs are regulatory molecules and suggested as non-invasive biomarkers for molecular diagnostics and prognostics. Altered expression levels of specific microRNAs are associated with hepatitis B virus infection and hepatocellular carcinoma. We previously identified differentially expressed microRNAs with liver-specific target genes in plasma from children with chronic hepatitis B. To further understand the biological role of these microRNAs in the pathogenesis of chronic hepatitis B, we have used the human liver cell line HepG2, with and without HBV replication, after transfection of hepatitis B virus expression vectors. RT-qPCR is the preferred method for microRNA studies, and a careful normalisation strategy, verifying the optimal set of reference genes, is decisive for correctly evaluating microRNA expression levels. The aim of this study was to provide valid reference genes for the human HCC-derived cell line HepG2., Results: A panel of 739 microRNAs was screened to identify the most stably expressed microRNAs, followed by a PubMed search identifying microRNAs previously used as reference genes. Sixteen candidate reference genes were validated by RT-qPCR. Reference gene stabilities were calculated first by standard deviations of ΔCt values and then by geNorm and NormFinder analyses, taking into account the amplification efficiency of each microRNA primer set. The optimal set of reference genes was verified by a target analysis using RT-qPCR on miR-215-5p., Conclusion: We identified miR-24-3p, miR-151a-5p, and miR-425-5p as the most valid combination of reference genes for microRNA RT-qPCR studies in our hepatitis B virus replicating HepG2 cell model.

Auricular pseudocyst is a benign idiopathic disease, which typically presents as painless spontaneous swelling over the anterior part of the auricle. It has usually been diagnosed in young healthy male patients. Its aetiology remains unknown, but hypothetically congenital dysplasia or chronic low-grade trauma could be the main causes. We present two case reports and a discussion of the auricle pseudocysts, their origin and treatment options.

The branchio-oto-renal (BOR) syndrome is an autosomal-dominant disorder characterized by hearing loss, branchial and renal anomalies. BOR is genetically heterogeneous and caused by mutations in EYA1 (8q13.3), SIX1 (14q23.1), SIX5 (19q13.3) and in an unidentified gene on 1q31. We examined six Danish families with BOR syndrome by assessing linkage to BOR loci, by performing EYA1 multiplex ligation-dependent probe amplification (MLPA) analysis for deletions and duplications and by sequencing of EYA1, SIX1 and SIX5. We identified four EYA1 mutations (c.920delG, IVS10-1G>A, IVS12+4A>G and p.Y591X) and one SIX1 mutation (p.W122R), providing a molecular diagnosis in five out of the six families (83%). The present, yet preliminary, observation that renal and temporal bone malformations are less frequent in SIX1-related disease suggests a slightly different clinical profile compared to EYA1-related disease. Unidentified mutations impairing mRNA expression or further genetic heterogeneity may explain the lack of mutation finding in one family despite LOD score indications of EYA1 involvement.

Aggressive sarcoidosis is seldom described with the middle ear as the only localisation. A case is described, and differential diagnoses, such as tuberculosis, histocytosis X, Wegener's granulomatosis, paraffinoma, sarcoidosis and necrotising sarcoid granulomatosis are discussed.

Background: Ischemic mitral regurgitation is caused by an imbalance of the entire mitral-ventricular complex. This interaction is mediated through the chordae tendineae force distribution, which may perturb several elements of the mitral valve apparatus. Our objective was to investigate the association between the mitral valvular 3-dimensional geometric perturbations and chordae tendineae force redistribution in a porcine model of acute ischemic mitral regurgitation., Methods: In 9 pigs, acute ischemic mitral regurgitation was induced by repeated microembolization of the left circumflex coronary artery. Mitral leaflet coaptation geometry was determined by 2-dimensional echocardiography and reconstructed 3-dimensionally. Leading edge chordal forces were measured by dedicated miniature force transducers at control and during ischemic mitral regurgitation., Results: During acute ischemic mitral regurgitation, there was a decreased tension of the primary chorda from the ischemic posterior left ventricular wall to the anterior leaflet (0.295 +/- 0.063 N vs 0.336 +/- 0.071 N [control]; P < .05). The tension of the chorda from the nonischemic anterior left ventricular wall to the anterior leaflet increased (0.375 +/- 0.066 N vs 0.333 +/- 0.071 N [control]; P < .05). In accordance, relative leaflet prolapse was observed at the ischemic commissural side, whereas there was an increase in the leaflet surface area at the nonischemic commissural side, indicating localized leaflet tethering., Conclusions: Acute ischemic mitral regurgitation due to posterior left ventricular wall ischemia was associated with focal chordal and leaflet tethering at the nonischemic commissural portion of the mitral valve and a paradoxical decrease of the chordal forces and relative prolapse at the ischemic site of the anterior mitral valve leaflet.

Three cases of auricular perichondritis caused by infection with Pseudomonas aeruginosa are described to draw attention to the high incidence of piercing-related infections and the risk of structural deformities to the outer ear. Various therapies and outcomes are described and current Danish legislation is highlighted.

We report an unusual case of Wegener's granulomatosis in a patient presenting with stridor and tracheal narrowing as the initial manifestation of the disease.

A case of perichondritis and necrosis of the cartilage of the outer ear after acupuncture of the ear is presented. Repeated cultures showed growth of Pseudomonas aeruginosa. Despite intensive antibiotic treatment and extensive surgical toilet, the patient developed a severely deformed outer ear.

By means of elective staining with PAS-alcian blue and a whole-mount technique, the entire mucosa from 10 macroscopically normal human adult larynges was investigated with a view to describing the morphology, number, and distribution of the intra-epithelial glands. The glands varied in shape from round to oblong and in size between 30 and 200 micron. The typical round intra-epithelial gland was found to consist of 15-30 mucus-secreting cells, radially arranged around a narrow lumen debouching in a narrow stoma surrounded by cilia. The glands were of irregular distribution in all regions of the larynx. In each larynx there were between 174 and 652 (median 309) intra-epithelial glands. The most striking finding was a pronounced fall in their number from the supraglottis to the subglottis. Two larynges contained intra-epithelial cyst-like structures measuring from 15 to 40 micron. In the supraglottis there was a significantly negative correlation between the occurrence of intra-epithelial and subepithelial glands. On the other hand, it was not possible to demonstrate a definite relationship between the active intra-epithelial glands and the cystic intra-epithelial structures.

From 12 clinically and macroscopically normal larynges from adult persons all the mucosa was prepared and stained with PAS-alcian blue to study the morphology, distribution, and density of the goblet cells. In each larynx goblet cells were counted in 600 fields of 0.01768 mm2 mucosal surface, distributed on 18, 18 and 24 localities in the subglottis, glottis with the sinus of Morgagni, and supraglottis respectively. The goblet cells form a continuous pattern, comprising the entire subglottis, the anterior commissure, the sinus of Morgagni, the false vocal cords, vestibule of the larynx, and reaching to the cranial part of the laryngeal surface of the epiglottis. Another continuous, but goblet cell-free area extended from a couple of mm posterior to the anterior commissure, posteriorad on the cranial surface of the vocal cords, ary regions, aryepiglottic fold, the edge of the epiglottis, and 4-5 mm of the laryngeal surface of the epiglottic top, epiglottic vallecula, piriform recess, and the postcricoid region. Between the pseudostratified, ciliated columnar epithelium with goblet cells and the goblet cell-free stratified squamous epithelium there is a transitional epithelium in which the goblet cells alter from the 40 micron tall goblet cells characteristic of the respiratory tract epithelium to being quite flattened in order to disappear completely in the stratified squamous epithelium. The goblet cell density is significantly lower in the subglottis, viz. 125 cells per field, than in the glottis with the sinus of Morgagni and supraglottis, where the median density is 166 and 161 cells respectively per field. A possible correlation between the influence of the respiratory air upon the density of goblet cells and the complex anatomy of the larynx is discussed.

Pieces of mucosa were removed from the subglottis, the entrance into the sinus of Morgagni, and from the ventricular fold in 82 patients with benign diseases of the larynx. The goblet cells in these specimens were studied morphologically and quantitatively by an elective staining method and a whole mount technique. In 38 patients, the clinical and histological findings indicated an inflammatory disease in the form of chronic laryngitis or laryngeal edema. In 44 patients, the diagnosis was a nonmalignant tumorous lesion, such as papilloma, polyp, or cyst. These findings were compared with a similar study of goblet cells in normal larynges. In the inflammatory group as well as in the tumor group, the goblet cell density in the subglottis and ventricular fold proved to be lower than in normal larynges, but significantly lower only on the ventricular fold. This finding is assumed to be a result of the increased turbulence of the respiratory air, due to the disease process in the glottic fissure, which causes a greater contact surface between the mucosa and the respiratory air. In the close vicinity of the tumorous lesion there was a reduced, but not significantly reduced, goblet cell density, possibly caused by a tumor-initiated metaplasia to an epithelium poor in goblet cells. There were no morphological differences in goblet cells between the two disease groups and the normal larynges.

A comparison between a body-worn hearing aid with a conventional earphone/earmould system, and a free-field dynamic headphone demonstrated the significance of the coupling between the hearing aid earphone and the subject's ear. Measurement of the insertion gain revealed destruction of the ear-canal resonance with a conventional earmould, whereas the ear-canal resonance was preserved by the use of a dynamic headphone. The use of a body-worn hearing aid connected to a dynamic headphone showed a greater amplification at low as well as at high frequencies. Furthermore, a significantly better discrimination score and an improved sound quality were demonstrated with the dynamic headphone.

The entire intra- and extralaryngeal mucosa from 10 adult persons having macroscopically normal larynges was examined by the whole-mount method and selective staining of the mucous elements by PAS-alcian blue in order to assess the number, distribution, and density of the subepithelial glands. After microdissection of the mucosa the glandular orifices were quantitated under the stereomicroscope. Glands were demonstrated in the various regions of all larynges. The total intralaryngeal number, when excluding the saccule, ranged between 603 and 1579 (median 890) glands. In 6 larynges, glands could be quantitated in the saccule, where their number ranged between 32 and 324 (median 89). In the extralaryngeal region the count for the total series ranged between 1 and 53 (median 9) glands. The median intralaryngeal density, excluding the saccule, was 0.27 gland/mm2, ranging between 0.23 and 0.47. The density was significantly greater in the glottic than in the sub- and supraglottic regions. The highest concentration of glands was observed in the saccule in which the median density was 0.82 gland/mm2. A correlation between gland density and age or sex was not demonstrable. On the basis of the results, the subepithelial glands must be considered a normal component of the mucociliary system in the laryngeal mucosa.

By a periodic acid-Schiff (PAS)-alcian blue, elective staining method, and a whole-mount technique, morphologic and quantitative studies of goblet cells and mucous glands were done of mucosal biopsies from the subglottis, the entrance to the sinus of Morgagni, and the false vocal cords in 60 smokers with cancer of the larynx. In 30 of the patients the mucosal biopsies were taken at the time of diagnosis, and in the other 30 after radiotherapy. In the nonirradiated group of smokers there was an increased goblet cell density in the sinus of Morgagni and in the subglottis compared with that in the same areas of the larynx from nonsmokers, but only of significance in the subglottis. There was a definite tendency to a lower goblet cell density in the irradiated group of smokers and an increased number of goblet cell-free areas because of epithelial metaplasia to stratified squamous epithelium. This finding was significant in the subglottis only. In the nonirradiated as well as in the irradiated smokers there was a common occurrence of subepithelial mucous glands with mild to appreciable degenerative changes. This morphologic difference between smokers and nonsmokers may represent the toxic action of tobacco on the laryngeal mucosa and may thus enter into the pathogenesis of laryngeal cancer.

From 10 macroscopically normal larynges from adult persons the mucosa was removed in toto, stained with PAS-alcian blue, and the morphology of the subepithelial glands were studied. The glands presented as simple, tubular, usually purely mucous, or as larger, branched, sero-mucous tubular-alveolar. Their orifices were in areas lined with squamous or columnar epithelium. Three different types of main excretory ducts were observed, the commonest of equal caliber throughout, the other two with ampullar or funnel-shaped distensions.

From 70 patients with laryngeal cancer, pieces of mucosa were taken from the subglottis, the entrance to the sinus of Morgagni, and from the ventricular fold. There were 35 taken at the time of diagnosis and the other 35 were taken after radiotherapy. By means of an elective staining method and a whole mount technique, morphological and quantitative studies of the goblet cells were performed. The findings were compared with corresponding investigations of goblet cells in normal larynges. The significantly lower density in the subglottis than in the sinus of Morgagni and ventricular fold observed in normal larynges was not found in the two groups of cancer patients. Instead, there was a tendency to an equalization of the goblet cell density in all 3 laryngeal regions. In the irradiated group the goblet cell density was lower, in all 3 regions, than in the nonirradiated cancer group and in normal larynges. In the close vicinity of the cancer the goblet cell density was reduced, possibly because the cancer process had initiated metaplasia to an epithelium which was poor in goblet cells. Between the two cancer groups and normal larynges there were no morphological differences in the goblet cells.

In 20 children, 21 ears with incipient adhesive otitis media were treated by mobilizing the thin, adherent tympanic membrane, strengthening it with a fascial graft, inserting Silastic on the promontory, and establishing middle-ear drainage. The evaluation criteria were adhesive, atrophic and retractive changes in the tympanic membrane. The median follow-up period was 19 months. At follow-up, the material showed a statistically significant improvement in hearing, with a median hearing-loss of 17 db. post-operatively against 30 db. pre-operatively. There was a pronounced improvement in the adhesive and atrophic, as well as in the retractive, changes in the tympanic membrane. The present method of otosurgery is taken to be justified by the threat that incipient adhesive otitis media will develop into total middle-ear atelectasis with maximum hearing-loss and a risk of cholesteatoma. A further justification is afforded by the favourable therapeutic results.