The life-saving benefit of dexamethasone in severe COVID-19 is linked to a reversal of monocyte dysregulation.

Dexamethasone is a life-saving treatment for severe COVID-19, yet its mechanism of action is unknown, and many patients deteriorate or die despite timely treatment initiation. Here, we identify dexamethasone treatment-induced cellular and molecular changes associated with improved survival in COVID-19 patients. We observed a reversal of transcriptional hallmark signatures in monocytes associated with severe COVID-19 and the induction of a monocyte substate characterized by the expression of glucocorticoid-response genes. These molecular responses to dexamethasone were detected in circulating and pulmonary monocytes, and they were directly linked to survival. Monocyte single-cell RNA sequencing (scRNA-seq)-derived signatures were enriched in whole blood transcriptomes of patients with fatal outcome in two independent cohorts, highlighting the potential for identifying non-responders refractory to dexamethasone. Our findings link the effects of dexamethasone to specific immunomodulation and reversal of monocyte dysregulation, and they highlight the potential of single-cell omics for monitoring in vivo target engagement of immunomodulatory drugs and for patient stratification for precision medicine approaches.

Complement activation induces excessive T cell cytotoxicity in severe COVID-19.

Severe COVID-19 is linked to both dysfunctional immune response and unrestrained immunopathology, and it remains unclear whether T cells contribute to disease pathology. Here, we combined single-cell transcriptomics and single-cell proteomics with mechanistic studies to assess pathogenic T cell functions and inducing signals. We identified highly activated CD16+ T cells with increased cytotoxic functions in severe COVID-19. CD16 expression enabled immune-complex-mediated, T cell receptor-independent degranulation and cytotoxicity not found in other diseases. CD16+ T cells from COVID-19 patients promoted microvascular endothelial cell injury and release of neutrophil and monocyte chemoattractants. CD16+ T cell clones persisted beyond acute disease maintaining their cytotoxic phenotype. Increased generation of C3a in severe COVID-19 induced activated CD16+ cytotoxic T cells. Proportions of activated CD16+ T cells and plasma levels of complement proteins upstream of C3a were associated with fatal outcome of COVID-19, supporting a pathological role of exacerbated cytotoxicity and complement activation in COVID-19.

Severe COVID-19 Is Marked by a Dysregulated Myeloid Cell Compartment.

Coronavirus disease 2019 (COVID-19) is a mild to moderate respiratory tract infection, however, a subset of patients progress to severe disease and respiratory failure. The mechanism of protective immunity in mild forms and the pathogenesis of severe COVID-19 associated with increased neutrophil counts and dysregulated immune responses remain unclear. In a dual-center, two-cohort study, we combined single-cell RNA-sequencing and single-cell proteomics of whole-blood and peripheral-blood mononuclear cells to determine changes in immune cell composition and activation in mild versus severe COVID-19 (242 samples from 109 individuals) over time. HLA-DRhiCD11chi inflammatory monocytes with an interferon-stimulated gene signature were elevated in mild COVID-19. Severe COVID-19 was marked by occurrence of neutrophil precursors, as evidence of emergency myelopoiesis, dysfunctional mature neutrophils, and HLA-DRlo monocytes. Our study provides detailed insights into the systemic immune response to SARS-CoV-2 infection and reveals profound alterations in the myeloid cell compartment associated with severe COVID-19.

Recognition of microbial viability via TLR8 drives TFH cell differentiation and vaccine responses.

Live attenuated vaccines are generally highly efficacious and often superior to inactivated vaccines, yet the underlying mechanisms of this remain largely unclear. Here we identify recognition of microbial viability as a potent stimulus for follicular helper T cell (TFH cell) differentiation and vaccine responses. Antigen-presenting cells (APCs) distinguished viable bacteria from dead bacteria through Toll-like receptor 8 (TLR8)-dependent detection of bacterial RNA. In contrast to dead bacteria and other TLR ligands, live bacteria, bacterial RNA and synthetic TLR8 agonists induced a specific cytokine profile in human and porcine APCs, thereby promoting TFH cell differentiation. In domestic pigs, immunization with a live bacterial vaccine induced robust TFH cell and antibody responses, but immunization with its heat-killed counterpart did not. Finally, a hypermorphic TLR8 polymorphism was associated with protective immunity elicited by vaccination with bacillus Calmette-Guérin (BCG) in a human cohort. We have thus identified TLR8 as an important driver of TFH cell differentiation and a promising target for TFH cell-skewing vaccine adjuvants.

SARS-CoV-2-reactive T cells in healthy donors and patients with COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the rapidly unfolding coronavirus disease 2019 (COVID-19) pandemic1,2. Clinical manifestations of COVID-19 vary, ranging from asymptomatic infection to respiratory failure. The mechanisms that determine such variable outcomes remain unresolved. Here we investigated CD4+ T cells that are reactive against the spike glycoprotein of SARS-CoV-2 in the peripheral blood of patients with COVID-19 and SARS-CoV-2-unexposed healthy donors. We detected spike-reactive CD4+ T cells not only in 83% of patients with COVID-19 but also in 35% of healthy donors. Spike-reactive CD4+ T cells in healthy donors were primarily active against C-terminal epitopes in the spike protein, which show a higher homology to spike glycoproteins of human endemic coronaviruses, compared with N-terminal epitopes. Spike-protein-reactive T cell lines generated from SARS-CoV-2-naive healthy donors responded similarly to the C-terminal region of the spike proteins of the human endemic coronaviruses 229E and OC43, as well as that of SARS-CoV-2. This results indicate that spike-protein cross-reactive T cells are present, which were probably generated during previous encounters with endemic coronaviruses. The effect of pre-existing SARS-CoV-2 cross-reactive T cells on clinical outcomes remains to be determined in larger cohorts. However, the presence of spike-protein cross-reactive T cells in a considerable fraction of the general population may affect the dynamics of the current pandemic, and has important implications for the design and analysis of upcoming trials investigating COVID-19 vaccines.

Single-cell RNA sequencing uncovers the nuclear decoy lincRNA PIRAT as a regulator of systemic monocyte immunity during COVID-19.

The systemic immune response to viral infection is shaped by master transcription factors, such as NF-κB, STAT1, or PU.1. Although long noncoding RNAs (lncRNAs) have been suggested as important regulators of transcription factor activity, their contributions to the systemic immunopathologies observed during SARS-CoV-2 infection have remained unknown. Here, we employed a targeted single-cell RNA sequencing approach to reveal lncRNAs differentially expressed in blood leukocytes during severe COVID-19. Our results uncover the lncRNA PIRAT (PU.1-induced regulator of alarmin transcription) as a major PU.1 feedback-regulator in monocytes, governing the production of the alarmins S100A8/A9, key drivers of COVID-19 pathogenesis. Knockout and transgene expression, combined with chromatin-occupancy profiling, characterized PIRAT as a nuclear decoy RNA, keeping PU.1 from binding to alarmin promoters and promoting its binding to pseudogenes in naïve monocytes. NF-κB-dependent PIRAT down-regulation during COVID-19 consequently releases a transcriptional brake, fueling alarmin production. Alarmin expression is additionally enhanced by the up-regulation of the lncRNA LUCAT1, which promotes NF-κB-dependent gene expression at the expense of targets of the JAK-STAT pathway. Our results suggest a major role of nuclear noncoding RNA networks in systemic antiviral responses to SARS-CoV-2 in humans.

Noncoding RNA MaIL1 is an integral component of the TLR4-TRIF pathway.

RNA has been proposed as an important scaffolding factor in the nucleus, aiding protein complex assembly in the dense intracellular milieu. Architectural contributions of RNA to cytosolic signaling pathways, however, remain largely unknown. Here, we devised a multidimensional gradient approach, which systematically locates RNA components within cellular protein networks. Among a subset of noncoding RNAs (ncRNAs) cosedimenting with the ubiquitin-proteasome system, our approach unveiled ncRNA MaIL1 as a critical structural component of the Toll-like receptor 4 (TLR4) immune signal transduction pathway. RNA affinity antisense purification-mass spectrometry (RAP-MS) revealed MaIL1 binding to optineurin (OPTN), a ubiquitin-adapter platforming TBK1 kinase. MaIL1 binding stabilized OPTN, and consequently, loss of MaIL1 blunted OPTN aggregation, TBK1-dependent IRF3 phosphorylation, and type I interferon (IFN) gene transcription downstream of TLR4. MaIL1 expression was elevated in patients with active pulmonary infection and was highly correlated with IFN levels in bronchoalveolar lavage fluid. Our study uncovers MaIL1 as an integral RNA component of the TLR4-TRIF pathway and predicts further RNAs to be required for assembly and progression of cytosolic signaling networks in mammalian cells.

The immunologic tumor microenvironment in endometrioid endometrial cancer in the morphomolecular context: mutual correlations and prognostic impact depending on molecular alterations.

OBJECTIVE: POLE-mutant, microsatellite-instable (MSI), p53-mutant and non-specific molecular profile (NSMP) are TCGA-defined molecular subgroups of endometrial cancer (EC). Hypothesizing that morphology and tumor immunology might differ depending on molecular background concerning composition and prognostic impact, we aimed to comprehensively interconnect morphologic, immunologic and molecular data. METHODS: TCGA-defined molecular groups were determined by immunohistochemistry and sequencing in n = 142 endometrioid EC. WHO-defined histopathological grading was performed. The immunologic microenvironment (iTME) was characterised by the quantification of intraepithelial and stromal populations of tumor-infiltrating lymphocytes (TIL: overall T-cells; T-Killer cells; regulatory T-cells (Treg)). Immunologic parameters were correlated with WHO-grading, TCGA-subgroups and prognosis. RESULTS: High density TIL were significantly more frequent in high-grade (G3) compared to low-grade (G1/2) EC in the whole cohort and in the subgroup of POLE-wildtype-/microsatellite-stable-EC. MSI was associated with high-level TIL-infiltration when taking into account the type of mismatch repair defect (MLH1/PMS2; MSH2/MSH6). Prognostic impact of biomarkers depended on molecular subgroups: In p53-mutant EC, Treg were independently prognostic, in NSMP, the unique independently prognostic biomarker was WHO-grading. CONCLUSIONS: EC morphology and immunology differ depending on genetics. Our study delineated two molecularly distinct subgroups of immunogenic EC characterized by high-density TIL-infiltration: MSI EC and high-grade POLE-wildtype/microsatellite-stable-EC. Prognostic impact of TIL-populations relied on TCGA-subgroups indicating specific roles for TIL depending on molecular background. In NSMP, histopathological grading was the only prognostic biomarker demonstrating the relevance of WHO-grading in an era of molecular subtyping.

Carotid endarterectomy significantly improves postoperative laryngeal sensitivity.

OBJECTIVE: Iatrogenic injury of the vagus nerve or its branches during carotid endarterectomy (CEA) can result in globus sensation, dysphagia, and even vocal fold immobility. Knowledge of morphologic and functional laryngopharyngeal outcomes after CEA is poor. The present study was performed to determine potential iatrogenic damage to the laryngeal innervation after CEA. An area of particular interest was the supraglottic sensory threshold, which was examined by Fiberoptic Endoscopic Evaluation of Swallowing With Sensory Testing (FEESST; Pentax Medical Company, Montvale, NJ), a validated and safe method for the determination of the motor and sensory components of swallowing. METHODS: FEESST was used preoperatively in 32 patients scheduled to undergo CEA and twice postoperatively to examine the motor and sensory components of swallowing. In this endolaryngeal examination, laryngopharyngeal sensory thresholds (in mm Hg) were defined as normal at <4.0 mm Hg air pulse pressure (APP), moderate deficit at 4.0 to 6.0 mm Hg APP, or severe deficit at >6.0 mm Hg APP, with a value >10.0 mm Hg APP indicating abolished laryngeal adductor reflex. Acoustic voice parameters were also analyzed for further functional changes of the larynx. RESULTS: The mean ± standard deviation preoperative FEESST measures showed no significant differences (P = .065) between the operated-on side (6.73 ± 1.73 mm Hg) and the opposite side (5.83 ± 1.68 mm Hg). At 2 days postoperatively, the threshold increased (P = .001) to 7.62 ± 1.98 mm Hg on the operated-on side. A laryngopharyngeal mucosal hematoma on the operated side was endoscopically detectable in eight patients (30.8%); in these patients, we found a markedly elevated (P = .021) measure of 9.50 ± 0.93 mm Hg. On the opposite (nonoperated-on) side of the laryngopharynx, the thresholds remained at the same level as preoperatively over all assessments (P >.05), whereas the differences between the operated and nonoperated-on sides and the hematoma and nonhematoma groups were highly significant (P = .004 and P = .001, respectively). Surprisingly, the sensory threshold on the operated-on side (6.08 ± 2.02 mm Hg) decreased significantly at the 6-week follow-up, even in relation to the preoperative measure (P = .022). With the exception of one patient with permanent unilateral vocal fold immobility, no signs of nerve injury were detected. CONCLUSIONS: In accordance with previous reports, injuries to the recurrent laryngeal nerve during CEA seem to be rare. In most patients, postoperative symptoms (globus, dysphagia, dysphonia) and signs fade within a few weeks without any specific therapeutic intervention. This study shows an improved long-term postoperative superior laryngeal nerve function with regard to laryngopharyngeal sensitivity.

Good outcome after liver transplantation for ALD without a 6 months abstinence rule prior to transplantation including post-transplant CDT monitoring for alcohol relapse assessment - a retrospective study.

Alcoholic liver disease (ALD) is the second most common indication for liver transplantation (LT). The utility of fixed intervals of abstinence prior to listing is still a matter of discussion. Furthermore, post-LT long-term observation is challenging, and biomarkers as carbohydrate-deficient transferrin (CDT) may help to identify alcohol relapse. We retrospectively analyzed data from patients receiving LT for ALD from 1996 to 2012. A defined period of alcohol abstinence prior to listing was not a precondition, and abstinence was evaluated using structured psychological interviews. A total of 382 patients received LT for ALD as main (n = 290) or secondary (n = 92) indication; median follow-up was 73 months (0-213). One- and five-year patient survival and graft survival rates were 82% and 69%, and 80% and 67%, respectively. A total of 62 patients (16%) experienced alcohol relapse. Alcohol relapse did not have a statistically significant effect on patient survival (P = 0.10). Post-transplant CDT measurements showed a sensitivity and specificity of 84% and 85%, respectively. In conclusion, this large single-center analysis showed good post-transplant long-term results in patients with ALD when applying structured psychological interviews before listing. Relapse rates were lower than those reported in the literature despite using a strict definition of alcohol relapse. Furthermore, post-LT CDT measurement proved to be a useful supplementary tool for detecting alcohol relapse.

Pathogen-triggered activation of plasmacytoid dendritic cells induces IL-10-producing B cells in response to Staphylococcus aureus.

Induction of polyclonal B cell activation is a phenomenon observed in many types of infection, but its immunological relevance is unclear. In this study we show that staphylococcal protein A induces T cell-independent human B cell proliferation by enabling uptake of TLR-stimulating nucleic acids via the V(H)3(+) BCR. We further demonstrate that Staphylococcus aureus strains with high surface protein A expression concomitantly trigger activation of human plasmacytoid dendritic cells (pDC). Sensitivity to chloroquine, cathepsin B inhibition, and a G-rich inhibitory oligodeoxynucleotide supports the involvement of TLR9 in this context. We then identify pDC as essential cellular mediators of B cell proliferation and Ig production in response to surface protein A-bearing S. aureus. The in vivo relevancy of these findings is confirmed in a human PBMC Nod/scid(Prkdc)/γc(-/-) mouse model. Finally, we demonstrate that co-operation of pDC and B cells enhances B cell-derived IL-10 production, a cytokine associated with immunosuppression and induction of IgG4, an isotype frequently dominating the IgG response to S. aureus. IL-10 release is partially dependent on TLR2-active lipoproteins, a hallmark of the Staphylococcus species. Collectively, our data suggest that S. aureus exploits pDC and TLR to establish B cell-mediated immune tolerance.

Recreational athletes during downhill-mountain biking (DMB) show high incidence of upper extremity fractures in combination with soft-tissue injuries.

Downhill-mountain biking (DMB) is a high-risk sport and often leads to several injuries, especially in non-professional athletes. We retrospectively analyzed the most common injuries and profiled the injury mechanism. Until now, there is no such analysis of injuries by non-professional mountain bike athletes. We collected patient data from patients who suffered from an injury during DMB. The inclusion criteria were (1) injury during the summer season of 2020 and 2021, (2) injury during off-road and downhill mountain bike sports activity, and (3) treatment at the Department of Traumatology of the Klinik Diakonissen Schladming. Patient data was analyzed regarding the type of injury, location of the injury, patient age and gender of the patients. Most patients with injury are at the age of 26-35. Second most are between 36 and 71 years old. The type of injury differs between age and gender. Mostly upper-extremity injuries occur with a high probability of shoulder injuries. In the elderly patients, we found additional injuries of the thorax and chest. To conclude, most common types of injuries are soft-tissue injuries, often in combination with fractures. The risk for injuries is higher for recreational athletes with different injury characteristics than professional athletes.

Manufacturing, characterization, and degradation of a poly(lactic acid) warp-knitted spacer fabric scaffold as a candidate for tissue engineering applications.

Three-dimensional bioabsorbable textiles represent a novel technology for the manufacturing of tissue engineering scaffolds. In the present study, 3D bioabsorbable poly(lactic acid) (PLA) spacer fabric scaffolds are fabricated by warp-knitting and their potential for tissue engineering is explored in vitro. Changes in physical properties and mechanical performance with different heat setting treatments are assessed. To characterize the microenvironment experienced by cells in the scaffolds, yarn properties are investigated prior to, and during, hydrolytic degradation. The differences in yarn morphology, thermal properties, infrared spectra, and mechanical properties are investigated and monitored during temperature accelerated in vitro degradation tests in phosphate buffered saline (PBS) solution at 58 °C and pH 7.4 for 55 days. Yarn and textile cytocompatibility are tested to assess the effect of materials employed, manufacturing conditions, post processing and sterilization on cell viability, together with the cytocompatibility of the textile degradation products. Results show that the heat setting process can be used to modify scaffold properties, such as thickness, porosity, pore size and stiffness within the range useful for tissue regeneration. Scaffold degradation rate in physiological conditions is estimated by comparing yarn degradation data with PLA degradation data from literature. This will potentially allow the prediction of scaffold mechanical stability in the long term and thus its suitability for the remodelling of different tissues. Mouse calvaria preosteoblast MC3T3-E1 cells attachment and proliferation are observed on the scaffold over 12 days of in vitro culture by 4',6-diamidino-2-phenylindole (DAPI) fluorescent staining and DNA quantification. The present work shows the potential of spacer fabric scaffolds as a versatile and scalable scaffold fabrication technique, having the ability to create a microenvironment with appropriate physical, mechanical, and degradation properties for 3D tissue engineering. The high control and tunability of spacer fabric properties makes it a promising candidate for the regeneration of different tissues in patient-specific applications.

The functional anatomy of the cricothyroid joint.

Postoperative long-term treatment problems of male-to-female transsexuals can be caused by the underestimated mobility of the cricothyroid joint. One hundred cadaveric larynges were analysed to reveal the morphological and functional anatomy of the CTJ focusing on possible horizontal and vertical gliding movements. Furthermore, the distance of the cricothyroid space for rotational movements was measured. The articular surface of the cricothyroid joint showed three different possibilities: a well-defined facet (Type 1 61%), no definable facet (Type 2 22%) or a flat surface with or without a tiny protuberance (Type 3 17%). Side different statements and intraindividual differences between male and female specimens were included. These different types affect horizontal and vertical gliding movements, besides rotational movements. The mobility between Type 1 and the others was highly significant (P < 0.001). Regarding different motions in the cricothyroid joint, rotational movements caused significant elongation of the vocal folds, which should be taken into consideration for phonosurgical methods.

Innate sensors that regulate vaccine responses.

Pattern recognition receptors (PRRs) control elemental functions of antigen presenting cells (APCs) and critically shape adaptive immune responses. Wielding a natural adjuvanticity, live attenuated vaccines elicit exceptionally efficient and durable immunity. Commonly used vaccine adjuvants target individual PRRs or bolster the immunogenicity of vaccines via indirect mechanisms of inflammation. Here, we review the impact of innate sensors on immune responses to live attenuated vaccines and commonly used vaccine adjuvants, with a focus on human vaccine responses. We discuss the unique potential of microbial nucleic acids and their corresponding sensing receptors to mimic live attenuated vaccines and promote protective immunity.

Toxicological risk at workplace and toxicity as Life Cycle Assessment impact category: Substitution of solvents as an example.

Substitution of hazardous substances against less hazardous ones is a central requirement of the European Chemical Regulation REACH (European Regulation 1907/2006/EC). Hazardous substances emitted from products may not only affect the worker; drift off and distribution in the environment may finally result in exposure of the general population. This potential threat to health is covered by the impact category "toxicity" in Life Cycle Assessments. In this paper, we present a case of a substitution of volatile organic compounds in a reactive varnish, and compare the "old" formulation with the "new" formulation against health risk to the worker, and concerning the Life Cycle Assessment impact category "toxicity". The "old" formulation contained Naphtha (petroleum), hydrodesulfurized, heavy and Solvent naphtha (petroleum), light, aromatic. In the new formulation, both naphthas were replaced by n-Butylacetate, 1-Ethoxy-2-propyl acetate and Ethyl-3-ethoxy propionate. In the European Union, the naphthas are classified as mutagens and carcinogens category 1, officially. However, if benzene is below 0.1 %, registrants in the EU proposed to omit this classification, and todays naptha products on the market obviously have benzene contents below 0.1 %. On a first glance, the improvement for workplace safety introduced by the substitution, therefore, is comparatively small, as it is for toxicity in Life Cycle Assessment. However, when background knowledge concerning chemical production processes of naphtha is included, benzene below a content of 0.1 % needs to be taken into consideration, and the benefit of substitution is more obvious.

Breast Cancer-Derived Lung Metastases Show Increased Pyruvate Carboxylase-Dependent Anaplerosis.

Cellular proliferation depends on refilling the tricarboxylic acid (TCA) cycle to support biomass production (anaplerosis). The two major anaplerotic pathways in cells are pyruvate conversion to oxaloacetate via pyruvate carboxylase (PC) and glutamine conversion to α-ketoglutarate. Cancers often show an organ-specific reliance on either pathway. However, it remains unknown whether they adapt their mode of anaplerosis when metastasizing to a distant organ. We measured PC-dependent anaplerosis in breast-cancer-derived lung metastases compared to their primary cancers using in vivo 13C tracer analysis. We discovered that lung metastases have higher PC-dependent anaplerosis compared to primary breast cancers. Based on in vitro analysis and a mathematical model for the determination of compartment-specific metabolite concentrations, we found that mitochondrial pyruvate concentrations can promote PC-dependent anaplerosis via enzyme kinetics. In conclusion, we show that breast cancer cells proliferating as lung metastases activate PC-dependent anaplerosis in response to the lung microenvironment.

Feasibility of piezoelectric endoscopic transsphenoidal craniotomy: a cadaveric study.

OBJECTIVE: Endoscopic transsphenoidal approach has become the gold standard for surgical treatment of treating pituitary adenomas or other lesions in that area. Opening of bony skull base has been performed with burrs, chisels, and hammers or standard instruments like punches and circular top knives. The creation of primary bone flaps-as in external craniotomies-is difficult.The piezoelectric osteotomes used in the present study allows creating a bone flap for endoscopic transnasal approaches in certain areas. The aim of this study was to prove the feasibility of piezoelectric endoscopic transnasal craniotomies. Study Design. Cadaveric study. METHODS: On cadaveric specimens (N = 5), a piezoelectric system with specially designed hardware for endonasal application was applied and endoscopic transsphenoidal craniotomies at the sellar floor, tuberculum sellae, and planum sphenoidale were performed up to a size of 3-5 cm(2). RESULTS: Bone flaps could be created without fracturing with the piezoosteotome and could be reimplanted. Endoscopic handling was unproblematic and time required was not exceeding standard procedures. CONCLUSION: In a cadaveric model, the piezoelectric endoscopic transsphenoidal craniotomy (PETC) is technically feasible. This technique allows the surgeon to create a bone flap in endoscopic transnasal approaches similar to existing standard transcranial craniotomies. Future trials will focus on skull base reconstruction using this bone flap.