CD200+ fibroblasts form a pro-resolving mesenchymal network in arthritis.

Fibroblasts are important regulators of inflammation, but whether fibroblasts change phenotype during resolution of inflammation is not clear. Here we use positron emission tomography to detect fibroblast activation protein (FAP) as a means to visualize fibroblast activation in vivo during inflammation in humans. While tracer accumulation is high in active arthritis, it decreases after tumor necrosis factor and interleukin-17A inhibition. Biopsy-based single-cell RNA-sequencing analyses in experimental arthritis show that FAP signal reduction reflects a phenotypic switch from pro-inflammatory MMP3+/IL6+ fibroblasts (high FAP internalization) to pro-resolving CD200+DKK3+ fibroblasts (low FAP internalization). Spatial transcriptomics of human joints indicates that pro-resolving niches of CD200+DKK3+ fibroblasts cluster with type 2 innate lymphoid cells, whereas MMP3+/IL6+ fibroblasts colocalize with inflammatory immune cells. CD200+DKK3+ fibroblasts stabilized the type 2 innate lymphoid cell phenotype and induced resolution of arthritis via CD200-CD200R1 signaling. Taken together, these data suggest a dynamic molecular regulation of the mesenchymal compartment during resolution of inflammation.

Evidence of innate lymphoid cell redundancy in humans.

Innate lymphoid cells (ILCs) have potent immunological functions in experimental conditions in mice, but their contributions to immunity in natural conditions in humans have remained unclear. We investigated the presence of ILCs in a cohort of patients with severe combined immunodeficiency (SCID). All ILC subsets were absent in patients with SCID who had mutation of the gene encoding the common γ-chain cytokine receptor subunit IL-2Rγ or the gene encoding the tyrosine kinase JAK3. T cell reconstitution was observed in patients with SCID after hematopoietic stem cell transplantation (HSCT), but the patients still had considerably fewer ILCs in the absence of myeloablation than did healthy control subjects, with the exception of rare cases of reconstitution of the ILC1 subset of ILCs. Notably, the ILC deficiencies observed were not associated with any particular susceptibility to disease, with follow-up extending from 7 years to 39 years after HSCT. We thus report here selective ILC deficiency in humans and show that ILCs might be dispensable in natural conditions, if T cells are present and B cell function is preserved.

Type I interferon restricts type 2 immunopathology through the regulation of group 2 innate lymphoid cells.

Viral respiratory tract infections are the main causative agents of the onset of infection-induced asthma and asthma exacerbations that remain mechanistically unexplained. Here we found that deficiency in signaling via type I interferon receptor led to deregulated activation of group 2 innate lymphoid cells (ILC2 cells) and infection-associated type 2 immunopathology. Type I interferons directly and negatively regulated mouse and human ILC2 cells in a manner dependent on the transcriptional activator ISGF3 that led to altered cytokine production, cell proliferation and increased cell death. In addition, interferon-γ (IFN-γ) and interleukin 27 (IL-27) altered ILC2 function dependent on the transcription factor STAT1. These results demonstrate that type I and type II interferons, together with IL-27, regulate ILC2 cells to restrict type 2 immunopathology.

Characterization of Vascular Niche in Systemic Sclerosis by Spatial Proteomics.

BACKGROUND: Systemic sclerosis (SSc) is a connective tissue disease that can serve as a model to study vascular changes in response to inflammation, autoimmunity, and fibrotic remodeling. Although microvascular changes are the earliest histopathologic manifestation of SSc, the vascular pathophysiology remains poorly understood. METHODS: We applied spatial proteomic approaches to deconvolute the heterogeneity of vascular cells at the single-cell level in situ and characterize cellular alterations of the vascular niches of patients with SSc. Skin biopsies of patients with SSc and control individuals were analyzed by imaging mass cytometry, yielding a total of 90 755 cells including 2987 endothelial cells and 4096 immune cells. RESULTS: We identified 7 different subpopulations of blood vascular endothelial cells (VECs), 2 subpopulations of lymphatic endothelial cells, and 3 subpopulations of pericytes. A novel population of CD34+;αSMA+ (α-smooth muscle actin);CD31+ VECs was more common in SSc, whereas endothelial precursor cells were decreased. Co-detection by indexing and tyramide signal amplification confirmed these findings. The microenvironment of CD34+;αSMA+;CD31+ VECs was enriched for immune cells and myofibroblasts, and CD34+;αSMA+;CD31+ VECs expressed markers of endothelial-to-mesenchymal transition. The density of CD34+;αSMA+;CD31+ VECs was associated with clinical progression of fibrosis in SSc. CONCLUSIONS: Using spatial proteomics, we unraveled the heterogeneity of vascular cells in control individuals and patients with SSc. We identified CD34+;αSMA+;CD31+ VECs as a novel endothelial cell population that is increased in patients with SSc, expresses markers for endothelial-to-mesenchymal transition, and is located in close proximity to immune cells and myofibroblasts. CD34+;αSMA+;CD31+ VEC counts were associated with clinical outcomes of progressive fibrotic remodeling, thus providing a novel cellular correlate for the crosstalk of vasculopathy and fibrosis.

PU.1 controls fibroblast polarization and tissue fibrosis.

Fibroblasts are polymorphic cells with pleiotropic roles in organ morphogenesis, tissue homeostasis and immune responses. In fibrotic diseases, fibroblasts synthesize abundant amounts of extracellular matrix, which induces scarring and organ failure. By contrast, a hallmark feature of fibroblasts in arthritis is degradation of the extracellular matrix because of the release of metalloproteinases and degrading enzymes, and subsequent tissue destruction. The mechanisms that drive these functionally opposing pro-fibrotic and pro-inflammatory phenotypes of fibroblasts remain unknown. Here we identify the transcription factor PU.1 as an essential regulator of the pro-fibrotic gene expression program. The interplay between transcriptional and post-transcriptional mechanisms that normally control the expression of PU.1 expression is perturbed in various fibrotic diseases, resulting in the upregulation of PU.1, induction of fibrosis-associated gene sets and a phenotypic switch in extracellular matrix-producing pro-fibrotic fibroblasts. By contrast, pharmacological and genetic inactivation of PU.1 disrupts the fibrotic network and enables reprogramming of fibrotic fibroblasts into resting fibroblasts, leading to regression of fibrosis in several organs.

Alternative splicing and intron retention: Their profiles and roles in cutaneous fibrosis of systemic sclerosis.

BACKGROUND: Alternative splicing (AS) and intron retention (IR) implicated in multiple pathophysiological processes, have rarely been reported in systemic sclerosis (SSc). METHODS: We integrated bulk RNA-seq and 4D label-free mass spectrometry to perform a multi-omics analysis of AS and IR in SSc skin tissue and fibroblasts. RMATS and iREAD were used to identify AS and IR, which were validated by real-time PCR. Spearman correlation and the LASSO method were employed to assess correlations among clinical features, introns, splicing factors (regulators of AS) and proteins. FINDINGS: AS profiles showed distinct alterations in SSc skin tissue, with the most pronounced changes occurring in IR. AS and IR were associated with total modified Rodnan skin score (mRSS) and local skin score. Upon TGF-ß stimulation, fibroblasts exhibited significant alterations in IR profiles, affecting genes related to fibroblast proliferation and collagen fibril organization. A comprehensive integrated analysis of introns, exons, and proteome profiles revealed that IR exerted a negative impact on protein expression, with certain changes being under intronic control. RT-PCR confirmed the presence of intron and exon-derived sequences of CTTN, OGA, MED16 and PHYKPL. Additionally, notable changes were observed in the regulatory network of splicing factors in SSc skin tissues. These factors are also involved in fibrosis pathways and correlated with clinical features. CONCLUSION: Totally, abnormal AS, IR profiles and splicing factors were identified in SSc, altered IRs and splicing factors participated in fibrosis-related pathways. IR exerted a negative impact on protein expression in TGF-ß-stimulated fibroblasts. Clarification of the IR mechanisms will provide new insights into the pathophysiology of SSc.

Translational control of the activation of transcription factor NF-κB and production of type I interferon by phosphorylation of the translation factor eIF4E.

Type I interferon is an integral component of the antiviral response, and its production is tightly controlled at the levels of transcription and translation. The eukaryotic translation-initiation factor eIF4E is a rate-limiting factor whose activity is regulated by phosphorylation of Ser209. Here we found that mice and fibroblasts in which eIF4E cannot be phosphorylated were less susceptible to virus infection. More production of type I interferon, resulting from less translation of Nfkbia mRNA (which encodes the inhibitor IκBα), largely explained this phenotype. The lower abundance of IκBα resulted in enhanced activity of the transcription factor NF-κB, which promoted the production of interferon-ß (IFN-ß). Thus, regulated phosphorylation of eIF4E has a key role in antiviral host defense by selectively controlling the translation of an mRNA that encodes a critical suppressor of the innate antiviral response.

Performance of serum biomarkers reflective of different pathogenic processes in systemic sclerosis-associated interstitial lung disease.

OBJECTIVE: Interstitial lung disease (ILD) is the leading cause of mortality in SSc. Novel biomarkers are crucial to improve outcomes in SSc-ILD. We aimed to compare the performance of potential serum biomarkers of SSc-ILD that reflect different pathogenic processes: KL-6 and SP-D (epithelial injury), CCL18 (type 2 immune response), YKL-40 (endothelial injury and matrix remodelling) and MMP-7 (ECM remodelling). METHODS: Baseline and follow-up serum samples from 225 SSc patients were analysed by ELISA. Progressive ILD was defined according to the 2022-ATS/ERS/JRS/ALAT guidelines. Linear mixed models and random forest models were used for statistical analyses. RESULTS: Serum levels of KL-6 [MD 35.67 (95% CI 22.44-48.89, P < 0.01)], SP-D [81.13 (28.46-133.79, P < 0.01)], CCL18 [17.07 (6.36-27.77, P < 0.01)], YKL-40 [22.81 (7.19-38.44, P < 0.01)] and MMP-7 [2.84 (0.88-4.80, P < 0.01)] were independently associated with the presence of SSc-ILD. A machine-learning model including all candidates classified patients with or without ILD with an accuracy of 85%. The combination of KL-6 and SP-D was associated with the presence [0.77 (0.53-1.00, P' <0.01)] and previous progression of SSc-ILD [OR 1.28 (1.01-1.61, P' =0.047)]. Higher baseline levels of KL-6 [OR 3.70 (1.52-9.03, P < 0.01)] or SP-D [OR 2.00 (1.06-3.78, P = 0.03)] increased the odds of future SSc-ILD progression, independent of other conventional risk factors, and the combination of KL-6 and SP-D [1.109 (0.665-1.554, P < 0.01)] showed improved performance compared with KL-6 and SP-D alone. CONCLUSION: All candidates performed well as diagnostic biomarkers for SSc-ILD. The combination of KL-6 and SP-D might serve as biomarker for the identification of SSc patients at risk of ILD progression.

S100A4-neutralizing monoclonal antibody 6B12 counteracts the established experimental skin fibrosis induced by bleomycin.

OBJECTIVES: Our previous studies have demonstrated that the Damage Associated Molecular Pattern (DAMP) protein, S100A4, is overexpressed in the involved skin and peripheral blood of patients with SSc. It is associated with skin and lung involvement, and disease activity. By contrast, lack of S100A4 prevented the development of experimental dermal fibrosis. Herein we aimed to evaluate the effect of murine anti-S100A4 mAb 6B12 in the treatment of preestablished experimental dermal fibrosis. METHODS: The effects of 6B12 were assessed at therapeutic dosages in a modified bleomycin-induced dermal fibrosis mouse model by evaluating fibrotic (dermal thickness, proliferation of myofibroblasts, hydroxyproline content, phosphorylated Smad3-positive cell count) and inflammatory (leukocytes infiltrating the lesional skin, systemic levels of selected cytokines and chemokines) outcomes, and transcriptional profiling (RNA sequencing). RESULTS: Treatment with 7.5 mg/kg 6B12 attenuated and might even reduce pre-existing dermal fibrosis induced by bleomycin as evidenced by reduction in dermal thickness, myofibroblast count and collagen content. These antifibrotic effects were mediated by the downregulation of TGF-ß/Smad signalling and partially by reducing the number of leukocytes infiltrating the lesional skin and decrease in the systemic levels of IL-1α, eotaxin, CCL2 and CCL5. Moreover, transcriptional profiling demonstrated that 7.5 mg/kg 6B12 also modulated several profibrotic and proinflammatory processes relevant to the pathogenesis of SSc. CONCLUSION: Targeting S100A4 by the 6B12 mAb demonstrated potent antifibrotic and anti-inflammatory effects on bleomycin-induced dermal fibrosis and provided further evidence for the vital role of S100A4 in the pathophysiology of SSc.

Germicidal efficacy of continuous and pulsed ultraviolet-C radiation on pathogen models and SARS-CoV-2.

Ultraviolet radiation's germicidal efficacy depends on several parameters, including wavelength, radiant exposure, microbial physiology, biological matrices, and surfaces. In this work, several ultraviolet radiation sources (a low-pressure mercury lamp, a KrCl excimer, and four UV LEDs) emitting continuous or pulsed irradiation were compared. The greatest log reductions in E. coli cells and B. subtilis endospores were 4.1 ± 0.2 (18 mJ cm-2) and 4.5 ± 0.1 (42 mJ cm-2) with continuous 222 nm, respectively. The highest MS2 log reduction observed was 2.7 ± 0.1 (277 nm at 3809 mJ cm-2). Log reductions of SARS-CoV-2 with continuous 222 nm and 277 nm were ≥ 3.4 ± 0.7, with 13.3 mJ cm-2 and 60 mJ cm-2, respectively. There was no statistical difference between continuous and pulsed irradiation (0.83-16.7% [222 nm and 277 nm] or 0.83-20% [280 nm] duty rates) on E. coli inactivation. Pulsed 260 nm radiation (0.5% duty rate) at 260 nm yielded significantly greater log reduction for both bacteria than continuous 260 nm radiation. There was no statistical difference in SARS-CoV-2 inactivation between continuous and pulsed 222 nm UV-C radiation and pulsed 277 nm radiation demonstrated greater germicidal efficacy than continuous 277 nm radiation. Greater radiant exposure for all radiation sources was required to inactivate MS2 bacteriophage. Findings demonstrate that pulsed irradiation could be more useful than continuous UV radiation in human-occupied spaces, but threshold limit values should be respected. Pathogen-specific sensitivities, experimental setup, and quantification methods for determining germicidal efficacy remain important factors when optimizing ultraviolet radiation for surface decontamination or other applications.

The novel molecular mechanism of pulmonary fibrosis: insight into lipid metabolism from reanalysis of single-cell RNA-seq databases.

Pulmonary fibrosis (PF) is a severe pulmonary disease with limited available therapeutic choices. Recent evidence increasingly points to abnormal lipid metabolism as a critical factor in PF pathogenesis. Our latest research identifies the dysregulation of low-density lipoprotein (LDL) is a new risk factor for PF, contributing to alveolar epithelial and endothelial cell damage, and fibroblast activation. In this study, we first integrative summarize the published literature about lipid metabolite changes found in PF, including phospholipids, glycolipids, steroids, fatty acids, triglycerides, and lipoproteins. We then reanalyze two single-cell RNA-sequencing (scRNA-seq) datasets of PF, and the corresponding lipid metabolomic genes responsible for these lipids' biosynthesis, catabolism, transport, and modification processes are uncovered. Intriguingly, we found that macrophage is the most active cell type in lipid metabolism, with almost all lipid metabolic genes being altered in macrophages of PF. In type 2 alveolar epithelial cells, lipid metabolic differentially expressed genes (DEGs) are primarily associated with the cytidine diphosphate diacylglycerol pathway, cholesterol metabolism, and triglyceride synthesis. Endothelial cells are partly responsible for sphingomyelin, phosphatidylcholine, and phosphatidylethanolamines reprogramming as their metabolic genes are dysregulated in PF. Fibroblasts may contribute to abnormal cholesterol, phosphatidylcholine, and phosphatidylethanolamine metabolism in PF. Therefore, the reprogrammed lipid profiles in PF may be attributed to the aberrant expression of lipid metabolic genes in different cell types. Taken together, these insights underscore the potential of targeting lipid metabolism in developing innovative therapeutic strategies, potentially leading to extended overall survival in individuals affected by PF.

Diagnostic Accuracy of a Mobile AI-Based Symptom Checker and a Web-Based Self-Referral Tool in Rheumatology: Multicenter Randomized Controlled Trial.

BACKGROUND: The diagnosis of inflammatory rheumatic diseases (IRDs) is often delayed due to unspecific symptoms and a shortage of rheumatologists. Digital diagnostic decision support systems (DDSSs) have the potential to expedite diagnosis and help patients navigate the health care system more efficiently. OBJECTIVE: The aim of this study was to assess the diagnostic accuracy of a mobile artificial intelligence (AI)-based symptom checker (Ada) and a web-based self-referral tool (Rheport) regarding IRDs. METHODS: A prospective, multicenter, open-label, crossover randomized controlled trial was conducted with patients newly presenting to 3 rheumatology centers. Participants were randomly assigned to complete a symptom assessment using either Ada or Rheport. The primary outcome was the correct identification of IRDs by the DDSSs, defined as the presence of any IRD in the list of suggested diagnoses by Ada or achieving a prespecified threshold score with Rheport. The gold standard was the diagnosis made by rheumatologists. RESULTS: A total of 600 patients were included, among whom 214 (35.7%) were diagnosed with an IRD. Most frequent IRD was rheumatoid arthritis with 69 (11.5%) patients. Rheport's disease suggestion and Ada's top 1 (D1) and top 5 (D5) disease suggestions demonstrated overall diagnostic accuracies of 52%, 63%, and 58%, respectively, for IRDs. Rheport showed a sensitivity of 62% and a specificity of 47% for IRDs. Ada's D1 and D5 disease suggestions showed a sensitivity of 52% and 66%, respectively, and a specificity of 68% and 54%, respectively, concerning IRDs. Ada's diagnostic accuracy regarding individual diagnoses was heterogenous, and Ada performed considerably better in identifying rheumatoid arthritis in comparison to other diagnoses (D1: 42%; D5: 64%). The Cohen κ statistic of Rheport for agreement on any rheumatic disease diagnosis with Ada D1 was 0.15 (95% CI 0.08-0.18) and with Ada D5 was 0.08 (95% CI 0.00-0.16), indicating poor agreement for the presence of any rheumatic disease between the 2 DDSSs. CONCLUSIONS: To our knowledge, this is the largest comparative DDSS trial with actual use of DDSSs by patients. The diagnostic accuracies of both DDSSs for IRDs were not promising in this high-prevalence patient population. DDSSs may lead to a misuse of scarce health care resources. Our results underscore the need for stringent regulation and drastic improvements to ensure the safety and efficacy of DDSSs. TRIAL REGISTRATION: German Register of Clinical Trials DRKS00017642; https://drks.de/search/en/trial/DRKS00017642.

Targeting of canonical WNT signaling ameliorates experimental sclerodermatous chronic graft-versus-host disease.

Chronic graft-versus-host disease (cGVHD) is a major life-threatening complication of allogeneic hematopoietic stem cell transplantation. The molecular mechanisms underlying cGVHD remain poorly understood, and targeted therapies for clinical use are not well established. Here, we examined the role of the canonical WNT pathway in sclerodermatous cGVHD (sclGVHD). WNT signaling was activated in human sclGVHD with increased nuclear accumulation of the transcription factor ß-catenin and a WNT-biased gene expression signature in lesional skin. Treatment with the highly selective tankryase inhibitor G007-LK, the CK1α agonist pyrvinium, or the LRP6 inhibitor salinomycin abrogated the activation of WNT signaling and protected against experimental cGVHD, without a significant impact on graft-versus-leukemia effect (GVL). Treatment with G007-LK, pyrvinium, or salinomycin almost completely prevented the development of clinical and histological features in the B10.D2 (H-2d) → BALB/c (H-2d) and LP/J (H-2b) → C57BL/6 (H-2b) models of sclGVHD. Inhibition of canonical WNT signaling reduced the release of extracellular matrix from fibroblasts and reduced leukocyte influx, suggesting that WNT signaling stimulates fibrotic tissue remodeling by direct effects on fibroblasts and by indirect inflammation-dependent effects in sclGVHD. Our findings may have direct translational potential, because pyrvinium is in clinical use, and tankyrase inhibitors are in clinical trials for other indications.

Assessment of myocardial fibrosis in patients with systemic sclerosis using [68Ga]Ga-FAPI-04-PET-CT.

PURPOSE: Myocardial fibrosis (MF) is a factor of poor prognosis in systemic sclerosis (SSc). Direct in-vivo visualization of fibroblast activation as early readout of MF has not been feasible to date. Here, we characterize 68Gallium-labeled-Fibroblast-Activation-Inhibitor-04 ([68Ga]Ga-FAPI-04)-PET-CT as a diagnostic tool in SSc-related MF. METHODS: In this proof-of-concept trial, six SSc patients with and eight without MF of the EUSTAR cohort Erlangen underwent [68Ga]Ga-FAPI-04-PET-CT and cardiac MRI (cMRI) and clinical and serologic investigations just before baseline and during follow-up between January 2020 and December 2020. Myocardial biopsy was performed as clinically indicated. RESULTS: [68Ga]Ga-FAPI-04 tracer uptake was increased in SSc-related MF with higher uptake in SSc patients with arrhythmias, elevated serum-NT-pro-BNP, and increased late gadolinium enhancement (LGE) in cMRI. Histologically, myocardial biopsies from cMRI- and [68Ga]Ga-FAPI-04-positive regions confirmed the accumulation of FAP+ fibroblasts surrounded by collagen deposits. We observed similar but not equal spatial distributions of [68Ga]Ga-FAPI-04 uptake and quantitative cMRI-based techniques. Using sequential [68Ga]Ga-FAPI-04-PET-CTs, we observed dynamic changes of [68Ga]Ga-FAPI-04 uptake associated with changes in the activity of SSc-related MF, while cMRI parameters remained stable after regression of molecular activity and rather indicated tissue damage. CONCLUSIONS: We present first in-human evidence that [68Ga]Ga-FAPI-04 uptake visualizes fibroblast activation in SSc-related MF and may be a diagnostic option to monitor cardiac fibroblast activity in situ.

Targeted prostaglandin E2 inhibition enhances antiviral immunity through induction of type I interferon and apoptosis in macrophages.

Aspirin gained tremendous popularity during the 1918 Spanish Influenza virus pandemic, 50 years prior to the demonstration of their inhibitory action on prostaglandins. Here, we show that during influenza A virus (IAV) infection, prostaglandin E2 (PGE2) was upregulated, which led to the inhibition of type I interferon (IFN) production and apoptosis in macrophages, thereby causing an increase in virus replication. This inhibitory role of PGE2 was not limited to innate immunity, because both antigen presentation and T cell mediated immunity were also suppressed. Targeted PGE2 suppression via genetic ablation of microsomal prostaglandin E-synthase 1 (mPGES-1) or by the pharmacological inhibition of PGE2 receptors EP2 and EP4 substantially improved survival against lethal IAV infection whereas PGE2 administration reversed this phenotype. These data demonstrate that the mPGES-1-PGE2 pathway is targeted by IAV to evade host type I IFN-dependent antiviral immunity. We propose that specific inhibition of PGE2 signaling might serve as a treatment for IAV.

A population-based projection of psoriatic arthritis in Germany until 2050: analysis of national statutory health insurance data of 65 million German population.

The population-based prevalence of psoriatic arthritis (PsA) is still unclear and not well described globally. The aim of this study was to conduct a population-based prevalence projection and provide long-term future estimations of PsA patients in Germany until 2050, using the illness-death model and based on historical data. We analyzed the national statutory health insurance data of 65 million population in the German Institute for Medical Documentation and Information between January 2009 and December 2012. We constructed an estimation of the PsA burden among the German population using the relevant epidemiological parameters to project the numbers of patients with PsA in Germany until 2050 under five possible scenarios by varying the incidence and mortality. The overall conservatively estimated prevalence of PsA in Germany in 2019 was 0.31% (95% CI 0.28-0.36%). Women contribute a higher prevalence than men in all five scenarios. In the assumed scenarios with increased incidence, the prevalence of PsA at 60 years of age could rise from 1% in 2019 to more than 3% in 2050 for both genders, with the increase particularly pronounced for women, reaching around 3.5%. However, in the assumed scenarios with decreasing incidence, the prevalence curve may flatten and begin a decreasing trend from 2035 to 2050 for both genders, achieving a prevalence of less than 1% in 2050. Our research is to generate assumed population-based data on PsA in Germany that can serve as a reference for public health stakeholders to prepare an optional intervention. We would expect worryingly high numbers in the coming decades if preventive strategies are not implemented. In the long term, it will be necessary to implement preventive strategies to identify predictors and treat psoriasis symptoms early in order to delay or even prevent the transition of psoriasis to PsA.

Diagnostic delay stages and pre-diagnostic treatment in patients with suspected rheumatic diseases before special care consultation: results of a multicenter-based study.

Early and effective discrimination (triage) of patients with inflammatory rheumatic diseases (IRD) and other diseases (non-IRD) is essential for successful treatment and preventing damage. The aim of this study was to investigate diagnostic delays and pre-diagnosis treatment in patients newly presenting to rheumatology outpatient clinics. A total of 600 patients newly presenting to one university hospital and two non-academic centers were included. Time from onset of symptoms to rheumatology consultation "total delay" as well as medical treatment before consultation were recorded. Median time from symptom onset to rheumatologist appointment (total delay) was 30 weeks. Median time to online search, first physician appointment request and first physician appointment was 2, 4 and 5 weeks, respectively. Total delay was significantly shorter for IRD patients compared to non-IRD patients, 26 vs 35 weeks (p = 0.007). Only 17.7% of all patients and 22.9% of IRD patients had a delay of less than 12 weeks. Total delay was significantly lower in patients seen in non-academic centers compared to the university center, 20 vs 50 weeks (p < 0.0001). 32.2% of IRD patients received medical treatment that eased their symptoms prior to the rheumatology appointment. These findings highlight the persistent diagnostic delays in rheumatology; however, they also suggest that current triage strategies effectively lead to earlier appointments for IRD patients. Improvement of triage methods and pre-diagnosis treatment could decrease overall burden of disease in IRD patients.

The Thermodynamic Stability of Membrane Proteins in Micelles and Lipid Bilayers Investigated with the Ferrichrom Receptor FhuA.

Extraction of integral membrane proteins into detergents for structural and functional studies often leads to a strong loss in protein stability. The impact of the lipid bilayer on the thermodynamic stability of an integral membrane protein in comparison to its solubilized form in detergent was examined and compared for FhuA from Escherichia coli and for a mutant, FhuAΔ5-160, lacking the N-terminal cork domain. Urea-induced unfolding was monitored by fluorescence spectroscopy to determine the effective free energies [Formula: see text] of unfolding. To obtain enthalpic and entropic contributions of unfolding of FhuA, [Formula: see text] were determined at various temperatures. When solubilized in LDAO detergent, wt-FhuA and FhuAΔ5-160 unfolded in a single step. The 155-residue cork domain stabilized wt-FhuA by [Formula: see text]~ 40 kJ/mol. Reconstituted into lipid bilayers, wt-FhuA unfolded in two steps, while FhuAΔ5-160 unfolded in a single step, indicating an uncoupled unfolding of the cork domain. For FhuAΔ5-160 at 35 °C, [Formula: see text] increased from ~ 5 kJ/mol in LDAO micelles to about ~ 20 kJ/mol in lipid bilayers, while the temperature of unfolding increased from TM ~ 49 °C in LDAO micelles to TM ~ 75 °C in lipid bilayers. Enthalpies [Formula: see text]were much larger than free energies [Formula: see text], for FhuAΔ5-160 and for wt-FhuA, and compensated by a large gain of entropy upon unfolding. The gain in conformational entropy is expected to be similar for unfolding of FhuA from micelles or bilayers. The strongly increased TM and [Formula: see text] observed for the lipid bilayer-reconstituted FhuA in comparison to the LDAO-solubilized forms, therefore, very likely arise from a much-increased solvation entropy of FhuA in bilayers.