Serum IL-17 and TNFα as prognostic biomarkers in systemic sclerosis patients: a prospective study.

Recent reports have demonstrated that endothelial injury is critical in the pathogenesis of systemic sclerosis (SSc) and is associated with increased levels of circulating inflammatory biomarkers. This study aims to analyze the serum concentrations of selected cytokines and evaluate their relationship with SSc clinics and the long-term course of the disease. This study included 43 SSc patients and 24 matched healthy controls. In both groups, we measured serum levels of inflammatory cytokines related to the inflammatory response, such as tumor necrosis factor (TNF)α, interferon (IFN)γ, interleukin (IL)-4, IL-6, IL-10, and IL-17, and fibroblast activation protein (FAP). Additionally, in SSc patients, we evaluated the presence of four single nucleotide polymorphisms (SNPs) located in the promotor region of the TNFA gene, namely rs361525, rs1800629, rs1799964, and rs1799724, which might be related to increased TNFα concentrations. The main aim consisted of associating inflammatory cytokines with (1) clinical disease characteristics and (2) longitudinal observation of survival and cancer prevalence. SSc patients were characterized by a 17% increase in serum TNFα. There was no other difference in serum cytokines between the studied groups and diffuse vs. limited SSc patients. As expected, evaluated serum cytokines correlated with inflammatory biomarkers (e.g., IL-6 and C-reactive protein). Interestingly, patients with higher IL-17 had decreased left ventricle ejection fraction. During the median 5-year follow-up, we recorded four cases of neoplastic diseases (lung cancer in two cases, squamous cell carcinoma of unknown origin, and breast cancer with concomitant multiple myeloma) and nine deaths. The causes of death included lung cancer (n = 2), renal crisis (n = 1), multiple-organ failure (n = 1), and unknown reasons in five cases. Surprisingly, higher TNFα was associated with an increased cancer prevalence, while elevated IL-17 with death risk in the follow-up. Furthermore, the AG rs361525 genotype referred to higher TNFα levels than GG carriers. Both AG rs361525 and CT rs1799964 genotypes were associated with increased cancer risk. Higher serum concentrations of TNFα characterize the SSc patients, with the highest values associated with cancer. On the other hand, increased IL-17 in peripheral blood might predict poor SSc prognosis. Further research is needed to validate these findings.

Low levels of endogenous anabolic androgenic steroids in females with severe asthma taking corticosteroids.

Rationale: Patients with severe asthma are dependent upon treatment with high doses of inhaled corticosteroids (ICS) and often also oral corticosteroids (OCS). The extent of endogenous androgenic anabolic steroid (EAAS) suppression in asthma has not previously been described in detail. The objective of the present study was to measure urinary concentrations of EAAS in relation to exogenous corticosteroid exposure. Methods: Urine collected at baseline in the U-BIOPRED (Unbiased Biomarkers for the Prediction of Respiratory Disease outcomes) study of severe adult asthmatics (SA, n=408) was analysed by quantitative mass spectrometry. Data were compared to that of mild-to-moderate asthmatics (MMA, n=70) and healthy subjects (HC, n=98) from the same study. Measurements and main results: The concentrations of urinary endogenous steroid metabolites were substantially lower in SA than in MMA or HC. These differences were more pronounced in SA patients with detectable urinary OCS metabolites. Their dehydroepiandrosterone sulfate (DHEA-S) concentrations were <5% of those in HC, and cortisol concentrations were below the detection limit in 75% of females and 82% of males. The concentrations of EAAS in OCS-positive patients, as well as patients on high-dose ICS only, were more suppressed in females than males (p<0.05). Low levels of DHEA were associated with features of more severe disease and were more prevalent in females (p<0.05). The association between low EAAS and corticosteroid treatment was replicated in 289 of the SA patients at follow-up after 12-18 months. Conclusion: The pronounced suppression of endogenous anabolic androgens in females might contribute to sex differences regarding the prevalence of severe asthma.

Activity of granulomatosis with polyangiitis and its correlation with mTOR phosphoproteomics in neutrophils.

Introduction: Granulomatosis with polyangiitis (GPA) is a small vessel vasculitis with a complex pathomechanism. Organ damage in GPA is also mediated by extracellular trap formation (NETosis). We analyzed the functional status of phosphoproteins modulating NETosis in neutrophils by the mammalian target of rapamycin (mTOR) pathway in GPA along with NETosis biomarkers. Methods: Phosphoproteins levels measured in isolated neutrophils from 42 patients with GPA (exacerbation n=21; remission n=21) and 21 healthy controls were compared to serum biomarkers of the disease. Results: Neutrophils in active disease manifested lowered levels of phosphorylated mTOR(Ser2448), PTEN(Ser380) and ULK1(Ser555), whereas phosphorylated GSK-3α/ß(Ser21/Ser9) was elevated. Exacerbation of GPA was characterized by elevated neutrophil dsDNA in serum, circulating mitochondrial DNA, and DNA-MPO complexes. A significant negative correlation between mTOR or PTEN phosphoproteins and biomarkers of GPA activity was also present, reflecting the clinical activity score of GPA. Positive correlations between phosphorylated GSK-3 α/ß and circulating mtDNA, DNA-MPO complexes, neutrophil-released dsDNA, or circulating proteins were also significant. Increased serum levels of IGFBP-2, TFF-3, CD147, and CHI3L1 accompanied GPA exacerbation, whereas DPP-IV levels were the lowest in active GPA. Using a principal component analysis basigin, PTEN and mTOR had the highest loadings on the discrimination function, allowing classification between active, remission, and control subjects with 98% performance. Conclusions: We present evidence that inhibited mTOR signaling accompanies NETosis in patients with GPA. The functional status of phosphoproteins suggests simultaneous activation of NETosis and autophagy. These results give rise to the study of autophagy as a mechanism underlying granuloma formation in GPA.

Association between clinical features and course of systemic sclerosis and serum interleukin-8, vascular endothelial growth factor, basic fibroblast growth factor, and interferon alpha.

BACKGROUND: Certain mediators, such as soluble growth factors and cytokines, among others, are implicated in the immunopathogenesis of systemic sclerosis (SSc). OBJECTIVES: This study aimed to examine the association between serum levels of vascular endothelial growth factor (VEGF), interleukin-8 (IL-8), interferon alpha (IFN-α), and basic fibroblast growth factor (bFGF) and the clinical presentation and course of SSc. MATERIAL AND METHODS: This longitudinal, observational study included 43 patients with SSc and 24 healthy subjects. Serum concentrations of VEGF, IL-8, IFN-α, and bFGF were measured at baseline in patients previously treated for SSc. Medical history of patients was analyzed retrospectively at the time of cytokine measurement to infer clinical correlations, and during follow-up for a median of 5 years, assessing the incidence of death or cancer. RESULTS: The bFGF and IFN-α concentrations differed between SSc patients and controls (p < 0.01). In turn, organ involvement and SSc phenotypes did not impact studied cytokine concentrations, similar to systemic steroid and/or immunosuppressant use at enrollment. However, we have documented a positive correlation between the current oral steroid dose and serum levels of IL-8 and bFGF. Furthermore, patients with a VEGF level ≥95.7 pg/mL and IFN-α level ≥3.6 pg/mL required cyclophosphamide therapy more often, currently or in the past (approx. 3-fold and 4-fold, respectively). Substantially elevated VEGF and IFN-α concentrations at baseline were associated with higher cancer occurrence (n = 4) during follow-up, while elevated circulating IL-8 level was associated with an increased risk of death (n = 9). CONCLUSIONS: The SSc group was characterized by higher serum concentrations of bFGF and IFN-α compared to healthy controls. Patients treated with cyclophosphamide or receiving higher systemic steroid doses, thus suffering from a more severe disease type, had increased cytokine levels. Elevated circulating IFN-α and VEGF levels might be correlated with cancer, whereas raised IL-8 levels may be associated with an increased risk of death. However, further research is needed to verify our findings.

Comprehensive Analysis of Circular RNAs in Endothelial Cells.

Non-coding RNAs constitute a heterogeneous group of molecules that lack the ability to encode proteins but retain the potential ability to influence cellular processes through a regulatory mechanism. Of these proteins, microRNAs, long non-coding RNAs, and more recently, circular RNAs have been the most extensively described. However, it is not entirely clear how these molecules interact with each other. For circular RNAs, the basics of their biogenesis and properties are also lacking. Therefore, in this study we performed a comprehensive analysis of circular RNAs in relation to endothelial cells. We identified the pool of circular RNAs present in the endothelium and showed their spectrum and expression across the genome. Using different computational strategies, we proposed approaches to search for potentially functional molecules. In addition, using data from an in vitro model that mimics conditions in the endothelium of an aortic aneurysm, we demonstrated altered expression levels of circRNAs mediated by microRNAs.

The role of inflammation in anxiety and depression in the European U-BIOPRED asthma cohorts.

BACKGROUND: Growing evidence indicates high comorbid anxiety and depression in patients with asthma. However, the mechanisms underlying this comorbid condition remain unclear. The aim of this study was to investigate the role of inflammation in comorbid anxiety and depression in three asthma patient cohorts of the Unbiased Biomarkers for the Prediction of Respiratory Disease Outcomes (U-BIOPRED) project. METHODS: U-BIOPRED was conducted by a European Union consortium of 16 academic institutions in 11 European countries. A subset dataset from subjects with valid anxiety and depression measures and a large blood biomarker dataset were analysed, including 198 non-smoking patients with severe asthma (SAn), 65 smoking patients with severe asthma (SAs), 61 non-smoking patients with mild-to-moderate asthma (MMA), and 20 healthy non-smokers (HC). The Hospital Anxiety and Depression Scale was used to measure anxiety and depression and a series of inflammatory markers were analysed by the SomaScan v3 platform (SomaLogic, Boulder, Colo). ANOVA and the Kruskal-Wallis test were used for multiple-group comparisons as appropriate. RESULTS: There were significant group effects on anxiety and depression among the four cohort groups (p < 0.05). Anxiety and depression of SAn and SAs groups were significantly higher than that of MMA and HC groups (p < 0.05. There were significant differences in serum IL6, MCP1, CCL18, CCL17, IL8, and Eotaxin among the four groups (p < 0.05). Depression was significantly associated with IL6, MCP1, CCL18 level, and CCL17; whereas anxiety was associated with CCL17 only (p < 0.05). CONCLUSIONS: The current study suggests that severe asthma patients are associated with higher levels of anxiety and depression, and inflammatory responses may underlie this comorbid condition.

Effectiveness and Safety of SARS-CoV-2 Vaccination in HIV-Infected Patients-Real-World Study.

The development of COVID-19 vaccines has been a triumph of biomedical research. However, there are still challenges, including assessment of their immunogenicity in high-risk populations, including PLWH. In the present study, we enrolled 121 PLWH aged >18 years, that were vaccinated against COVID-19 in the Polish National Vaccination Program. Patients filled in questionnaires regarding the side effects of vaccination. Epidemiological, clinical, and laboratory data were collected. The efficacy of COVID-19 vaccines was evaluated with an ELISA that detects IgG antibodies using a recombinant S1 viral protein antigen. The interferon-gamma release assay (IGRA) was applied to quantitate interferon-gamma (IFN-γ) to assess cellular immunity to SARS-CoV-2. In total, 87 patients (71.9%) received mRNA vaccines (BNT162b2-76 (59.5%), mRNA-1273- 11 (9.1%)). A total of 34 patients (28.09%) were vaccinated with vector-based vaccines (ChAdOx Vaxzevria- 20 (16.52%), Ad26.COV2.S- 14 (11.6%)). A total of 95 (78.5%) of all vaccinated patients developed a protective level of IgG antibodies. Only eight PLWH (6.6%) did not develop cellular immune response. There were six patients (4.95%) that did not develop a cellular and humoral response. Analysis of variance proved that the best humoral and cellular response related to the administration of the mRNA-1273 vaccine. COVID-19 vaccines were found to be immunogenic and safe in PLWH. Vaccination with mRNA vaccines were related to better humoral and cellular responses.

Rhinovirus-induced epithelial RIG-I inflammasome suppresses antiviral immunity and promotes inflammation in asthma and COVID-19.

Rhinoviruses and allergens, such as house dust mite are major agents responsible for asthma exacerbations. The influence of pre-existing airway inflammation on the infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is largely unknown. We analyse mechanisms of response to viral infection in experimental in vivo rhinovirus infection in healthy controls and patients with asthma, and in in vitro experiments with house dust mite, rhinovirus and SARS-CoV-2 in human primary airway epithelium. Here, we show that rhinovirus infection in patients with asthma leads to an excessive RIG-I inflammasome activation, which diminishes its accessibility for type I/III interferon responses, leading to their early functional impairment, delayed resolution, prolonged viral clearance and unresolved inflammation in vitro and in vivo. Pre-exposure to house dust mite augments this phenomenon by inflammasome priming and auxiliary inhibition of early type I/III interferon responses. Prior infection with rhinovirus followed by SARS-CoV-2 infection augments RIG-I inflammasome activation and epithelial inflammation. Timely inhibition of the epithelial RIG-I inflammasome may lead to more efficient viral clearance and lower the burden of rhinovirus and SARS-CoV-2 infections.

Non-eosinophilic asthma in nonsteroidal anti-inflammatory drug exacerbated respiratory disease.

BACKGROUND: The cellular inflammatory pattern of nonsteroidal anti-inflammatory drug-exacerbated respiratory disease (N-ERD) is heterogeneous. However, data on the heterogeneity of non-eosinophilic asthma (NEA) with aspirin hypersensitivity are scanty. By examination of N-ERD patients based on clinical data and eicosanoid biomarkers we aimed to identify NEA endotypes potentially guiding clinical management. METHODS: Induced sputum was collected from patients with N-ERD. Sixty six patients (49.6% of 133 N-ERD) with NEA were included in the hierarchical cluster analysis based on clinical and laboratory data. The quality of clustering was evaluated using internal cluster validation with different indices and a practical decision tree was proposed to simplify stratification of patients. RESULTS: The most frequent NEA pattern was paucigranulocytic (PGA; 75.8%), remaining was neutrophilic asthma (NA; 24.2%). Four clusters were identified. Cluster #3 included the highest number of NEA patients (37.9%) with severe asthma and PGA pattern (96.0%). Cluster #1 (24.2%) included severe only asthma, with a higher prevalence of NA (50%). Cluster #2 (25.8%) comprised well-controlled mild or severe asthma (PGA; 76.5%). Cluster #4 contained only 12.1% patients with well-controlled moderate asthma (PGA; 62.5%). Sputum prostaglandin D2 levels distinguished cluster #1 from the remaining clusters with an area under the curve of 0.94. CONCLUSIONS: Among identified four NEA subtypes, clusters #3 and #1 represented N-ERD patients with severe asthma but a different inflammatory signatures. All the clusters were discriminated by sputum PGD2 levels, asthma severity, and age of patients. The heterogeneity of non-eosinophilic N-ERD suggests a need for novel targeted interventions.

Stratification of asthma by lipidomic profiling of induced sputum supernatant.

BACKGROUND: Asthma is a chronic respiratory disease with significant heterogeneity in its clinical presentation and pathobiology. There is need for improved understanding of respiratory lipid metabolism in asthma patients and its relation to observable clinical features. OBJECTIVE: We performed a comprehensive, prospective, cross-sectional analysis of the lipid composition of induced sputum supernatant obtained from asthma patients with a range of disease severities, as well as from healthy controls. METHODS: Induced sputum supernatant was collected from 211 adults with asthma and 41 healthy individuals enrolled onto the U-BIOPRED (Unbiased Biomarkers for the Prediction of Respiratory Disease Outcomes) study. Sputum lipidomes were characterized by semiquantitative shotgun mass spectrometry and clustered using topologic data analysis to identify lipid phenotypes. RESULTS: Shotgun lipidomics of induced sputum supernatant revealed a spectrum of 9 molecular phenotypes, highlighting not just significant differences between the sputum lipidomes of asthma patients and healthy controls, but also within the asthma patient population. Matching clinical, pathobiologic, proteomic, and transcriptomic data helped inform the underlying disease processes. Sputum lipid phenotypes with higher levels of nonendogenous, cell-derived lipids were associated with significantly worse asthma severity, worse lung function, and elevated granulocyte counts. CONCLUSION: We propose a novel mechanism of increased lipid loading in the epithelial lining fluid of asthma patients resulting from the secretion of extracellular vesicles by granulocytic inflammatory cells, which could reduce the ability of pulmonary surfactant to lower surface tension in asthmatic small airways, as well as compromise its role as an immune regulator.

Decreased protein C activity, lower ADAMTS13 antigen and free protein S levels accompanied by unchanged thrombin generation potential in hospitalized COVID-19 patients.

INTRODUCTION: COVID-19 is associated with an increased thromboembolic risk. However, the mechanisms triggering clot formation in those patients remain unknown. PATIENTS AND METHODS: In 118 adult Caucasian severe but non-critically ill COVID-19 patients (median age 58 years; 73 % men) and 46 controls, we analyzed in vitro plasma thrombin generation profile (calibrated automated thrombogram [CAT assay]) and investigated thrombophilia-related factors, such as protein C and antithrombin activity, free protein S level, presence of antiphospholipid antibodies and factor V Leiden R506Q and prothrombin G20210A mutations. We also measured circulating von Willebrand factor (vWF) antigen and a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) antigen and activity. In patients, blood samples were collected on admission to the hospital before starting any therapy, including heparin. Finally, we examined the relationship between observed alterations and disease follow-up, such as thromboembolic complications. RESULTS: COVID-19 patients showed 17 % lower protein C activity, 22 % decreased free protein S levels, and a higher prevalence of positive results for IgM anticardiolipin antibodies. They also had 151 % increased vWF, and 27 % decreased ADAMTS13 antigens compared with controls (p < 0.001, all). On the contrary, thrombin generation potential was similar to controls. In the follow-up, pulmonary embolism (PE) occurred in thirteen (11 %) patients. They were characterized by a 55 % elevated D-dimer (p = 0.04) and 2.7-fold higher troponin I (p = 0.002) during hospitalization and 29 % shorter time to thrombin peak in CAT assay (p = 0.009) compared to patients without PE. CONCLUSIONS: In COVID-19, we documented prothrombotic abnormalities of peripheral blood. PE was characterized by more dynamic thrombin generation growth in CAT assay performed on admittance to the hospital.

Monocyte exposure to fine particulate matter results in miRNA release: A link between air pollution and potential clinical complication.

Chronic exposure to PM2.5 contributes to the pathogenesis of numerous disorders, although the underlying mechanisms remain unknown. The study investigated whether exposure of human monocytes to PM2.5 is associated with alterations in miRNAs. Monocytes were exposed in vitro to PM2.5 collected during winter and summer, followed by miRNA isolation from monocytes. Additionally, in 140 persons chronically exposed to air pollution, some miRNA patterns were isolated from serum seasonally. Between-season differences in chemical PM2.5 composition were observed. Some miRNAs were expressed both in monocytes and in human serum. MiR-34c-5p and miR-223-5p expression was more pronounced in winter. Bioinformatics analyses showed that selected miRNAs were involved in the regulation of several pathways. The expression of the same miRNA species in monocytes and serum suggests that these cells are involved in the production of miRNAs implicated in the development of disorders mediated by inflammation, oxidative stress, proliferation, and apoptosis after exposure to PM2.5.

Chemerin as a Potential Marker of Resolution of Inflammation in COVID-19 Infection.

Chemerin is one of the specialized pro-resolving mediators that participate in the early phase of inflammation and contribute to the initiation of the pro-resolving response. There is a paucity of data regarding the time course of chemerin during acute infections. We aimed to evaluate the sequence of inflammatory responses in the acute COVID-19 phase throughout onset and resolution of inflammation. We evaluated changes in selected biomarkers in COVID-19 survivors on the 7-day and 28-day follow up. Chemerin was lower in patients with baseline moderate/severe disease at day 7 compared with asymptomatic patients and individuals with mild illness (7265 [5526−9448] vs. 8730 [6888−11,058] pg/mL; p = 0.03). Only in patients with moderate/severe disease, but not in those with mild symptoms, were chemerin concentrations decreased one week after infection onset compared with baseline (7265 [5526−9448] vs. 8866 [6383−10,690] pg/mL; p < 0.05) with a subsequent increase on the 28-day follow up (9313 [7353−11,033] pg/mL; p < 0.05). Resolution of inflammation in the group of moderate/severe SARS-CoV2 infection was associated with increasing serum concentrations of chemerin, contrary to pro-inflammatory cytokines and adipokines (pentraxin 3, TNFα, resistin, leptin). A similar pattern of angiopoietin-2 dynamics may suggest signs of enhanced vascularization as a consequence of acute SARS-CoV2 infection.

Machine learning in the diagnosis of asthma phenotypes during coronavirus disease 2019 pandemic.

Background: During the coronavirus disease 2019 (COVID-19) pandemic, it has become a pressing need to be able to diagnose aspirin hypersensitivity in patients with asthma without the need to use oral aspirin challenge (OAC) testing. OAC is time consuming and is associated with the risk of severe hypersensitive reactions. In this study, we sought to investigate whether machine learning (ML) based on some clinical and laboratory procedures performed during the pandemic might be used for discriminating between patients with aspirin hypersensitivity and those with aspirin-tolerant asthma. Methods: We used a prospective database of 135 patients with non-steroidal anti-inflammatory drug (NSAID)-exacerbated respiratory disease (NERD) and 81 NSAID-tolerant (NTA) patients with asthma who underwent OAC. Clinical characteristics, inflammatory phenotypes based on sputum cells, as well as eicosanoid levels in induced sputum supernatant and urine were extracted for the purpose of applying ML techniques. Results: The overall best ML model, neural network (NN), trained on a set of best features, achieved a sensitivity of 95% and a specificity of 76% for diagnosing NERD. The 3 promising models (i.e., multiple logistic regression, support vector machine, and NN) trained on a set of easy-to-obtain features including only clinical characteristics and laboratory data achieved a sensitivity of 97% and a specificity of 67%. Conclusions: ML techniques are becoming a promising tool for discriminating between patients with NERD and NTA. The models are easy to use, safe, and achieve very good results, which is particularly important during the COVID-19 pandemic.

The interplay between the airway epithelium and tissue macrophages during the SARS-CoV-2 infection.

The first line of antiviral immune response in the lungs is secured by the innate immunity. Several cell types take part in this process, but airway macrophages (AMs) are among the most relevant ones. The AMs can phagocyte infected cells and activate the immune response through antigen presentation and cytokine release. However, the precise role of macrophages in the course of SARS-CoV-2 infection is still largely unknown. In this study, we aimed to evaluate the role of AMs during the SARS-CoV-2 infection using a co-culture of fully differentiated primary human airway epithelium (HAE) and human monocyte-derived macrophages (hMDMs). Our results confirmed abortive SARS-CoV-2 infection in hMDMs, and their inability to transfer the virus to epithelial cells. However, we demonstrated a striking delay in viral replication in the HAEs when hMDMs were added apically after the epithelial infection, but not when added before the inoculation or on the basolateral side of the culture. Moreover, SARS-CoV-2 inhibition by hMDMs seems to be driven by cell-to-cell contact and not by cytokine production. Together, our results show, for the first time, that the recruitment of macrophages may play an important role during the SARS-CoV-2 infection, limiting the virus replication and its spread.

Dynamic in vitro hemocompatibility of oligoproline self-assembled monolayer surfaces.

The blood compatibility of self-assembled monolayers (SAMs) of oligoproline, a nonionic antifouling peptide, was investigated using the cone-and-plate assay imitating arterial blood flow conditions. End-capped oligoprolines composed of 6 and 9 proline residues (Pro6 and Pro9) and a Cys residue were synthesized for preparing SAMs (Pro-SAMs) on Au-sputtered glass. The surface of Pro-SAMs indicated hydrophilic property with a smooth topology. The adsorption of blood components and the adhesion of blood cells, including leukocytes and platelets, were strongly suppressed on Pro-SAMs. Moreover, Pro9-SAM did not trigger the activation of platelets (i.e., the conformational change of GPIIb/IIIa and P-selectin (CD62P) expression on platelets and the formation of aggregates). Our results demonstrate that Pro9-SAM completely inhibited acute thrombogenic responses and the activation of platelets under dynamic conditions.