The coronavirus disease 2019 (COVID-19) is related to enhanced production of NETs, and autoimmune/autoinflammatory phenomena. We evaluated the proportion of low-density granulocytes (LDG) by flow cytometry, and their capacity to produce NETs was compared with that of conventional neutrophils. NETs and their protein cargo were quantified by confocal microscopy and ELISA. Antinuclear antibodies (ANA), anti-neutrophil cytoplasmic antibodies (ANCA) and the degradation capacity of NETs were addressed in serum. MILLIPLEX assay was used to assess the cytokine levels in macrophages’ supernatant and serum. We found a higher proportion of LDG in severe and critical COVID-19 which correlated with severity and inflammatory markers. Severe/critical COVID-19 patients had higher plasmatic NE, LL-37 and HMGB1-DNA complexes, whilst ISG-15-DNA complexes were lower in severe patients. Sera from severe/critical COVID-19 patients had lower degradation capacity of NETs, which was reverted after adding hrDNase. Anti-NET antibodies were found in COVID-19, which correlated with ANA and ANCA positivity. NET stimuli enhanced the secretion of cytokines in macrophages. This study unveils the role of COVID-19 NETs as inducers of pro-inflammatory and autoimmune responses. The deficient degradation capacity of NETs may contribute to the accumulation of these structures and anti-NET antibodies are related to the presence of autoantibodies.
Background Lactobacillus is a genus of Gram-positive non-spore-forming rods usually found in the microbiota of the oral cavity, gastrointestinal tract, and female genitourinary tract. Also, they are commonly used in the food industry as supplements and probiotics. Lactobacilli are normally considered non-pathogenic to the human body, however, under certain circumstances such as immunosuppression, they can cause severe infections, with only a few cases of bacteremia, infective endocarditis, pneumonia, meningitis, and intra-abdominal infections reported. Among these presentations, a pyogenic liver abscess is rather rare. Case presentation We describe the case of a 59-year-old man with a history of diabetes mellitus and multiple abdominal surgeries with the latest being in 2014 presenting with bacteremia and multiple large pyogenic liver abscesses due to Lactobacillus gasseri, which did not appear to be related to the use of probiotics or immunosuppression. Conclusions Given the high prevalence of diabetes mellitus and the increased use of probiotics, it is expected that in the future we will see an increase in infections caused by Lactobacilli. Medical management with antibiotics and percutaneous drainage were successful strategies for the treatment of this unusual case of pyogenic liver abscesses and bacteremia caused by Lactobacillus gasseri.
Background/Objective: Biomarkers for disease activity and damage accrual in idiopathic inflammatory myopathies (IIMs) are currently lacking. The purpose of this cross-sectional study is to analyze the relationship among low-density granulocytes (LDGs), neutrophil extracellular traps (NETs), and clinical and immunological features of patients with IIM. Methods:We assessed disease activity, damage accrual, amount of LDGs, NETs, expression of LL-37, and serum cytokines in 65 adult patients with IIM. Differences between groups and correlations were assessed by Kruskal-Wallis, Mann-Whitney U, and Spearman ρ tests. The association between LDGs, NETs, disease activity, calcinosis, and cutaneous ulcers was assessed by logistic regression. To address the capacity of LDGs and NETs to diagnose disease activity, we used receiving operating characteristic curves.Results: Low-density granulocytes were higher in patients with active disease, ulcers, calcinosis, and anti-MDA5 antibodies, which correlated with serum levels of IL-17A and IL-18. Neutrophil extracellular traps were higher in patients with calcinosis, elevated titers of antinuclear antibodies, and positive anti-PM/Scl75 tests. The combination of a high proportion of both total LDGs and NETs was associated with the presence of calcinosis and cutaneous ulcers. LL-37 was higher in NETs originating from LDGs. Normal-density neutrophils were elevated in patients with active dermatomyositis.Conclusions: Low-density granulocytes and NETs containing LL-37 are increased in patients with IIM and active disease, and correlate with proinflammatory cytokines. Both total and CD10 + LDGs are potential biomarkers for disease activity and, in combination with NETs, have the potential to detect patients who are at risk for cutaneous ulcers and calcinosis.
Background: Monocytes and toll-like receptors (TLR) have been found in the inflammatory infiltrate of muscle biopsies in patients with idiopathic inflammatory myopathies (IIM), suggesting an important role of these cells in the pathogenesis of myositis. The monocyte subsets, their TLR expression in peripheral blood and their relationship with the clinical characteristics of patients with IIM has not been addressed. Methods:We recruited 45 patients with IIM diagnosis and 15 age and sex-adjusted healthy controls. We assessed the disease activity and damage, performed a nailfold capillaroscopy and registered the cardio-pulmonary parameters from the medical charts. Monocyte subsets, their expression of TLR2 and TLR4 and the serum Th1/Th2/Th17 cytokines levels were evaluated by flow cytometry. We expressed quantitative variables as medians and interquartile ranges (IQR) or minimum and maximum (min-max). Differences between groups were assessed with Mann-Whitney U and the Kruskal-Wallis tests. Correlation between quantitative variables was assessed with Spearman Rho.Results: Twenty-nine patients were women (64.4%) and 32 (71.1%) had dermatomyositis. In comparison to healthy controls, patients with active IIM had a higher percentage of intermediate monocytes and lower amounts of classical monocytes. Patients with IIM had a higher expression of TLR4 in all their monocyte subsets, regardless of disease activity and prednisone treatment. Serum IL-6 correlated with the TLR2 expression in every monocyte subset and the expression of TLR2 in intermediate monocytes was higher among patients with dysphagia. Subjects with nailfold capillaroscopy abnormalities had a higher amount of TLR2+ classical and non-classical monocytes and those with interstitial lung disease (ILD) had a higher percentage of TLR4+ non-classical monocytes. The classical and intermediate monocytes from patients with anti Mi2 antibodies had a higher expression of TLR4. The percentage of intermediate monocytes and the expression of TLR4 in all monocyte subsets showed a good diagnostic capacity in patients with IIM. Conclusion:Patients with IIM have a differential pool of monocyte subsets with an enhanced expression of TLR2 and TLR4, which correlates with disease activity and distinctive clinical features including dysphagia, ILD, vasculopathy, and pro-inflammatory cytokines. These immunological features might be useful as a potential diagnostic tool as well as novel disease activity biomarkers in IIM.
Background Neutrophil extracellular traps (NETs) from patients with systemic lupus erythematosus (SLE) are characterized by lower ubiquitylation and myeloperoxidase (MPO) as a substrate. The structural and functional effect of such modification and if there are additional post-translational modifications (PTMs) are unknown. Methods To assess the expression and functional role of PTMs in NETs of patients with SLE; reactivation, proliferation and cytokine production was evaluated by flow cytometry using co-cultures with dendritic cells (DC) and CD4+ from SLE patients and healthy controls. The impact of ubiquitylation on MPO was assessed by molecular dynamics. The expression of ISG15 in NETs was evaluated by immunofluorescence and Western Blot. Results Fifteen patients with SLE and ten healthy controls were included. In the co-cultures of CD4+ lymphocytes with DC stimulated with ubiquitylated MPO or recombinant MPO, a higher expression of IFNγ and IL-17A was found in CD4+ from SLE patients (p < 0.05). Furthermore, with DC stimulated with ubiquitylated MPO a trend towards increased expression of CD25 and Ki67 was found in lupus CD4+ lymphocytes, while the opposite was documented in controls (p < 0.05). Through molecular dynamics we found the K129-K488-K505 residues of MPO as susceptible to ubiquitylation. Ubiquitylation affects the hydration status of the HEME group depending on the residue to which it is conjugated. R239 was found near by the HEME group when the ubiquitin was in K488-K505. In addition, we found greater expression of ISG15 in the SLE NETs vs controls (p < 0.05), colocalization with H2B (r = 0.81) only in SLE samples and increased production of IFNγ in PBMCs stimulated with lupus NETs compared to healthy controls NETs. Conclusion The ubiquitylated MPO has a differential effect on the induction of reactivation of CD4+ lymphocytes in patients with SLE, which may be related to structural changes by ubiquitylation at the catalytic site of MPO. Besides a lower ubiquitylation pattern, NETs of patients with SLE are characterized by the expression of ISG15, and the induction of IFNγ by Th1 cells.
Background: Most COVID-19 mortality scores were developed in the early months of the pandemic and now available evidence-based interventions have helped reduce its lethality. It has not been evaluated if the original predictive performance of these scores holds true nor compared it against Clinical Gestalt predictions. We tested the current predictive accuracy of six COVID-19 scores and compared it with Clinical Gestalt predictions. Methods: 200 COVID-19 patients were enrolled in a tertiary hospital in Mexico City between September and December 2020. Clinical Gestalt predictions of death (as a percentage) and LOW-HARM, qSOFA, MSL-COVID-19, NUTRI-CoV and NEWS2 were obtained at admission. We calculated the AUC of each score and compared it against Clinical Gestalt predictions and against their respective originally reported value. Results: 106 men and 60 women aged 56+/-9 and with confirmed COVID-19 were included in the analysis. The observed AUC of all scores was significantly lower than originally reported; LOW-HARM 0.96 (0.94-0.98) vs 0.76 (0.69-0.84), qSOFA 0.74 (0.65-0.81) vs 0.61 (0.53-0.69), MSL-COVID-19 0.72 (0.69-0.75) vs 0.64 (0.55-0.73) NUTRI-CoV 0.79 (0.76-0.82) vs 0.60 (0.51-0.69), NEWS2 0.84 (0.79-0.90) vs 0.65 (0.56-0.75), Neutrophil-Lymphocyte ratio 0.74 (0.62-0.85) vs 0.65 (0.57-0.73). Clinical Gestalt predictions were non-inferior to mortality scores (AUC=0.68 (0.59-0.77)). Adjusting the LOW-HARM score with locally derived likelihood ratios did not improve its performance. However, some scores performed better than Clinical Gestalt predictions when clinician's confidence of prediction was <80%. Conclusion: No score was significantly better than Clinical Gestalt predictions. Despite its subjective nature, Clinical Gestalt has relevant advantages for predicting COVID-19 clinical outcomes.
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