Proinflammatory pattern in the lower airways of non-asthmatic obese adolescents.

BACKGROUND: Obesity is known to be a pro-inflammatory condition affecting multiple organs. Obesity as a systemic pro-inflammatory state, might be associated with bronchial inflammation in non-smoking adolescents with a BMI ≥ 30 kg/m2 without evidence of concomitant chronic diseases. MATERIALS AND METHODS: We studied non-asthmatic obese patients (n = 20; median age 15.8 years; BMI 35.0 kg/m2) compared to age matched healthy control subjects (n = 20; median age 17.5 years; BMI 21.5 kg/m2). Induced sputum differential cell counts and sputum mRNA levels were assessed for all study subjects. Serum levels of CRP, IL-6, and IL-8 were measured. Further, IL-5, IL-6, IL-8, IL-13, IL-17, TNF-α, IFN-γ, and IP-10 protein levels were analyzed in induced sputum was. RESULTS: Serum CRP levels, sputum inflammatory cell load and sputum eosinophils differed significantly between obese and non-obese subjects, for sputum neutrophils, a correlation was shown with BMI ≥ 30 kg/m2. Differences were also observed for sputum mRNA expression of IL6, IL8, IL13, IL17, IL23, and IFN-γ, as well as the transcription factors T-bet, GATA3, and FoxP3. CONCLUSIONS: Increased bronchial inflammation, triggered by systemic or local inflammatory effects of obesity itself, may account for the higher rates of airway disease in obese adolescents.

MicroRNA Profiling of the Inflammatory Response after Early and Late Asthmatic Reaction.

A high proportion of house dust mite (HDM)-allergic asthmatics suffer from both an early asthmatic reaction (EAR) and a late asthmatic reaction (LAR) which follows it. In these patients, allergic inflammation is more relevant. MiRNAs have been shown to play an important role in the regulation of asthma's pathology. The aim of this study was to analyze the miRNA profile in patients with mild asthma and an HDM allergy after bronchial allergen provocation (BAP). Seventeen patients with EAR/no LAR and 17 patients with EAR plus LAR, determined by a significant fall in FEV1 after BAP, were differentially analyzed. As expected, patients with EAR plus LAR showed a more pronounced allergic inflammation and FEV1 delta drop after 24 h. NGS-miRNA analysis identified the down-regulation of miR-15a-5p, miR-15b-5p, and miR-374a-5p after BAP with the highest significance in patients with EAR plus LAR, which were negatively correlated with eNO and the maximum decrease in FEV1. These miRNAs have shared targets like CCND1, VEGFA, and GSK3B, which are known to be involved in airway remodeling, basement membrane thickening, and Extracellular Matrix deposition. NGS-profiling identified miRNAs involved in the inflammatory response after BAP with HDM extract, which might be useful to predict a LAR.

Endothelial dysfunction and atherosclerosis related miRNA-expression in patients with haemophilia.

INTRODUCTION: Elevated markers of endothelial dysfunction and inflammation indicate worse endothelial function in the aging haemophilia population. MicroRNAs (miRNAs) regulate gene expression post-transcriptionally. Several miRNAs have been shown to be involved in the process of endothelial dysfunction and atherosclerosis. AIM: The aim of this study was to determine the underlying molecular pathways of endothelial dysfunction and inflammation in haemophilia patients. METHODS: A total of 25 patients with severe or moderate haemophilia A (20 patients) or B (5 patients), 14 controls and 18 patients with coronary artery disease (CAD) after myocardial infarction were included in this study. Expression of miRNA-126, -155, -222, -1, -let7a, -21 and -197 were analysed using a real time polymerase chain reaction. Network-based visualisation and analysis of the miRNA-target interactions were performed using the MicroRNA ENrichment TURned NETwork (MIENTURNET). RESULTS: Expression of miRNA-126 (p < .05) and miRNA-let7a (p < .05) were significantly higher in CAD patients compared to haemophilia patients and controls. MiRNA-21 (p < .05) was significantly elevated in CAD patients compared to controls. MiRNA-155 (p < .05), miRNA-1 (p < .05) and miRNA-197 (p < .05) were significantly higher expressed in CAD and haemophilia patients compared to controls and showed a strong correlation with increased levels of interleukin-6 (IL-6) and soluble intercellular adhesion molecule-1 (sICAM-1). The network analysis revealed interactions in the cytokine signalling, focal adhesion and VEGFA-VEGFR2 pathway (Vascular endothelial growth factor, -receptor). CONCLUSION: This study characterises miRNA expression in haemophilia patients in comparison to CAD patients and healthy controls. The results imply comparable biological processes in CAD and haemophilia patients.

Aerosol measurement identifies SARS-CoV 2 PCR positive adults compared with healthy controls.

BACKGROUND: SARS-CoV-2 is spread primarily through droplets and aerosols. Exhaled aerosols are generated in the upper airways through shear stress and in the lung periphery by 'reopening of collapsed airways'. Aerosol measuring may detect highly contagious individuals ("super spreaders or super-emitters") and discriminate between SARS-CoV-2 infected and non-infected individuals. This is the first study comparing exhaled aerosols in SARS-CoV-2 infected individuals and healthy controls. DESIGN: A prospective observational cohort study in 288 adults, comprising 64 patients testing positive by SARS CoV-2 PCR before enrollment, and 224 healthy adults testing negative (matched control sample) at the University Hospital Frankfurt, Germany, from February to June 2021. Study objective was to evaluate the concentration of exhaled aerosols during physiologic breathing in SARS-CoV-2 PCR-positive and -negative subjects. Secondary outcome measures included correlation of aerosol concentration to SARS-CoV-2 PCR results, change in aerosol concentration due to confounders, and correlation between clinical symptoms and aerosol. RESULTS: There was a highly significant difference in respiratory aerosol concentrations between SARS-CoV-2 PCR-positive (median 1490.5/L) and -negative subjects (median 252.0/L; p < 0.0001). There were no significant differences due to age, sex, smoking status, or body mass index. ROC analysis showed an AUC of 0.8918. CONCLUSIONS: Measurements of respiratory aerosols were significantly elevated in SARS-CoV-2 positive individuals, which helps to understand the spread and course of respiratory viral infections, as well as the detection of highly infectious individuals.

Association between Polyunsaturated Fatty Acid Profile and Bronchial Inflammation in Bronchiolitis Obliterans.

Introduction: Bronchiolitis obliterans (BO) is a chronic lung disease, which occurs after an insult to the lower airways, in particular after airway infections or after stem cell transplantation, and which results in persistent inflammation. N-3 and n-6 polyunsaturated fatty acids (PUFA) have been shown to influence the inflammatory processes in chronic inflammatory conditions. Since BO is maintained by persistent pulmonary inflammation, a disbalanced n-6/n-3 fatty acid profile could support the inflammatory process in patients with BO and therefore, could become an approach to new therapeutic options. Methods: Twenty-five patients with BO (age: 13; 7-39) and 26 healthy controls (age: 19; 7-31) participated in the study. Lung function (forced viral capacity (FVC), forced expiratory volume 1 (FEV1), residual volume (RV)), and lung clearance index (LCI) were measured. Induced sputum was analyzed for cytology and cytokine levels (IL-1ß, IL-6, IL-8, TNF-α) using cytometric bead array (CBA). The PUFA profile was determined in the serum and induced sputum by gas chromatography. Results: Patients presented with significantly lower FVC and FEV1 as well as higher RV and LCI measurements compared to the control group. Apart from a massive airway inflammation indicated by elevated numbers of total cells and neutrophils, the CBA analysis showed significantly increased levels of IL-1ß, IL-6, and IL-8. The analysis of PUFA in sputum and serum revealed a significant difference in the ratio between the n-6 PUFA arachidonic acid (AA) and the n-3 PUFA docosahexaenoic acid (DHA) (AA : DHA). Furthermore, the AA : DHA ratio significantly correlated with the inflammatory cytokines in induced sputum. Conclusion: Lung function in BO is significantly impaired and associated with uncontrolled neutrophil-dominated airway inflammation. Furthermore, the imbalance in the AA/DHA ratio in favor of n-6 PUFA demonstrates a pro-inflammatory microenvironment in the cell membrane, which correlates with the inflammatory cytokines in induced sputum and might be an option for an anti-inflammatory therapy in BO.

In Vitro Models of Tissue and Organ Regeneration.

The recovery of cells after tissue and organ injury is a complex process [...].

microRNA Expression of Renal Proximal Tubular Epithelial Cells and Their Extracellular Vesicles in an Inflammatory Microenvironment In Vitro.

Renal proximal tubular epithelial cells (PTCs) are central players during renal inflammation. In response to inflammatory signals, PTCs not only self-express altered mRNAs, microRNAs (miRNAs), proteins, and lipids, but also release altered extracellular vesicles (EVs). These EVs also carry inflammation-specific cargo molecules and are key players in cell-cell-communication. Understanding the precise molecular and cellular mechanisms that lead to inflammation in the kidney is the most important way to identify early targets for the prevention or treatment of acute kidney injury. Therefore, highly purified human PTCs were used as an in vitro model to study the cellular response to an inflammatory microenvironment. A cytokine-induced inflammatory system was established to analyze different miRNA expression in cells and their EVs. In detail, we characterized the altered miR expression of PTCs and their released EVs during induced inflammation and showed that 12 miRNAs were significantly regulated in PTCs (6 upregulated and 6 downregulated) and 9 miRNAs in EVs (8 upregulated and 1 downregulated). We also showed that only three of the miRNAs were found to overlap between cells and EVs. As shown by the KEGG pathway analysis, these three miRNAs (miR-146a-5p, miR-147b, and miR-155-5p) are functionally involved in the regulation of the Toll-like receptor signaling pathway and significantly correlated with the inflammatory mediators IL6 and ICAM1 released by stimulated PTCs. Especially with regard to a possible clinical use of miRs as new biomarkers, an accurate characterization of the miR expression altered during inflammatory processes is of enormous importance.

Gliflozins Have an Anti-Inflammatory Effect on Renal Proximal Tubular Epithelial Cells in a Diabetic and Inflammatory Microenvironment In Vitro.

Inflammation is intimately involved in the pathogenesis of diabetic kidney disease. Inhibition of SGLT-2 by a specific class of drugs, gliflozins, has been shown to reduce inflammation and attenuate the progression of diabetic nephropathy, in addition to its main effect of inhibiting renal glucose reabsorption. We used highly purified human renal proximal tubular epithelial cells (PTCs) as an in vitro model to study the cellular response to a diabetic (high glucose) and inflammatory (cytokines) microenvironment and the effect of gliflozins. In this context, we investigated the influence of SGLT-2 inhibition by empa- and dapagliflozin (500 nM) on the expression of pro-inflammatory factors (IL-1ß, IL-6, TNF-α, MCP-1, and ICAM-1). The results clearly indicate an anti-inflammatory effect of both gliflozins. Although induced expression of the four cytokines was only slightly attenuated, there was a clear effect on the expression of the adhesion molecule ICAM-1, a master regulator of cellular responses in inflammation and injury resolution. The induced expression of ICAM-1 mRNA was significantly reduced by approximately 13.5% by empagliflozin and also showed an inhibitory trend with dapagliflozin. However, induced ICAM-1 protein expression was significantly inhibited from 24.71 ± 1.0 ng/mL to 18.81 ± 3.9 (empagliflozin) and 19.62 ± 2.1 ng/mL (dapagliflozin). In conclusion, an additional anti-inflammatory effect of empa- and dapagliflozin in therapeutically observed concentrations was demonstrated in primary human PTCs in vitro.

Bronchial inflammation biomarker patterns link humoral immunodeficiency with bronchiectasis-related small airway dysfunction.

BACKGROUND: The progression of chronic destructive lung disease in patients with humoral immunodeficiency (ID) and concomitant development of bronchiectasis is difficult to prevent. Lung function tests in these patients typically show bronchial obstruction of the small airways in combination with increased air trapping in the distal airways, which is consistent with small airway dysfunction. OBJECTIVE: The objective was to assess the grade of chronic lower airway inflammation and small airway dysfunction from induced sputum and the corresponding local pro-inflammatory mediator pattern to discriminate patients affected by bronchiectasis-related Small Airway Dysfunction (SAD). METHODS: In a prospective design, 22 patients with ID (14 CVID, 3 XLA, 3 hyper-IgM syndrome, 1 hyper-IgE syndrome and low IgG levels due to treatment with rituximab and 1 SCID after BMT and persistent humoral defect) and 21 healthy controls were examined. Lung function, Fraction Expiratory Nitric Oxide (FeNO) and pro-inflammatory cytokine levels were compared in subsets of patients with (ID + BE) and without bronchiectasis (ID) pre-stratified using high-resolution computed tomography (HRCT) scans and control subjects. RESULTS: Analysis of induced sputum showed significantly increased total cell counts and severe neutrophilic inflammation in ID. The concomitant SAD revealed higher total cell numbers compared to ID. Bronchial inflammation in ID is clearly mirrored by pro-inflammatory mediators IL-1ß, IL-6 and CXCL-8, whilst TNF-α revealed a correlation with lung function parameters altered in the context of bronchiectasis-related Small Airway Dysfunction. CONCLUSIONS: In spite of immunoglobulin substitution, bronchial inflammation was dominated by neutrophils and was highly increased in patients with ID + BE. Notably, the pro-inflammatory cytokines in patients with ID were significantly increased in induced sputum. The context-dependent cytokine pattern in relation to the presence of concomitant bronchiectasis associated with SAD in ID patients could be helpful in delimiting ID patient subgroups and individualizing therapeutic approaches.

Regulatory B cells in patients suffering from inborn errors of immunity with severe immune dysregulation.

BACKGROUND: Immune dysregulation as a result of an inborn error of immunity (IEI) leads to the complicated symptoms of refractory multi-organ immune dysregulation. B lymphocytes with immune regulatory capacity (Breg) are activated by environmental triggers and act as regulators of the immune response as observed in several autoimmune diseases. OBJECTIVE: We sought to investigate the Breg profile and the CD21low expressing B cells of patients with LRBA deficiency (N = 6) and non-LRBA deficiency IEI (N = 13) with overlapping clinical symptoms of immune dysregulation. Normal values for Breg subpopulations were obtained from patients age-matched healthy cohorts (N = 48). Furthermore, we investigated the impact of abatacept treatment in LRBA deficient patients receiving biweekly abatacept (N = 5). METHODS: Using a flow cytometric approach with a pre-formulated antibody panel in peripheral blood samples, Breg subsets including plasmablasts (CD27+CD38hi), transitional B cells (CD24hiCD38hi), and B10 cells (CD24hiCD27+), and additionally the CD21low B cells (CD21lowCD38low) were analyzed. Breg function was assessed by the interleukin-10 expression within the CD19+ population. Additionally, B cell cytokines were measured in cell culture supernatants. RESULTS: We observe significant alterations of B cell/Breg subpopulations in the LRBA deficient cohort including a severe lack of memory B cells (P = 0.031) and B10 cells (P = 0.031) as well as a tendency towards higher CD21low B cells (P = 0.063). Within the non-LRBA deficient cohort, we observe a significant expansion of the plasmablasts (P = 0.012), and a tendency towards elevated levels of CD21low expressing B cells (P = 0.063). The treatment with abatacept ameliorated disease symptoms in the LRBA deficient cohort and led to an effective decrease in CD21low B cells over time (P = 0.021). Furthermore, there was a significantly increased level of B cell-activating factor (BAFF; P = 0.02) and lower IL-12p70 secretion upon stimulation (P = 0.020) in the LRBA cohort. CONCLUSION: Aberrant maturation of Breg subsets and the pathological expansion of CD21low B cells in patients with IEI may have therapeutic implications. Patients suffering from LRBA deficiency show a lack of memory B cells, insufficient expansion of B10 cells, increased BAFF levels as well as an increase in circulating CD21low B cells. Abatacept treatment results in a steady decrease in CD21low B cells.

Vitreous cytokine levels following the administration of a single 0.19 mg fluocinolone acetonide (ILUVIEN®) implant in patients with refractory diabetic macular edema (DME)-results from the ILUVIT study.

PURPOSE: To investigate the changes in vitreous inflammatory and angiogenic cytokine levels, primarily interleukin-(IL)-6, following intravitreal injection of the 0.19 mg fluocinolone acetonide (FAc, ILUVIEN®) implant in patients with diabetic macular edema. METHODS: A single-center phase IV study involving 12 patients' eyes with diabetic macular edema. Vitreous fluid samples were obtained prior to intravitreal injection of the fluocinolone acetonide implant and then again over a 6-month period. Vitreous samples were examined using a cytometric bead array to measure IL-6, IL-8, IP-10, MCP-1, VEGF, and CD54. PIGF and PEDF were measured using an enzyme-linked immunosorbent assay. Changes in the cytokine and chemokine expression patterns were analyzed. Clinical parameters such as BCVA and center point thickness (CPT) were also examined. RESULTS: There were mean reductions in all parameters between baseline and month 6. Significant changes (p < 0.05 versus baseline) were observed in the expression of IL-6, IP-10, MCP-1, and CD54 following the administration of fluocinolone acetonide implant. VEGF and PIGF increased at month 1 before declining at month 6, though this trend was not significant. CPT decreased rapidly between screening and the first follow-up visit, and this decrease was sustained. BCVA remained relatively stable throughout. CONCLUSION: This study demonstrated changes in vitreous inflammatory and angiogenic cytokine levels following intravitreal injection of the FAc implant in patients with diabetic macular edema. Data show that the fluocinolone acetonide implant led to rapid and sustained reductions of some inflammatory cytokines with improvement of the overall clinical picture.

Effects of Hypoxia on RNA Cargo in Extracellular Vesicles from Human Adipose-Derived Stromal/Stem Cells.

Mesenchymal stromal/stem cells and their derivates are the most promising cell source for cell therapies in regenerative medicine. The application of extracellular vesicles (EVs) as cell-free therapeuticals requires particles with a maximum regenerative capability to enhance tissue and organ regeneration. The cargo of mRNA and microRNA (miR) in EVs after hypoxic preconditioning has not been extensively investigated. Therefore, the aim of our study was the characterization of mRNA and the miR loading of EVs. We further investigated the effects of the isolated EVs on renal tubular epithelial cells in vitro. We found 3131 transcripts to be significantly regulated upon hypoxia. Only 15 of these were downregulated, but 3116 were up-regulated. In addition, we found 190 small RNAs, 169 of these were miRs and 21 were piwi-interacting RNAs (piR). However, only 18 of the small RNAs were significantly altered, seven were miRs and 11 were piRs. Interestingly, all seven miRs were down-regulated after hypoxic pretreatment, whereas all 11 piRs were up-regulated. Gene ontology term enrichment and miR-target enrichment analysis of the mRNAs and miR were also performed in order to study the biological background. Finally, the therapeutic effect of EVs on human renal tubular epithelial cells was shown by the increased expression of three anti-inflammatory molecules after incubation with EVs from hypoxic pretreatment. In summary, our study demonstrates the altered mRNA and miR load in EVs after hypoxic preconditioning, and their anti-inflammatory effect on epithelial cells.

The incidence and type of cancer in patients with ataxia-telangiectasia via a retrospective single-centre study.

Ataxia-telangiectasia (A-T) is a hereditary immune system disorder with neurodegeneration. Its first neurologic symptoms include ataxic gait in early childhood, with slowly progressive cerebellar ataxia, oculomotor apraxia, oculocutaneous telangiectasia, and progressive muscle weakness. Neonatal screening for severe T-cell deficiency was recently found to diagnose A-T patients with a significantly reduced naïve T-cell pool. Our study includes 69 A-T patients between 8 January 2002 and 1 December 2019. Nineteen cases of cancer were diagnosed in 17 patients (25%), with a median overall survival [OS; 95% cumulative indcidence (CI)] of 26·9 years for the entire cohort. The 15-year OS of 82·5% (72-95%) was significantly decreased among A-T patients with malignancies, who had a median OS of 2·11 years, with a two-year-estimated OS of 50·7% (31-82%). Haematological malignancies were the major causes of death within the initial years of life with a 15 times increased risk for death [HR (95% CI): 6·9 (3·1-15.2), P < 0·001] upon malignancy diagnosis. Male patients with A-T are at a higher cancer risk than their female counterparts. This manuscript highlights the need for cancer surveillance and prevention, as well as optimal treatment in this cohort.

Characterization of Extracellular Vesicles from Preconditioned Human Adipose-Derived Stromal/Stem Cells.

Cell-free therapy using extracellular vesicles (EVs) from adipose-derived mesenchymal stromal/stem cells (ASCs) seems to be a safe and effective therapeutic option to support tissue and organ regeneration. The application of EVs requires particles with a maximum regenerative capability and hypoxic culture conditions as an in vitro preconditioning regimen has been shown to alter the molecular composition of released EVs. Nevertheless, the EV cargo after hypoxic preconditioning has not yet been comprehensively examined. The aim of the present study was the characterization of EVs from hypoxic preconditioned ASCs. We investigated the EV proteome and their effects on renal tubular epithelial cells in vitro. While no effect of hypoxia was observed on the number of released EVs and their protein content, the cargo of the proteins was altered. Proteomic analysis showed 41 increased or decreased proteins, 11 in a statistically significant manner. Furthermore, the uptake of EVs in epithelial cells and a positive effect on oxidative stress in vitro were observed. In conclusion, culture of ASCs under hypoxic conditions was demonstrated to be a promising in vitro preconditioning regimen, which alters the protein cargo and increases the anti-oxidative potential of EVs. These properties may provide new potential therapeutic options for regenerative medicine.

The MiR-320 Family Is Strongly Downregulated in Patients with COVID-19 Induced Severe Respiratory Failure.

A high incidence of thromboembolic events associated with high mortality has been reported in severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infections with respiratory failure. The present study characterized post-transcriptional gene regulation by global microRNA (miRNA) expression in relation to activated coagulation and inflammation in 21 critically ill SARS-CoV-2 patients. The cohort consisted of patients with moderate respiratory failure (n = 11) and severe respiratory failure (n = 10) at an acute stage (day 0-3) and in the later course of the disease (>7 days). All patients needed supplemental oxygen and severe patients were defined by the requirement of positive pressure ventilation (intubation). Levels of D-dimers, activated partial thromboplastin time (aPTT), C-reactive protein (CRP), and interleukin (IL)-6 were significantly higher in patients with severe compared with moderate respiratory failure. Concurrently, next generation sequencing (NGS) analysis demonstrated increased dysregulation of miRNA expression with progression of disease severity connected to extreme downregulation of miR-320a, miR-320b and miR-320c. Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis revealed involvement in the Hippo signaling pathway, the transforming growth factor (TGF)-ß signaling pathway and in the regulation of adherens junctions. The expression of all miR-320 family members was significantly correlated with CRP, IL-6, and D-dimer levels. In conclusion, our analysis underlines the importance of thromboembolic processes in patients with respiratory failure and emphasizes miRNA-320s as potential biomarkers for severe progressive SARS-CoV-2 infection.

Immune competence and respiratory symptoms in patients with ataxia telangiectasia: A prospective follow-up study.

Ataxia telangiectasia is a multi-system disorder characterized by progressive cerebellar ataxia, malignancies, chronic pulmonary disease and immunodeficiency. The aim of our study was to determine the immune competence and prevalence of respiratory infections and/or chronic cough in classical A-T patients compared to age-matched healthy controls. STUDY DESIGN: We recruited 20 classical A-T not treated by immunoglobulins and 21 healthy age-matched control patients. The caregivers were advised to keep a daily diary with the following items (daytime and nighttime cough, runny nose, fever), number of cold episodes, number of antibiotic treatments. RESULTS: Patients with A-T showed significant differences compared to healthy controls in symptom score, daytime and nighttime cough, days with symptoms and missed days in kindergarten/school. Severe infections with hospitalization occurred rarely. Respiratory symptoms did not correlate with immunoglobulin levels in A-T patients. CONCLUSIONS: Mild symptoms like chronic cough were present in A-T patients, possibly indicating ongoing silent crippling disease.

Clinical relevance of Aspergillus fumigatus sensitization in cystic fibrosis.

RATIONALE: The clinical relevance of sensitization to Aspergillus (A) fumigatus in cystic fibrosis (CF) is unclear. Some researchers propose that specific A fumigatus IgE is an innocent bystander, whereas others describe it as the major cause of TH-2-driven asthma-like disease. OBJECTIVES: Lung function parameters in mild CF patients may be different in patients with and without A fumigatus sensitization. We aimed to ascertain whether allergen exposure to A fumigatus by bronchial allergen provocation (BAP) induces TH-2 inflammation comparable to an asthma-like disease. METHODS: A total of 35 patients, aged 14.8 ± 8.5 years, and 20 healthy controls were investigated prospectively. The patients were divided into two groups: group 1 (n = 18): specific (s)IgE negative, and group 2 (n = 17): sIgE positive (≥0.7 KU/L) for A fumigatus. Lung function, exhaled NO, and induced sputum were analysed. All sensitized patients with an FEV1 > 75% (n = 13) underwent BAP with A fumigatus, and cell counts, and the expression of IL-5, IL-13, INF-γ, and IL-8 as well as transcription factors T-bet, GATA-3, and FoxP3, were measured. RESULTS: Lung function parameters decreased significantly compared to controls, but not within the CF patient group. After BAP, 8 of 13 patients (61%) had a significant asthmatic response and increased eNO 24 hours later. In addition, marked TH-2-mediated inflammation involving eosinophils, IL-5, IL-13, and FoxP3 became apparent in induced sputum cells. CONCLUSION: Our study demonstrated the clinical relevance of A fumigatus for the majority of sensitized CF patients. A distinct IgE/TH-2-dominated inflammation was found in induced sputum after A fumigatus exposure.

No Cytotoxic and Inflammatory Effects of Empagliflozin and Dapagliflozin on Primary Renal Proximal Tubular Epithelial Cells under Diabetic Conditions In Vitro.

Gliflozins are inhibitors of the renal proximal tubular sodium-glucose co-transporter-2 (SGLT-2), that inhibit reabsorption of urinary glucose and they are able to reduce hyperglycemia in patients with type 2 diabetes. A renoprotective function of gliflozins has been proven in diabetic nephropathy, but harmful side effects on the kidney have also been described. In the current project, primary highly purified human renal proximal tubular epithelial cells (PTCs) have been shown to express functional SGLT-2, and were used as an in vitro model to study possible cellular damage induced by two therapeutically used gliflozins: empagliflozin and dapagliflozin. Cell viability, proliferation, and cytotoxicity assays revealed that neither empagliflozin nor dapagliflozin induce effects in PTCs cultured in a hyperglycemic environment, or in co-medication with ramipril or hydro-chloro-thiazide. Oxidative stress was significantly lowered by dapagliflozin but not by empagliflozin. No effect of either inhibitor could be detected on mRNA and protein expression of the pro-inflammatory cytokine interleukin-6 and the renal injury markers KIM-1 and NGAL. In conclusion, empa- and dapagliflozin in therapeutic concentrations were shown to induce no direct cell injury in cultured primary renal PTCs in hyperglycemic conditions.

Impact of Antifungal Compounds on Viability and Anti-Aspergillus Activity of Human Natural Killer Cells.

Despite the availability of new antifungal compounds, invasive aspergillosis carries high morbidity and mortality in hematopoietic stem cell transplant recipients. In vitro studies and animal models suggest that the adoptive transfer of natural killer (NK) cells might be a promising immunotherapeutic option in this setting. As it is unclear whether the viability and function of human NK cells are affected by common antifungal agents, we analyzed the interaction of various concentrations of amphotericin B deoxycholate (AmB-D), liposomal amphotericin B, caspofungin, fluconazole, voriconazole, and posaconazole with human NK cells. When adding NK cells to therapeutic concentrations of antifungal agents, a significant increase in the antifungal effect was seen for caspofungin and voriconazole, whereas NK cells significantly decreased the hyphal damage of escalated doses of AmB-D. In contrast, therapeutic concentrations of all antifungal compounds tested did not have a negative effect on proliferation, viability, and the release of soluble immunomodulatory molecules of NK cells. These data indicate that therapeutic concentrations of the antifungal agents tested do not negatively affect the functional properties of human NK cells, which is a prerequisite for further studies evaluating NK cells as antifungal immunotherapy in immunocompromised patients suffering from invasive aspergillosis.

Ataxia telangiectasia alters the ApoB and reelin pathway.

Autosomal recessive ataxia telangiectasia (A-T) is characterized by radiosensitivity, immunodeficiency, and cerebellar neurodegeneration. A-T is caused by inactivating mutations in the ataxia telangiectasiamutated (ATM) gene, a serine-threonine protein kinase involved in DNA damage response and excitatory neurotransmission. The selective vulnerability of cerebellar Purkinje neurons (PN) to A-T is not well understood. Employing global proteomic profiling of cerebrospinal fluid from patients at ages around 15 years, we detected reduced calbindin, reelin, cerebellin-1, cerebellin-3, protocadherin fat 2, sempahorin 7A, and increased apolipoprotein B and J peptides. Bioinformatic enrichment was observed for pathways of lipoproteins, endocytosis, extracellular matrix receptor interaction, peptidase activity, adhesion, calcium binding, and complement immunity. This seemed important since secretion of reelin from glutamatergic afferent axons is crucial for PN lipoprotein receptor endocytosis and lipid signaling. Reelin expression is downregulated by irradiation and reelin/ApoB mutations are known causes of ataxia. Validation efforts in 2-month-old Atm-/- mice before onset of motor deficits confirmed cerebellar transcript reductions for reelin receptors Apoer2/Vldlr with increases for their ligands Apoe/Apoh and cholesterol 24-hydroxylase Cyp46a1. Concomitant dysregulations were found for Vglut2/Sema7a as climbing fiber markers, glutamate receptors like Grin2b, and calcium homeostasis factors (Atp2b2, Calb1, Itpr1), while factors involved in DNA damage, oxidative stress, neuroinflammation, and cell adhesion were normal at this stage. Quantitative immunoblots confirmed ApoB and ApoJ increases and VLDLR reduction in cerebellar tissue at the age of 2 months. These findings show that ApoB excess and reelin signaling deficits reflect the neurodegeneration in A-T in a sensitive and specific way. As extracellular factors, apolipoproteins and their cargo such as vitamin E may be useful for neuroprotective interventions.