Differential Lysotracker Uptake Defines Two Populations of Distal Epithelial Cells in Idiopathic Pulmonary Fibrosis

Idiopathic lung fibrosis (IPF) is a progressive and fatal degenerative lung disease of unknown etiology. Although in its final stages it implicates in a reactive manner all lung cell types, the initial damage involves the alveolar epithelial compartment, in particular the alveolar epithelial type 2 cells (AEC2s). AEC2s serve dual progenitor and surfactant secreting functions, both of which are deeply impacted in IPF. Thus, we hypothesize that the size of the surfactant processing compartment, as measured by Lysotracker incorporation, allows the identification of different epithelial states in the IPF lung. Flow cytometry analysis of epithelial Lysotracker incorporation delineates two populations (Lysohigh and Lysolow) of AEC2s which behave in a compensatory manner during bleomycin injury and in the donor/IPF lung. Employing flow cytometry and transcriptomic analysis of cells isolated from donor and IPF lungs, we demonstrate that the Lysohigh population expresses all classical AEC2 markers and is drastically diminished in IPF. The Lysolow population, which is increased in proportion in IPF, co-expresses AEC2s and basal cell markers resembling the phenotype of the previously identified intermediate AEC2 population in the IPF lung. In that regard, we provide an in-depth flow-cytometry characterization of Lysotracker uptake, HTII-280, proSP-C, mature SP-B, NGFR, KRT5 and CD24 expression in human lung epithelial cells. Combining functional analysis with extra- and intra- cellular marker expression and transcriptomic analysis, we advance the current understanding of epithelial cell behavior and fate in lung fibrosis.

A Randomized, Placebo-Controlled Trial Assessing the Effect of VISBIOME ES Probiotic in People With HIV on Antiretroviral Therapy

Background A5350, a phase II, randomized, double-blind study, evaluated the safety and tolerability of the probiotic Visbiome Extra Strength (ES) over 24 weeks and measured effects on inflammation and intestinal barrier function. Methods The primary outcome was change in soluble CD14 (sCD14) levels; secondary outcomes included safety and tolerability, markers of inflammation and cellular activation, and microbiome. In a substudy, gut permeability was assessed by paired colonic biopsies measuring the area of lamina propria occupied by CD4+ cells, interleukin (IL)-17+ cells, and myeloperoxidase (MPO). Changes between arms were compared with the 2-sample t test with equal variance or the Wilcoxon rank-sum test. For safety, the highest graded adverse events (AEs) were compared between arms using the Fisher exact test. Results Overall, 93 participants enrolled: 86% male, median age 51 years, median CD4 count 712 cells/mm3. Visbiome ES was safe and well tolerated. There was no difference in mean change in sCD14 from baseline to week 25/26 between placebo (mean change, 92.3 µg/L; 95% CI, –48.5 to 233 µg/L) and Visbiome ES (mean change, 41.0 µg/L; 95% CI, –94.1 to 176.2 µg/L; P=.60). Similarly, no statistically significant differences between arms in inflammatory marker changes were identified. In substudy participants, no statistical differences between arms for change in cellular marker expression or gut permeability were observed (P>.05 for all). The microbiome demonstrated increased probiotic species and a significant decrease in Gammaproteobacteria (P=.044) in the Visbiome ES arm. Conclusions Visbiome ES was safe and altered the microbiome but demonstrated no effect on systemic inflammatory markers, pathology, or gut permeability in antiretroviral therapy–treated people with HIV.

Evaluation of B-Cell Kinetics After Acellular Pertussis Vaccination in Four Cohorts of Different Age and Priming Background

Pertussis is a vaccine-preventable disease caused by the bacterium Bordetella pertussis. Over the past years, the incidence and mortality of pertussis increased significantly. A possible cause is the switch from whole cell to acellular pertussis vaccines, although other factors may also contribute. To develop future vaccines and improve current vaccination strategies, it is critical to understand factors influencing the generation of immunological memory. We applied high-dimensional flow cytometry to investigate changes in B cells in individuals of different ages and distinct priming backgrounds upon administration of an acellular pertussis booster vaccine. These findings were correlated to vaccine-specific plasma cells and serum Ig levels. Expansion and maturation of plasma cells 7 days post-vaccination was the most prominent cellular change in all age groups, and was most pronounced for more mature IgG1+ plasma cells. Cellular responses were stronger in individuals primed with whole cell vaccine than in individuals primed with acellular vaccine. Moreover, IgG1+ plasma cell expansion weakly correlated with Prn- and PT- specific serum IgG levels. Our study points at plasma cells as a potential early cellular marker of an immune response and contributes to understanding differences in immune responses between age groups and priming backgrounds.

Altered synaptic connectivity and brain function in mice lacking microglial adapter protein Iba1

Growing evidence indicates that microglia impact brain function by regulating synaptic pruning and formation as well as synaptic transmission and plasticity. Iba1 (ionized Ca+2-binding adapter protein 1), encoded by the Allograft inflammatory factor 1 (Aif1) gene, is an actin-interacting protein in microglia. Although Iba1 has long been used as a cellular marker for microglia, its functional role remains unknown. Here, we used global, Iba1-deficient (Aif1−/−) mice to characterize microglial activity, synaptic function, and behavior. Microglial imaging in acute hippocampal slices and fixed tissues from juvenile mice revealed that Aif1−/− microglia display reductions in ATP-induced motility and ramification, respectively. Biochemical assays further demonstrated that Aif1−/− brain tissues exhibit an altered expression of microglial-enriched proteins associated with synaptic pruning. Consistent with these changes, juvenile Aif1−/− mice displayed deficits in the excitatory synapse number and synaptic drive assessed by neuronal labeling and whole-cell patch-clamp recording in acute hippocampal slices. Unexpectedly, microglial synaptic engulfment capacity was diminished in juvenile Aif1−/− mice. During early postnatal development, when synapse formation is a predominant event in the hippocampus, the excitatory synapse number was still reduced in Aif1−/− mice. Together, these findings support an overall role of Iba1 in excitatory synaptic growth in juvenile mice. Lastly, postnatal synaptic deficits persisted in adulthood and correlated with significant behavioral changes in adult Aif1−/− mice, which exhibited impairments in object recognition memory and social interaction. These results suggest that Iba1 critically contributes to microglial activity underlying essential neuroglia developmental processes that may deeply influence behavior.

Novel Biomarkers of Endothelial Dysfunction in Cardiovascular Diseases

The review analyzes the role of assessing the state of the endothelium in the onset and progression of cardiovascular diseases, stratification of their risks, since endothelial dysfunction (ED) is a crucial predictor of this pathologies. In this regard, this paper presents the modern understanding of the methods for assessing ED, presents the advantages and disadvantages of various techniques. Despite the fact that flow-mediated dilation is widely used as a classical method for studying endothelial function, this technique depends on the physiological state of sensory nerves and calcium-activated potassium channels, cardiac output. This review focuses on new biomarkers for ED such as endothelial microparticles, endoglin and endocan, and discusses the relevance of the criteria for their use in clinical practice. Based on current scientific advances, the authors concluded that among these three newest biomarkers, today, endocan can be considered a more informative and reliable cellular marker of ED. Moreover, the authors have shown that when measured separately, many of the studied classical circulating biomarkers do not provide reliable information about the state of the endothelium, since the endothelial function has a complex physiological nature which therefore raises the question of the advisability of considering a combination of classical and new biomarkers for improving the assessment of the endothelial state.

Lipid and nucleocapsid N-protein accumulation in COVID-19 patient lung and infected cells

The trafficking of coronaviruses in lung of COVID-19 patients is not well understood and virus particles are difficult to find. Here we have visualized virus particles in SARS-CoV-2 infected cells by focusing on viral protein detection, in combination with ultrastructure. We studied how the virus is altering the cell morphology and determined that in Vero cells, lipid filled compartments contained various viral proteins. In these cells, also membrane enclosed multi-virus bodies were visible that contain a different set of viral proteins. We demonstrated that lipid filled compartments are novel viral induced compartments, as no known cellular marker such as lipid droplet or lysosomal marker was present. Using this knowledge, we then studied lung tissue from patients with a fatal SARS-Cov-2 infection, processed in a similar manner. Again we detected lipid filled compartments with viral proteins nsp4 and the stable nucleocapsid N-protein. The presence of these lipid filled compartments with viral proteins induced by SARS-CoV-2 infections, could be why the immune response of the COVID-19 patients is so strong, resulting in a fatal infection, and should be considered for new therapeutic strategies.

Impact of Snoring on Telomere Shortening in Adolescents with Atopic Diseases

Atopic diseases can impose a significant burden on children and adolescents. Telomere length is a cellular marker of aging reflecting the impact of cumulative stress exposure on individual health. Since elevated oxidative stress and inflammation burden induced by chronic atopy and snoring may impact telomere length, this study aimed to investigate whether snoring would moderate the relationship between atopic diseases and telomere length in early adolescence. We surveyed 354 adolescents and their parents. Parents reported the adolescents’ history of atopic diseases, recent snoring history as well as other family sociodemographic characteristics. Buccal swab samples were also collected from the adolescents for telomere length determination. Independent and combined effects of atopic diseases and snoring on telomere length were examined. Among the surveyed adolescents, 174 were reported by parents to have atopic diseases (20 had asthma, 145 had allergic rhinitis, 53 had eczema, and 25 had food allergy). Shorter TL was found in participants with a history of snoring and atopic diseases (β = −0.34, p = 0.002) particularly for asthma (β = −0.21, p = 0.007) and allergic rhinitis (β = −0.22, p = 0.023). Our findings suggest that snoring in atopic patients has important implications for accelerated telomere shortening. Proper management of atopic symptoms at an early age is important for the alleviation of long-term health consequences at the cellular level.

Assessment of salivary pro inflammatory cytokines profile level in patients treated with labial and lingual fixed orthodontic appliances

The secretions of certain cytokines, chemokines and growth factors are triggered by orthodontic appliances, which often affect the remodelling of periodontal tissues. Critical cumulative forces are applied by various types of orthodontic appliances to the periodontium. The secretion of such molecules is probably responsible, through molecular and cellular communications, for the optimal resorption of hard tissues in the periodontal setting, which therefore enables the coordination of multiple movements of tooth. This study assessed and compared a wide range of cytokines, cellular marker analysis and defensins present in the saliva samples of human subjected to orthodontic treatment with two different treatment modalities, i.e., conventional lingual and labial fixed orthodontic appliances. A total 40 samples of saliva were obtained, of which 20 were treated with traditional lingual appliances and 20 were treated with labial fixed appliances. After 21 days of treatment, all salivary samples were collected from the subjects. In order to analyse a broad range of soluble cytokine levels in saliva by flow cytometry, a bead-based immunoassay was performed. Cell surface markers were analysed by flow cytometry. Protein levels of saliva for defensins were quantified by ELISA. Non-significant differences were observed in the cytokine levels in the saliva except for the significant effects for CCL2, IL-17A and IL-6. Cellular markers CD45 and CD326 showed high percentage in conventional lingual samples. Defensin levels were found to be lower in conventional lingual patients. Subjects with conventional lingual appliances had significantly higher salivary protein levels of IL-1β, CCL2, IL17A, and IL-6, higher CD45+ and CD326+ cells and lower defensin levels than subjects with fixed labial appliances. The current study provided a clear basis for the development of innovative methods to aid in the improvement of various procedural treatments and orthodontic equipment of next generation.

The comparison of amniotic fluid nuclearfactor-kappa B levels in pregnant women who underwent cesarean section or normal vaginal labor

Objective Inflammatory changes in the amniotic membranes during prenatal period have a critical important in the rupture of membranes and the onset of labor. The inflammatory changes in the membranes caused by normal vaginal labors and cesarean sections (C/S) are different than each other. Nuclear factor-kappa B (Nf- kB) is the basic cellular marker of the inflammation change in biological fluids and tissues. We planned this study to compare amniotic fluid NF-kB (AF-NF-kB) concentrations in normal vaginal labor cases and those who underwent non-emergency C/S. Methods One-hundred singleton term pregnant women who did not have any serious fetal and maternal problems were included in the study. The pregnant women were separated into two groups, which contained 50 cases each. The week of gestation was calculated according to the last menstrual period and ultrasonography measurements. The groups included 50 patients who did not have the history of clinical chorioamnionitis and preterm premature rupture of membranes and were decided to have normal vaginal labor, and 50 term pregnant women who were decided to have cesarean section due to maternal or perinatal reasons. The amniotic fluid samples were collected during cesarean section or normal vaginal labor. After opening the membranes by scalpel in C/S cases and following spontaneous or artificial membrane rupture in vaginal labor cases, the amniotic fluid samples were collected. AF-NF-kB concentrations were evaluated by ELISA method. Results AF-NF-kB levels of the patients in the group of normal vaginal labor were significantly higher than AF-NF-kB levels of the patients in C/S group. AF-NF-kB levels were about 2 times higher in the group of normal vaginal labor (1.44±0.40 ng/ mL vs. 0.71±2.60 ng/mL, p<0.001). There was a positive but insignificant correlation between the fetal birth weight and AF-NF-kB levels in the patients who underwent normal vaginal labor. There was no significant correlation between AF-NF-kB levels and demographic and clinical characteristics of the patients who underwent labor by C/S. Conclusion Normal vaginal labor is associated with the increased AF-NF-kB concentrations compared to C/S cases. AF-NF-kB levels seem to be a potential predictor for the spontaneous fetal membrane rupture.

Ovotestes suggest cryptic genetic influence in a reptile model for temperature-dependent sex determination

Sex determination and differentiation in reptiles is complex. Temperature-dependent sex determination (TSD), genetic sex determination (GSD) and the interaction of both environmental and genetic cues (sex reversal) can drive the development of sexual phenotypes. The jacky dragon ( Amphibolurus muricatus ) is an attractive model species for the study of gene–environment interactions because it displays a form of Type II TSD, where female-biased sex ratios are observed at extreme incubation temperatures and approximately 50 : 50 sex ratios occur at intermediate temperatures. This response to temperature has been proposed to occur due to underlying sex determining loci, the influence of which is overridden at extreme temperatures. Thus, sex reversal at extreme temperatures is predicted to produce the female-biased sex ratios observed in A. muricatus . The occurrence of ovotestes during development is a cellular marker of temperature sex reversal in a closely related species Pogona vitticeps . Here, we present the first developmental data for A. muricatus , and show that ovotestes occur at frequencies consistent with a mode of sex determination that is intermediate between GSD and TSD. This is the first evidence suggestive of underlying unidentified sex determining loci in a species that has long been used as a model for TSD.