Rapid discovery of monoclonal antibodies by microfluidics-enabled FACS of single pathogen-specific antibody-secreting cells.

Monoclonal antibodies are increasingly used to prevent and treat viral infections and are pivotal in pandemic response efforts. Antibody-secreting cells (ASCs; plasma cells and plasmablasts) are an excellent source of high-affinity antibodies with therapeutic potential. Current methods to study antigen-specific ASCs either have low throughput, require expensive and labor-intensive screening or are technically demanding and therefore not widely accessible. Here we present a straightforward technology for the rapid discovery of monoclonal antibodies from ASCs. Our approach combines microfluidic encapsulation of single cells into an antibody capture hydrogel with antigen bait sorting by conventional flow cytometry. With our technology, we screened millions of mouse and human ASCs and obtained monoclonal antibodies against severe acute respiratory syndrome coronavirus 2 with high affinity (<1 pM) and neutralizing capacity (<100 ng ml-1) in 2 weeks with a high hit rate (>85% of characterized antibodies bound the target). By facilitating access to the underexplored ASC compartment, the approach enables efficient antibody discovery and immunological studies into the generation of protective antibodies.

The Role of Semaphorin 6D (Sema6D) in Non-Muscle-Invasive Bladder Cancer-A Preliminary Study on Human Plasma and Urine.

The incidence of bladder cancer worldwide in the last three decades has been increasing in both men and women. So far, there is no established non-invasive bladder cancer biomarker in daily clinical practice. Semaphorin 6D (sema6D) is a transmembrane protein that belongs to the class VI semaphorins. The aim of this study was to evaluate for the first time the potential role of sema6D in bladder cancer. The study group consisted of 40 patients with non-muscle-invasive bladder cancer (NMIBC) and the control group of 20 patients without malignancies. There was a statistically significantly higher urinary sema6D concentration in patients than controls (p < 0.05) but no significant difference in plasma 6D. There were no statistically significant differences in urinary or plasma concentration of sema6D between low- or high-grade cancer and according to the tumor stage in TNM classification. There was a statistically significant negative correlation between plasma sema6D and age of patients (R = -0.6; p = 0.019). Plasma sema6D does not seem to be useful in the clinical practice at this point. However, the urinary sema6D concentration could potentially serve as a marker of NMIBC used for diagnostic purposes, monitoring, and early relapse detection or the assessment of the treatment efficacy. Urinary sema6D is probably not associated with the grading or staging of NMIBC, so it cannot be used for the prediction of disease prognosis.

Neutrophils and galectin-3 defend mice from lethal bacterial infection and humans from acute respiratory failure.

Respiratory infection by Pseudomonas aeruginosa, common in hospitalized immunocompromised and immunocompetent ventilated patients, can be life-threatening because of antibiotic resistance. This raises the question of whether the host's immune system can be educated to combat this bacterium. Here we show that prior exposure to a single low dose of lipopolysaccharide (LPS) protects mice from a lethal infection by P. aeruginosa. LPS exposure trained the innate immune system by promoting expansion of neutrophil and interstitial macrophage populations distinguishable from other immune cells with enrichment of gene sets for phagocytosis- and cell-killing-associated genes. The cell-killing gene set in the neutrophil population uniquely expressed Lgals3, which encodes the multifunctional antibacterial protein, galectin-3. Intravital imaging for bacterial phagocytosis, assessment of bacterial killing and neutrophil-associated galectin-3 protein levels together with use of galectin-3-deficient mice collectively highlight neutrophils and galectin-3 as central players in LPS-mediated protection. Patients with acute respiratory failure revealed significantly higher galectin-3 levels in endotracheal aspirates (ETAs) of survivors compared to non-survivors, galectin-3 levels strongly correlating with a neutrophil signature in the ETAs and a prognostically favorable hypoinflammatory plasma biomarker subphenotype. Taken together, our study provides impetus for harnessing the potential of galectin-3-expressing neutrophils to protect from lethal infections and respiratory failure.

Sexual dysfunctions in psoriatic patients.

Introduction: Psoriasis is one the most common skin diseases associated with a great decrease in the quality of patients' lives. Methods: We aimed to study sexual dysfunctions in psoriatic patients using the Female Sexual Function Index (FSFI) for women and the International Index of Erectile Function (IIEF) for men via an anonymous online survey. The study included 80 psoriatic patients and 75 controls without dermatoses. Results: There was a downward trend in the total IIEF score in psoriatic men compared to controls. 58% of male patients and 76% of controls had a normal IIEF score. There was no significant difference in IIEF between patients treated and not with systemic agents. 62% of female patients had a decreased FSFI score, whereas in the control group, the majority of subjects (54%) had a normal FSFI score. There was no significant difference in FSFI score between patients and controls. Female patients treated with systemic antipsoriatic agents had significantly worse lubrication, satisfaction with sexual life, and pain. Discussion: Our study has shown that the majority of questioned female psoriatic patients had sexual dysfunction according to FSFI, particularly they had worse satisfaction with sexual life and less sexual desire compared to women without psoriasis. The majority of male patients did not have sexual dysfunction according to IIEF, however, they had significantly worse overall satisfaction with sexual life and confidence to keep an erection. Systemic antipsoriatic treatment does not probably influence sexual dysfunctions in men but it does in women although we were not able to assess the severity or resolution of lesions after those treatments. However embarrassing, psoriatic patients should be questioned about their sexual lives by dermatologists, and more studies are needed to explore this matter.

Ultrahigh Throughput Evolution of Tryptophan Synthase in Droplets via an Aptamer Sensor.

Tryptophan synthase catalyzes the synthesis of a wide array of noncanonical amino acids and is an attractive target for directed evolution. Droplet microfluidics offers an ultrahigh throughput approach to directed evolution (up to 107 experiments per day), enabling the search for biocatalysts in wider regions of sequence space with reagent consumption minimized to the picoliter volume (per library member). While the majority of screening campaigns in this format on record relied on an optically active reaction product, a new assay is needed for tryptophan synthase. Tryptophan is not fluorogenic in the visible light spectrum and thus falls outside the scope of conventional droplet microfluidic readouts, which are incompatible with UV light detection at high throughput. Here, we engineer a tryptophan DNA aptamer into a sensor to quantitatively report on tryptophan production in droplets. The utility of the sensor was validated by identifying five-fold improved tryptophan synthases from ∼100,000 protein variants. More generally, this work establishes the use of DNA-aptamer sensors with a fluorogenic read-out in widening the scope of droplet microfluidic evolution.

Hemoglobin scavenger receptor CD163 as a potential biomarker of hemolysis-induced hepatobiliary injury in sickle cell disease.

Sickle cell disease (SCD)-associated chronic hemolysis promotes oxidative stress, inflammation, and thrombosis leading to organ damage, including liver damage. Hemoglobin scavenger receptor CD163 plays a protective role in SCD by scavenging both hemoglobin-haptoglobin complexes and cell-free hemoglobin. A limited number of studies in the past have shown a positive correlation of CD163 expression with poor disease outcomes in patients with SCD. However, the role and regulation of CD163 in SCD-related hepatobiliary injury have not been fully elucidated yet. Here we show that chronic liver injury in SCD patients is associated with elevated levels of hepatic membrane-bound CD163. Hemolysis and increase in hepatic heme, hemoglobin, and iron levels elevate CD163 expression in the SCD mouse liver. Mechanistically we show that heme oxygenase-1 (HO-1) positively regulates membrane-bound CD163 expression independent of nuclear factor erythroid 2-related factor 2 (NRF2) signaling in SCD liver. We further demonstrate that the interaction between CD163 and HO-1 is not dependent on CD163-hemoglobin binding. These findings indicate that CD163 is a potential biomarker of SCD-associated hepatobiliary injury. Understanding the role of HO-1 in membrane-bound CD163 regulation may help identify novel therapeutic targets for hemolysis-induced chronic liver injury.

Droplet microfluidic system for high throughput and passive selection of bacteria producing biosurfactants.

Traditional methods for the enrichment of microorganisms rely on growth in a selective liquid medium or on an agar plate, followed by tedious characterization. Droplet microfluidic techniques have been recently used to cultivate microorganisms and preserve enriched bacterial taxonomic diversity. However, new methods are needed to select droplets comprising not only growing microorganisms but also those exhibiting specific properties, such as the production of value-added compounds. We describe here a droplet microfluidic screening technique for the functional selection of biosurfactant-producing microorganisms, which are of great interest in the bioremediation and biotechnology industries. Single bacterial cells are first encapsulated into picoliter droplets for clonal cultivation and then passively sorted at high throughput based on changes in interfacial tension in individual droplets. Our method expands droplet-based microbial enrichment with a novel approach that reduces the time and resources needed for the selection of surfactant-producing bacteria.

Dry eye syndrome symptoms in patients with psoriasis.

BACKGROUND: Psoriasis is one of the most common dermatoses associated with a variety of comorbidities. There have been some reports on its possible association with ocular disorders however dry eye syndrome (DES) in such patients has been poorly investigated. OBJECTIVES: To investigate the frequency of DES symptoms in psoriatic patients, also regarding psoriasis severity in PASI, manifestation and therapy. METHODS: 40 patients with psoriasis and 40 volunteers without dermatoses were enrolled in the study. They completed Ocular Surface Disease Index (OSDI) questionnaire and were objectively examined by IDRA® device to perform automatic interferometry, automatic meibography of lower eyelid glands, non-invasive break-up time (NIBUT), blink quality and tear meniscus height. RESULTS: Patients with psoriasis had statistically significantly thicker lipid layer (p = 0.0042 left eye, p = 0.0313 right eye) and greater loss of Meibomian glands compared to controls (p = 0.0128 left eye, p = 0.048 right eye). The patients had lower, although insignificantly, eye blink quality and tear meniscus height than the control group, as well as shorter NIBUT and higher score in OSDI. After the division of patients into two groups-with or without nails involvement/psoriatic arthritis/systemic treatment- we did not observe any significant differences between the groups. PASI did not correlate with any DES parameter. CONCLUSIONS: This is the first study of DES symptoms with an objective IDRA® analyzer. We managed to observe that patients with psoriasis have thicker lipid layer and higher Meibomian glands' loss in lower eyelids. Based on all assessed objective and subjective parameters psoriatics do not seem to have an increased risk of DES. The presence of psoriatic arthritis or nail involvement does not seem to be a predisposing factor for DES development. PASI probably cannot be a prognostic factor for any of the DES-associated parameters. Nevertheless, DES in psoriasis requires further research on bigger samples to establish reliable recommendations.