Monocyte Anisocytosis Changes in Patients after Major Burn Injuries.

The recovery of patients after severe burns is a long and complex process. Recently, genomic analysis of white blood cells from burn and trauma patients revealed excessive and prolonged innate immune activation in patients with complicated outcomes. However, translating this knowledge into practical biomarkers has not been possible yet. Although several biomarkers for monitoring burn patients have been proposed, their ability to accurately distinguish between inflammation stemming from initial tissue destruction, infections, and organ failure complications is limited. Here, we focused on monocytes, critical innate immune cells in the response to burn injured tissues. We measured the monocyte anisocytosis (quantified as monocyte distribution width, MDW, a recently emerged marker of sepsis) throughout the recovery of patients from the time of burn injury until the end of the hospital stay. We observed that MDW increases in patients during the first week after major burns. Among the patients with major burns who survive, MDW starts decreasing in the second week and normalizes by the end of the hospital stay. The duration of hospital stay appears to be proportional to how fast MDW decreases during the second week after the injury. We also found that MDW decreases significantly in most patients after excision and debridement surgeries but not after allo- and auto-graft surgeries. Moreover, high MDW values correlated with a higher rate of positive microbiology blood culture samples and respiratory infections. These findings underscore the importance of monitoring MDW as a potential biomarker for the risk of complications during burn patient recovery.

Microfluidic Assays for Probing Neutrophil-Borrelia Interactions in Blood During Lyme Disease.

Human neutrophils are highly sensitive to the presence of Borrelia burgdorferi (Bb), the agent of Lyme disease (LD), in tissues. Although Bb is also found in the blood of LD patients, far less is known about how neutrophils respond to Bb in the presence of blood. In this study, we employed microfluidic tools to probe the interaction between human neutrophils and Bb and measured the activation of human neutrophils in blood samples from patients. We found that neutrophils migrate vigorously toward Bb in the presence of serum, and this process was complement-dependent. Preventing complement factor 5 cleavage or blocking complement receptors decreased neutrophil's ability to interact with Bb. We also found that spiking Bb directly into the blood from healthy donors induced spontaneous neutrophil motility. This response to Bb was also complement-dependent. Preventing complement factor 5 cleavage decreased spontaneous neutrophil motility in Bb-spiked blood. Moreover, we found that neutrophils in blood samples from acute LD patients displayed spontaneous motility patterns similar to those observed in Bb-spiked samples. Neutrophil motility was more robust in blood samples from LD patients than that measured in healthy and ill controls, validating the utility of the microfluidic assay for the study of neutrophil-Bb interactions in the presence of blood.

Human Neutrophils Respond to Complement Activation and Inhibition in Microfluidic Devices.

Complement activation is key to anti-microbial defenses by directly acting on microbes and indirectly by triggering cellular immune responses. Complement activation may also contribute to the pathogenesis of numerous inflammatory and immunological diseases. Consequently, intense research focuses on developing therapeutics that block pathology-causing complement activation while preserving anti-microbial complement activities. However, the pace of research is slowed down significantly by the limitations of current tools for evaluating complement-targeting therapeutics. Moreover, the effects of potential therapeutic agents on innate immune cells, like neutrophils, are not fully understood. Here, we employ microfluidic assays and measure chemotaxis, phagocytosis, and swarming changes in human neutrophils ex vivo in response to various complement-targeting agents. We show that whereas complement factor 5 (C5) cleavage inhibitor eculizumab blocks all neutrophil anti-microbial functions, newer compounds like the C5 cleavage inhibitor RA101295 and C5a receptor antagonist avacopan inhibit chemotaxis and swarming while preserving neutrophil phagocytosis. These results highlight the utility of microfluidic neutrophil assays in evaluating potential complement-targeting therapeutics.

Neutrophil dysfunction in cystic fibrosis.

BACKGROUND: Excessive neutrophil inflammation is the hallmark of cystic fibrosis (CF) airway disease. Novel technologies for characterizing neutrophil dysfunction may provide insight into the nature of these abnormalities, revealing a greater mechanistic understanding and new avenues for CF therapies that target these mechanisms. METHODS: Blood was collected from individuals with CF in the outpatient clinic, CF individuals hospitalized for a pulmonary exacerbation, and non-CF controls. Using microfluidic assays and advanced imaging technologies, we characterized 1) spontaneous neutrophil migration using microfluidic motility mazes, 2) neutrophil migration to and phagocytosis of Staphylococcal aureus particles in a microfluidic arena, 3) neutrophil swarming on Candida albicans clusters, and 4) Pseudomonas aeruginosa-induced neutrophil transepithelial migration using micro-optical coherence technology (µOCT). RESULTS: Participants included 44 individuals: 16 Outpatient CF, 13 Hospitalized CF, and 15 Non-CF individuals. While no differences were seen with spontaneous migration, CF neutrophils migrated towards S. aureus particles more quickly than non-CF neutrophils (p < 0.05). CF neutrophils, especially Hospitalized CF neutrophils, generated significantly larger aggregates around S. aureus particles over time. Hospitalized CF neutrophils were more likely to have dysfunctional swarming (p < 0.01) and less efficient clearing of C. albicans (p < 0.0001). When comparing trans-epithelial migration towards Pseudomonas aeruginosa epithelial infection, Outpatient CF neutrophils displayed an increase in the magnitude of transmigration and adherence to the epithelium (p < 0.05). CONCLUSIONS: Advanced technologies for characterizing CF neutrophil function reveal significantly altered migratory responses, cell-to-cell clustering, and microbe containment. Future investigations will probe mechanistic basis for abnormal responses in CF to identify potential avenues for novel anti-inflammatory therapeutics.

ß-Dispersion of blood during sedimentation.

Aggregation of human red blood cells (RBC) is central to various pathological conditions from bacterial infections to cancer. When left at low shear conditions or at hemostasis, RBCs form aggregates, which resemble stacks of coins, known as 'rouleaux'. We experimentally examined the interfacial dielectric dispersion of aggregating RBCs. Hetastarch, an RBC aggregation agent, is used to mimic conditions leading to aggregation. Hetastrach concentration is incrementally increased in blood from healthy donors to measure the sensitivity of the technique. Time lapse electrical impedance measurements were conducted as red blood cells form rouleaux and sediment in a PDMS chamber. Theoretical modeling was used for obtaining complex permittivity of an effective single red blood cell aggregate at various concentrations of hetastarch. Time response of red blood cells' impedance was also studied to parametrize the time evolution of impedance data. Single aggregate permittivity at the onset of aggregation, evolution of interfacial dispersion parameters, and sedimentation kinetics allowed us to distinguish differential aggregation in blood.

Measuring spontaneous neutrophil motility signatures from a drop of blood using microfluidics.

Neutrophils play an essential role in the protection against infection, as they are the most numerous circulating white blood cell population and the first responders to injury. Their numbers in blood are frequently measured in the clinic and used as an indicator of ongoing infections. During inflammation and sepsis, the ability of neutrophils to migrate is disrupted, which may increase the risk of infection, even when the neutrophil count is normal. However, measurements of neutrophil migration in patients are rarely performed because of the challenges of performing the migration assays in a clinical setting. Here, we describe a microfluidic assay that measures the spontaneous neutrophil migration signatures associated with sepsis. The assay uses one droplet of patient's blood in a microfluidic device, which circumvents the need for neutrophil isolation from blood. This assay may also be useful for the study of the effect of various immune modulators on neutrophil migration behavior from healthy volunteers and patients.

Multiplex cell microarrays for high-throughput screening.

Microarray technology was developed in the early 1990s to measure the transcription levels of thousands of genes in parallel. The basic premise of high-density arraying has since been expanded to create cell microarrays. Cells on chip are powerful experimental tools for high-throughput and multiplex screening of samples or cellular functions. Miniaturization increases assay throughput while reducing both reagent consumption and cell population heterogeneity effect, making these systems attractive for a wide range of assays, from drug discovery to toxicology, stem cell research and therapy. It is usual to functionalize the surface of a substrate to design cell microarrays. One form of cell microarrays, the transfected cell microarray, wherein plasmid DNA or siRNA spotted on the surface of a substrate is reverse-transfected locally into adherent cells, has become a standard tool for parallel cell-based analysis. With the advent of technology, cells can also be directly spotted onto functionalized surfaces using robotic fluid-dispensing devices or printed directly on bio-ink material. We are providing herein an overview of the latest developments in optical cell microarrays allowing high-throughput and high-content analysis.