Altered transcriptomic immune responses of maintenance hemodialysis patients to the COVID-19 mRNA vaccine.

Background: End-stage renal disease (ESRD) patients experience immune compromise characterized by complex alterations of both innate and adaptive immunity, and results in higher susceptibility to infection and lower response to vaccination. This immune compromise, coupled with greater risk of exposure to infectious disease at hemodialysis (HD) centers, underscores the need for examination of the immune response to the COVID-19 mRNA-based vaccines. Methods: The immune response to the COVID-19 BNT162b2 mRNA vaccine was assessed in 20 HD patients and cohort-matched controls. RNA sequencing of peripheral blood mononuclear cells was performed longitudinally before and after each vaccination dose for a total of six time points per subject. Anti-spike antibody levels were quantified prior to the first vaccination dose (V1D0) and 7 d after the second dose (V2D7) using anti-spike IgG titers and antibody neutralization assays. Anti-spike IgG titers were additionally quantified 6 mo after initial vaccination. Clinical history and lab values in HD patients were obtained to identify predictors of vaccination response. Results: Transcriptomic analyses demonstrated differing time courses of immune responses, with prolonged myeloid cell activity in HD at 1 wk after the first vaccination dose. HD also demonstrated decreased metabolic activity and decreased antigen presentation compared to controls after the second vaccination dose. Anti-spike IgG titers and neutralizing function were substantially elevated in both controls and HD at V2D7, with a small but significant reduction in titers in HD groups (p<0.05). Anti-spike IgG remained elevated above baseline at 6 mo in both subject groups. Anti-spike IgG titers at V2D7 were highly predictive of 6-month titer levels. Transcriptomic biomarkers after the second vaccination dose and clinical biomarkers including ferritin levels were found to be predictive of antibody development. Conclusions: Overall, we demonstrate differing time courses of immune responses to the BTN162b2 mRNA COVID-19 vaccination in maintenance HD subjects comparable to healthy controls and identify transcriptomic and clinical predictors of anti-spike IgG titers in HD. Analyzing vaccination as an in vivo perturbation, our results warrant further characterization of the immune dysregulation of ESRD. Funding: F30HD102093, F30HL151182, T32HL144909, R01HL138628. This research has been funded by the University of Illinois at Chicago Center for Clinical and Translational Science (CCTS) award UL1TR002003.

Megakaryocytes respond during sepsis and display innate immune cell behaviors.

Megakaryocytes (MKs) are precursors to platelets, the second most abundant cells in the peripheral circulation. However, while platelets are known to participate in immune responses and play significant functions during infections, the role of MKs within the immune system remains largely unexplored. Histological studies of sepsis patients identified increased nucleated CD61+ cells (MKs) in the lungs, and CD61+ staining (likely platelets within microthrombi) in the kidneys, which correlated with the development of organ dysfunction. Detailed imaging cytometry of peripheral blood from patients with sepsis found significantly higher MK counts, which we predict would likely be misclassified by automated hematology analyzers as leukocytes. Utilizing in vitro techniques, we show that both stem cell derived MKs (SC MKs) and cells from the human megakaryoblastic leukemia cell line, Meg-01, undergo chemotaxis, interact with bacteria, and are capable of releasing chromatin webs in response to various pathogenic stimuli. Together, our observations suggest that MK cells display some basic innate immune cell behaviors and may actively respond and play functional roles in the pathophysiology of sepsis.

Efficient Front-Rear Coupling in Neutrophil Chemotaxis by Dynamic Myosin II Localization.

Efficient chemotaxis requires rapid coordination between different parts of the cell in response to changing directional cues. Here, we investigate the mechanism of front-rear coordination in chemotactic neutrophils. We find that changes in the protrusion rate at the cell front are instantaneously coupled to changes in retraction at the cell rear, while myosin II accumulation at the rear exhibits a reproducible 9-15-s lag. In turning cells, myosin II exhibits dynamic side-to-side relocalization at the cell rear in response to turning of the leading edge and facilitates efficient turning by rapidly re-orienting the rear. These manifestations of front-rear coupling can be explained by a simple quantitative model incorporating reversible actin-myosin interactions with a rearward-flowing actin network. Finally, the system can be tuned by the degree of myosin regulatory light chain (MRLC) phosphorylation, which appears to be set in an optimal range to balance persistence of movement and turning ability.

Microfluidic arenas for war games between neutrophils and microbes.

Measurements of neutrophil activities such as cell migration and phagocytosis are generally performed using low-content bulk assays, which provide little detail activity at the single cell level, or flow cytometry methods, which have the single cell resolution but lack perspective on the kinetics of the process. Here, we present a microfluidic assay for measuring the essential functions that contribute to the antimicrobial activity of neutrophils: migration towards the target, and killing of microbes. The assay interrogates the interactions between isolated human neutrophils and populations of live, proliferating microbes. The outcome is measured in a binary mode that is reflective of in vivo infections, which are either cleared or endure the host response. The outcome of the interactions is also characterized at single cell resolution for both the neutrophils and the microbes. We applied the assay to test the response of neutrophils from intensive care patients to live Staphylococcus aureus, and observed alterations of antimicrobial neutrophil activity in patients, including those with sepsis. By directly measuring neutrophil activity against live targets at high spatial and temporal resolution, this assay provides unique insights into the life-or-death contest shaping the outcome of interactions between populations of neutrophils and microbes.

A New Test for the Detection of Direct Oral Anticoagulants (Rivaroxaban and Apixaban) in the Emergency Room Setting.

Determining whether a patient has taken a direct oral anticoagulant (DOAC) is critical during the periprocedural and preoperative period in the emergency department. However, the inaccessibility of complete medical records, along with the generally inconsistent sensitivity of conventional coagulation tests to these drugs, complicates clinical decision making and puts patients at risk of uncontrollable bleeding. In this study, we evaluate the utility of inhibitor-II-X (i-II-X), a novel, microfluidics-based diagnostic assay for the detection and identification of Factor Xa inhibitors (FXa-Is) in an acute care setting. DESIGN: First-in-human, 91-patient, single-center retrospective pilot study. SETTING: Emergency room. PATIENTS: Adult patients admitted into the emergency department, which received any clinician-ordered coagulation test requiring a 3.2% buffered sodium citrate blood collection tube. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Plasma samples from patients admitted to the emergency department were screened for the use of FXa-Is, including apixaban and rivaroxaban, within the past 24 hours using our new i-II-X microfluidic test. i-II-X results were then compared with results from conventional coagulation tests, including prothrombin time (PT) and international normalized ratio (INR), which were ordered by treating clinicians, and an anti-Xa assay for rivaroxaban. The i-II-X test detected DOACs in samples collected from the emergency department with 95.20% sensitivity and 100.00% specificity. Unlike PT and INR, i-II-X reliably identified patients who had prolonged clotting times secondary to the presence of a FXa-I. CONCLUSIONS: The i-II-X test overcomes the limitations of currently available coagulation tests and could be a useful tool by which to routinely screen patients for DOACs in emergency and critical care settings. Our new diagnostic approach is particularly relevant in clinical situations where medical records may be unavailable, or where precautions need to be taken prior to invasive interventions, such as specific reversal agent administration.

Diagnosis of sepsis from a drop of blood by measurement of spontaneous neutrophil motility in a microfluidic assay.

Current methods for the diagnosis of sepsis have insufficient precision, causing regular misdiagnoses. Microbiological tests can help diagnose sepsis but are usually too slow to have an impact on timely clinical-decision making. Neutrophils have high sensitivity to infections, yet measurements of neutrophil surface markers, genomic changes, and phenotype alterations have had only a marginal effect on sepsis diagnosis. Here, we report a microfluidic assay that measures the spontaneous motility of neutrophils in the context of plasma, in one droplet of blood. We measured the performance of the assay in two independent cohorts of critically ill patients suspected of sepsis. In the first cohort, we developed a machine-learning-based scoring system (sepsis score) that segregated patients with sepsis from those without sepsis. In the second cohort, we validated the sepsis score in a double-blinded, prospective case-control study. For the 42 patients across the two cohorts, the assay identified sepsis patients with 97% sensitivity and 98% specificity. The neutrophil assay could potentially be used to accurately diagnose and monitor sepsis in larger populations of at-risk patients.

Progressive mechanical confinement of chemotactic neutrophils induces arrest, oscillations, and retrotaxis.

Neutrophils reach the sites of inflammation and infection in a timely manner by navigating efficiently through mechanically complex interstitial spaces, following the guidance of chemical gradients. However, our understanding of how neutrophils that follow chemical cues overcome mechanical obstacles in their path is restricted by the limitations of current experimental systems. Observations in vivo provide limited insights due to the complexity of the tissue environment. Here, we developed microfluidic devices to study the effect of progressive mechanical confinement on the migration patterns of human neutrophils toward chemical attractants. Using these devices, we identified four migration patterns: arrest, oscillation, retrotaxis, and persistent migration. The proportion of these migration patterns is different in patients receiving immunosuppressant treatments after kidney transplant, patients in critical care, and neonatal patients with infections and is distinct from that in healthy donors. The occurrence of these migration patterns is independent of the nuclear lobe number of the neutrophils and depends on the integrity of their cytoskeletal components. Our study highlights the important role of mechanical cues in moving neutrophils and suggests the mechanical constriction-induced migration patterns as potential markers for infection and inflammation.

Rheology of transgenic switchgrass reveals practical aspects of biomass processing.

BACKGROUND: Mechanical properties of transgenic switchgrass have practical implications for biorefinery technologies. Presented are fundamentals for simple (thermo)mechanical measurements of genetically transformed switchgrass. Experimental basics are provided for the novice, where the intention is to promote collaboration between plant biologists and materials scientists. RESULTS: Stem sections were subjected to two stress modes: (1) torsional oscillation in the linear response region, and (2) unidirectional torsion to failure. Specimens were analyzed while submerged/saturated in ethylene glycol, simulating natural hydration and allowing experimental temperatures above 100 °C for an improved view of the lignin glass transition. Down-regulation of the 4-Coumarate:coenzyme A ligase gene (reduced lignin content and altered monomer composition) generally resulted in less stiff and weaker stems. These observations were associated with a reduction in the temperature and activation energy of the lignin glass transition, but surprisingly with no difference in the breadth and intensity of the tan Î´ signal. The results showed promise in further investigations of how rheological methods relate to stem lignin content, composition, and functional properties in the field and in bioprocessing. CONCLUSIONS: Measurements such as these are complicated by small specimen size; however, torsional rheometers (relatively common in polymer laboratories) are well suited for this task. As opposed to the expense and complication of relative humidity control, solvent-submersion rheological methods effectively reveal fundamental structure/property relationships in plant tissues. Demonstrated are low-strain linear methods, and also nonlinear yield and failure analysis; the latter is very uncommon for typical rheological equipment.

Convergent and Divergent Migratory Patterns of Human Neutrophils inside Microfluidic Mazes.

Neutrophils are key cellular components of the innate immune response and characteristically migrate from the blood towards and throughout tissues. Their migratory process is complex, guided by multiple chemoattractants released from injured tissues and microbes. How neutrophils integrate the various signals in the tissue microenvironment and mount effective responses is not fully understood. Here, we employed microfluidic mazes that replicate features of interstitial spaces and chemoattractant gradients within tissues to analyze the migration patterns of human neutrophils. We find that neutrophils respond to LTB4 and fMLF gradients with highly directional migration patterns and converge towards the source of chemoattractant. We named this directed migration pattern convergent. Moreover, neutrophils respond to gradients of C5a and IL-8 with a low-directionality migration pattern and disperse within mazes. We named this alternative migration pattern divergent. Inhibitors of MAP kinase and PI-3 kinase signaling pathways do not alter either convergent or divergent migration patterns, but reduce the number of responding neutrophils. Overlapping gradients of chemoattractants conserve the convergent and divergent migration patterns corresponding to each chemoattractant and have additive effects on the number of neutrophils migrating. These results suggest that convergent and divergent neutrophil migration-patterns are the result of simultaneous activation of multiple signaling pathways.

Trapped Chromatin Fibers Damage Flowing Red Blood Cells.

Neutrophils are the most abundant white blood cells in the circulation and serve antimicrobial functions. One of their antimicrobial mechanisms involves the release of neutrophil extracellular traps (NETs), long chromatin fibers decorated with antimicrobial granular proteins that contribute to the elimination of pathogens. However, the release of NETs has also been associated with disease processes. While recent research has focused on biochemical reactions catalyzed by NETs, significantly less is known about the mechanical effect of NETs in circulation. Here, microfluidic devices and biophysical models are employed to study the consequences of the interactions between NETs trapped in channels and red blood cells (RBCs) flowing in blood over the NETs. It has been found that the RBCs can be deformed and ruptured after interactions with NETs, generating RBC fragments. Significant increases in the number of RBC fragments have also been found in the circulation of patients with conditions in which NETs have been demonstrated to be present in circulation, including sepsis and kidney transplant. Further studies will probe the potential utility of RBC fragments in the diagnostic, monitoring, and treatment of diseases associated with the presence of NETs in circulation.

Neutrophil chemotaxis and transcriptomics in term and preterm neonates.

Neutrophils play a crucial role in combating life-threatening bacterial infections in neonates. Previous studies investigating neonatal cell function have been limited because of restricted volume sampling. Here, using novel microfluidic approaches, we provide the first description of neutrophil chemotaxis and transcriptomics from whole blood of human term and preterm neonates, as well as young adults. Ex vivo percent cell migration, neutrophil velocity, and directionality to N-formylmethionyl-leucyl-phenylalanine were measured from whole blood using time-lapse imaging of microfluidic chemotaxis. Genome-wide expression was also evaluated in CD66b+ cells using microfluidic capture devices. Neutrophils from preterm neonates migrated in fewer numbers compared to term neonates (preterm 12.3%, term 30.5%, P = 0.008) and at a reduced velocity compared to young adults (preterm 10.1 µm/min, adult 12.7 µm/min, P = 0.003). Despite fewer neutrophils migrating at slower velocities, neutrophil directionality from preterm neonates was comparable to adults and term neonates. 3607 genes were differentially expressed among the 3 groups (P < 0.001). Differences in gene expression between neutrophils from preterm and term neonates were consistent with reduced pathogen recognition and antimicrobial activity but not neutrophil migration, by preterm neonates. In summary, preterm neonates have significant disturbances in neutrophil chemotaxis compared to term neonates and adults, and these differences in phenotype appear at the transcriptional level to target inflammatory pathways in general, rather than in neutrophil migration and chemotaxis.

Temporal gradients limit the accumulation of neutrophils towards sources of chemoattractant.

Neutrophil trafficking during inflammation is a highly orchestrated process, coordinating neutrophil recruitment, sterilization of the wound, and inflammation resolution. Although the chemotactic signals guiding neutrophil recruitment to sites of inflammation are relatively well understood, mechanisms controlling cessation of neutrophil recruitment and return to normal tissue physiology remain undefined. To gain insights into these processes, we designed a microfluidic device with an array of chemoattractant reservoirs, which mimics the microenvironment in infected tissues, when multiple clusters of microbes are present. We monitored the temporal dynamics of neutrophil recruitment towards the chemoattractant reservoirs at single cell resolution, for 3 hours. We observed robust neutrophil recruitment that reached a plateau after 1.5 hours, despite the continuous presence of robust chemoattractant gradients around the reservoirs. The timing of the plateau was dependent on the geometry of the devices and was independent from the number of neutrophils. Based on these observations, we ruled out sub-population sensitivity, chemoattractant scavenging, and production of a self-limiting stop signal as potential mechanisms underpinning the plateau in neutrophil recruitment. We found a strong correlation between the temporal stabilization of concentration changes and the plateau in neutrophils recruitment. These results suggest that dynamic aspects of chemoattractant gradients are key for maximizing recruitment during the acute phase of infections and limiting the accumulation of neutrophils as soon as the infection is contained.

Neutrophil Interactions Stimulate Evasive Hyphal Branching by Aspergillus fumigatus.

Invasive aspergillosis (IA), primarily caused by Aspergillus fumigatus, is an opportunistic fungal infection predominantly affecting immunocompromised and neutropenic patients that is difficult to treat and results in high mortality. Investigations of neutrophil-hypha interaction in vitro and in animal models of IA are limited by lack of temporal and spatial control over interactions. This study presents a new approach for studying neutrophil-hypha interaction at single cell resolution over time, which revealed an evasive fungal behavior triggered by interaction with neutrophils: Interacting hyphae performed de novo tip formation to generate new hyphal branches, allowing the fungi to avoid the interaction point and continue invasive growth. Induction of this mechanism was independent of neutrophil NADPH oxidase activity and neutrophil extracellular trap (NET) formation, but could be phenocopied by iron chelation and mechanical or physiological stalling of hyphal tip extension. The consequence of branch induction upon interaction outcome depends on the number and activity of neutrophils available: In the presence of sufficient neutrophils branching makes hyphae more vulnerable to destruction, while in the presence of limited neutrophils the interaction increases the number of hyphal tips, potentially making the infection more aggressive. This has direct implications for infections in neutrophil-deficient patients and opens new avenues for treatments targeting fungal branching.

Technical Advance: Changes in neutrophil migration patterns upon contact with platelets in a microfluidic assay.

Neutrophils are traditionally regarded as the "first responders" of the immune system. However, recent observations revealed that platelets often respond earlier to recruit and activate neutrophils within sites of injury and inflammation. Currently, platelet-neutrophil interactions are studied by intravital microscopy. Although such studies provide exceptional, physiologic in vivo data, they are also laborious and have low throughput. To accelerate platelet-neutrophil interaction studies, we have developed and optimized an ex vivo microfluidic platform with which the interactions between platelets and moving neutrophils are measured at single-cell level in precise conditions and with high throughput. With the use of this new assay, we have evaluated changes in neutrophil motility upon direct contact with platelets. Motility changes include longer distances traveled, frequent changes in direction, and faster neutrophil velocities compared with a standard motility response to chemoattractant fMLP. We also found that the neutrophil-platelet direct interactions are transient and mediated by CD62P-CD162 interactions, localized predominantly at the uropod of moving neutrophils. This "crawling," oscillatory neutrophil behavior upon platelet contact is consistent with previous in vivo studies and validates the use of this new test for the exploration of this interactive relationship.

Open Versus Endovascular or Hybrid Thoracic Aortic Aneurysm Repair.

Thoracic aortic aneurysms are associated with significant morbidity and mortality. There are multiple underlying etiologies, including genetic abnormalities, that have important implications in their natural history. The variable histologic, anatomic, and clinical presentations necessitate careful consideration of available treatment options. Surgical repair of these aneurysms has been the mainstay of treatment; however, these approaches can carry a relatively high risk of morbidity and mortality. Endovascular approaches have now become first-line therapy for descending thoracic aneurysms, and with advancements in graft technology, endovascular approaches are being increasingly employed for hybrid repairs of the aortic arch and even the ascending aorta. However, to date, clinical outcomes from randomized trials and long-term follow-up are limited. As technology continues to advance, there is the potential for further integration of surgical and endovascular treatments so that patients have the best opportunity for a favorable outcome.

Hydrocephalus and Ventriculoperitoneal Shunts: Modes of Failure and Opportunities for Improvement.

Between 0.5 and 4 of every 1000 children are born with hydrocephalus. Hydrocephalus is an over-accumulation of cerebrospinal fluid (CSF) in the ventricles of the brain, which can affect cognitive function, vision, appetite, and cranial nerve function. Left untreated, hydrocephalus can result in death. The current treatment for hydrocephalus uses ventriculoperitoneal (VP) shunts with valves to redirect CSF from the ventricles into the peritoneum. Shunt technology is limited by a number of complications, which include infection after implantation, shunt obstruction due to clot formation or catheter obstruction by scar tissue or choroid plexus, disconnection and tubing migration, and overdrainage or underdrainage of CSF due to valve malfunction. While modifications to surgical procedures and shunt design have been introduced, only modest improvements in outcomes have been observed. Here we provide an overview of hydrocephalus, VP shunts, and their modes of failure, and we identify numerous areas of opportunity for biomedical engineers and physicians to collaborate to improve the performance of VP shunts.