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Infected macrophages transition into aerobic glycolysis, a metabolic program crucial for control of bacterial infection. However, antimicrobial mechanisms supported by aerobic glycolysis are unclear. Methylglyoxal is a highly toxic aldehyde that modifies proteins and DNA and is produced as a side-product of glycolysis. Here we show that despite the toxicity of this aldehyde, infected macrophages generate high levels of methylglyoxal during aerobic glycolysis while downregulating the detoxification system. We use targeted mutations in mice to modulate methylglyoxal generation and show that reducing methylglyoxal production by the host promotes survival of Listeria monocytogenes and Mycobacterium tuberculosis , whereas increasing methylglyoxal levels improves control of bacterial infection. Furthermore, we show that bacteria that are unable to detoxify methylglyoxal are avirulent and experience up to 1000-fold greater genomic mutation frequency during infection. Taken together, these results suggest that methylglyoxal is an antimicrobial innate immune effector that defends the host against bacterial pathogens.

Background: Education about the prevalent chronic disease of obesity is still minimal and variable in medical school curricula. In a student-led effort with faculty support, the authors designed and implemented an obesity medicine elective at Case Western Reserve University School of Medicine (CWRU). The 10-week elective, taught by seven physicians and one dietitian, was offered in January 2023 to medical students and included: weekly lectures, an interactive session with a patient, shadowing in obesity medicine practices, attendance at a distance-learning intensive behavioral lifestyle program, student presentations, and a final written reflection. The purpose of this study was to analyze the elective reflections and identify themes about the elective's value and areas to improve., Methods: The authors analyzed reflections from the 20 medical students that completed the elective via qualitative thematic analysis. The analysis was performed using the Braun and Clarke six-phase framework: (1) become familiar with the data, (2) generate initial codes, (3) search for themes, (4) review themes, (5) define themes, and (6) write-up., Results: The themes identified were improved: (1) understanding of obesity as a chronic disease, (2) knowledge about treatment options for obesity (3) confidence in compassionate obesity counseling skills, and (4) skills to confront weight bias. Theme (5) consisted of highlights (hearing from experts, practicing evidence-based medicine, and interacting with patients), and areas to improve (session length, presentation format, more peer-to-peer interaction, and more diverse patient interactions)., Conclusions: Medical student assessments of a new obesity medicine elective described improved attitudes, knowledge, and skills to address obesity and obesity bias. Students were very satisfied and contributed ideas for improvements. This elective structure and evaluation method is a feasible model to provide medical students with meaningful experiences related to obesity., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Objective: Continuous glucose monitoring (CGM) has demonstrated benefits in managing inpatient diabetes. We initiated this single-arm pilot feasibility study during the COVID-19 pandemic in 11 patients with diabetes to determine the feasibility and accuracy of real-time CGM in patients who underwent cardiac surgery and whose care was being transitioned from the intensive care unit., Methods: A Clarke error grid analysis was used to compare CGM and point-of-care measurements. The mean absolute relative difference (MARD) of the paired measurements was calculated to assess the accuracy of CGM for glucose measurements during the first 24 hours on CGM, the remaining time on CGM, and for different chronic kidney disease (CKD) strata., Results: Overall MARD between point-of-care and CGM measurements was 14.80%. MARD for patients without CKD IV and V with an estimated glomerular filtration rate (eGFR) of ≥20 mL/min/1.73 m 2 was 12.13%. Overall, 97% of the CGM values were within the no-risk zone of the Clarke error grid analysis. For the first 24 hours, a sensitivity analysis of the overall MARD for all patients and those with an eGFR of ≥20 mL/min/1.73 m 2 was 15.42% ± 14.44% and 12.80% ± 7.85%, respectively. Beyond the first 24 hours, overall MARD for all patients and those with an eGFR of ≥20 mL/min/1.73 m 2 was 14.54% ± 13.21% and 11.86% ± 7.64%, respectively., Conclusion: CGM has shown great promise in optimizing inpatient diabetes management in the noncritical care setting and after the transition of care from the intensive care unit with high clinical reliability and accuracy. More studies are needed to further assess CGM in patients with advanced CKD., (Copyright © 2022 AACE. Published by Elsevier Inc. All rights reserved.)

Tuberculosis consistently causes more deaths worldwide annually than any other single pathogen, making new effective vaccines an urgent priority for global public health. Among potential adjuvants, STING-activating cyclic dinucleotides (CDNs) uniquely stimulate a cytosolic sensing pathway activated only by pathogens. Recently, we demonstrated that a CDN-adjuvanted protein subunit vaccine robustly protects against tuberculosis infection in mice. In this study, we delineate the mechanistic basis underlying the efficacy of CDN vaccines for tuberculosis. CDN vaccines elicit CD4 T cells that home to lung parenchyma and penetrate into macrophage lesions in the lung. Although CDNs, like other mucosal vaccines, generate B cell-containing lymphoid structures in the lungs, protection is independent of B cells. Mucosal vaccination with a CDN vaccine induces Th1, Th17, and Th1-Th17 cells, and protection is dependent upon both IL-17 and IFN-γ. Single-cell RNA sequencing experiments reveal that vaccination enhances a metabolic state in Th17 cells reflective of activated effector function and implicate expression of Tnfsf8 (CD153) in vaccine-induced protection. Finally, we demonstrate that simply eliciting Th17 cells via mucosal vaccination with any adjuvant is not sufficient for protection. A vaccine adjuvanted with deacylated monophosphoryl lipid A (MPLA) failed to protect against tuberculosis infection when delivered mucosally, despite eliciting Th17 cells, highlighting the unique promise of CDNs as adjuvants for tuberculosis vaccines., (Copyright © 2022 by The American Association of Immunologists, Inc.)

Cholesterol-dependent cytolysins (CDCs) are essential virulence factors for many human pathogens like Streptococcus pneumoniae (pneumolysin, PLY), Streptococcus pyogenes (streptolysin O, SLO), and Listeria monocytogenes (Listeriolysin, LLO) and induce cytolysis and inflammation. Recently, we identified that pneumococcal PLY interacts with the mannose receptor (MRC-1) on specific immune cells thereby evoking an anti-inflammatory response at sublytic doses. Here, we identified the interaction sites between MRC-1 and CDCs using computational docking. We designed peptides from the CTLD4 domain of MRC-1 that binds to PLY, SLO, and LLO, respectively. In vitro, the peptides blocked CDC-induced cytolysis and inflammatory cytokine production by human macrophages. Also, they reduced PLY-induced damage of the epithelial barrier integrity as well as blocked bacterial invasion into the epithelium in a 3D lung tissue model. Pre-treatment of human DCs with peptides blocked bacterial uptake via MRC-1 and reduced intracellular bacterial survival by targeting bacteria to autophagosomes. In order to use the peptides for treatment in vivo, we developed calcium phosphate nanoparticles (CaP NPs) as peptide nanocarriers for intranasal delivery of peptides and enhanced bioactivity. Co-administration of peptide-loaded CaP NPs during infection improved survival and bacterial clearance in both zebrafish and mice models of pneumococcal infection. We suggest that MRC-1 peptides can be employed as adjunctive therapeutics with antibiotics to treat bacterial infections by countering the action of CDCs., (© 2020 The Authors. Published under the terms of the CC BY 4.0 license.)

Intercellular communication through the secretion of soluble factors plays a vital role in a wide range of biological processes (e.g., homeostasis, immune response), yet identification and quantification of many of these factors can be challenging due to their degradation or sequestration in cell culture media prior to analysis. Here, we present a customizable bead-based system capable of simultaneously binding to live cells (through antibody-mediated cell tethering) and capturing cell-secreted molecules. Our functionalized beads capture secreted molecules (e.g., hepatocyte growth factor secreted by fibroblasts) that are diminished when sampled via traditional supernatant analysis techniques ( p < 0.05), effectively rescuing a reduced signal in the presence of neutralizing components in the cell culture media. Our system enables capture and analysis of molecules integral to chemical communication that would otherwise be markedly decreased prior to analysis.

Chronic lung diseases are a leading cause of morbidity and mortality across the globe, encompassing a diverse range of conditions from infections with pathogenic microorganisms to underlying genetic disorders. The respiratory tract represents an active interface with the external environment having the primary immune function of resisting pathogen intrusion and maintaining homeostasis in response to the myriad of stimuli encountered within its microenvironment. To perform these vital functions and prevent lung disorders, a chemical and biological cross-talk occurs in the complex milieu of the lung that mediates and regulates the numerous cellular processes contributing to lung health. In this review, we will focus on the role of cross-talk in chronic lung infections, and discuss how different cell types and signaling pathways contribute to the chronicity of infection(s) and prevent effective immune clearance of pathogens. In the lung microenvironment, pathogens have developed the capacity to evade mucosal immunity using different mechanisms or virulence factors, leading to colonization and infection of the host; such mechanisms include the release of soluble and volatile factors, as well as contact dependent (juxtracrine) interactions. We explore the diverse modes of communication between the host and pathogen in the lung tissue milieu in the context of chronic lung infections. Lastly, we review current methods and approaches used to model and study these host-pathogen interactions in vitro , and the role of these technological platforms in advancing our knowledge about chronic lung diseases., (Copyright © 2020 Berry, Haack, Theberge, Brighenti and Svensson.)

Tuberculosis (TB) is one of the most potent infectious diseases in the world, causing more deaths than any other single infectious agent. TB infection is caused by inhalation of Mycobacterium tuberculosis (Mtb) and subsequent phagocytosis and migration into the lung tissue by innate immune cells (e.g., alveolar macrophages, neutrophils, and dendritic cells), resulting in the formation of a fused mass of immune cells known as the granuloma. Considered the pathological hallmark of TB, the granuloma is a complex microenvironment that is crucial for pathogen containment as well as pathogen survival. Disruption of the delicate granuloma microenvironment via numerous stimuli, such as variations in cytokine secretions, nutrient availability, and the makeup of immune cell population, can lead to an active infection. Herein, we present a novel in vitro model to examine the soluble factor signaling between a mycobacterial infection and its surrounding environment. Adapting a newly developed suspended microfluidic platform, known as Stacks, we established a modular microscale infection model containing human immune cells and a model mycobacterial strain that can easily integrate with different microenvironmental cues through simple spatial and temporal "stacking" of each module of the platform. We validate the establishment of suspended microscale (4 μL) infection cultures that secrete increased levels of proinflammatory factors IL-6, VEGF, and TNFα upon infection and form 3D aggregates (granuloma model) encapsulating the mycobacteria. As a proof of concept to demonstrate the capability of our platform to examine soluble factor signaling, we cocultured an in vitro angiogenesis model with the granuloma model and quantified morphology changes in endothelial structures as a result of culture conditions ( P < 0.05 when comparing infected vs. uninfected coculture systems). We envision our modular in vitro granuloma model can be further expanded and adapted for studies focusing on the complex interplay between granulomatous structures and their surrounding microenvironment, as well as a complementary tool to augment in vivo signaling and mechanistic studies., (Copyright © 2020 Berry, Gower, Su, Seshadri and Theberge.)

Background: In 2016, HIV-2 nucleic acid testing (NAT) was added to a shared service program that conducts HIV-1 NAT for public health laboratories performing the recommended algorithm for diagnosing HIV. Here, we evaluate the usefulness of HIV-2 NAT in this program as compared with HIV-1 NAT., Methods: Specimens eligible for HIV-1 NAT were reactive on an HIV-1/2 antibody or antigen/antibody initial test and nonreactive or indeterminate on a supplemental antibody test or were reactive for HIV-1 antigen-only on an HIV-1/2 antigen/antibody initial test. Specimens eligible for HIV-2 NAT were reactive on an initial test, HIV-2 indeterminate or HIV indeterminate on a supplemental antibody test and had no detectable HIV-1 RNA or were reactive for HIV-2 antibody on an HIV-1/2 antigen/antibody test, and this reactivity was not confirmed with a supplemental antibody assay. All specimens were tested in a reference laboratory using APTIMA HIV-1 qualitative RNA and/or a validated qualitative HIV-2 RNA real-time PCR assay., Results: During 2016 to 2019, HIV-1 RNA was detected in 234 (14%) of 1731 specimens tested. HIV-2 RNA was not detected in 52 specimens tested. Median time from specimen collection to reporting of HIV-1 and HIV-2 NAT results by year ranged from 9 to 10 days and from 22 to 27 days, respectively. Two specimens with HIV-2 indeterminate results on a supplemental antibody test had detectable HIV-1 RNA., Conclusions: A shared service model for HIV-1 NAT is both feasible and beneficial for public health laboratories. However, because no HIV-2 infections were detected, our data suggest that this program should reconsider the usefulness of HIV-2 NAT testing.

Droplet-based microfluidics enables compartmentalization and controlled manipulation of small volumes. Open microfluidics provides increased accessibility, adaptability, and ease of manufacturing compared to closed microfluidic platforms. Here, we begin to build a toolbox for the emerging field of open channel droplet-based microfluidics, combining the ease of use associated with open microfluidic platforms with the benefits of compartmentalization afforded by droplet-based microfluidics. We develop fundamental microfluidic features to control droplets flowing in an immiscible carrier fluid within open microfluidic systems. Our systems use capillary flow to move droplets and carrier fluid through open channels and are easily fabricated through 3D printing, micromilling, or injection molding; further, droplet generation can be accomplished by simply pipetting an aqueous droplet into an empty open channel. We demonstrate on-chip incubation of multiple droplets within an open channel and subsequent transport (using an immiscible carrier phase) for downstream experimentation. We also present a method for tunable droplet splitting in open channels driven by capillary flow. Additional future applications of our toolbox for droplet manipulation in open channels include cell culture and analysis, on-chip microscale reactions, and reagent delivery., Competing Interests: Conflicts of Interest: The authors acknowledge the following potential conflicts of interest in companies pursuing open microfluidic technologies: EB: Tasso, Inc., Salus Discovery, LLC, and Stacks to the Future, LLC; ABT: Stacks to the Future, LLC.

Extubation failure is associated with considerable morbidity and mortality in postoperative patients with congenital heart disease (CHD). The study purpose was to investigate initial extubation success utilizing neurally adjusted ventilatory assist (NAVA) compared with pressure-regulated volume controlled, synchronized intermittent mandatory ventilation with pressure support (SIMV-PRVC + PS) for ventilatory weaning in patients who required prolonged mechanical ventilation (MV). Also, total days on MV, inotropes, sedation, analgesia, and pediatric intensive care unit (PICU) length of stay (LOS) between both groups were compared. This was a non-randomized pilot study utilizing historical controls (SIMV-PRVC + PS; n  = 40) compared with a prospective study population (NAVA; n  = 35) in a Level I PICU and was implemented to help future trial designs. All patients ( n  = 75) required prolonged MV ≥96 hours due to their complex postoperative course. Ventilator weaning initiation and management was standardized between both groups. Ninety-seven percent of the NAVA group was successfully extubated on the initial attempt, while 80% were in the SIMV-PRVC + PS group ( p  = 0.0317). Patients placed on NAVA were eight times more likely to have successful initial extubation (odds ratio [OR]: 8.50, 95% confidence interval [CI]: 1.01, 71.82). The NAVA group demonstrated a shorter median duration on MV (9.0 vs. 11.0 days, p  = 0.032), PICU LOS (9.0 vs. 13.5 days, p  < 0.0001), and shorter median duration of days on dopamine (8.0 vs. 11.0 days, p  = 0.0022), milrinone (9.0 vs. 12.0 days, p  = 0.0002), midazolam (8.0 vs. 12.0 days, p  < 0.0001), and fentanyl (9.0 vs. 12.5 days, p  < 0.0001) compared with the SIMV-PRVC + PS group. NAVA compared with SIMV-PRVC + PS was associated with a greater initial extubation success rate. NAVA should be considered as a mechanical ventilator weaning strategy in postoperative congenital heart disease (CHD) patients and warrants further investigation.

Background: The capacity of HIV Antigen/Antibody (Ag/Ab) immunoassays (IA) to detect HIV-1 p24 antigen has resulted in improved detection of HIV-1 infections in comparison to Ab-only screening assays. Since its introduction in the US, studies have shown that the Determine HIV-1/2 Ag/Ab Combo assay (Determine Ag/Ab) detects HIV infection earlier than laboratory-based IgM/IgG-sensitive IAs, but its sensitivity for HIV-1 p24 Ag detection is reduced compared to laboratory-based Ag/Ab assays. However, further evaluation is needed to assess its capacity to detect acute HIV-1 infection., Objective: To assess the performance of Determine Ag/Ab in serum from acute HIV-1 infections., Study Design: Select serum specimens that screened reactive on a laboratory-based Ag/Ab IA or IgM/IgG Ab-only IA, with a negative or indeterminate supplemental antibody test and detectable HIV-1 RNA were retrospectively tested with Determine Ag/Ab. Results were compared with those of the primary screening immunoassay to evaluate concordance within this set of algorithm-defined acute infections., Results: Of 159 algorithm-defined acute HIV-1 specimens, Determine Ag/Ab was reactive for 105 resulting in 66.0% concordance. Of 125 that were initially detected by a laboratory-based Ag/Ab IA, 81 (64.8%) were reactive by Determine Ag/Ab. A total of 34 acute specimens were initially detected by a laboratory-based IgM/IgG Ab-only IA and 24 (70.6%) of those were reactive by Determine Ag/Ab., Conclusions: Due to their enhanced sensitivity, laboratory-based Ag/Ab IAs continue to be preferred over the Determine Ag/Ab as the screening method used by laboratories conducting HIV diagnostic testing on serum and plasma specimens., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Cellular communication between multiple cell types is a ubiquitous process that is responsible for vital physiological responses observed in vivo (e.g., immune response, organ function). Many in vitro coculture strategies have been developed, both in traditional culture and microscale systems, and have shown the potential to recreate some of the physiological behaviors of organs or groups of cells. A fundamental limitation of current systems is the difficulty of reconciling the additional engineering requirements for creating soluble factor signaling systems (e.g., segregated cell culture) with the use of well-characterized materials and platforms that have demonstrated successful results and biocompatibility in assays. We present a new open-microfluidic platform, the Monorail Device, that is placed in any existing well plate or Petri dish and enables patterning of segregated coculture regions, thereby allowing the direct upgrade of monoculture experiments into multiculture assays. Our platform patterns biocompatible hydrogel walls via microfluidic spontaneous capillary flow (SCF) along a rail insert set inside commercially available cultureware, creating customized pipette-accessible cell culture chambers that require fewer cells than standard macroscale culture. Importantly, the device allows the use of native surfaces without additional modification or treatments, while creating permeable dividers for the diffusion of soluble factors. Additionally, the ease of patterning afforded by our platform makes reconfiguration of the culture region as simple as changing the rail insert. We demonstrate the ability of the device to pattern flows on a variety of cell culture surfaces and create hydrogel walls in complex and precise shapes. We characterize the physical parameters that enable a reproducible SCF-driven flow and highlight specialized design features that increase the ease of use of the device and control of the open microfluidic flow. Further, we present the performance of our platform according to useful coculture criteria, including permeability and integrity of our hydrogel walls and surface-sensitive cell culture. Lastly, we show the potential of this type of platform to create modular multikingdom culture systems that can be used to study soluble factor signaling between mammalian cells, bacteria, and fungi, as well as the potential for adaptation of this technology by researchers across multiple fields.

The quantification of blood cells provides critical information about a patient's health status. Sophisticated analytical equipment, such as hematology analyzers, have been developed to perform these measurements, but limited-resource settings often lack the infrastructure required to purchase, operate, and maintain instrumentation. To address these practical challenges, paper-based microfluidic devices have emerged as a platform to develop diagnostic assays specifically for use at the point-of-care. To date, paper-based microfluidic devices have been used broadly in diagnostic assays that apply immunoassay, clinical chemistry, and electrochemistry techniques. The analysis of cells, however, has been largely overlooked. In this communication, we demonstrate a paper-based microfluidic device that enables the controlled transport of red blood cells (RBCs) and the measurement of the hematocrit-the ratio of RBC packed cell volume to total volume of whole blood. The properties of paper, device treatment, and device geometry affect the overall extent and reproducibility of transport of RBCs. Ultimately, we developed an inexpensive (US$0.03 per device) thermometer-styled device where the distance traveled by RBCs is proportional to the hematocrit. These results provide a foundation for the design of paper-based microfluidic devices that enable the separation and detection of cells in limited-resource settings.