A high-throughput lysosome trafficking assay guides ligand selection and elucidates differences in CD22-targeted nanodelivery.

Targeted nanoparticles offer potential to selectively deliver therapeutics to cells; however, their subcellular fate following endocytosis must be understood to properly design mechanisms of drug release. Here we describe a nanoparticle platform and associated cell-based assay to observe lysosome trafficking of targeted nanoparticles in live cells. The nanoparticle platform utilizes two fluorescent dyes loaded onto PEG-poly(glutamic acid) and PEG-poly(Lysine) block co-polymers that also comprise azide reactive handles on PEG termini to attach antibody-based targeting ligands. Fluorophores were selected to be pH-sensitive (pHrodo Red) or pH-insensitive (Alexafluor 488) to report when nanoparticles enter low pH lysosomes. Dye-labelled block co-polymers were further assembled into polyion complex micelle nanoparticles and crosslinked through amide bond formation to form stable nano-scaffolds for ligand attachment. Cell binding and lysosome trafficking was determined in live cells by fluorescence imaging in 96-well plates and quantification of red- and green-fluorescence signals over time. The platform and assay was validated for selection of optimal antibody-derived targeting ligands directed towards CD22 for nanoparticle delivery. Kinetic analysis of uptake and lysosome trafficking indicated differences between ligand types and the ligand with the highest lysosome trafficking efficiency translated into effective DNA delivery with nanoparticles bearing the optimal ligand.

The ability of this pH-sensitive reporter platform to rapidly screen ligands in nanoparticle format will enable identification and production of targeted NPs with desired lysosome trafficking properties.

Multiomics Characterization of a Less Invasive Microfluidic-Based Cell Sorting Technique.

Fluorescence-activated cell sorting (FACS) is a specialized technique to isolate specific cell subpopulations with a high level of recovery and accuracy. However, the cell sorting procedure can impact the viability and metabolic state of cells. Here, we performed a comparative study and evaluated the impact of traditional high-pressure charged droplet-based and microfluidic chip-based sorting on the metabolic and phosphoproteomic profile of different cell types. While microfluidic chip-based sorted cells more closely resembled the unsorted control group for most cell types tested, the droplet-based sorted cells showed significant metabolic and phosphoproteomic alterations. In particular, greater changes in redox and energy status were present in cells sorted with the droplet-based cell sorter along with larger shifts in proteostasis. 13C-isotope tracing analysis on cells recovering postsorting revealed that the sorter-induced suppression of mitochondrial TCA cycle activity recovered faster in the microfluidic chip-based sorted group. Apart from this, amino acid and lipid biosynthesis pathways were suppressed in sorted cells, with minimum impact and faster recovery in the microfluidic chip-based sorted group. These results indicate microfluidic chip-based sorting has a minimum impact on metabolism and is less disruptive compared to droplet-based sorting.

Learning from Power Signals: An Automated Approach to Electrical Disturbance Identification within a Power Transmission System.

As power quality becomes a higher priority in the electric utility industry, the amount of disturbance event data continues to grow. Utilities do not have the required personnel to analyze each event by hand. This work presents an automated approach for analyzing power quality events recorded by digital fault recorders and power quality monitors operating within a power transmission system. The automated approach leverages rule-based analytics to examine the time and frequency domain characteristics of the voltage and current signals. Customizable thresholds are set to categorize each disturbance event. The events analyzed within this work include various faults, motor starting, and incipient instrument transformer failure. Analytics for fourteen different event types have been developed. The analytics were tested on 160 signal files and yielded an average accuracy of 99%. Continuous nominal signal data analysis was performed using an approach called the cyclic histogram. The cyclic histogram process is intended to be integrated into the digital fault recorders themselves in order to facilitate the detection of subtle signal variations that are too small to trigger a disturbance event and that can occur over hours or days. In addition to reducing memory requirements by a factor of 320, it is anticipated that cyclic histogram processing will aid in identifying incipient events and identifiers. This project is expected to save engineers time by automating the classification of disturbance events and increasing the reliability of the transmission system by providing near real-time detection and identification of disturbances as well as prevention of problems before they occur.

Fentanyl overdose: Temporal effects and prognostic factors in SKH1 mice.

Fentanyl exposure and overdose are growing concerns in public health and occupational safety. This study aimed to establish parameters of fentanyl lethality in SKH1 mice for future overdose research. Lethality was determined using the up-down procedure, with subjects monitored post-administration using pulse oximetry (5 min) and then whole-body plethysmography (40 min). Following the determination of subcutaneous dose-response, [18F]Fluorodeoxyglucose positron emission tomography (18 F-FDG PET) was performed after LD10 fentanyl at 40 min, 6 h, 24 h or 7 days post-dose. LD10 and LD50 were observed to be 110 and 135 mg/kg, respectively, and consistent with four-parameter logistic fit values of 111.2 and 134.6 mg/kg (r2  = 0.9996). Overdose (LD10 or greater) yielded three distinct cardiovascular groups: survival, non-survival with blood oxygen saturation (SpO2) minimum ≥37% and non-survival with SpO2 <37%. Breaths per minute, minute volume and inspiratory quotient were significantly different between surviving and non-surviving animals for up to 40 min post-injection. 18 F-FDG PET revealed decreased glucose uptake in the heart, lungs and brain for up to 24 h. These findings provide critical insights into fentanyl lethality in SKH1 mice, including non-invasive respiratory effects and organ-specific impacts that are invaluable for future translational studies investigating the temporal effects of fentanyl overdose.

Opioids and Vitamin C: Known Interactions and Potential for Redox-Signaling Crosstalk.

Opioids are among the most widely used classes of pharmacologically active compounds both clinically and recreationally. Beyond their analgesic efficacy via µ opioid receptor (MOR) agonism, a prominent side effect is central respiratory depression, leading to systemic hypoxia and free radical generation. Vitamin C (ascorbic acid; AA) is an essential antioxidant vitamin and is involved in the recycling of redox cofactors associated with inflammation. While AA has been shown to reduce some of the negative side effects of opioids, the underlying mechanisms have not been explored. The present review seeks to provide a signaling framework under which MOR activation and AA may interact. AA can directly quench reactive oxygen and nitrogen species induced by opioids, yet this activity alone does not sufficiently describe observations. Downstream of MOR activation, confounding effects from AA with STAT3, HIF1α, and NF-κB have the potential to block production of antioxidant proteins such as nitric oxide synthase and superoxide dismutase. Further mechanistic research is necessary to understand the underlying signaling crosstalk of MOR activation and AA in the amelioration of the negative, potentially fatal side effects of opioids.

Short-term effects of electronic cigarettes on cerebrovascular function: A time course study.

NEW FINDINGS: What is the central question of this study? Acute exposure to electronic cigarettes (Ecigs) triggers abnormal vascular responses in systemic arteries; however, effects on cerebral vessels are poorly understood and time for recovery is not known. We hypothesized that exposure to cigarettes or Ecigs would trigger rapid (<4 h) impairment of the middle cerebral artery (MCA) but that this would resolve by 24 h. What is the main finding and its importance? Cigarettes and Ecigs caused similar degree and duration of MCA impairment. We find it takes ~72 hours after exposure for MCA function to return to normal. This suggests that Ecig use is likely to produce similar adverse vascular health outcomes to those seen with cigarette smoke. ABSTRACT: Temporal influences of electronic cigarettes (Ecigs) on blood vessels are poorly understood. In this study, we evaluated a single episode of cigarette versus Ecig exposure on middle cerebral artery (MCA) reactivity and determined how long after the exposure MCA responses took to return to normal. We hypothesized that cigarette and Ecig exposure would induce rapid (<4 h) reduction in MCA endothelial function and would resolve within 24 h. Sprague-Dawley rats (4 months old) were exposed to either air (n = 5), traditional cigarettes (20 puffs, n = 16) or Ecigs (20-puff group, n = 16; or 60-puff group, n = 12). Thereafter, the cigarette and Ecig groups were randomly assigned for postexposure vessel myography testing on day 0 (D0, 1-4 h postexposure), day 1 (D1, 24-28 h postexposure), day 2 (D2, 48-52 h postexposure) and day 3 (72-76 h postexposure). The greatest effect on endothelium-dependent dilatation was observed within 24 h of exposure (∼50% decline between D0 and D1) for both cigarette and Ecig groups, and impairment persisted with all groups for up to 3 days. Changes in endothelium-independent dilatation responses were less severe (∼27%) and shorter lived (recovering by D2) compared with endothelium-dependent dilatation responses. Vasoconstriction in response to serotonin (5-HT) was similar to endothelium-independent dilatation, with greatest impairment (∼45% for all exposure groups) at D0-D1, returning to normal by D2. These data show that exposure to cigarettes and Ecigs triggers a similar level/duration of cerebrovascular dysfunction after a single exposure. The finding that Ecig (without nicotine) and cigarette (with nicotine) exposure produce the same effects suggesting that nicotine is not likely to be triggering MCA dysfunction, and that vaping (with/without nicotine) has potential to produce the same vascular harm and/or disease as smoking.

Novel mechanisms of action contributing to benralizumab's potent anti-eosinophilic activity.

BACKGROUND: Benralizumab is a humanised, anti-interleukin-5 receptor α monoclonal antibody with anti-eosinophilic activity. Lack of fucose (afucosylation) increases its affinity to CD16a and significantly enhances antibody-dependent cell-mediated cytotoxicity by natural killer (NK) cells. Although benralizumab proved clinically efficacious in clinical trials for patients with severe asthma and hypereosinophilic syndrome, in-depth characterisation of its anti-eosinophilic mechanisms of action remains elusive. METHODS: Here, we further investigated the mechanisms involved in benralizumab's anti-eosinophilic activities by employing relevant primary human autologous cell co-cultures and real-time-lapse imaging combined with flow cytometry. RESULTS: In the presence of NK cells, benralizumab induced potent eosinophil apoptosis as demonstrated by the upstream induction of Caspase-3/7 and upregulation of cytochrome c. In addition, we uncovered a previously unrecognised mechanism whereby benralizumab can induce eosinophil phagocytosis/efferocytosis by macrophages, a process called antibody-dependent cellular phagocytosis. Using live cell imaging, we unravelled the stepwise processes leading to eosinophil apoptosis and uptake by activated macrophages. Through careful observations of cellular co-culture assays, we identified a novel role for macrophage-derived tumour necrosis factor (TNF) to further enhance benralizumab-mediated eosinophil apoptosis through activation of TNF receptor 1 on eosinophils. TNF-induced eosinophil apoptosis was associated with cytochrome c upregulation, mitochondrial membrane depolarisation and increased Caspase-3/7 activity. Moreover, activated NK cells were found to amplify this axis through the secretion of interferon-γ, subsequently driving TNF expression by macrophages. CONCLUSIONS: Our data provide deeper insights into the timely appearance of events leading to benralizumab-induced eosinophil apoptosis and suggest that additional mechanisms may contribute to the potent anti-eosinophilic activity of benralizumab in vivo. Importantly, afucosylation of benralizumab strongly enhanced its potency for all mechanisms investigated.

Corticosterone and chlorpyrifos oxon exposure elicits spatiotemporal MAPK phosphoprotein signaling in a mouse brain.

Chlorpyrifos (CPF) is one of the most widely-used pesticides globally for agricultural purposes. Certain occupations (e.g., farmers, military) are at an increased risk for high-dose exposure to CPF, which can lead to seizures and irreversible brain injury. Workers with the highest risk of exposure typically experience increased circulating cortisol levels, which is related to physiological stress. To better represent this exposure scenario, a mouse model utilized exogenous administration of corticosterone (CORT; high physiologic stress mimic) in combination with chlorpyrifos oxon (CPO; oxon metabolite of CPF); this combination increases neuroinflammation post-exposure. In the present study adult male C57BL/6J mice were given CORT (200 µg/mL) in drinking water for seven days followed by a single intraperitoneal injection of CPO (8.0 mg/kg) on day eight, and euthanized 0.5, 2, and 24 h post-injection. Ten post-translationally modified proteins were measured in the frontal cortex and striatum to evaluate brain region-specific effects. The spatiotemporal response to CORT + CPO sequentially activated phosphoproteins (p-ERK1/2, p-MEK1/2, p-JNK) involved in mitogen-activated protein kinase (MAPK) signaling. Observed p-ZAP70 responses further integrated MAPK signaling and provided a spatiotemporal connection between protein phosphorylation and neuroinflammation. This study provides insight into the spatiotemporal cellular signaling cascade following CORT + CPO exposure that represent these vulnerable populations.

Differential phosphoprotein signaling in the cortex in mouse models of Gulf War Illness using corticosterone and acetylcholinesterase inhibitors.

AIMS: Veterans from the 1990-91 Gulf War were exposed to acetylcholinesterase inhibitors (AChEIs), and, following service, an estimated one-third began suffering from a medically unexplained, multi-symptom illness termed Gulf War Illness (GWI). Previous research has developed validated rodent models that include exposure to exogenous corticosterone (CORT) and AChEIs to simulate high stress and chemical exposures encountered in theater. This combination of exposures in mice resulted in a marked increase in neuroinflammation, which is a common symptom of veterans suffering from GWI. To further elucidate the mechanisms associated with these mouse models of GWI, an investigation into intracellular responses in the cortex were performed to characterize the early cellular signaling changes associated with this exposure-initiated neuroinflammation. MAIN METHODS: Adult male C57BL/6J mice were exposed to CORT in the drinking water (200 µg/mL) for 7 days followed by a single intraperitoneal injection of diisopropyl fluorophosphate (DFP; 4.0 mg/kg) or chlorpyrifos oxon (CPO; 8.0 mg/kg), on day 8 and euthanized 0.5, 2, and 24 h post-injection. Eleven post-translationally modified protein targets were measured using a multiplexed ELISA. KEY FINDINGS: Phosphoprotein responses were found to be exposure specific following AChEI insult, with and without CORT. Specifically, CORT + CPO exposure was found to sequentially activate several phosphoproteins involved in mitogen activated protein kinase signaling (p-MEK1/2, p-ERK1/2, and p-JNK). DFP alone similarly increased proteins in this pathway (p-RPS6, and p-JNK), but the addition of CORT ameliorated these affects. SIGNIFICANCE: The results of this study provide insight into differentially activated pathways depending on AChEI in these GWI models.

Tissue-level cytokines in a rodent model of chronic implant-associated infection.

Systemic cytokine concentrations have been extensively studied in implant-associated infections, providing sensitive diagnostic markers. However, less is known about the relationships of tissue-level cytokines surrounding the joint. The aim of this study was to define the cytokine profiles of tissues to investigate the use of these cytokines as markers of debridement in chronic joint infection. Using a rodent model, muscle samples were obtained from rats following Kirschner wire implantation and infection with Staphylococcus aureus to determine if: (1) differences exist in cytokine concentrations with proximity to infection, and (2) localized infection-specific markers can be identified on a tissue level to potentially serve as debridement markers in the future. Samples were collected from 4 distinct locations, and the concentrations of interleukin(IL)-1α, IL-1ß, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-13, granulocyte-macrophage colony-stimulating factor, interferon-γ, and tumor necrosis factor-α were quantified in each sample, relative to the amount of tissue. Cytokine concentrations differed with proximity to the joint when implant or infection was present, and tissues at the operative knee joint showed the highest levels of most cytokines. Additionally, IL-1ß, IL-4, and IL-6 showed promise, beyond diagnostics, as tissue-level indicators of infection response. Ultimately, this study illustrated that tissue-level evaluation provided insight into infection-specific response, and these markers may be useful for guiding the debridement of implant-associated infections.

Electronic Cigarettes and Vaping-Associated Lung Injury (EVALI): A Rural Appalachian Experience.

Background: Electronic cigarette use has increased dramatically since their introduction in 2007. Respiratory complications, particularly lipoid pneumonia, have been reported as early as 2012. An outbreak of pulmonary injury in 2019 has been reported in patients using vaping products.Research Question: To describe a rural Appalachian tertiary center's experience of EVALI and to identify novel mechanisms of pulmonary injury patterns.Study Design and Methods: We present a consecutive case series of 17 patients admitted to our rural, academic, tertiary care institution with EVALI from August 2019 to March 2020. Demographics, baseline characteristics, co-morbidities, vaping behavior, and hospital course were recorded. Broncho-alveolar lavage specimens were assessed for lipid-laden macrophages and hemosiderin-laden macrophages with stains for Oil-Red-O (n = 15) and Prussian Blue (n = 14) respectively.The patient volunteered e-liquid materials (n = 6), and vapors were analyzed using a proton transfer reaction time-of-flight mass spectrometer (PTR-TOF-MS) to describe the chemical profile. Post-discharge interviews were conducted.Results: The most common CT finding was bilateral ground-glass opacities with a predilection for lower lung zones. The most frequent pulmonary injury pattern was lipoid pneumonia. The majority of EVALI patients were critically ill requiring ventilation or ECMO. The most severely ill patients were noted to be positive for iron stains in macrophages and showed higher volatile organic compound (VOC) levels in chemical analysis.Interpretation: Based on our experience, EVALI in rural Appalachia presented with relatively severe respiratory failure. Worse outcomes appear to be correlated to high levels of VOCs, iron deposition in lungs, and concomitant infection.

Localized cytokine responses to total knee arthroplasty and total knee revision complications.

BACKGROUND: The study of localized immune-related factors has proven beneficial for a variety of conditions, and one area of interest in the field of orthopaedics is the impact of implants and localized infections on immune response. Several cytokines have shown increased systemic concentrations (in serum/plasma) in response to implants and infection, but tissue-level cytokines have not been investigated as thoroughly. METHODS: This exploratory study investigated tissue-level cytokines in a cohort of patients (N = 17) in response to total knee arthroplasty and total knee revision to better understand the immune response to implants and localized infection (e.g., prosthetic joint infection). The overall goal of this study was to provide insight into the localized cytokine response of tissues and identify tissue-level markers specific to inflammation caused by implants vs. inflammation caused by infection. Tissues were collected across several anatomical locations and assayed with a panel of 20 human inflammatory cytokines to understand spatial differences in cytokine levels. RESULTS: In this study, six cytokines were elevated in implanted joints, as compared to native joints: IL-10, IL-12p70, IL-13, IL-17A, IL-4, and TNF-α (p < 0.05). Seven cytokines showed infection-dependent increases in localized tissues: IL-1α, IL-1ß, IL-6, IL-8, MCP-1, MIP-1α, and MIP-1ß (p < 0.05). CONCLUSIONS: This study demonstrated that differences exist in tissue-level cytokines in response to presence of implant, and some cytokines were specifically elevated for infection; these responses may be informative of overall tissue health. These results highlight the utility of investigating localized cytokine concentrations to offer novel insights for total knee arthroplasty and total knee revision procedures, as well as their complications. Ultimately, this information could provide additional, quantitative measurements of tissue to aid clinical decision making and patient treatment options.

Metabolite-Based Modification of Poly(l-lysine) for Improved Gene Delivery.

Synthetic gene delivery systems employ multiple functions to enable safe and effective transport of DNA to target cells. Here, we describe metabolite-based poly(l-lysine) (PLL) modifiers that improve transfection by imparting both pH buffering and nanoparticle stabilization functions within a single molecular unit. PLL modifiers were based on morpholine (M), morpholine and niacin (MN), or thiomorpholine (TM). PLL modification with (MN) or (TM) imparted buffering function over the pH range of 5-7 both in solution and live cells and enhanced the stability of PLL DNA nanoparticles, which exhibited higher resistance to polyanion exchange and prolonged blood circulation. These properties translated into increased transfection efficiency in vitro coupled with reduced toxicity compared to unmodified PLL and PLL(M). Furthermore, PEG-PLL(MN) DNA nanoparticles transfected muscle tissue in vivo for >45 days following intramuscular injection. These polymer modifiers demonstrate the successful design of multifunctional units that improve transfection of synthetic gene delivery systems while maintaining biocompatibility.

Optical properties of metasurfaces infiltrated with liquid crystals.

Optical metasurfaces allow the ability to precisely manipulate the wavefront of light, creating many interesting and exotic optical phenomena. However, they generally lack dynamic control over their optical properties and are limited to passive optical elements. In this work, we report the nontrivial infiltration of nanostructured metalenses with three respective nematic liquid crystals of different refractive index and birefringence. The optical properties of the metalens are evaluated after liquid-crystal infiltration to quantify its effect on the intended optical design. We observe a significant modification of the metalens focus after infiltration for each liquid crystal. These optical changes result from modification of local refractive index surrounding the metalens structure after infiltration. We report qualitative agreement of the optical experiments with finite-difference time-domain solver (FDTD) simulation results. By harnessing the tunability inherent in the orientation dependent refractive index of the infiltrated liquid crystal, the metalens system considered here has the potential to enable dynamic reconfigurability in metasurfaces.

Impact of Cytokines and Phosphoproteins in Response to Chronic Joint Infection.

The early cellular response to infection has been investigated extensively, generating valuable information regarding the mediators of acute infection response. Various cytokines have been highlighted for their critical roles, and the actions of these cytokines are related to intracellular phosphorylation changes to promote infection resolution. However, the development of chronic infections has not been thoroughly investigated. While it is known that wound healing processes are disrupted, the interactions of cytokines and phosphoproteins that contribute to this dysregulation are not well understood. To investigate these relationships, this study used a network centrality approach to assess the impact of individual cytokines and phosphoproteins during chronic inflammation and infection. Tissues were taken from patients undergoing total knee arthroplasty (TKA) and total knee revision (TKR) procedures across two tissue depths to understand which proteins are contributing most to the dysregulation observed at the joint. Notably, p-c-Jun, p-CREB, p-BAD, IL-10, IL-12p70, IL-13, and IFN-γ contributed highly to the network of proteins involved in aseptic inflammation caused by implants. Similarly, p-PTEN, IL-4, IL-10, IL-13, IFN-γ, and TNF-α appear to be central to signaling disruptions observed in septic joints. Ultimately, the network centrality approach provided insight into the altered tissue responses observed in chronic inflammation and infection.

Antibacterial Monoclonal Antibodies Do Not Disrupt the Intestinal Microbiome or Its Function.

Antibiotics revolutionized the treatment of infectious diseases; however, it is now clear that broad-spectrum antibiotics alter the composition and function of the host's microbiome. The microbiome plays a key role in human health, and its perturbation is increasingly recognized as contributing to many human diseases. Widespread broad-spectrum antibiotic use has also resulted in the emergence of multidrug-resistant pathogens, spurring the development of pathogen-specific strategies such as monoclonal antibodies (MAbs) to combat bacterial infection. Not only are pathogen-specific approaches not expected to induce resistance in nontargeted bacteria, but they are hypothesized to have minimal impact on the gut microbiome. Here, we compare the effects of antibiotics, pathogen-specific MAbs, and their controls (saline or control IgG [c-IgG]) on the gut microbiome of 7-week-old, female, C57BL/6 mice. The magnitude of change in taxonomic abundance, bacterial diversity, and bacterial metabolites, including short-chain fatty acids (SCFA) and bile acids in the fecal pellets from mice treated with pathogen-specific MAbs, was no different from that with animals treated with saline or an IgG control. Conversely, dramatic changes were observed in the relative abundance, as well as alpha and beta diversity, of the fecal microbiome and bacterial metabolites in the feces of all antibiotic-treated mice. Taken together, these results indicate that pathogen-specific MAbs do not alter the fecal microbiome like broad-spectrum antibiotics and may represent a safer, more-targeted approach to antibacterial therapy.

Bioenergetic Analyses of In Vitro and In Vivo Samples to Guide Toxicological Endpoints.

Toxicology is a broad field that requires the translation of biochemical responses to xenobiotic exposures into useable information to ensure the safety of the public. Modern techniques are improving rapidly, both quantitatively and qualitatively, to provide the tools necessary to expand available toxicological datasets and refine our ability to translate that data into relevant information via bioinformatics. These new techniques can, and do, impact many of the current critical roles in toxicology, including the environmental, forensic, preclinical/clinical, and regulatory realms. One area of rapid expansion is our understanding of bioenergetics, or the study of the transformation of energy in living organisms, and new mathematical approaches are needed to interpret these large datasets. As bioenergetics are intimately involved in the regulation of how and when a cell responds to xenobiotics, monitoring these changes (i.e., metabolic fluctuations) in cells/tissues post-exposure provides an approach to define the temporal scale of pharmacodynamic responses, which can be used to guide additional toxicological techniques (e.g., "omics"). This chapter will summarize important in vitro assays and in vivo imaging techniques to take real-time measurements. Using this information, our laboratory has utilized bioenergetics to identify significant time points of pharmacodynamic relevance as well as forecast the cell's eventual fate.

Salivary cytokines as a biomarker of social stress in a mock rescue mission.

Using salivary inflammatory markers as a noninvasive biomonitoring technique within natural social contexts has become increasingly important to link social and biological responses. Many studies have associated circulating cytokines to distinct aspects of physical activity and social/emotional behavior; however, they have not been linked to success and failure in a naturalistic setting for military personnel performing tasks. In this study, salivary cytokines were studied in a group of fifteen Air Force Reserve Officers' Training Corps (ROTC; 14 males, 1 female) subjects performing three mock hostage rescue missions, designed to prompt responses associated with baseline, success, and failure. Each subject completed the tasks of the mission individually and again in randomly assigned teams. Participants were outfitted via direct skin contact with comfortable external Zephyr™ sensors to monitor heart rate, breathing rate, and activity while completing each task. Saliva samples were collected before and after the completion of each mission, and cytokine levels were quantified using enzyme-labelled immunoassay (ELISA) beads. These biomarkers were used to describe the body's immune response to success and failure when performing a mock rescue mission individually and in a team. All measured cytokine levels increased following failed missions performed individually, compared to cytokine levels associated with successful missions. When completing the tasks as a team, there were no significant differences in cytokine response between success and failure; however, being in a team stimulated an increased pre-mission cytokine response, suggesting the concept of teamwork and performing with peers for the first time had a more significant impact than the notion of failing. Additionally, none of the cytokines tested for individual missions correlated to physical activity markers (heart rate, breathing rate, activity) measured during performance. These results indicate a potentially new noninvasive method of determining social stress levels under taxing conditions.

Targeted drug delivery via caveolae-associated protein PV1 improves lung fibrosis.

Systemic administration of bio-therapeutics can result in only a fraction of drug reaching targeted tissues, with the majority of drug being distributed to tissues irrelevant to the drug's site of action. Targeted delivery to specific organs may allow for greater accumulation, better efficacy, and improved safety. We investigated how targeting plasmalemma vesicle-associated protein (PV1), a protein found in the endothelial caveolae of lungs and kidneys, can promote accumulation in these organs. Using ex vivo fluorescence imaging, we show that intravenously administered αPV1 antibodies localize to mouse lungs and kidneys. In a bleomycin-induced idiopathic pulmonary fibrosis (IPF) mouse model, αPV1 conjugated to Prostaglandin E2 (PGE2), a known anti-fibrotic agent, significantly reduced collagen content and fibrosis whereas a non-targeted PGE2 antibody conjugate failed to slow fibrosis progression. Our results demonstrate that PV1 targeting can be utilized to deliver therapeutics to lungs and this approach is potentially applicable for various lung diseases.