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To mount a robust immune response, naïve T cells must differentiate into cytokine producing effector T cells. However, if T cell activation and differentiation is poorly controlled, excess inflammation can occur, resulting in allergy or autoimmune disease. Thus, protective immunity is dependent on a fine tuning of differentiation and cytokine production by T cells. This can be accomplished by E3 Ubiquitin Ligases, as these enzymes regulate the levels of cellular proteins. We performed a screen to identify Cullin E3 ubiquitin ligases that were active in T cells and identified Cullin 5 (Cul 5) as being the most dynamically activated Cullin during T cell activation. While it is known that Cul 5 aids in the formation of the CRL5 complex (for Cullin Ring Ligase), whether and how Cul 5 regulates immune system function is unknown. We have now generated mice that lack Cul 5 in T cells. Initial characterization of these Cul 5 fl/fl CD4-Cre mice revealed that Cul 5 is critical in preventing spontaneous and allergen-induced inflammation in the lungs by inhibiting Th2 differentiation in CD4 T cells. To see if this protective effect of Cullin 5 was important at other mucosal surfaces, we employed a colitis model, known as DSS-induced colitis to experimentally mimic Inflammatory Bowel Disease (IBD). We found that mice that lack Cul5 in T cells showed significantly more weight loss and colon pathology than their wild type counterparts. Surprisingly, the mice did not show increased cytokine production by CD4 T cells, rather, they showed high numbers of CD8+ T cells, that while activated, were less likely to produce IFN-γ. Taken together, our preliminary data support that Cullin 5 may regulate CD8 T cells responses either by limiting cytotoxic activity or by promoting IFN-γ production to facilitate macrophage bactericidal activity. Our data support that Cul 5 prevents colitis by regulating CD8 T cell numbers and/or function.

AMA Research Challenge 2023

Cullin 5 Limits Disease Severity in DSS-Colitis

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Pseudomonas aeruginosa (PA) is a gram-negative pathogen that causes bacteremia and septicemia in severely burned patients; a situation that often leads to multiorgan failure and death. Despite our extensive knowledge of PA wound infection, there are several gaps in our understanding of the PA response to the blood environment. We recently showed that upon its growth in human blood or human serum (HS), PA strain PAO1 alters the expression of more than 1,000 genes. Many of these alterations may occur through the PA-outer membrane proteins (PA-OMPs). OMPs contribute significantly to the pathogenesis of gram-negative pathogens. We hypothesized that HS alters the production of PA-OMPs. To test this, we grew PAO1, which causes sepsis and death in the murine model of thermal injury, in Luria-Bertani broth (LBB) or LBB containing 10% HS, extracted the OMPs, and analyzed their profile using SDS-polyacrylamide gel electrophoresis. Analysis of the silver-stained gels revealed that 10% HS repressed the production of at least six OMPs with molecular weights (MW) of 68.2, 34.5, 23.8, 17.9, 13.7, and 11.9 kDa but induced the production of at least five others (MW 66.1, 41, 28.5, 22.8, and 22 kDa). Similar to PA-PAOI1, PA-PA14 causes sepsis and death. However, its genome is larger than that of PAO1 and it carries several additional virulence genes. Therefore, we also compared the OMP profile of PAO1 and PA14. Although the two strains share multiple common proteins, each strain has its own unique ones. As with PAO1, HS manipulated the production of several PA14 OMPs. These results suggest that 1) in response to different components of HS, PA varies the production of its OMPs, 2) virulent PA strains differ in their OMPs, and 3) HS-induced OMPs may be targeted in future investigations designed to produce potential serum therapies for PA infected/severely burned patients. 


Human serum alters the production of several Pseudomonas aeruginosa outer membrane proteins.

Background:
Annually, over 300 million patients undergo surgery requiring the administration of a combination of anesthetic pharmacologic compounds (APCs). Surgery is associated with significant tissue trauma, triggering a complex inflammatory response that engages the innate and adaptive branches of the immune system. Complications, including end-organ damage, infections, and protracted recovery, arise as pro-inflammatory and immune-suppressive responses tilt out of balance. Accumulating evidence suggests that, in addition to desired anesthetic properties, many APCs have immune-modulatory properties that can positively or negatively impact surgical recovery. However, the effect of APCs on specific innate and
adaptive immune cell functions warrants further exploration to personalize anesthetic choice based on individual patient and surgery needs. We hypothesize that APCs alter critical immune cell responses and effector functions implicated in the human immune response to trauma and injury.

Methods:
We developed a high-throughput multiplex mass-tag barcoding (MCB) assay to screen 16 commonly administered APCs for their capacity to modulate trauma-related immune and adaptive immune responses. These include general and local anesthetics, narcotic and benzodiazepine analgesics, neuromuscular blocking agents, and NSAIDs. We also selected ten additional compounds frequently used in anesthesia settings: antiemetics, diuretics, antidotes, catecholamines, and beta blockers. Using single-cell mass cytometry, we quantify the effect of each APC on 1500 intracellular signaling responses in 25 innate and adaptive immune cell subsets in whole blood samples (500µL) across a range of drug concentrations and stimulation conditions, including lipopolysaccharide (LPS), interferon alpha (IFNa), and a cocktail of interleukins 2, 4, and 6.

Results:
A first MCB screen was completed using flunisolide, a glucocorticoid compound with a known immunosuppressive effect, allowing for assay optimization. This identified marked changes in protein phosphorylation of inflammatory pathways between immune cells exposed and not exposed to flunisolide. These results show that flunisolide inhibited adaptive immune cell subsets to a greater extent than innate immune cell subsets in a cell-type and signaling response-specific manner.

Conclusion:
Having established the MCB experimental framework, the next experiments will systematically evaluate the immune-modulatory properties of the proposed library of APCs. Eventually, the MCB assay could be utilized clinically to determine the immune-modulatory effect of a complex anesthesia regimen – a first step toward personalized anesthesia.

A High-Throughput Mass Cytometry Assay to Investigate Immune Cell Modulation of Anesthetic Drugs

Background: Pulmonary hypertension (PH) is a fatal disease associated with pulmonary vascular remodeling that leads to right ventricular (RV) failure and death. While miR-10b-5p exerts pro-proliferative effects in various cancers via inhibition of its targets cell adhesion molecule 1 (CADM1) and apoptotic peptidase activating factor 1 (APAF1), its role in PH is elusive. We hypothesize that PH is associated with upregulation of lung miR10b-5p and downregulation of its targets CADM1 and APAF1. Further, miR10b-5p inhibition may rescue pre-existing PH.

Methods: PH was induced in male rats by monocrotaline (MCT; 60mg/kg sc; n=4; followed for 30-days) or Sugen/Hypoxia (SuHx; SU5416; 20mg/kg sc and 10% O2 hypoxia for 3-wks followed by normoxia for 2-wks; n=3). In vivo inhibition of miR10b-5p was performed from day14-30 after MCT (5 intratracheal injections of antagomir every 3-4 days; 5nM/ injection; n=3). PBS treated MCT rats served as controls (n=5). Rats were followed by echo. RV catheterization was performed terminally, and RV hypertrophy index was calculated. MiRNA microarray was performed to assess miR10b in rat lungs. Lungs were stained to quantify vascular remodeling. RT-PCR was performed to assess gene expression. Human pulmonary artery smooth muscle cells (HPASMCs) were incubated with miR10b mimic for 24h to assess proliferation. Data are mean±SEM. 

Results: MCT and SuHx rats developed severe PH. MiRNA microarray revealed a 3-fold increase in miR10b in MCT rat lungs. PCR validated increased miR10b in MCT and SuHx rat lungs. Further, there was a significant downregulation of miR10b targets CADM1 (MCT 1.64-fold; SuHx 1.48-fold) and APAF1 (MCT 1.23-fold; SuHx 1.21-fold) in lungs. In vivo knockdown of miR10b resulted in rescue of PH in MCT rats (RVSP: 50.7±4.42 in antagomir vs. 95.81±3.70 mmHg in placebo; p<0.005; RV hypertrophy index: 0.52±0.02 in antagomir vs. 0.80±0.07; p<0.05; RVID: diastolic 2.44±0.68 in antagomir vs. 4.55±0.42 mm; p<0.05). PCR demonstrated 30% decrease in lung miR10b and histology revealed reduced pulmonary arteriolar medial wall thickness (32.72±1.12 in antagomir vs. 42.24±3.26% in placebo, p<0.005) in rats treated with antagomir. Finally, miR10b overexpression resulted in HPASMC proliferation in a dose-dependent manner.

Conclusion: Pre-clinical PH is associated with an increase in miR10b-5p expression and decrease in its targets CADM1 and APAF1. miR10b-5p inhibition rescues experimental PH and may serve as a novel therapeutic target.

MicroRNA10b-5p Inhibition as a Novel Therapeutic Strategy for Severe Pulmonary Hypertension

Background
Ventricular arrhythmias such as ventricular tachycardia (VT) and ventricular fibrillation (VF) are dangerous heart rhythms given their potential to lead to sudden cardiac death. Ventricular arrhythmias are most often associated with ischemic heart disease, but may also arise from heart failure or ion channel abnormalities. Why some hearts affected with such risk factors develop VT or VF while others do not is an open question. Many risk factors for arrhythmia such as ischemia, heart failure, hypertension, diabetes, and aging are associated with increased reactive oxygen species (ROS). Mitochondria are tightly intertwined with the regulation of ROS, and are a prominent organelle in the energy-intensive myocardium. In a mouse model of atrial fibrillation (AF), mitochondria in the atria show swelling, reduced cristae density, and increased mitochondrial degradation known as mitophagy when compared to controls. Reducing ROS and mitophagy leads to a 50-fold reduction in AF burden. Herein, we evaluated whether ventricular arrhythmias are associated with mitochondrial changes in the ventricle. 

Methods
The lab has established transgenic mice expressing a F1795A mutation in the human cardiac Nav1.5 channel gene SCN5A, leading to persistent Na+ current, and demonstrated spontaneous VT and VF in these mice. Wild-type (n=2) and double transgenic (dTG) mice (n=4) expressing human F1759A-Nav1.5 channels as well as reverse tet-transactivator protein had their ventricles sectioned and underwent transmission electron microscopy. Images acquired at 15000x and 50000x magnification were quantified using ImageJ. 

Results
Mitochondria in transgenic F1759A mice ventricles were of significantly smaller area than mitochondria of control mice (345080  4982 nm2 vs. 409409  9516 nm2, p<0.0001) (n=2191, 801 mitochondria respectively). Ventricles of transgenic mice also show greater number of mitochondria per field when compared to controls (73.03  2.884 vs. 58.46  4.32, p=0.008) (n=13, 30 fields). Furthermore, imaging revealed a population of mitochondria with membrane disruption and absence of cristae consistent with mitophagy. The percentage of mitochondria undergoing mitophagy was significantly increased in F1759A mice compared to controls (11.80%  2.256 vs. 0.33%  0.33, p=0.0019).

Conclusion
Transmission electron microscopy of the ventricles of F1759A mice with spontaneous ventricular arrhythmias reveals smaller and increased number of mitochondria undergoing mitophagy. These results suggest mitochondria may be involved in the pathogenesis of VT and VF. Further work will be needed to determine if mitochondrial changes are upstream of ventricular arrhythmogenesis and whether targeting mitochondrial quality can prevent arrhythmias.

A transgenic Na+ channel mouse model of spontaneous ventricular arrhythmias demonstrates ventricular mitochondrial structural alterations

Background. In addition to providing the energy for cardiac muscle contraction and building blocks for cellular homeostasis, nutrient metabolism also generates signaling molecules for the regulation of cardiac functional and structural remodeling under various conditions. While the alteration of these metabolic signals has been linked to the development of heart failure, the molecular mechanisms involved remain incompletely understood. Olfactory receptors (ORs) are G-protein-coupled receptors which play a pivotal role in the body’s response to chemical stimuli from the external environment. Although initially believed to be exclusively located at the olfactory epithelium, ORs have been detected in many other tissue and organs where they allow for cellular adaptation to changes in metabolite levels in the bloodstream. Recent work from our lab and others has identified OR51E2 as a cardiac OR. 
Hypothesis. We hypothesize that cardiac expression of OR51E2 is altered by the development of metabolic diseases and heart failure. We further anticipate that that the dysregulation of OR51E2 expression plays a role in cardiac adaptation to stress. 
Methods. In order to determine the impact of metabolic derangements on cardiac OR51E2 expression, OR51E2 mRNA levels were quantified by real-time PCR in the normal mouse heart (n=4) and compared to that of hearts from mouse models of obesity and type 2 diabetes (ob/ob mice, n=4; db/db mice, n=4). The same method was used to quantify cardiac OR51E2 mRNA levels in normal rats (n=8) and rats rendered diabetic via intraperitoneal injection of streptozotocin (STZ; n=10). Western blotting was used to correlate mRNA data with OR51E2 protein expression levels in the mouse hearts. Next, OR51E2 mRNA levels were quantified in hearts from mice subjected to transverse aortic constriction (TAC)-induced pressure overload (n=10) in order to determine whether the development of heart failure affects OR51E2 expression. 
Results. The expression of OR51E2 increased by 36-43% in hearts of ob/ob mice (p=0.010) and STZ-treated rats (p=0.024) when compared to hearts from non-obese and non-diabetic animals. While no changes were detected at the mRNA level for hearts of db/db mice, OR51E2 protein levels were found to increase by 83% (p=0.001). Lastly, the failing heart of mice subjected to TAC-induced pressure overload also exhibited higher OR51E2 mRNA levels when compared to sham-operated mice (p=0.001). 
Conclusions. Our results demonstrate that cardiac OR51E2 is upregulated in states of metabolic dysregulation and hypertrophic heart failure. Future investigations will aim to identify the nature of the signals transduced by OR51E2 in the mammalian heart, and the consequences of their dysregulations for cardiac structure and function during disease states.

A Potential Role for the Olfactory Receptor OR51E2 in Cardiac Adaptation to 
Metabolic and Hemodynamic Stress

Background
Atherosclerosis is a chronic inflammatory disease that contributes to over 50% of deaths in western societies. Adult men are at a unique risk because estrogen provides a well-documented atheroprotective effect for premenopausal women. Therefore, this study focuses on development of lesions in adult male mice. Atherosclerosis is characterized by the formation of plaques, lipid-filled deposits on arterial walls. Late stage plaques are susceptible to rupture, leading to life-threatening events like stroke and myocardial infarction. Plaques are primarily composed of foam cells, or macrophages laden with cholesterol. The formation of foam cells is mediated by various receptors to engage in endocytosis. One key enzyme in this process is Protein Kinase C epsilon (PKCε), which catalyzes the addition of membrane lipids to a forming phagosome. In vitro, PKCε deficient macrophages are unable to efficiently phagocytose and thus PKCε has a demonstrated potential to be atheroprotective. To study this potential in vivo, C57Bl/6 mice underwent Cre-Lox recombination to create a mεKO strain, which lacks PKCε in myeloid cells. We predict that the mεKO mice, which are PKCε deficient, will accumulate more plaque burden than WT mice.

Methods
At 8 weeks, mice were injected with the PCSK-AAV8 virus to induce hypercholesterolemia by inhibiting LDL receptors in the liver. This state was further reinforced by a high fat (“Western”) diet. Serum was collected during this time to confirm hypercholesterolemia. 16 weeks post injection, the aortic arch and descending thoracic aortas of WT and mεKO mice were collected. These samples were pinned flat, stained with Oil Red O, a fat soluble dye, to mark areas of plaque development, and photographed. Using ImageJ, sample area was standardized across all aortas and a color threshold was determined as a double-blinded method to calculate percent lesion coverage or “plaque burden.”

Results
WT mice (n = 8) had an average plaque burden of 1.393% (+/- 1.141%) while mεKO mice (n=7) had a significantly higher average plaque burden of 5.820% (+/- 2.411%). The p-value of a Student’s t-test is <0.001 (***).

Conclusions
The results confirm our hypothesis and demonstrate that PKCε may play a role in preventing lesion development in atherosclerosis. Additionally, current research suggests that plaque composition may be as important as plaque area in determining prognosis; mεKO mice developed visibility more opaque plaques than WT mice, and analyzing differences in composition may be productive.

Deletion of myeloid protein kinase C epsilon exacerbates atherosclerosis

Pregnancy requires significantly increased cardiac output (~30%) achieved through peripheral vasodilation and increased stroke volume, due to increased end-diastolic volume. Most women resolve these physiological changes within 6 months of delivery; however, some women progress to heart failure late in pregnancy or in the immediate post-partum period. Peri-partum cardiomyopathy (PPCM) is a rare condition of largely unknown etiology which, at its most-severe and life-threatening, requires cardiac transplantation. Pre-eclampsia occurs at much higher rates in the PPCM population (~40%) compared with the generally pregnant population (~2-5%). We have previously shown that transgenic mice expressing the human-specific isoform of the TBXA2R gene (TPβ) spontaneously develop pre-eclampsia accompanied by all the associated hallmarks expected from the human disease. We now show TPβ transgenic (TPβ-Tg) mice are a model of PPCM. Using trans-thoracic echocardiographic imaging, TPβ-Tg mice had no basal phenotype; however, unlike C57/Bl6J (WT) mice that resolved the pregnancy-induced cardiac remodeling, TPβ-Tg mice had exaggerated cardiac dilatation at term (E18.5; P<0.01) which increased in severity over the subsequent 6 months (P<0.005). In addition, TP-Tg mice exhibited reduced ejection fraction (22±13% vs 67% in WT), significant LV wall thinning (1.12±0.16 mm vs 1.6±0.17 mm WT), and loss of LV mass (66.55±9 mg vs 95±11mg WT). Analysis of myocardium of human PPCM patients undergoing heart transplant (n=12) compared to healthy myocardium from non-diseased donors (n=42) by RT-PCR and IHC showed highly expressed TPβ in PPCM patients. In human PPCM samples, there was a significant (p<0.01) dysregulation of genes associated with circadian rhythm as determined by RNAseq analysis. Quantitative PCR analysis of hearts from non-pregnant (WT=8; TPβ-Tg=4) and pregnant (WT=10; TPβ-Tg=13) mice found similar dysregulation of multiple genes associated with circadian rhythm compared to WT mice (p<0.01). This indicates that PPCM may be associated with failure to terminate CLOCK signaling as a result of TPβ activation. Our results support dysregulation of TBXA2R splicing as part of the pathogenesis of PPCM and identify new therapeutic targets for this condition.

Characterization of a Novel Clinically Relevant Mouse Model of Postpartum Cardiomyopathy

Background: Cardiomyocytes are the contractile muscle cells of the heart. A key developmental feature of mammalian cardiomyocytes is their terminal differentiation early in life and inability to proliferate following myocardial injury. As cardiomyocyte function is directly correlated to cellular metabolism and mitochondrial respiration, we aim to explore their respiration early in life when the cells have proliferative capabilities. Based on previous work in the lab, we hypothesize that our dual cellular ATP indicator and continuous oxygen monitoring sensor will be useful in analyzing the mitochondrial respiration changes in early cardiomyocytes.
Methods: To test the hypothesis, this study used isolated neonatal mouse cardiomyocytes which are useful cell models as the cells undergo proliferation for 7 days following their isolation. To culture these cells, hearts from mouse pups were harvested at an age of 1 to 3 days. The tissue was lysed and enzymatically digested to isolate the cardiomyocytes. Following isolation, the cells were cultured for around 7 days. A novel genetically encoded fluorescent ATP indicator of cytosolic and mitochondrial ATP (smacATPi) was transduced in the cultured cardiomyocytes to monitor the dynamic changes of ATP in response to metabolic inhibitors. Continuous recording of oxygen consumption of the cardiomyocytes was achieved using a Recipher cultured cell oxygen sensing system. The drug 2-deoxy-D-glucose (2-DG), a glycolysis inhibitor, was applied to the cells.
Results: Evaluating changes in mitochondrial respiration and ATP production was performed by live cell imaging using a Cytation 5 imaging system. Cardiomyocytes with green and red fluorescence were identified, indicating the feasibility of the adenovirus-mediated smacATPi expression in cultured cardiomyocytes. Furthermore, the respective changes in green (cytosolic ATP) and red (mitochondrial ATP) fluorescence in response to 2-DG were observed. The oxygen consumption rate of the cultured cardiomyocytes was continuously monitored over a 7-day period using the online oxygen sensing device. Analysis of the data collected by the above methods demonstrates the predicted changes in mitochondrial respiration following drug treatment in that mitochondrial respiration decreases in response to the metabolic inhibitor, 2-DG.
Conclusion: Experimentation demonstrates that the methods employed in this study provide insight into mitochondrial respiration changes in early cardiomyocytes and how cellular respiration responds to different metabolic inhibitors. For future investigations, evaluating real-time spatiotemporal cellular ATP dynamics and cellular respiration may help identify novel insights into previously unknown biochemical mechanisms in cardiomyocytes for the development of effective treatments of cardiac disorders.

Oxygen Consumption and ATP Production in Neonatal Mouse Cardiomyocytes

Reactive oxygen species (ROS) are a heterogeneous group of highly reactive ions and molecules derived from molecular oxygen (O2), which can cause DNA damage and lead to skin cancer. NADPH oxidase 1 (Nox1) is a major producer of ROS in the skin upon exposure to ultraviolet light. Functionally, Nox1 forms a holoenzyme complex that generates two superoxide molecules and reduces NADPH. The signaling activation occurs when the organizer subunit Noxo1 translocates to the plasma membrane bringing a cytochrome p450 through interaction with Cyba. We propose to design inhibitors that prevent Cyba-Noxo1 binding as a topical application to reduce UV-generated ROS in human skin cells. 

Inhibitor design started from an apocynin backbone structure to generate a small molecule to serve as an anchor point, inhibitor 1, followed by adding a polyethylene glycol linked biotin in inhibitor 2. Further in vitro experiments using isothermal calorimetric (ITC) were used to quantify the binding affinity (KD). ITC and MALDI (TOF) MS was also used to show if binding of a Cyba peptide to Noxo1 was blocked. In vivo experiments were performed using donated skin explants with topical application of the two inhibitors while exposing the samples to UV. 

Inhibitors 1 and 2 were nontoxic in human cells, and successfully increased cell viability after UV exposure. Biophysical using ITC and MALDI revealed biotin as not necessary for inhibition while confirming affinity between inhibitor 2 and Noxo1. Further in vitro experiments using isothermal calorimetric binding quantification showed that inhibitor 2 bound a Noxo1 peptide with a KD of 2nM. Both using isothermal calorimetric binding and MALDI (TOF) MS showed that the binding of a Cyba peptide to Noxo1 was blocked. Experiments show that in primary skin explant models with ultraviolet light exposure, inhibitor 2 reduced the amount of cyclobutene pyrimidine dimers in DNA, a molecule known to lead to carcinogenesis. 

These findings demonstrate a novel inhibition of Nox1, which serves as a basis for further research to determine if this inhibitor can be added to topical sunscreens for public use in hopes of decreasing UVR-induced DNA damage and subsequent carcinogenesis. In addition, this novel sunscreen formulation could be a non-toxic alternative to chemical based barrier sunscreens that are currently on the market. 


Serendipitous Identification of a NOXO1 Inhibitor By Addition of a Polyethylene Glycol Chain

Background
Ceramides play an essential role in the formation of the stratum corneum, and include a long-chain sphingoid base in an amide linkage with one or more fatty acids (FAs). Skin contains many unique FAs, particularly esterified omega (EO)-hydroxy(OH)ceramides with ultra-long FA chains, which are critical to the CLE.
Atopic Dermatitis, a common pediatric skin disorder with cutaneous inflammation and barrier impairment, is recognized to have a deficiency of ceramides, including EO-OH ceramides, based on targeted lipidomic analyses. Untargeted lipidomics (Q Exactive HF OrbiTrap) is a new technology that, as RNA-Seq or proteomics, allows comprehensive comparison of diseased vs healthy tissues but has yet to be described for skin.

Methods
We extracted lipids from tape strips of lesional atopic dermatitis (AD-L) and nonlesional (NL) skin from 31 AD patients (0.2-17.3 yrs old, mean 7 yrs; 51% female; 58% White) and 14 age- and site-matched healthy controls (C) for untargeted lipidomic analysis.

Results
PCA plots showed distinct separation between AD-L and C skin, regardless of sex or age. 637 distinct ceramide species were detected in AD and 616 in C skin, with 291 decreased in AD-L vs. C and 242 reduced in AD-L vs NL skin. Total normalized signal response of ceramides decreased 84% in AD-L and 51% in NL skin vs C (p < 0.001). Most ceramide classes decreased by >80% in AD-L vs C skin (EO by 88-93%; ODS/ADS 91%; NP 90%; NH 87%; NSD 83%; OH/AH 83%; and OSD/ASD 82%).
We found a new ceramide class (m), overall decreased in AD-L by 81%. A few species were increased in EOSD, OH/AH, OSD/ASD, NDS, NH, NP, and NS classes. Ceramide changes in NL skin were all in AD-L. Decreased AD-L ceramides correlated with eczema severity indices: SCORAD and EASI scores (Spearman r= –0.4 to -0.57, p < 0.001); m-ceramides with EASI and Pruritus ADQ scores (r= -0.43 and r= -0.44, p < 0.01). Increases of NDS in AD-L correlated with EASI (r=0.48, p < 0.01); and NH, NP, and NS with Itch VAS (r= 0.73 to 0.93, all p < 0.05).

Conclusion
The discovery of a new ceramide class (m), which is found to be decreased in AD-L highlights the value of untargeted lipidomic analyses for understanding skin disorders and in clinical trials that can target specific ceramides in the metabolic pathway.

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Human serum alters the production of several Pseudomonas aeruginosa outer membrane proteins.

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