Hematopoietic prostaglandin D synthase defines a proeosinophilic pathogenic effector human T(H)2 cell subpopulation with enhanced function.

BACKGROUND: IL-5(+) pathogenic effector T(H)2 (peT(H)2) cells are a T(H)2 cell subpopulation with enhanced proinflammatory function that has largely been characterized in murine models of allergic inflammation. OBJECTIVE: We sought to identify phenotype markers for human peT(H)2 cells and characterize their function in patients with allergic eosinophilic inflammatory diseases. METHODS: Patients with eosinophilic gastrointestinal disease (EGID), patients with atopic dermatitis (AD), and nonatopic healthy control (NA) subjects were enrolled. peT(H)2 and conventional T(H)2 (cT(H)2) cell phenotype, function, and cytokine production were analyzed by using flow cytometry. Confirmatory gene expression was measured by using quantitative RT-PCR. Prostaglandin D2 levels were measured with ELISA. Gut T(H)2 cells were obtained by means of esophagogastroduodenoscopy. RESULTS: peT(H)2 cells were identified as chemoattractant receptor-homologous molecule expressed on T(H)2 cells-positive (CRTH2(+)), hematopoietic prostaglandin D synthase-positive CD161(hi) CD4 T cells. peT(H)2 cells expressed significantly greater IL-5 and IL-13 than did hematopoietic prostaglandin D synthase-negative and CD161(-) cT(H)2 cells. peT(H)2 cells were highly correlated with blood eosinophilia (r = 0.78-0.98) and were present in 30- to 40-fold greater numbers in subjects with EGID and those with AD versus NA subjects. Relative to cT(H)2 cells, peT(H)2 cells preferentially expressed receptors for thymic stromal lymphopoietin, IL-25, and IL-33 and demonstrated greater responsiveness to these innate pro-TH2 cytokines. peT(H)2 but not cT(H)2 cells produced prostaglandin D2. In patients with EGID and those with AD, peT(H)2 cells expressed gut- and skin-homing receptors, respectively. There were significantly greater numbers of peT(H)2 cells in gut tissue from patients with EGID versus NA subjects. CONCLUSION: peT(H)2 cells are the primary functional proinflammatory human T(H)2 cell subpopulation underlying allergic eosinophilic inflammation. The unambiguous phenotypic identification of human peT(H)2 cells provides a powerful tool to track these cells in future pathogenesis studies and clinical trials.

Trisomy 21 Alters Cell Proliferation and Migration of iPSC-Derived Cardiomyocytes on Type VI Collagen.

Purpose: Individuals with Down syndrome (DS) are 2000 times more likely to develop a congenital heart defect (CHD) than the typical population Freeman et al. in Am J Med Genet 80:213-217 (1998). The majority of CHDs in individuals with DS characteristically involve the atrioventricular (AV) canal, including the valves and the atrial or ventricular septum. Type VI collagen (COLVI) is the primary structural component in the developing septa and endocardial cushions, with two of the three genes encoding for COLVI located on human chromosome 21 and upregulated in Down syndrome (von Kaisenberg et al. in Obstet Gynecol 91:319-323, 1998; Gittenberger-De Groot et al. in Anatom Rec Part A 275:1109-1116, 2023). Methods: To investigate the effect of COLVI dosage on cardiomyocytes with trisomy 21, induced pluripotent stem cells (iPSC) from individuals with DS and age- and sex-matched controls were differentiated into cardiomyocytes (iPSC-CM) and plated on varying concentrations of COLVI. Results: Real time quantitative PCR showed decreased expression of cardiac-specific genes of DS iPSC-CM lines compared to control iPSC-CM. As expected, DS iPSC-CM had increased expression of genes on chromosome 21, including COL6A1, COL6A2, as well as genes not located on chromosome 21, namely COL6A3, HAS2 and HYAL2. We found that higher concentrations of COLVI result in decreased proliferation and migration of DS iPSC-CM, but not control iPSC-CM. Conclusions: These results suggest that the increased expression of COLVI in DS may result in lower migration-driven elongation of endocardial cushions stemming from lower cell proliferation and migration, possibly contributing to the high incidence of CHD in the DS population. Supplementary Information: The online version contains supplementary material available at 10.1007/s12195-023-00791-x.

Testing the Reliability of Optical Coherence Tomography to Measure Epidermal Thickness and Distinguish Volar and Nonvolar Skin.

In persons with limb loss, prosthetic devices cause skin breakdown, largely because residual limb skin (nonvolar) is not intended to bear weight such as palmoplantar (volar) skin. Before evaluation of treatment efficacy to improve skin resiliency, efforts are needed to establish normative data and assess outcome metric reliability. The purpose of this study was to use optical coherence tomography to (i) characterize volar and nonvolar skin epidermal thickness and (ii) examine the reliability of optical coherence tomography. Four orientations of optical coherence tomography images were collected on 33 volunteers (6 with limb loss) at 2 time points, and the epidermis was traced to quantify thickness by 3 evaluators. Epidermal thickness was greater (P < .01) for volar skin (palm) (265.1 ± 50.9 µm, n = 33) than for both nonvolar locations: posterior thigh (89.8 ± 18.1 µm, n = 27) or residual limb (93.4 ± 27.4 µm, n = 6). The inter-rater intraclass correlation coefficient was high for volar skin (0.887-0.956) but low for nonvolar skin (thigh: 0.292-0.391, residual limb: 0.211-0.580). Correlation improved when comparing only 2 evaluators who used the same display technique (palm: 0.827-0.940, thigh: 0.633-0.877, residual limb: 0.213-0.952). Despite poor inter-rater agreement for nonvolar skin, perhaps due to challenges in identifying the dermal-epidermal junction, this study helps to support the utility of optical coherence tomography to distinguish volar from nonvolar skin.

Functional and physical interaction between the mismatch repair and FA-BRCA pathways.

Fanconi anemia (FA) is a rare genetic disorder characterized by bone marrow failure and an increased risk for leukemia and cancer. Fifteen proteins thought to function in the repair of DNA interstrand crosslinks (ICLs) comprise what is known as the FA-BRCA pathway. Activation of this pathway leads to the monoubiquitylation and chromatin localization of FANCD2 and FANCI. It has previously been shown that FANCJ interacts with the mismatch repair (MMR) complex MutLα. Here we show that FANCD2 interacts with the MMR proteins MSH2 and MLH1. FANCD2 monoubiquitylation, foci formation and chromatin loading are greatly diminished in MSH2-deficient cells. Human or mouse cells lacking MSH2 or MLH1 display increased sensitivity and radial formation in response to treatment with DNA crosslinking agents. Studies in human cell lines and Drosophila mutants suggest an epistatic relationship between FANCD2, MSH2 and MLH1 with regard to ICL repair. Surprisingly, the interaction between MSH2 and MLH1 is compromised in multiple FA cell lines, and FA cell lines exhibit deficient MMR. These results suggest a significant role for MMR proteins in the activation of the FA pathway and repair of ICLs. In addition, we provide the first evidence for a defect in MMR in FA cell lines.

Running Gait Training Improves Outcomes at United States Air Force Basic Military Training.

INTRODUCTION: The aim is to investigate the impact of large-group, motor learning-based running gait training on injury risk in United States Air Force (USAF) Basic Military Training (BMT). DESIGN: A prospective quasi-experimental program evaluation is used. MATERIALS AND METHODS: Medical providers taught running gait form to groups of trainees in the first week of training of BMT from August 2020 to March 2021. The main outcome measures included risk ratio of reported injuries, removal from training because of injury, and separation from service because of injury. RESULTS: Of BMT trainees, 2,205 underwent group, motor learning-based running gait training; this was compared with two intake groups (nA = 3,941 and nB = 2,041) who were only given introductions to sports medicine staff in a classroom setting. Reported pain complaints increased (χ2 = 27.4A and 20.83B, P < .001). Risk ratios for more severe injuries necessitating time out of training or separation from USAF were reduced, although these were statistically not significant (13%, P = .48 and 22%, P = .29, respectively). Leadership implemented gait training across BMT, and data from the following 8 weeks of intake (n = 6,223) demonstrated similar trends in increases in patient reports of pain (χ2 = 67.25, P < .001) but significantly reduced risk ratios of removal from training (32%, χ2 = 16.35, P < .001) or separation (32%, χ2 = 12.54, P < .001). CONCLUSIONS: While not previously shown to mitigate injury, large-group, running gait training was associated with a significant reduction in injury severity defined by training delays and separation from service in USAF BMT.

Pathogenic Effector Th2 Cells in Allergic Eosinophilic Inflammatory Disease.

There is an absolute requirement for Th2 cells in the pathogenesis of allergen-driven eosinophil-rich type 2 inflammation. Although Th2 cells are generally regarded as a homogeneous population, in the past decade there has been increasing evidence for a minority subpopulation of IL-5+ Th2 cells that have enhanced effector function. This IL-5+ Th2 subpopulation has been termed pathogenic effector Th2 (peTh2), as it exhibits greater effector function and disease association than conventional Th2 cells. peTh2 cells have a different expression profile, differentially express transcription factors, and preferentially use specific signaling pathways. As such, peTh2 cells are a potential target in the treatment of allergic eosinophilic inflammation. This review examines peTh2 cells, both in mouse models and human disease, with an emphasis on their role in the pathogenesis of allergic eosinophilic inflammation.

Detection of Intracellular Cytokines by Flow Cytometry.

Intracellular cytokine staining (ICCS), employing fluorescently labeled MAbs detected by flow cytometry, has emerged as the premier technique for studying cytokine expression at the single-cell level. Advances in polychromatic flow cytometry have dramatically enhanced the sophistication of ICCS investigations. ICCS can simultaneously measure multiple cytokines within a single cell, allowing the detection of complex cytokine phenotypes. Additionally, cytokines can be measured with a variety of other analytes, including transcription factors, proliferation dilution dyes, activation markers, and viability dyes. This capability, combined with the high throughput inherent in the instrumentation, gives ICCS an enormous advantage over other single-cell techniques such as ELISPOT, limiting dilution, and T cell cloning. The unit describes intracellular staining of cells that have already been stimulated in vitro and fixed. Methods for in vitro activation by PMA and ionomycin or antigens, fixation of cell suspensions, and cell surface staining are also described.