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Type 1 Natural Killer T-cells (NKT1 cells) play a critical role in mediating hepatic ischemia-reperfusion injury (IRI). Although hepatic steatosis is a major risk factor for preservation type injury, how NKT cells impact this is understudied. Given NKT1 cell activation by phospholipid ligands recognized presented by CD1d, we hypothesized that NKT1 cells are key modulators of hepatic IRI because of the increased frequency of activating ligands in the setting of hepatic steatosis. We first demonstrate that IRI is exacerbated by a high-fat diet (HFD) in experimental murine models of warm partial ischemia. This is evident in the evaluation of ALT levels and Phasor-Fluorescence Lifetime (Phasor-FLIM) Imaging for glycolytic stress. Polychromatic flow cytometry identified pronounced increases in CD45+CD3+NK1.1+NKT1 cells in HFD fed mice when compared to mice fed a normal diet (ND). This observation is further extended to IRI, measuring ex vivo cytokine expression in the HFD and ND. Much higher interferon-gamma (IFN-γ) expression is noted in the HFD mice after IRI. We further tested our hypothesis by performing a lipidomic analysis of hepatic tissue and compared this to Phasor-FLIM imaging using “long lifetime species”, a byproduct of lipid oxidation. There are higher levels of triacylglycerols and phospholipids in HFD mice. Since N-acetylcysteine (NAC) is able to limit hepatic steatosis, we tested how oral NAC supplementation in HFD mice impacted IRI. Interestingly, oral NAC supplementation in HFD mice results in improved hepatic enhancement using contrast-enhanced magnetic resonance imaging (MRI) compared to HFD control mice and normalization of glycolysis demonstrated by Phasor-FLIM imaging. This correlated with improved biochemical serum levels and a decrease in IFN-γ expression at a tissue level and from CD45+CD3+CD1d+ cells. Lipidomic evaluation of tissue in the HFD+NAC mice demonstrated a drastic decrease in triacylglycerol, suggesting downregulation of the PPAR-γ pathway.

Innate lymphoid cells (ILCs), the most recently described family of lymphoid cells, play fundamental roles in tissue homeostasis through the production of key cytokine. Group 1 ILCs, comprised of conventional natural killer cells (cNKs) and type 1 ILCs (ILC1s), have been implicated in regulating immune-mediated inflammatory diseases. However, the role of ILC1s in nonalcoholic fatty liver disease (NAFLD) and ischemia-reperfusion injury (IRI) is unclear. Here, we investigated the role of ILC1 and cNK cells in a high-fat diet (HFD) murine model of partial warm IRI. We demonstrated that hepatic steatosis results in more severe IRI compared to non-steatotic livers. We further elicited that HFD-IRI mice show a significant increase in the ILC1 population, whereas the cNK population was unchanged. Since ILC1 and cNK are major sources of IFN-γ and TNF-α, we measured the level of ex vivo cytokine expression in normal diet (ND)-IRI and HFD-IRI conditions. We found that ILC1s in HFD-IRI mice produce significantly more IFN-γ and TNF-α when compared to ND-IRI. To further assess whether ILC1s are key proinflammatory effector cells in hepatic IRI of fatty livers, we studied both Rag1 −/− mice, which possess cNK cells, and a substantial population of ILC1s versus the newly generated Rag1 −/− Tbx21 −/− double knockout (Rag1-Tbet DKO) mice, which lack type 1 ILCs, under HFD IRI conditions. Importantly, HFD Rag1-Tbet DKO mice showed significant protection from hepatic injury upon IRI when compared to Rag1 −/− mice, suggesting that T-bet-expressing ILC1s play a role, at least in part, as proinflammatory effector cells in hepatic IRI under steatotic conditions.

Pulmonary Hypertension (PH) is a pathophysiologic condition characterized by hypoxemia and right ventricular strain. Proliferation of fibroblasts, smooth muscle cells, and endothelial cells is central to the pathology of PH in animal models and in humans. Methionine aminopeptidase-2 (MetAP2) regulates proliferation in a variety of cell types including endothelial cells, smooth muscle cells, and fibroblasts. MetAP2 is inhibited irreversibly by the angiogenesis inhibitor fumagillin. We have previously found that inhibition of MetAP2 with fumagillin in bleomycin-injured mice decreased pulmonary fibrosis by selectively decreasing the proliferation of lung myofibroblasts. In this study, we investigated the role of fumagillin as a potential therapy in experimental PH. In vivo, treatment of rats with fumagillin early after monocrotaline injury prevented PH and right ventricular remodeling by decreasing the thickness of the medial layer of the pulmonary arteries. Treatment with fumagillin beginning two weeks after monocrotaline injury did not prevent PH but was associated with decreased right ventricular mass and decreased cardiomyocyte hypertrophy, suggesting a direct effect of fumagillin on right ventricular remodeling. Incubation of rat pulmonary artery smooth muscle cells (RPASMC) with fumagillin and MetAP2-targeting siRNA inhibited proliferation of RPASMC in vitro. Platelet-derived growth factor, a growth factor that is important in the pathogenesis of PH and stimulates proliferation of fibroblasts and smooth muscle cells, strongly increased expression of MetP2. By immunohistochemistry, we found that MetAP2 was expressed in the lesions of human pulmonary arterial hypertension. We propose that fumagillin may be an effective adjunctive therapy for treating PH in patients.

Mycoplasma-induced rash and mucositis (MIRM) is a variant of Stevens-Johnson Syndrome (SJS) gered by pneumoniae. It is characterized by sparse/absent cutaneous involvement and moderate/severe involvement of two or more mucosal sites. Esophageal findings in MIRM have not been described and rarely identified in SJS as endoscopy is not routine. A 16-year-old male with Ehlers Danlos syndrome, PAI-1 mutation and Chiari malformation, presented with fever, URI symptoms and rapidly progressing oral blisters for three days. M. pneumoniae PCR was positive and HSV PCR was negative. He then developed eye and urogenital involvement, as well as severe odynophagia. Three years prior, a similar episode required multi-specialty evaluation and the lesions resolved without therapy or definitive diagnosis; however, viral etiology was suspected. Given the extent of oral lesions, he underwent endoscopy which showed mild ulcerations in the mid and distal esophagus. Biopsies showed intraepidermal acantholysis and a focal suprabasilar cleft. Repeat endoscopy during his second episode revealed erythema, friability and white mucosal protrusions, suggestive of pervasive exfoliative esophagitis. Esophageal biopsies described similar findings with increased severity. CMV, HSV and fungal staining were negative. The stomach and duodenum were normal both times. He was empirically treated for Mycoplasma and required nutrition via NG tube. Swallowed topical fluticasone was initiated and continued until follow-up endoscopy two months later. Due to concern for slow clinical response, he received two doses of high-dose IVIG administered three days after starting fluticasone and five days after completing a course of azithromycin. Rapid clinical improvement was noted. Two months later, he had complete endoscopic and histologic resolution of esophagitis. Fluticasone therapy was discontinued and he has remained asymptomatic. Digestive tract lesions are rare manifestations of MIRM or SJS. Limited cases have been reported and primarily in association with SJS. This is the first case with endoscopic confirmation of severe unique esophageal pathology associated with MIRM. It illustrates the extent to which esophageal mucosa can be affected in MIRM and provides reassurance that full recovery can be achieved once the underlying infection is treated. The efficacy of swallowed topical fluticasone in resolving esophagitis cannot be confirmed as additional concurrent therapies were given.

This study explored second year undergraduates' academic motivators and blockers at multiple ecological system levels. A total of 47 second year undergraduate students from Engineering, Nursing and Psychology in an Australian city university took part in participant-led interviews regarding their academic experience beyond their foundational semester. Using an ecological system approach as the overarching theoretical framework, Tinto's model of student retention/persistence and Self-Determination Theory (SDT) were merged within this framework to identify potential motivators and blockers across individual, microsystem, mesosystem and macrosystem levels. Results revealed that academic motivators and blockers existed at multiple ecological levels and wielded direct as well as indirect impacts on student persistence. Cohort and context-specific challenges were also identified and this demonstrated the need to better understand the contributions of an ecological approach to student persistence experience. [Author abstract]

Health professional students from high-income countries increasingly participate in short-term experiences in global health (STEGH) conducted abroad. One common criticism of STEGH is the inherent power differential that exists between visiting learners and the local community. To highlight this power differential, this paper explores perceived benefits as described by volunteer and community respondents and applies Maslow's hierarchy of needs to commonly identified themes in each respondent group. A semistructured survey was used to collect qualitative responses from both volunteers and community members located in a Dominican Republic community, that is, a hotspot for traditionally conducted STEGH. Thematic analysis identified themes of perceived benefits from both respondent groups; each group's common themes were then classified and compared within Maslow's hierarchy of needs. Each respondent group identified resource provision as a perceived benefit of STEGH, but volunteer respondents primarily focused on the provision of highly-skilled, complex resources while community respondents focused on basic necessities (food, water, etc.) Volunteer respondents were also the only group to also mention spiritual/religious/life experiences, personal skills development, and relationships as perceived benefits. Applying Maslow's hierarchy thus demonstrates a difference in needs: community respondents focused on benefits that address deficiency needs at the bottom of the hierarchy while volunteers focused on benefits addressing self-transcendence/actualization needs at the top of the hierarchy. The perceived difference in needs met by STEGH between volunteers and the host community within Maslow's hierarchy may drive an inherent power differential. Refocusing STEGH on the relationship level of the hierarchy (i.e., focusing on partnerships) might help mitigate this imbalance and empower host communities.

BackgroundReproducible immune profiling is crucial for translational and clinical research and for developing accurate prognoses and effective treatments. Reproducibly identifying low-expressing targets and rare functional populations is challenging with fluorescence flow cytometry due to spectral spillover, autofluorescence and unmixing errors, issues not present with mass cytometry. Further, multiple workflows in mass cytometry provide flexibility and minimize technical variation, including the abilities to freeze metal-tagged antibody cocktails, barcode samples and freeze stained samples for future acquisition. The goal of this study was to assess the consistency and robustness of mass cytometry across various staining and acquisition workflows.MethodsHuman whole blood and PBMC samples were stained with antibody panels containing up to 50 surface and intracellular markers for phenotyping and functional profiling via checkpoint markers and cytokines. Stained samples were frozen and acquired on a later date using three CyTOF™ XT PRO and three CyTOF XT systems to assess repeatability and reproducibility, and to ensure data quality was not compromised when samples were acquired at 2x and 4x speeds with the CyTOF XT PRO system.ResultsBoth systems were highly repeatable and reproducible across multiple instruments. Cell population frequencies and signal intensities were not significantly different between the two CyTOF systems. Both systems had high sensitivity and dynamic range for abundant and low-abundance markers to accurately identify T helper cell populations. Furthermore, opt-SNE analysis revealed that the enhanced throughput of the CyTOF XT PRO system retained clear visualization of major immune subsets and striking functional diversity in high-dimensional space.ConclusionsOverall, these studies demonstrate that CyTOF XT and CyTOF XT PRO systems generate highly repeatable and reproducible data. Moreover, the CyTOF XT PRO system enables acquisition at up to 4x increased event rate without compromising data quality. Automated acquisition by the CyTOF XT PRO system enables researchers to accurately and reproducibly streamline human immunophenotyping and functional profiling, leading to accelerated biological insights and discoveries.For Research Use Only. Not for use in diagnostic procedures.

Aims We investigated titration patterns of angiotensin‐converting enzyme inhibitors (ACEis)/angiotensin receptor blockers (ARBs) and beta‐blockers, quality of life (QoL) over 6 months, and associated 1 year outcome [all‐cause mortality/heart failure (HF) hospitalization] in a real‐world population with HF with reduced ejection fraction (HFrEF). Methods and results Participants with HFrEF (left ventricular ejection fraction <40%) from a prospective multi‐centre study were examined for use and dose [relative to guideline‐recommended maintenance dose (GRD)] of ACEis/ARBs and beta‐blockers at baseline and 6 months. ‘Stay low’ was defined as <50% GRD at both time points, ‘stay high’ as ≥50% GRD, and ‘up‐titrate’ and ‘down‐titrate’ as dose trajectories. Among 1110 patients (mean age 63 ± 13 years, 16% women, 26% New York Heart Association Class III/IV), 714 (64%) were multi‐ethnic Asians from Singapore and 396 were from New Zealand (mainly European ethnicity). Baseline use of either ACEis/ARBs or beta‐blockers was high (87%). Loop diuretic was prescribed in >80% of patients, mineralocorticoid receptor antagonist in about half of patients, and statins in >90% of patients. At baseline, only 11% and 9% received 100% GRD for each drug class, respectively, with about half (47%) achieving ≥50% GRD for ACEis/ARBs or beta‐blockers. At 6 months, a large majority remained in the ‘stay low’ category, one third remained in ‘stay high’, whereas 10–16% up‐titrated and 4–6% down‐titrated. Patients with lower (vs. higher) N‐terminal pro‐beta‐type natriuretic peptide levels were more likely to be up‐titrated or be in ‘stay high’ for ACEis/ARBs and beta‐blockers (P = 0.002). Ischaemic aetiology, prior HF hospitalization, and enrolment in Singapore (vs. New Zealand) were independently associated with higher odds of ‘staying low’ (all P < 0.005) for prescribed doses of ACEis/ARBs and beta‐blockers. Adjusted for inverse probability weighting, ≥100% GRD for ACEis/ARBs [hazard ratio (HR) = 0.42; 95% confidence interval (CI) 0.24–0.73] and ≥50% GRD for beta‐blockers (HR = 0.58; 95% CI 0.37–0.90) (vs. Nil) were associated with lower hazards for 1 year composite outcome. Country of enrolment did not modify the associations of dose categories with 1 year composite outcome. Higher medication doses were associated with greater improvements in QoL. Conclusions Although HF medication use at baseline was high, most patients did not have these medications up‐titrated over 6 months. Multiple clinical factors were associated with changes in medication dosages. Further research is urgently needed to investigate the causes of lack of up‐titration of HF therapy (and its frequency), which could inform strategies for timely up‐titration of HF therapy based on clinical and biochemical parameters.