Somatic mutations associate with clonal expansion of CD8+ T cells.

Somatic mutations in T cells can cause cancer but also have implications for immunological diseases and cell therapies. The mutation spectrum in nonmalignant T cells is unclear. Here, we examined somatic mutations in CD4+ and CD8+ T cells from 90 patients with hematological and immunological disorders and used T cell receptor (TCR) and single-cell sequencing to link mutations with T cell expansions and phenotypes. CD8+ cells had a higher mutation burden than CD4+ cells. Notably, the biggest variant allele frequency (VAF) of non-synonymous variants was higher than synonymous variants in CD8+ T cells, indicating non-random occurrence. The non-synonymous VAF in CD8+ T cells strongly correlated with the TCR frequency, but not age. We identified mutations in pathways essential for T cell function and often affected lymphoid neoplasia. Single-cell sequencing revealed cytotoxic TEMRA phenotypes of mutated T cells. Our findings suggest that somatic mutations contribute to CD8+ T cell expansions without malignant transformation.

Somatic compensation of inherited bone marrow failure.

Inherited bone marrow failure syndromes (IBMFS) are a heterogeneous group of genetic disorders characterized by insufficient blood cell production and increased risk of transformation to myeloid malignancies. While genetically diverse, IBMFS are collectively defined by a cell-intrinsic hematopoietic stem cell (HSC) fitness defect that impairs HSC self-renewal and hematopoietic differentiation. In IBMFS, HSCs frequently acquire mutations that improve cell fitness, a phenomenon known as somatic compensation. Somatic compensation can occur via distinct genetic processes such as loss of the germline mutation or somatic alterations in pathways affected by the disease-causing gene. While the clinical implications of somatic compensation in IBMFS remain to be fully discovered, understanding these mutational processes can help understand disease pathophysiology and may inform future diagnostic and therapeutic approaches. In this review, we highlight current understanding about somatic compensation in IBMFS.

Nursing Students' Perceptions on Healthcare-Associated Infection Control and Prevention Teaching and Learning Experience in Portugal.

Healthcare-associated infections (HAI) are one of the major concerns worldwide, posing significant challenges to healthcare professionals' education and training. This study intended to measure nursing students' perceptions regarding their learning experiences on HAI prevention and control. In the first phase of the study, a cross-sectional and descriptive study with a convenience sample composed of undergraduate nursing students from Portugal, Spain, Poland, and Finland was conducted to develop the InovSafeCare questionnaire. In the second phase, we applied the InovSafeCare scale in a sample of nursing students from two Portuguese higher education institutions to explore which factors impact nursing students' adherence to HAI prevention and control measures in clinical settings. In phase one, the InovSafeCare questionnaire was applied to 1326 students internationally, with the instrument presenting adequate psychometric qualities with reliability results in 14 dimensions. During phase two, the findings supported that Portuguese nursing students' adherence to HAI prevention and control measures is influenced not only by the curricular offerings and resources available in academic settings, but also by the standards conveyed by nursing tutors during clinical placements. Our findings support the need for a dedicated curricular focus on HAI prevention and control learning, not only through specific classroom modules, innovative resources, and pedagogical approaches, but also through a complementary and coordinated liaison between teachers and tutors in academic and clinical settings.

Nursing Students' Perceptions on Healthcare-Associated Infection Control and Prevention Teaching and Learning Experience: Development and Validation of a Scale in Four European Countries.

Healthcare-associated infections are one of the major concerns worldwide. This study presents the development and the validation process of the InovSafeCare scale and aimed at identifying and measuring the ecosystem variables related to healthcare-associated infection (HCAI) prevention and control practices in European nurse students. Qualitative and quantitative approaches were used to (1) elaborate an item pool related to the educational environment, the healthcare setting environment, and the attitudes, beliefs, and performance of the nursing students regarding HCAI prevention and control and (2) analyze psychometric properties of the scale using factor analysis. The validated InovSafeCare scale was applied to undergraduate nursing students of five European Higher Education Institutions. The partial least square structural equation modeling (PLS-SEM) method with SMART-PLS3 software was used. The study sample consists of 657 nursing students, who responded a self-report inventory. From the analyzed data were identified 14 factors. The InovSafeCare scale reveals good validity and reliability of the dimensions in different European countries.

STAT3 activation in large granular lymphocyte leukemia is associated with cytokine signaling and DNA hypermethylation.

Large granular lymphocyte leukemia (LGLL) is characterized by somatic gain-of-function STAT3 mutations. However, the functional effects of STAT3 mutations on primary LGLL cells have not been studied in detail. In this study, we show that CD8+ T cells isolated from STAT3 mutated LGLL patients have high protein levels of epigenetic regulators, such as DNMT1, and are characterized by global hypermethylation. Correspondingly, treatment of healthy CD8+ T cells with IL-6, IL-15, and/or MCP-1 cytokines resulted in STAT3 activation, increased DNMT1, EZH2, c-MYC, l-MYC, MAX, and NFκB levels, increased DNA methylation, and increased oxidative stress. Similar results were discovered in KAI3 NK cells overexpressing gain-of-function STAT3Y640F and STAT3G618R mutants compared to KAI3 NK cells overexpressing STAT3WT. Our results also confirm that STAT3 forms a direct complex with DNMT1, EZH2, and HDAC1. In STAT3 mutated LGLL cells, DNA methyltransferase (DNMT) inhibitor azacitidine abrogated the activation of STAT3 via restored SHP1 expression. In conclusion, STAT3 mutations cause DNA hypermethylation resulting in sensitivity to DNMT inhibitors, which could be considered as a novel treatment option for LGLL patients with resistance to standard treatments.

The clinical and functional effects of TERT variants in myelodysplastic syndrome.

Germline pathogenic TERT variants are associated with short telomeres and an increased risk of developing myelodysplastic syndrome (MDS) among patients with a telomere biology disorder. We identified TERT rare variants in 41 of 1514 MDS patients (2.7%) without a clinical diagnosis of a telomere biology disorder who underwent allogeneic transplantation. Patients with a TERT rare variant had shorter telomere length (P < .001) and younger age at MDS diagnosis (52 vs 59 years, P = .03) than patients without a TERT rare variant. In multivariable models, TERT rare variants were associated with inferior overall survival (P = .034) driven by an increased incidence of nonrelapse mortality (NRM; P = .015). Death from a noninfectious pulmonary cause was more frequent among patients with a TERT rare variant. Most variants were missense substitutions and classified as variants of unknown significance. Therefore, we cloned all rare missense variants and quantified their impact on telomere elongation in a cell-based assay. We found that 90% of TERT rare variants had severe or intermediate impairment in their capacity to elongate telomeres. Using a homology model of human TERT bound to the shelterin protein TPP1, we inferred that TERT rare variants disrupt domain-specific functions, including catalysis, protein-RNA interactions, and recruitment to telomeres. Our results indicate that the contribution of TERT rare variants to MDS pathogenesis and NRM risk is underrecognized. Routine screening for TERT rare variants in MDS patients regardless of age or clinical suspicion may identify clinically inapparent telomere biology disorders and improve transplant outcomes through risk-adapted approaches.

Short telomere length predicts nonrelapse mortality after stem cell transplantation for myelodysplastic syndrome.

Allogeneic hematopoietic stem cell transplantation is the only potentially curative treatment for patients with myelodysplastic syndrome (MDS), but long-term survival is limited by the risk of transplant-related complications. Short telomere length, mediated by inherited or acquired factors, impairs cellular response to genotoxic and replicative stress and could identify patients at higher risk for toxicity after transplantation. We measured relative telomere length in pretransplant recipient blood samples in 1514 MDS patients and evaluated the association of telomere length with MDS disease characteristics and transplantation outcomes. Shorter telomere length was significantly associated with older age, male sex, somatic mutations that impair the DNA damage response, and more severe pretransplant cytopenias, but not with bone marrow blast count, MDS treatment history, or history of prior cancer therapy. Among 1267 patients ≥40 years old, telomere length in the shortest quartile was associated with inferior survival (P < .001) because of a high risk of nonrelapse mortality (NRM; P = .001) after adjusting for significant clinical and genetic variables. The adverse impact of shorter telomeres on NRM was independent of recipient comorbidities and was observed selectively among patients receiving more intensive conditioning, including myeloablative regimens and higher dose melphalan-based reduced-intensity regimens. The effect of shorter telomeres on NRM was prominent among patients who developed severe acute graft-versus-host disease, suggesting that short telomere length may limit regenerative potential of mucosal tissues after acute injury. MDS patients with shorter telomere length, who have inferior survival driven by excess toxicity, could be considered for strategies focused on minimizing toxic effects of transplantation.

Somatic mTOR mutation in clonally expanded T lymphocytes associated with chronic graft versus host disease.

Graft versus host disease (GvHD) is the main complication of allogeneic hematopoietic stem cell transplantation (HSCT). Here we report studies of a patient with chronic GvHD (cGvHD) carrying persistent CD4+ T cell clonal expansion harboring somatic mTOR, NFKB2, and TLR2 mutations. In the screening cohort (n = 134), we detect the mTOR P2229R kinase domain mutation in two additional cGvHD patients, but not in healthy or HSCT patients without cGvHD. Functional analyses of the mTOR mutation indicate a gain-of-function alteration and activation of both mTORC1 and mTORC2 signaling pathways, leading to increased cell proliferation and decreased apoptosis. Single-cell RNA sequencing and real-time impedance measurements support increased cytotoxicity of mutated CD4+ T cells. High throughput drug-sensitivity testing suggests that mutations induce resistance to mTOR inhibitors, but increase sensitivity for HSP90 inhibitors. Our findings imply that somatic mutations may contribute to aberrant T cell proliferations and persistent immune activation in cGvHD, thereby paving the way for targeted therapies.

GABAB receptor positive allosteric modulators with different efficacies affect neuroadaptation to and self-administration of alcohol and cocaine.

Drugs of abuse induce widespread synaptic adaptations in the mesolimbic dopamine (DA) neurons. Such drug-induced neuroadaptations may constitute an initial cellular mechanism eventually leading to compulsive drug-seeking behavior. To evaluate the impact of GABAB receptors on addiction-related persistent neuroplasticity, we tested the ability of orthosteric agonist baclofen and two positive allosteric modulators (PAMs) of GABAB receptors to suppress neuroadaptations in the ventral tegmental area (VTA) and reward-related behaviors induced by ethanol and cocaine. A novel compound (S)-1-(5-fluoro-2,3-dihydro-1H-inden-2-yl)-4-methyl-6,7,8,9-tetrahydro-[1,2,4]triazolo[4,3-a]quinazolin-5(4H)-one (ORM-27669) was found to be a GABAB PAM of low efficacy as agonist, whereas the reference compound (R,S)-5,7-di-tert-butyl-3-hydroxy-3-trifluoromethyl-3H-benzofuran-2-one (rac-BHFF) had a different allosteric profile being a more potent PAM in the calcium-based assay and an agonist, coupled with potent PAM activity, in the [35 S] GTPγS binding assay in rat and human recombinant receptors. Using autoradiography, the high-efficacy rac-BHFF and the low-efficacy ORM-27669 potentiated the effects of baclofen on [35 S] GTPγS binding with identical brain regional distribution. Treatment of mice with baclofen, rac-BHFF, or ORM-27669 failed to induce glutamate receptor neuroplasticity in the VTA DA neurons. Pretreatment with rac-BHFF at non-sedative doses effectively reversed both ethanol- and cocaine-induced plasticity and attenuated cocaine i.v. self-administration and ethanol drinking. Pretreatment with ORM-27669 only reversed ethanol-induced neuroplasticity and attenuated ethanol drinking but had no effects on cocaine-induced neuroplasticity or self-administration. These findings encourage further investigation of GABAB receptor PAMs with different efficacies in addiction models to develop novel treatment strategies for drug addiction.

Exploring effects of remote ischemic preconditioning in a pig model of hypothermic circulatory arrest.

OBJECTIVES: During aortic and cardiac surgery, risks for mortality and morbidity are inevitable. Surgical setups involving deep hypothermic circulatory arrest (DHCA) are effective to achieve organ protection against ischemic injury. The aim of this study was to identify humoural factors mediating additive protective effects of remote ischemic preconditioning (RIPC) in a porcine model of DHCA. DESIGN: Twenty-two pigs were randomized into the RIPC group (n = 11) and the control group (n = 11). The RIPC group underwent four 5-minute hind limb ischemia-reperfusion cycles prior to cardiopulmonary bypass and DHCA. All animals underwent identical surgical procedures including 60 min DHCA at 18 °C. Blood samples were collected from vena cava and sagittal sinus at several time points. After the 8-hour follow-up period, the brain, heart, and kidney tissue samples were collected for tissue analyses. RESULTS: Serum levels of brain damage marker S100B recovered faster in the RIPC group, after 4 hours of the arrest, (p < .05). Systemic lactate levels were lower and cardiac index was higher in the RIPC group postoperatively. Immunohistochemical cerebellum regional scores of antioxidant response regulator Nrf2 were better in the RIPC group (mean: 1.1, IQR: 0.0-2.5) compared with the control group (mean: 0.0, IQR: 0.0-0.0), reaching borderline statistical significance (p = .064). RIPC induced detectable modulations of plasma proteome and metabolites. CONCLUSIONS: The faster recovery of S100B, lower systemic lactate levels and favourable regional antioxidant response suggest possible neuronal cellular and mitochondrial protection by RIPC, whereas better cardiac index underlines functional effects of RIPC. The exact humoural factor remains unclear.

Notch Downregulation and Extramedullary Erythrocytosis in Hypoxia-Inducible Factor Prolyl 4-Hydroxylase 2-Deficient Mice.

Erythrocytosis is driven mainly by erythropoietin, which is regulated by hypoxia-inducible factor (HIF). Mutations in HIF prolyl 4-hydroxylase 2 (HIF-P4H-2) (PHD2/EGLN1), the major downregulator of HIFα subunits, are found in familiar erythrocytosis, and large-spectrum conditional inactivation of HIF-P4H-2 in mice leads to severe erythrocytosis. Although bone marrow is the primary site for erythropoiesis, spleen remains capable of extramedullary erythropoiesis. We studied HIF-P4H-2-deficient (Hif-p4h-2gt/gt) mice, which show slightly induced erythropoiesis upon aging despite nonincreased erythropoietin levels, and identified spleen as the site of extramedullary erythropoiesis. Splenic hematopoietic stem cells (HSCs) of these mice exhibited increased erythroid burst-forming unit (BFU-E) growth, and the mice were protected against anemia. HIF-1α and HIF-2α were stabilized in the spleens, while the Notch ligand genes Jag1, Jag2, and Dll1 and target Hes1 became downregulated upon aging HIF-2α dependently. Inhibition of Notch signaling in wild-type spleen HSCs phenocopied the increased BFU-E growth. HIFα stabilization can thus mediate non-erythropoietin-driven splenic erythropoiesis via altered Notch signaling.

HIF-P4H-2 deficiency protects against skeletal muscle ischemia-reperfusion injury.

We show here that mice hypomorphic for hypoxia-inducible factor prolyl 4-hydroxylase-2 (HIF-P4H-2) (Hif-p4h-2 (gt/gt)), the main regulator of the stability of the HIFα subunits, have normoxic stabilization of HIF-1α and HIF-2α in their skeletal muscles. The size of the capillaries, but not their number, was increased in the skeletal muscles of the Hif-p4h-2 (gt/gt) mice, whereas the amount of glycogen was reduced. The expression levels of genes for glycolytic enzymes, glycogen branching enzyme 1 and monocarboxylate transporter 4, were increased in the Hif-p4h-2 (gt/gt) skeletal muscles, whereas no significant increases were detected in the levels of any vasculature-influencing factor studied. Serum lactate levels of the Hif-p4h-2 (gt/gt) mice recovered faster than those of the wild type following exercise. The Hif-p4h-2 (gt/gt) mice had elevated hepatic phosphoenolpyruvate carboxykinase activity, which may have contributed to the faster clearance of lactate. The Hif-p4h-2 (gt/gt) mice had smaller infarct size following limb ischemia-reperfusion injury. The increased capillary size correlated with the reduced infarct size. Following ischemia-reperfusion, glycogen content and ATP/ADP and CrP/Cr levels of the skeletal muscle of the Hif-p4h-2 (gt/gt) mice were higher than in the wild type. The higher glycogen content correlated with increased expression of phosphofructokinase messenger RNA (mRNA) and the increased ATP/ADP and CrP/Cr levels with reduced apoptosis, suggesting that HIF-P4H-2 deficiency supported energy metabolism during ischemia-reperfusion and protection against injury. Key messages: HIF-P4H-2 deficiency protects skeletal muscle from ischemia-reperfusion injury. The mechanisms involved are mediated via normoxic HIF-1α and HIF-2α stabilization. HIF-P4H-2 deficiency increases capillary size but not number. HIF-P4H-2 deficiency maintains energy metabolism during ischemia-reperfusion.

A genetic mechanism for Tibetan high-altitude adaptation.

Tibetans do not exhibit increased hemoglobin concentration at high altitude. We describe a high-frequency missense mutation in the EGLN1 gene, which encodes prolyl hydroxylase 2 (PHD2), that contributes to this adaptive response. We show that a variant in EGLN1, c.[12C>G; 380G>C], contributes functionally to the Tibetan high-altitude phenotype. PHD2 triggers the degradation of hypoxia-inducible factors (HIFs), which mediate many physiological responses to hypoxia, including erythropoiesis. The PHD2 p.[Asp4Glu; Cys127Ser] variant exhibits a lower K(m) value for oxygen, suggesting that it promotes increased HIF degradation under hypoxic conditions. Whereas hypoxia stimulates the proliferation of wild-type erythroid progenitors, the proliferation of progenitors with the c.[12C>G; 380G>C] mutation in EGLN1 is significantly impaired under hypoxic culture conditions. We show that the c.[12C>G; 380G>C] mutation originated ∼8,000 years ago on the same haplotype previously associated with adaptation to high altitude. The c.[12C>G; 380G>C] mutation abrogates hypoxia-induced and HIF-mediated augmentation of erythropoiesis, which provides a molecular mechanism for the observed protection of Tibetans from polycythemia at high altitude.

3-Heterocycle-phenyl N-alkylcarbamates as FAAH inhibitors: design, synthesis and 3D-QSAR studies.

Carbamates are a well-established class of fatty acid amide hydrolase (FAAH) inhibitors. Here we describe the synthesis of meta-substituted phenolic N-alkyl/aryl carbamates and their in vitro FAAH inhibitory activities. The most potent compound, 3-(oxazol-2yl)phenyl cyclohexylcarbamate (2 a), inhibited FAAH with a sub-nanomolar IC(50) value (IC(50)=0.74 nM). Additionally, we developed and validated three-dimensional quantitative structure-activity relationships (QSAR) models of FAAH inhibition combining the newly disclosed carbamates with our previously published inhibitors to give a total set of 99 compounds. Prior to 3D-QSAR modeling, the degree of correlation between FAAH inhibition and in silico reactivity was also established. Both 3D-QSAR methods used, CoMSIA and GRID/GOLPE, produced statistically significant models with coefficient of correlation for external prediction (R(2) (PRED)) values of 0.732 and 0.760, respectively. These models could be of high value in further FAAH inhibitor design.

Chiral 3-(4,5-dihydrooxazol-2-yl)phenyl alkylcarbamates as novel FAAH inhibitors: Insight into FAAH enantioselectivity by molecular docking and interaction fields.

Fatty acid amide hydrolase (FAAH) and monoglyceride lipase (MGL) are the main enzymes responsible for the hydrolysis of endogenous cannabinoids N-arachidonoylethanolamide (AEA) and 2-arachidonoylglycerol (2-AG), respectively. Phenyl alkylcarbamates are FAAH inhibitors with anxiolytic and analgesic activities in vivo. Herein we present for the first time the synthesis and biological evaluation of a series of chiral 3-(2-oxazoline)-phenyl N-alkylcarbamates as FAAH inhibitors. Furthermore, the structural background of chirality on the FAAH inhibition is explored by analyzing the protein-ligand interactions. Remarkably, 10-fold difference in potency was observed for (R)- and (S)-derivatives of 3-(5-methyl-4,5-dihydrooxazol-2-yl)phenyl cyclohexylcarbamate (6a vs. 6b). Molecular modelling indicated an important interaction between the oxazoline nitrogen and FAAH active site.

The synthesis and biological evaluation of para-substituted phenolic N-alkyl carbamates as endocannabinoid hydrolyzing enzyme inhibitors.

A series of para-substituted phenolic N-alkyl carbamates were evaluated for their FAAH and MGL inhibitory activities. The compounds were generally selective for FAAH, with IC(50) values in the nM range, whereas inhibition of MGL required concentrations three orders of magnitude higher. The most potent compounds, dodecylcarbamic acid 4-(4,5-dihydrothiazol-2-yl)phenyl (12) and 4-(1,2,3-thiadiazol-4-yl)phenyl (26) esters, inhibited FAAH and MGL with IC(50) values at the low-nanomolar (IC(50)s; 0.0063 and 0.012 microM) and the low-micromolar ranges (IC(50)s; 2.1 and 1.0 microM), respectively. Compound 26 also inhibited both FAAH-dependent AEA uptake and AEA hydrolysis (IC(50); 0.082 microM) by intact RBL2H3 cells, and could also reduce 2-AG hydrolysis by these cells at concentrations >or=0.030 microM.

Design, synthesis, and in vitro evaluation of carbamate derivatives of 2-benzoxazolyl- and 2-benzothiazolyl-(3-hydroxyphenyl)-methanones as novel fatty acid amide hydrolase inhibitors.

Fatty acid amide hydrolase (FAAH) is an intracellular serine hydrolase, which catalyzes the hydrolysis of the endocannabinoid N-arachidonoylethanolamide to arachidonic acid and ethanolamine. FAAH also hydrolyzes another endocannabinoid, 2-arachidonoylglycerol (2-AG). However, 2-AG has been assumed to be hydrolyzed mainly by monoacylglycerol lipase (MAGL) or a MAGL-like enzyme. Inhibition of FAAH or MAGL activity might lead to beneficial effects in many physiological disorders such as pain, inflammation, and anxiety due to increased endocannabinoid-induced activation of cannabinoid receptors CB1 and CB2. In the present study, a total of 34 novel compounds were designed, synthesized, characterized, and tested against FAAH and MAGL-like enzyme activity. Altogether, 16 compounds were found to inhibit FAAH with half-maximal inhibition concentrations (IC50) between 28 and 380 nM. All the active compounds belong to the structural family of carbamates. Compounds 14 and 18 were found to be the most potent FAAH inhibitors, which may serve as lead structures for novel FAAH inhibitors.