Tγδ LGLL identifies a subset with more symptomatic disease: analysis of an international cohort of 137 patients.

Tγδ large granular lymphocyte leukemia (LGLL) is a rare variant of T-cell LGLL (T-LGLL) that has been less investigated as compared with the more frequent Tαß LGLL, particularly in terms of frequency of STAT3 and STAT5b mutations. In this study, we characterized the clinical and biological features of 137 patients affected by Tγδ LGLL; data were retrospectively collected from 1997 to 2020 at 8 referral centers. Neutropenia and anemia were the most relevant clinical features, being present in 54.2% and 49.6% of cases, respectively, including severe neutropenia and anemia in ∼20% of cases each. Among the various treatments, cyclosporine A was shown to provide the best response rates. DNA samples of 97 and 94 cases were available for STAT3 and STAT5b mutation analysis, with 38.1% and 4.2% of cases being mutated, respectively. Clinical and biological features of our series of Tγδ cases were also compared with a recently published Tαß cohort including 129 cases. Though no differences in STAT3 and STAT5b mutational frequency were found, Tγδ cases more frequently presented with neutropenia (P = .0161), anemia (P < .0001), severe anemia (P = .0065), and thrombocytopenia (P = .0187). Moreover, Vδ2- cases displayed higher frequency of symptomatic disease. Overall, Tγδ cases displayed reduced survival with respect to Tαß cases (P = .0017). Although there was no difference in STAT3 mutation frequency, our results showed that Tγδ LGLL represents a subset of T-LGLL characterized by more frequent symptoms and reduced survival as compared with Tαß LGLL.

Distinguishing pathophysiological features of heart failure with reduced and preserved ejection fraction: A comparative analysis of two mouse models.

Heart failure (HF) is a heterogeneous condition that can be categorized according to the left ventricular ejection fraction (EF) into HF with reduced (HFrEF) or preserved (HFpEF) EF. Although HFrEF and HFpEF share some common clinical manifestations, the mechanisms underlying each phenotype are often found to be distinct. Identifying shared and divergent pathophysiological features might expand our insights on HF pathophysiology and assist the search for therapies for each HF subtype. In this study, we evaluated and contrasted two new murine models of non-ischaemic HFrEF and cardiometabolic HFpEF in terms of myocardial structure, left ventricular function, gene expression, cardiomyocyte calcium handling, mitochondrial polarization and protein acetylation in a head-to-head fashion. We found that in conditions of similar haemodynamic stress, the HFrEF myocardium underwent a more pronounced hypertrophic and fibrotic remodelling, whereas inflammation was greater in the HFpEF myocardium. We observed opposing features on calcium release, which was diminished in the HFrEF cardiomyocyte but enhanced in the HFpEF cardiomyocyte. Mitochondria were less polarized in both HFrEF and HFpEF cardiomyocytes, reflecting similarly impaired metabolic capacity. Hyperacetylation of cardiac proteins was observed in both models, but it was more accentuated in the HFpEF heart. Despite shared features, unique triggering mechanisms (neurohormonal overactivation in HFrEF vs. inflammation in HFpEF) appear to determine the distinct phenotypes of HF. The findings of the present research stress the need for further exploration of the differential mechanisms underlying each HF subtype, because they might require specific therapeutic interventions. KEY POINTS: The mechanisms underlying heart failure with either reduced (HFrEF) or preserved (HFpEF) ejection fraction are often found to be different. Previous studies comparing pathophysiological traits between HFrEF and HFpEF have been conducted on animals of different ages and strains. The present research contrasted two age-matched mouse models of non-ischaemic HFrEF and cardiometabolic HFpEF to uncover divergent and shared features. We found that upon similar haemodynamic stress, the HFrEF heart experienced a more pronounced hypertrophic and fibrotic remodelling, whereas inflammation appeared to be greater in the HFpEF myocardium. Calcium release was diminished in the HFrEF cardiomyocyte and enhanced in the HFpEF cardiomyocyte. Mitochondria were comparably less polarized in both HFrEF and HFpEF myocytes. Hyperacetylation of proteins was common to both models, but stronger in the HFpEF heart. Casting light on common and distinguishing features might ease the quest for phenotype-specific therapies for heart failure patients.

Discovery of 9H-pyrimido[4,5-b]indole derivatives as dual RET/TRKA inhibitors.

Aberrant RET kinase signaling is activated in numerous cancers including lung, thyroid, breast, pancreatic, and prostate. Recent approvals of selective RET inhibitors, pralsetinib and selpercatinib, has shifted the focus of RET kinase drug discovery programs towards the development of selective inhibitors. However, selective inhibitors invariably lose efficacy as the selective nature of the inhibitor places Darwinian-like pressure on the tumor to bypass treatment through the selection of novel oncogenic drivers. Further, selective inhibitors are restricted for use in tumors with specific genetic backgrounds that do not encompass diverse patient classes. Here we report the identification of a pyrimido indole RET inhibitor found to also have activity against TRK. This selective dual RET/TRK inhibitor can be utilized in tumors with both RET and TRK genetic backgrounds and can also provide blockade of NTRK-fusions that are selected for from RET inhibitor treatments. Efforts towards developing dual RET/TRK inhibitors can be beneficial in terms of encompassing more diverse patient classes while also achieving blockade against emerging resistance mechanisms.

Immune cell kinetics and antibody response in COVID-19 patients with low-count monoclonal B-cell lymphocytosis.

Low-count monoclonal B-cell lymphocytosis (MBLlo ) has been associated with an underlying immunodeficiency and has recently emerged as a new risk factor for severe COVID-19. Here, we investigated the kinetics of immune cell and antibody responses in blood during COVID-19 of MBLlo versus non-MBL patients. For this study, we analyzed the kinetics of immune cells in blood of 336 COVID-19 patients (74 MBLlo and 262 non-MBL), who had not been vaccinated against SARS-CoV-2, over a period of 43 weeks since the onset of infection, using high-sensitivity flow cytometry. Plasma levels of anti-SARS-CoV-2 antibodies were measured in parallel by ELISA. Overall, early after the onset of symptoms, MBLlo COVID-19 patients showed increased neutrophil, monocyte, and particularly, plasma cell (PC) counts, whereas eosinophil, dendritic cell, basophil, and lymphocyte counts were markedly decreased in blood of a variable percentage of samples, and with a tendency toward normal levels from week +5 of infection onward. Compared with non-MBL patients, MBLlo COVID-19 patients presented higher neutrophil counts, together with decreased pre-GC B-cell, dendritic cell, and innate-like T-cell counts. Higher PC levels, together with a delayed PC peak and greater plasma levels of anti-SARS-CoV-2-specific antibodies (at week +2 to week +4) were also observed in MBLlo patients. In summary, MBLlo COVID-19 patients share immune profiles previously described for patients with severe SARS-CoV-2 infection, associated with a delayed but more pronounced PC and antibody humoral response once compared with non-MBL patients.

TNFSF13B rs9514828 gene polymorphism and soluble B cell activating factor levels: Association with apical periodontitis.

AIM: The aim of this case-control study was to evaluate the association between the TNFSF13B rs9514828 (-871 C > T) polymorphism and soluble BAFF (sBAFF) in apical periodontitis (AP) patients. METHODOLOGY: Two hundred and sixty one healthy subjects (HS) and 158 patients with AP classified as: 46 acute apical abscess (AAA), 81 primary AP (pAP) and 31 secondary AP (sAP) patients were included. Genomic DNA (gDNA) was extracted from peripheral blood cells according to the salting out method. The TNFSF13B rs9514828 (NC_000013.11:g.108269025C > T) were identified using polymerase chain reaction (PCR) followed by restriction fragment length polymorphisms (RFLP). Serum sBAFF levels were measured by ELISA test. The chi-squared or Fisher's exact test was performed. Odds ratios (OR) and 95% confidence intervals (95% CI) were calculated to evaluate the risk of AP associated with the rs9514828. The Mann-Whitney U test and Kruskal-Wallis analysis were used for non-normally distributed data. Differences were considered significant with a p-value <.05. RESULTS: No differences in the genotype/allele frequencies were shown between HS and patients with AAA. However, the TT genotype (OR = 2.68, 95% CI: 1.10-6.53; p = .025) and T allele (OR = 1.46, 95% CI: 1.00-2.12; p = .045) were associated with increased risk of pAP. In contrast, the minor allele T significantly decreased the risk of sAP (OR = 0.49, 95% CI: 0.024-0.99; p = .043). sBAFF serum levels were increased in AAA and pAP compared with HS (p < .01 and p = .021, respectively). The AAA patients had higher sBAFF serum levels than pAP (p = .034) and sAP (p < .01). CONCLUSIONS: These results suggest that the TNFSF13B rs9514828 (-871 C > T) polymorphism is associated with pAP susceptibility and that BAFF is a cytokine that might be involved in acute and chronic AP. The future exploration of the rs9514828 polymorphism in other AP cohorts is recommended.

Metastatic TNBC is closely associated with a fused mitochondrial morphology and a glycolytic and lipogenic metabolism.

Mitochondria modify their function and morphology to satisfy the bioenergetic demand of the cells. Cancer cells take advantage of these features to sustain their metabolic, proliferative, metastatic, and survival necessities. Understanding the morphological changes to mitochondria in the different grades of triple-negative breast cancer (TNBC) could help to design new treatments. Consequently, this research explored mitochondrial morphology and the gene expression of some proteins related to mitochondrial dynamics, as well as proteins associated with oxidative and non-oxidative metabolism in metastatic and non-metastatic TNBC. We found that mitochondrial morphology and metabolism are different in metastatic and non-metastatic TNBC. In metastatic TNBC, there is overexpression of genes related to mitochondrial dynamics, fatty-acid metabolism, and glycolysis. These features are accompanied by a fused mitochondrial morphology. By comparison, in non-metastatic TNBC, there is a stress-associated mitochondrial morphology with hyperfragmented mitochondria, accompanied by the upregulated expression of genes associated with the biogenesis of mitochondria; both of which are characteristics related to the higher production of reactive oxygen species observed in this cell line. These differences between metastatic and non-metastatic TNBC should provide a better understanding of metastasis and contribute to the development of improved specific and personalized therapies for TNBC.

Modifications in GPR21 and GPR82 genes expression as a consequence of metabolic syndrome etiology.

Metabolic syndrome (MS) has been related with alterations in expression levels of orphan G protein coupled receptors (GPCRs) such as GPR21 and GPR82, which could be involved in some of the elements that characterizes the metabolic syndrome. The aim of this work was to evaluate changes in GPR21 and GPR82 receptors expression in two models of metabolic syndrome: one genetic (Zucker rats), and the other based on a diet (70% fructose for 9 weeks). GPR21 and GPR82 gene expressions were evaluated in brain, heart, aorta, liver and kidney by RT-qPCR. Rats with a high fructose diet, as well as obese Zucker rats, showed initial stages of pancreatic damage and alterations in some biochemical parameters related to the model consistent with the classification of MS. GPR21 and GPR82 receptors expressed in all tissues. The expression of GPR21 decreased in heart, aorta and kidney, but in liver the expression was different: decreased in diet model and increased in genetic model. In contrast, GPR82 expression depended of tissue and metabolic syndrome model. The results highlight the possible role of GPR21 and GPR82 receptors in the development MS. We conclude that the expression of GPR21 and GPR82 in different tissues is related with MS and depend of the origin of the syndrome, so they could be a therapeutic target for that syndrome.

Mitochondrial Hyperacetylation in the Failing Hearts of Obese Patients Mediated Partly by a Reduction in SIRT3: The Involvement of the Mitochondrial Permeability Transition Pore.

BACKGROUND/AIMS: Cyclophilin D (CypD) mediates the mitochondrial permeability transition pore (mPTP) opening that contributes to mitochondrial dysfunction. CypD is regulated by its acetylation/deacetylation state that depends on Sirtuin-3 (SIRT3) mitochondrial deacetylase. Since obesity and metabolic syndrome decrease SIRT3 activity and expression, we tested the hypothesis that CypD hyperacetylation promotes mitochondrial dysfunction under this pathophysiological state, which is associated with ventricular dysfunction and heart failure. METHODS: Myocardial tissue samples from patients with left ventricular heart failure, with either obesity or normal weight, were processed for the expression of SIRT3 and acetylation profile by Western Blot (WB). In addition, a rat model of obesity and metabolic syndrome induced by 30% (w/v) of sucrose was conducted. The WB analysis was used to determine the levels of mitochondrial expression of SIRT3, Adenine Nucleotide Translocator (ANT), CypD and the acetylation profile, as well as immunoprecipitation to establish the acetylation levels of CypD. Mitochondrial function was assessed by oxygen consumption analysis and maximum Ca2+ retention capacity. Oxidative stress was assessed by aconitase activity, protein carbonyl and thiol groups content. RESULTS: SIRT3 expression in the biopsies of the failing human hearts showed a 46% decrease in the expression levels of obese patients in comparison to the non-obese patients (p=0.0219). Remarkably, body mass index was associated with protein acetylation (0.627; p = 0.035), suggesting that the acetylation profiles of the failing hearts of obese patients are partly mediated by a reduction in SIRT3, which is also associated with higher BNP levels, indicating a more severe ventricular dysfunction (-0.636; p = 0.043). Accordingly, obese rats demonstrated a SIRT3 mitochondrial expression decrease of 22% concomitantly with a hyperacetylated mitochondrial profile, including CypD. Cardiac mitochondria from obese animals were 2.5-fold more prone to mPTP opening than the controls. CONCLUSION: Our results indicate that obesity reduces SIRT3 expression and that CypD hyperacetylation increases mPTP opening, suggesting that the activation of SIRT3 might be a potential target to decrease ventricular dysfunction and slow the progression of heart failure.

Large-Scale Assessment of Extractables and Leachables in Single-Use Bags for Biomanufacturing.

Single-use technologies (SUTs) are widely used during biopharmaceutical manufacture as disposable bioreactors or media and buffer storage bags. Despite their advantages, the risk of release of extractable and leachable (E&Ls) substances is considered an important drawback in adopting disposables in the biomanufacturing process. E&Ls may detrimentally affect cell viability or productivity or may persist during purification and present a risk to the patient if remaining in the final drug product. In this study, 34 plastic films from single-use bags (SUBs) for cell cultivation were extracted with selected solvents that represent reasonable worst-case conditions for most typical biomanufacturing applications. SUBs were incubated at small-scale under accelerated-aging conditions that represented standard operational conditions of use. Leachables analysis was performed following dispersive liquid-liquid microextraction (DLLME) for analyte preconcentration and removal of matrix interference. Resulting extracts were characterized by GC-headspace for volatiles, high resolution GC-Orbitrap-MS/MS for semivolatiles, high resolution LC-Orbitrap-MS/MS for nonvolatiles, and ICP-MS for trace elemental analysis. Multivariate statistical analysis of the analytical data revealed significant correlations between the type and concentration of compounds and bags features including brand, manufacturing date and polymer type. The analytical data demonstrates that, over recent years, the nature of E&Ls has been altered due to the implementation of manufacturing changes and new types of polymers and may change further with the future advent of regulations that will limit or ban the use of certain raw materials and additives. The broad E&L database generated herein facilitates toxicological assessments from a biomanufacturing standpoint and provides practical guidelines for confident determination of E&Ls to enable screening and elimination of nonsatisfactory films for single use bioprocessing.

Silica nanoparticles induce cardiotoxicity interfering with energetic status and Ca2+ handling in adult rat cardiomyocytes.

Recent evidence has shown that nanoparticles that have been used to improve or create new functional properties for common products may pose potential risks to human health. Silicon dioxide (SiO2) has emerged as a promising therapy vector for the heart. However, its potential toxicity and mechanisms of damage remain poorly understood. This study provides the first exploration of SiO2-induced toxicity in cultured cardiomyocytes exposed to 7- or 670-nm SiO2 particles. We evaluated the mechanism of cell death in isolated adult cardiomyocytes exposed to 24-h incubation. The SiO2 cell membrane association and internalization were analyzed. SiO2 showed a dose-dependent cytotoxic effect with a half-maximal inhibitory concentration for the 7 nm (99.5 ± 12.4 µg/ml) and 670 nm (>1,500 µg/ml) particles, which indicates size-dependent toxicity. We evaluated cardiomyocyte shortening and intracellular Ca2+ handling, which showed impaired contractility and intracellular Ca2+ transient amplitude during ß-adrenergic stimulation in SiO2 treatment. The time to 50% Ca2+ decay increased 39%, and the Ca2+ spark frequency and amplitude decreased by 35 and 21%, respectively, which suggest a reduction in sarcoplasmic reticulum Ca2+-ATPase (SERCA) activity. Moreover, SiO2 treatment depolarized the mitochondrial membrane potential and decreased ATP production by 55%. Notable glutathione depletion and H2O2 generation were also observed. These data indicate that SiO2 increases oxidative stress, which leads to mitochondrial dysfunction and low energy status; these underlie reduced SERCA activity, shortened Ca2+ release, and reduced cell shortening. This mechanism of SiO2 cardiotoxicity potentially plays an important role in the pathophysiology mechanism of heart failure, arrhythmias, and sudden death.NEW & NOTEWORTHY Silica particles are used as novel nanotechnology-based vehicles for diagnostics and therapeutics for the heart. However, their potential hazardous effects remain unknown. Here, the cardiotoxicity of silica nanoparticles in rat myocytes has been described for the first time, showing an impairment of mitochondrial function that interfered directly with Ca2+ handling.

Evidence of alterations in the expression of orphan receptors GPR26 and GPR39 due to the etiology of the metabolic syndrome.

AIMS: Metabolic syndrome (MS) is composed of several metabolic abnormalities that increase the risk of cardiovascular diseases and diabetes. Although there are treatments for the components of MS, this pathology maintains a high mortality, suggesting that there are other mechanisms in which orphan receptors such as GPR26 and GPR39 may be involved. For this reason, the aim of this work was to evaluate the expression of GPR26 and GPR39 orphan receptors in two models of MS (diet and genetics). MATERIALS AND METHODS: We used male Wistar rats, which received 70% fructose in drinking water for 9 weeks, and obese Zucker rats. We measured weight, blood pressure, glucose, triglycerides, total cholesterol, HDL cholesterol, LDL cholesterol to determine the MS and the expression of the orphan receptors GPR26 and GPR39 in brain, heart, aorta, liver, and kidney by RT-PCR. RESULTS: The analysis of the expression of the orphan receptors GPR26 and GPR39 showed that the receptors are expressed in some tissues, but the expression of the GPR26 tends to decrease in the heart and aorta, whereas in the brain, no changes were observed, this receptor is not expressed in the liver and kidney of both strains. The expression of GPR39 isoforms depends on the tissue and MS model. CONCLUSIONS: We conclude that the orphan receptors GPR26, GPR39v1, and GPR39v2 are expressed in different tissues and their profile expression is dependent on the etiology of the MS.

Antineoplastic copper coordinated complexes (Casiopeinas) uncouple oxidative phosphorylation and induce mitochondrial permeability transition in cardiac mitochondria and cardiomyocytes.

Copper-based drugs, Casiopeinas (Cas), exhibit antiproliferative and antineoplastic activities in vitro and in vivo, respectively. Unfortunately, the clinical use of these novel chemotherapeutics could be limited by the development of dose-dependent cardiotoxicity. In addition, the molecular mechanisms underlying Cas cardiotoxicity and anticancer activity are not completely understood. Here, we explore the potential impact of Cas on the cardiac mitochondria energetics as the molecular mechanisms underlying Cas-induced cardiotoxicity. To explore the properties on mitochondrial metabolism, we determined Cas effects on respiration, membrane potential, membrane permeability, and redox state in isolated cardiac mitochondria. The effect of Cas on the mitochondrial membrane potential (Δψm) was also evaluated in isolated cardiomyocytes by confocal microscopy and flow cytometry. Cas IIIEa, IIgly, and IIIia predominately inhibited maximal NADH- and succinate-linked mitochondrial respiration, increased the state-4 respiration rate and reduced membrane potential, suggesting that Cas also act as mitochondrial uncouplers. Interestingly, cyclosporine A inhibited Cas-induced mitochondrial depolarization, suggesting the involvement of mitochondrial permeability transition pore (mPTP). Similarly to isolated mitochondria, in isolated cardiomyocytes, Cas treatment decreased the Δψm and cyclosporine A treatment prevented mitochondrial depolarization. The production of H2O2 increased in Cas-treated mitochondria, which might also increase the oxidation of mitochondrial proteins such as adenine nucleotide translocase. In accordance, an antioxidant scavenger (Tiron) significantly diminished Cas IIIia mitochondrial depolarization. Cas induces a prominent loss of membrane potential, associated with alterations in redox state, which increases mPTP opening, potentially due to thiol-dependent modifications of the pore, suggesting that direct or indirect inhibition of mPTP opening might reduce Cas-induced cardiotoxicity.

Role of SERCA and the sarcoplasmic reticulum calcium content on calcium waves propagation in rat ventricular myocytes.

In Ca(2+)-overloaded ventricular myocytes, SERCA is crucial to steadily achieve the critical sarcoplasmic reticulum (SR) Ca(2+) level to trigger and sustain Ca(2+) waves, that propagate at constant rate (ʋwave). High luminal Ca(2+) sensitizes RyR2, thereby increasing Ca(2+) sparks frequency, and the larger RyR2-mediated SR Ca(2+) flux (dF/dt) sequentially activates adjacent RyR2 clusters. Recently, it was proposed that rapid SERCA Ca(2+) reuptake, ahead of the wave front, further sensitizes RyR2, increasing ʋwave. Nevertheless, this is controversial because rapid cytosolic Ca(2+) removal could instead impair RyR2 activation. We assessed whether rapid SR Ca(2+) uptake enhances ʋwave by changing SERCA activity (ҡDecay) over a large range (∼175%). We used normal (Ctrl) and hyperthyroid rat (HT; reduced phospholamban by ∼80%) myocytes treated with thapsigargin or isoproterenol (ISO). We found that ʋwave and dF/dt had a non-linear dependency with ҡDecay, while Ca(2+) waves amplitude was largely unaffected. Furthermore, SR Ca(2+) also showed a non-linear dependency with ҡDecay, however, the relationships ʋwave vs. SR Ca(2+) and ʋwave vs. dF/dt were linear, suggesting that high steady state SR Ca(2+) determines ʋwave, while rapid SERCA Ca(2+) uptake does not. Finally, ISO did not increase ʋwave in HT cells, therefore, ISO-enhanced ʋwave in Ctrl depended on high SR Ca(2+).

Impaired oxidative metabolism and calcium mishandling underlie cardiac dysfunction in a rat model of post-acute isoproterenol-induced cardiomyopathy.

Stress-induced cardiomyopathy, triggered by acute catecholamine discharge, is a syndrome characterized by transient, apical ballooning linked to acute heart failure and ventricular arrhythmias. Rats receiving an acute isoproterenol (ISO) overdose (OV) suffer cardiac apex ischemia-reperfusion damage and arrhythmia, and then undergo cardiac remodeling and dysfunction. Nevertheless, the subcellular mechanisms underlying cardiac dysfunction after acute damage subsides are not thoroughly understood. To address this question, Wistar rats received a single ISO injection (67 mg/kg). We found in vivo moderate systolic and diastolic dysfunction at 2 wk post-ISO-OV; however, systolic dysfunction recovered after 4 wk, while diastolic dysfunction worsened. At 2 wk post-ISO-OV, cardiac function was assessed ex vivo, while mitochondrial oxidative metabolism and stress were assessed in vitro, and Ca(2+) handling in ventricular myocytes. These were complemented with sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA), phospholamban (PLB), and RyR2 expression studies. Ex vivo, basal mechanical performance index (MPI) and oxygen consumption rate (MVO2) were unchanged. Nevertheless, upon increase of metabolic demand, by ß-adrenergic stimulation (1-100 nM ISO), the MPI versus MVO2 relation decreased and shifted to the right, suggesting MPI and mitochondrial energy production uncoupling. Mitochondria showed decreased oxidative metabolism, membrane fragility, and enhanced oxidative stress. Myocytes presented systolic and diastolic Ca(2+) mishandling, and blunted response to ISO (100 nM), and all these without apparent changes in SERCA, PLB, or RyR2 expression. We suggest that post-ISO-OV mitochondrial dysfunction may underlie decreased cardiac contractility, mainly by depletion of ATP needed for myofilaments and Ca(2+) transport by SERCA, while exacerbated oxidative stress may enhance diastolic RyR2 activity.

Changes in T-Tubules and Sarcoplasmic Reticulum in Ventricular Myocytes in Early Cardiac Hypertrophy in a Pressure Overload Rat Model.

BACKGROUND/AIMS: Pressure-overload (PO) causes cardiac hypertrophy (CH), and eventually leads to heart failure (HF). HF ventricular myocytes present transverse-tubules (TT) loss or disarrangement and decreased sarcoplasmic reticulum (SR) density, and both contribute to altered Ca2+ signaling and heart dysfunction. It has been shown that TT remodeling precedes HF, however, it is unknown whether SR structural and functional remodeling also starts early in CH. METHODS: Using confocal microscopy, we assessed TT (with Di-8-ANNEPS) and SR (with SR-trapped Mag-Fluo-4) densities, as well as SR fluorophore diffusion (fluorescence recovery after photobleach; FRAP), cytosolic Ca2+ signaling and ex vivo cardiac performance in a PO rat hypertrophy model induced by abdominal aortic constriction (at 6 weeks). RESULTS: Rats developed CH, while cardiac performance, basal and upon ß-adrenergic stimulation, remained unaltered. TT density decreased by ∼14%, without spatial disarrangement, while SR density decreased by ∼7%. More important, FRAP was ∼30% slower, but with similar maximum recovery, suggesting decreased SR interconnectivity. Systolic and diastolic Ca2+ signaling and SR Ca2+ content were unaltered. CONCLUSION: SR remodeling is an early CH event, similar to TT remodeling, appearing during compensated hypertrophy. Nevertheless, myocytes can withstand those moderate structural changes in SR and TT, preserving normal Ca2+ signaling and contractility.

Surgical Efficiency Comparison Between Two Phacoemulsification Systems.

Purpose: To assess the surgical efficiency in cataract surgery comparing two phacoemulsification systems. Methods: Prospective, consecutive-comparative study in a two-site private practice. Three hundred and one eyes undergoing standard or femtosecond laser-assisted (FLACS) cataract surgery with either the R-Evo Smart (BVI, Waltham, USA) and/or the Centurion Vision System (Alcon, Fort Worth, USA). Preoperative eye characteristics (degree of cataract using the lens opacities classification system LOCS III grading) and intraoperative outcomes (total ultrasound time and total estimated fluid aspirated/drainage bag weighting) were registered in all cases. Results: One hundred and fifty-five eyes undergone cataract surgery with the R-Evo Smart and 146 eyes with the Centurion Vision System phacoemulsification systems. Mean cataract grade was 3.07 ± 0.78 and 2.96 ± 0.85 for the R-Evo Smart and Centurion Vision System groups, respectively (p = 0.12). Mean total ultrasound time was 18.99 ± 12.85 and 40.24 ± 21.91 seconds for the R-Evo Smart and Centurion Vision System groups, respectively (p < 0.01). Mean total estimated fluid aspirated/drainage bag weighting was 53.00 ± 14.56 g and 54.33 ± 14.88 cc for the R-Evo Smart and Centurion Vision System groups, respectively (p = 0.21). Considering non-FLACS surgery (98 eyes with the R-Evo Smart and 63 eyes with the Centurion Vision System), mean cataract grade was 2.95 ± 0.74 and 2.97 ± 0.91 for the R-Evo Smart and Centurion Vision System groups, respectively (p = 0.44). Mean total ultrasound time was 19.96 ± 11.20 and 42.84 ± 28.35 seconds for the R-Evo Smart and Centurion Vision System groups, respectively (p < 0.01). Mean total estimated fluid aspirated/drainage bag weighting was 55.95 ± 14.76 g and 55.97 ± 13.62 cc for the R-Evo Smart and Centurion Vision System groups, respectively (p = 0.49). No adverse events were found in the two groups of eyes. Conclusion: The objective measurement of surgical efficiency through total ultrasound time during lens removal and fluid consumption during both lens removal and irrigation/aspiration proved R-Evo Smart to be an efficient phacoemulsification platform, in comparison with the current standard of care Centurion Vision System.

High Interleukin 21 Levels in Patients with Systemic Lupus Erythematosus: Association with Clinical Variables and rs2221903 Polymorphism.

Background: Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by autoantibody production and diverse tissue and organ inflammatory affections. Interleukin 21 (IL-21) is implicated in B cell survival, proliferation, differentiation, class switching, and immunoglobulin production; therefore, it is considered a key cytokine in the pathogenesis of SLE. However, its association with disease activity and clinical phenotypes remains unclear. We aimed to evaluate the association of IL-21 levels with the disease activity and clinical phenotypes in patients with SLE. Also, we analyzed the IL21 polymorphisms associated with increased IL-21 levels. Methods: The IL-21 serum levels were determined using the enzyme-linked immunosorbent assay (ELISA) method. The rs2221903 and rs2055979 polymorphisms were assessed in 300 healthy controls (HCs) and 300 patients with SLE by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. The levels of IL-21 were monitored during follow-up visits in 59 patients with SLE. Results: The patients with SLE showed higher IL-21 levels compared to the HCs. The IL-21 levels did not correlate with Mex-SLEDAI and were not different in patients with inactive, mild-moderate, and severe disease. The IL-21 levels were increased in patients with hematological affection. The ROC curve analysis revealed that the IL-21 levels had good predictive power in discriminating among patients with SLE and HCs. In a follow-up analysis, the levels of IL-21 remained higher in the patients with SLE even when the patients were in remission. Also, the rs2221903 polymorphism was associated with increased IL-21 levels. Conclusions: This study highlights the importance of IL-21 as a key cytokine in SLE. IL-21 levels are higher in patients with SLE and remain increased regardless of disease activity. According to the ROC analysis, IL-21 is a potential biomarker of SLE. Further longitudinal studies are needed to explore the relationship between IL-21 and the clinical phenotypes of SLE.

A novel NKp80-based strategy for universal identification of normal, reactive and tumor/clonal natural killer-cells in blood.

Purpose: Natural killer (NK) cells are traditionally identified by flow cytometry using a combination of markers (CD16/CD56/CD3), because a specific NK-cell marker is still missing. Here we investigated the utility of CD314, CD335 and NKp80, compared to CD16/CD56/CD3, for more robust identification of NK-cells in human blood, for diagnostic purposes. Methods: A total of 156 peripheral blood (PB) samples collected from healthy donors (HD) and patients with diseases frequently associated with loss/downregulation of classical NK-cell markers were immunophenotyped following EuroFlow protocols, aimed at comparing the staining profile of total blood NK-cells for CD314, CD335 and NKp80, and the performance of distinct marker combinations for their accurate identification. Results: NKp80 showed a superior performance (vs. CD314 and CD335) for the identification of NK-cells in HD blood. Besides, NKp80 improved the conventional CD16/CD56/CD3-based strategy to identify PB NK-cells in HD and reactive processes, particularly when combined with CD16 for further accurate NK-cell-subsetting. Although NKp80+CD16 improved the identification of clonal/tumor NK-cells, particularly among CD56- cases (53%), aberrant downregulation of NKp80 was observed in 25% of patients, in whom CD56 was useful as a complementary NK-cell marker. As NKp80 is also expressed on T-cells, we noted increased numbers of NKp80+ cytotoxic T-cells at the more advanced maturation stages, mostly in adults. Conclusion: Here we propose a new robust approach for the identification of PB NK-cells, based on the combination of NKp80 plus CD16. However, in chronic lymphoproliferative disorders of NK-cells, addition of CD56 is recommended to identify clonal NK-cells, due to their frequent aberrant NKp80- phenotype.

A dual role for receptor-interacting protein kinase 2 (RIP2) kinase activity in nucleotide-binding oligomerization domain 2 (NOD2)-dependent autophagy.

Autophagy is triggered by the intracellular bacterial sensor NOD2 (nucleotide-binding, oligomerization domain 2) as an anti-bacterial response. Defects in autophagy have been implicated in Crohn's disease susceptibility. The molecular mechanisms of activation and regulation of this process by NOD2 are not well understood, with recent studies reporting conflicting requirements for RIP2 (receptor-interacting protein kinase 2) in autophagy induction. We examined the requirement of NOD2 signaling mediated by RIP2 for anti-bacterial autophagy induction and clearance of Salmonella typhimurium in the intestinal epithelial cell line HCT116. Our data demonstrate that NOD2 stimulates autophagy in a process dependent on RIP2 tyrosine kinase activity. Autophagy induction requires the activity of the mitogen-activated protein kinases MEKK4 and p38 but is independent of NFκB signaling. Activation of autophagy was inhibited by a PP2A phosphatase complex, which interacts with both NOD2 and RIP2. PP2A phosphatase activity inhibited NOD2-dependent autophagy but not activation of NFκB or p38. Upon stimulation of NOD2, the phosphatase activity of the PP2A complex is inhibited through tyrosine phosphorylation of the catalytic subunit in a process dependent on RIP2 activity. These findings demonstrate that RIP2 tyrosine kinase activity is not only required for NOD2-dependent autophagy but plays a dual role in this process. RIP2 both sends a positive autophagy signal through activation of p38 MAPK and relieves repression of autophagy mediated by the phosphatase PP2A.