Distinct and Overlapping Effector Functions of Expanded Human CD4+, CD8α+ and CD4-CD8α- Invariant Natural Killer T Cells

CD1d-restricted invariant natural killer T (iNKT) cells have diverse immune stimulatory/regulatory activities through their ability to release cytokines and to kill or transactivate other cells. Activation of iNKT cells can protect against multiple diseases in mice but clinical trials in humans have had limited impact. Clinical studies to date have targeted polyclonal mixtures of iNKT cells and we proposed that their subset compositions will influence therapeutic outcomes. We sorted and expanded iNKT cells from healthy donors and compared the phenotypes, cytotoxic activities and cytokine profiles of the CD4+, CD8α+ and CD4−CD8α− double-negative (DN) subsets. CD4+ iNKT cells expanded more readily than CD8α+ and DN iNKT cells upon mitogen stimulation. CD8α+ and DN iNKT cells most frequently expressed CD56, CD161 and NKG2D and most potently killed CD1d+ cell lines and primary leukemia cells. All iNKT subsets released Th1 (IFN-γ and TNF-α) and Th2 (IL-4, IL-5 and IL-13) cytokines. Relative amounts followed a CD8α>DN>CD4 pattern for Th1 and CD4>DN>CD8α for Th2. All iNKT subsets could simultaneously produce IFN-γ and IL-4, but single-positivity for IFN-γ or IL-4 was strikingly rare in CD4+ and CD8α+ fractions, respectively. Only CD4+ iNKT cells produced IL-9 and IL-10; DN cells released IL-17; and none produced IL-22. All iNKT subsets upregulated CD40L upon glycolipid stimulation and induced IL-10 and IL-12 secretion by dendritic cells. Thus, subset composition of iNKT cells is a major determinant of function. Use of enriched CD8α+, DN or CD4+ iNKT cells may optimally harness the immunoregulatory properties of iNKT cells for treatment of disease

The Changing Landscape for Stroke\ua0Prevention in AF: Findings From the GLORIA-AF Registry Phase 2

Background GLORIA-AF (Global Registry on Long-Term Oral Antithrombotic Treatment in Patients with Atrial Fibrillation) is a prospective, global registry program describing antithrombotic treatment patterns in patients with newly diagnosed nonvalvular atrial fibrillation at risk of stroke. Phase 2 began when dabigatran, the first non\u2013vitamin K antagonist oral anticoagulant (NOAC), became available. Objectives This study sought to describe phase 2 baseline data and compare these with the pre-NOAC era collected during phase 1. Methods During phase 2, 15,641 consenting patients were enrolled (November 2011 to December 2014); 15,092 were eligible. This pre-specified cross-sectional analysis describes eligible patients\u2019 baseline characteristics. Atrial fibrillation disease characteristics, medical outcomes, and concomitant diseases and medications were collected. Data were analyzed using descriptive statistics. Results Of the total patients, 45.5% were female; median age was 71 (interquartile range: 64, 78) years. Patients were from Europe (47.1%), North America (22.5%), Asia (20.3%), Latin America (6.0%), and the Middle East/Africa (4.0%). Most had high stroke risk (CHA2DS2-VASc [Congestive heart failure, Hypertension, Age  6575 years, Diabetes mellitus, previous Stroke, Vascular disease, Age 65 to 74 years, Sex category] score  652; 86.1%); 13.9% had moderate risk (CHA2DS2-VASc = 1). Overall, 79.9% received oral anticoagulants, of whom 47.6% received NOAC and 32.3% vitamin K antagonists (VKA); 12.1% received antiplatelet agents; 7.8% received no antithrombotic treatment. For comparison, the proportion of phase 1 patients (of N = 1,063 all eligible) prescribed VKA was 32.8%, acetylsalicylic acid 41.7%, and no therapy 20.2%. In Europe in phase 2, treatment with NOAC was more common than VKA (52.3% and 37.8%, respectively); 6.0% of patients received antiplatelet treatment; and 3.8% received no antithrombotic treatment. In North America, 52.1%, 26.2%, and 14.0% of patients received NOAC, VKA, and antiplatelet drugs, respectively; 7.5% received no antithrombotic treatment. NOAC use was less common in Asia (27.7%), where 27.5% of patients received VKA, 25.0% antiplatelet drugs, and 19.8% no antithrombotic treatment. Conclusions The baseline data from GLORIA-AF phase 2 demonstrate that in newly diagnosed nonvalvular atrial fibrillation patients, NOAC have been highly adopted into practice, becoming more frequently prescribed than VKA in Europe and North America. Worldwide, however, a large proportion of patients remain undertreated, particularly in Asia and North America. (Global Registry on Long-Term Oral Antithrombotic Treatment in Patients With Atrial Fibrillation [GLORIA-AF]; NCT01468701

Omecamtiv mecarbil in chronic heart failure with reduced ejection fraction, GALACTIC‐HF: baseline characteristics and comparison with contemporary clinical trials

Aims: The safety and efficacy of the novel selective cardiac myosin activator, omecamtiv mecarbil, in patients with heart failure with reduced ejection fraction (HFrEF) is tested in the Global Approach to Lowering Adverse Cardiac outcomes Through Improving Contractility in Heart Failure (GALACTIC‐HF) trial. Here we describe the baseline characteristics of participants in GALACTIC‐HF and how these compare with other contemporary trials. Methods and Results: Adults with established HFrEF, New York Heart Association functional class (NYHA) ≥ II, EF ≤35%, elevated natriuretic peptides and either current hospitalization for HF or history of hospitalization/ emergency department visit for HF within a year were randomized to either placebo or omecamtiv mecarbil (pharmacokinetic‐guided dosing: 25, 37.5 or 50 mg bid). 8256 patients [male (79%), non‐white (22%), mean age 65 years] were enrolled with a mean EF 27%, ischemic etiology in 54%, NYHA II 53% and III/IV 47%, and median NT‐proBNP 1971 pg/mL. HF therapies at baseline were among the most effectively employed in contemporary HF trials. GALACTIC‐HF randomized patients representative of recent HF registries and trials with substantial numbers of patients also having characteristics understudied in previous trials including more from North America (n = 1386), enrolled as inpatients (n = 2084), systolic blood pressure < 100 mmHg (n = 1127), estimated glomerular filtration rate < 30 mL/min/1.73 m2 (n = 528), and treated with sacubitril‐valsartan at baseline (n = 1594). Conclusions: GALACTIC‐HF enrolled a well‐treated, high‐risk population from both inpatient and outpatient settings, which will provide a definitive evaluation of the efficacy and safety of this novel therapy, as well as informing its potential future implementation

Characterization of the molecular diversity of brazilian snakes Bothrops cotiara and Crotalus durissus terrificus venom

A variabilidade composicional das peçonhas de serpentes retrata a evolução toxinológica e a adaptação a diferentes nichos ecológicos. Essa variabilidade tem sido observada em relação a diferentes fatores, como idade, dieta, gênero, sazonalidade, e distribuição geográfica. As grandes famílias de toxinas reproduzem a diversificação por meio de adaptação evolutiva, neo-funcionalização e retenção de múltiplos genes parálogos, manifestando-se como variações quantitativas e qualitativas. Em oposição a alterações arbitrárias, a diversificação composicional aparenta ser inerente às estruturas biológicas. No intuito de se investigar a variabilidade e diversidade da peçonha de duas serpentes brasileiras, o repertório de toxinas foi avaliado através das abordagens peptidômica e proteômica. No Capítulo 1, diversos novos peptídeos foram caracterizados por análise peptidômica no veneno da serpente Bothrops cotiara. Dentre os peptídeos identificados, um novo fragmento críptico de uma metaloproteinase apresentou inibição da enzima conversora de angiotensina (ECA), com constantes de inibição abaixo de 1 µM. Este inibidor não canônico da ECA não atende ao consenso estrutural de peptídeos potenciadores de bradicinina de serpentes, e apresenta semelhanças estruturais com a bradicinina e peptídeos relacionados à bradicinina. Desta forma, os resultados contribuem para o repertório de peptídeos biologicamente ativos derivados de venenos de serpentes capazes de inibir a ECA para além das estruturas e precursores conhecidos. No Capítulo 2 foi realizada uma análise abrangente da peçonha de Crotalus durissus terrificus, revelando mutações e dimorfismo sexual em níveis peptidômico e proteômico. A análise de mutações revelou novas toxinas com alinhamento estrutural predito condizente com estruturas homólogas/parálogas determinadas experimentalmente. Uma vez que as variações de resíduos não ocorreram em aminoácidos centrais de interação das toxinas, os resultados sugerem que as mutações observadas podem não impactar significativamente a estabilidade e funções dessas proteínas, sugerindo a conservação estrutural de toxinas evolutivamente relacionadas. O conhecimento acerca da diversidade toxinológica pode contribuir com o avanço no tratamento adequado de acidentes ofídicos, e consequentemente, para o combate dessa doença tropical negligenciada, bem como pode fundamentar o desenvolvimento de novos fármacos através da prospecção destas vastas bibliotecas de toxinas.The compositional variability of snake venom depicts a dynamic representation of toxinological evolution to adapt to different ecological niches. This variability has been reported at several levels, such as age, gender, seasonality, geographic, dietary, interfamily, intergenus, and intraspecies. The large families of toxins reproduce diversification through evolutionary adaptation, neofunctionalization, and retention of multiple paralogous genes, manifesting itself as a natural quantitative and qualitative variation. Rather than arbitrary deviations, compositional diversification appears to be inherent within specimens’ biological framework. To investigate venom diversity and variability of two Brazilian snakes, the toxin repertoire was assessed through peptidomic and proteomic approach. In chapter 1, several peptides were characterized through peptidomic analysis of Bothrops cotiara snake venom. Among the identified peptides, a novel cryptide fragment of a metalloproteinase was capable of inhibiting angiotensin converting enzyme (ACE), with inhibition constants below 1 µM. This non-canonical ACE inhibitor presented structural similarities do bradykinin and bradykinin-related peptides and did not fulfill the primary structure consensus of bradykinin potentiating peptides, thus contributing to the repertoire of biologically active peptides capable of inhibiting ACE beyond the current known motifs and precursors. In chapter 2 a comprehensive analysis of Crotalus durissus terrificus venom was performed, uncovering sexual dimorphism at both peptidomic and protemic levels. Furthermore, mutation analysis unraveled novel toxins with consistent structural alignment to homologous/paralogous experimentally determined structures. As mutational variants were not detected in key contact residues of venom toxins, these results suggest that the observed mutations may not significantly impact the overall stability and toxic activities of these proteins, underscoring the conservation of structural features in evolutionary related toxins. The knowledge regarding toxinological diversity represents a critical advance in the proper treatment of snakebites, making substantial contributions to addressing the burden of this neglected tropical disease, as well as underpins the potential for drug development through bioprospecting these vast toxins libraries.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)140689/2019-

ArtinM Cytotoxicity in B Cells Derived from Non-Hodgkin’s Lymphoma Depends on Syk and Src Family Kinases

Receptors on the immune cell surface have a variety of glycans that may account for the immunomodulation induced by lectins, which have a carbohydrate recognition domain (CRD) that binds to monosaccharides or oligosaccharides in a specific manner. ArtinM, a D-mannose-binding lectin obtained from Artocarpus heterophyllus, has affinity for the N-glycans core. Immunomodulation by ArtinM toward the Th1 phenotype occurs via its interaction with TLR2/CD14 N-glycans on antigen-presenting cells, as well as recognition of CD3γ N-glycans on murine CD4+ and CD8+ T cells. ArtinM exerts a cytotoxic effect on Jurkat human leukemic T-cell line and human myeloid leukemia cell line (NB4). The current study evaluated the effects of ArtinM on murine and human B cells derived from non-Hodgkin’s lymphoma. We found that murine B cells are recognized by ArtinM via the CRD, and the ArtinM stimulus did not augment the proliferation rate or production of IL-2. However, murine B cell incubation with ArtinM augmented the rate of apoptosis, and this cytotoxic effect of ArtinM was also seen in human B cell-lines sourced from non-Hodgkin’s lymphoma Raji cell line. This cytotoxic effect was inhibited by the phosphatase activity of CD45 on Lck, and the protein kinases of the Src family contribute to cell death triggered by ArtinM

Proteomics and life-history variability of Endogenous Phospholipases A2 Inhibitors (PLIs) in Bothrops jararaca plasma.

In Brazil, the genus Bothrops is responsible for most ophidian accidents. Snake venoms have a wide variety of proteins and peptides exhibiting a broad repertoire of pharmacological and toxic effects that elicit systemic injury and characteristic local effects. The snakes' natural resistance to envenomation caused by the presence of inhibitory compounds on their plasma have been extensively studied. However, the presence of these inhibitors in different developmental stages is yet to be further discussed. The aim of this study was to evaluate the ontogeny of Bothrops jararaca plasma inhibitor composition and, to this end, plasma samples of B. jararaca were obtained from different developmental stages (neonates, youngs, and adults) and sexes (female and male). SDS-PAGE, Western blotting, affinity chromatography, and mass spectrometry were performed to analyze the protein profile and interaction between B. jararaca plasma and venom proteins. In addition, the presence of γBjPLI, a PLA2 inhibitor previously identified and characterized in B. jararaca serum, was confirmed by Western blotting. According to our results, 9-17% of plasma proteins were capable of binding to venom proteins in the three developmental stages. The presence of different endogenous inhibitors and, more specifically, different PLA2 inhibitor (PLI) classes and antihemorrhagic factors were confirmed in specimens of B. jararaca from newborn by mass spectrometry. For the first time, the αPLI and βPLI were detected in B. jararaca plasma, although low or no ontogenetic and sexual correlation were found. The γPLI were more abundant in adult female, than in neonate and young female, but similar to neonate, young and adult male according to the results of mass spectrometry analysis. Our results suggest that there are proteins in the plasma of these animals that can help counteract the effects of self-envenomation from birth