Rosetta FlexPepDock to predict peptide-MHC binding: An approach for non-canonical amino acids.

Computation methods that predict the binding of peptides to MHC-I are important tools for screening and identifying immunogenic antigens and have the potential to accelerate vaccine and drug development. However, most available tools are sequence-based and optimized only for peptides containing the twenty canonical amino acids. This omits a large number of peptides containing non-canonical amino acids (NCAA), or residues that undergo varied post-translational modifications such as glycosylation or phosphorylation. These modifications fundamentally alter peptide immunogenicity. Similarly, existing structure-based methods are biased towards canonical peptide backbone structures, which may or may not be preserved when NCAAs are present. Rosetta FlexPepDock ab-initio is a structure-based computational protocol able to evaluate peptide-receptor interaction where no prior information of the peptide backbone is known. We benchmarked FlexPepDock ab-initio for docking canonical peptides to MHC-I, and illustrate for the first time the method's ability to accurately model MHC-I bound epitopes containing NCAAs. FlexPepDock ab-initio protocol was able to recapitulate near-native structures (≤1.5Å) in the top lowest-energy models for 20 out of 25 cases in our initial benchmark. Using known experimental binding affinities of twenty peptides derived from an influenza-derived peptide, we showed that FlexPepDock protocol is able to predict relative binding affinity as Rosetta energies correlate well with experimental values (r = 0.59, p = 0.006). ROC analysis revealed 80% true positive and a 40% false positive rate, with a prediction power of 93%. Finally, we demonstrate the protocol's ability to accurately recapitulate HLA-A*02:01 bound phosphopeptide backbone structures and relative binding affinity changes, the theoretical structure of the lymphocytic choriomeningitis derived glycosylated peptide GP392 bound to MHC-I H-2Db, and isolevuglandin-adducted peptides. The ability to use non-canonical amino acids in the Rosetta FlexPepDock protocol may provide useful insight into critical amino acid positions where the post-translational modification modulates immunologic responses.

Vascular Damage in Resistant Hypertension: TNF-Alpha Inhibition Effects on Endothelial Cells.

Inflammatory cytokines have been associated with the pathophysiology of hypertension and target organ damage (TOD). Resistant hypertensive patients (RHTN) are characterized by poor blood pressure control and higher prevalence of TOD. This study evaluated the relationship between plasma levels of TNF-α and arterial stiffness (pulse wave velocity-PWV) in 32 RHTN and 19 normotensive subjects. Moreover, we investigated the effect of TNF-α inhibition on human endothelial cells (HUVECs) incubated with serum from RHTN and normotensive subjects. HUVECs containing serum obtained from normotensive (n = 8) and hypertensive (n = 8) individuals were treated with TNF-α inhibitor (infliximab). Cell suspensions were used for measurement of DNA fragmentation and reactive oxygen species (ROS) content. RHTN patients showed higher levels of TNF-α compared to normotensive subjects, as well as higher PWV. Positive correlation was found between TNF-α levels and PWV measures in the whole group. HUVECs incubated with serum from RHTN showed increased cell apoptosis and higher ROS content compared to normotensive subjects. Infliximab attenuated the apoptosis of HUVECs incubated with serum from RHTN, but no effect in ROS production was observed. Our findings suggest that TNF-α might mediate, at least in part, vascular damage in resistant hypertension.

Refractory and resistant hypertension: characteristics and differences observed in a specialized clinic.

Resistant hypertension (RH) is defined as uncontrolled blood pressure (BP) despite the use of ≥3 anti-hypertensive drugs, or controlled requiring use of ≥4 drugs. Recently, a new definition for an extreme phenotype of RH (uncontrolled BP using at least five drugs) has emerged-the refractory hypertension (RfH). Although characteristics of RH are well established, little is known about this newly described subgroup. For this work, 116 subjects with RH were enrolled from a specialized clinic and divided into RH (n = 80) and RfH (n = 36). Subjects were submitted to echocardiography, 24-hour ambulatory BP measurement and biochemical analyses. Logistic regression analysis demonstrated that: (1) white-coat effect (odds ratio [OR], 3.23; 95% confidence interval [CI], 1.12-9.27; P = .03), (2) black race (OR, 6.67; 95% CI, 1.99-16.16; P < .001), and (3) left ventricular mass index (OR, 1.02; 95% CI, 1.01-1.03; P = .04) were independent predictors of refractoriness. In conclusion, RfH and RH present different patient characteristics, and these phenotypic aspects can be useful for better understanding this harder-to-treat subgroup.

Increased arterial stiffness in resistant hypertension is associated with inflammatory biomarkers.

BACKGROUND: Increased levels of inflammatory biomarkers such as interleukin-6 (IL-6), 10 (IL-10), 1ß (IL-1ß), tumor necrosis factor-α (TNF-α) high-sensitivity C-reactive protein (hs-CRP) are associated with arterial stiffness in hypertension. Indeed, resistant hypertension (RHTN) leads to unfavorable prognosis attributed to poor blood pressure (BP) control and target organ damage. This study evaluated the potential impact of inflammatory biomarkers on arterial stiffness in RHTN. METHODS: In this cross-sectional study, 32 RHTN, 20 mild hypertensive (HTN) and 20 normotensive (NT) patients were subjected to office BP and arterial stiffness measurements assessed by pulse wave velocity (PWV). Inflammatory biomarkers were measured in plasma samples. RESULTS: PWV was increased in RHTN compared with HTN and NT (p < 0.05). TNF-α levels were significantly higher in RHTN and HTN than NT patients. No differences in IL-6 levels were observed. RHTN patients had a higher frequency of subjects with increased levels of IL-10 and IL-1ß compared with HTN and NT patients. Finally, IL-1ß was independently associated with PWV (p < 0.001; R(2) = 0.5; ß = 0.077). CONCLUSION: RHTN subjects have higher levels of inflammatory cytokines (TNF-α, IL-1ß and IL-10) as well as increased arterial stiffness, and detectable IL-1ß levels are associated arterial stiffness. These findings suggest that inflammation plays a possible role in the pathophysiology of RHTN.

Plasma 8-isoprostane levels are associated with endothelial dysfunction in resistant hypertension.

BACKGROUND: Impaired endothelial function and arterial stiffness are associated with hypertension and are important risk factors for cardiovascular events. Reactive oxygen species reduce nitric oxide bioavailability and have a pivotal role in endothelial function. Resistant hypertension (RHTN) is characterized by blood pressure (BP) above goal (140/90mmHg) in spite of the concurrent use of ≥3 antihypertensive drugs of different classes. This study evaluated the association between 8-isoprostane levels, an oxidative stress marker, endothelial function and arterial stiffness, in RHTN. METHODS: Ninety-four RHTN and 55 well-controlled hypertensive (HT) patients were included. Plasma 8-isoprostane levels were determined by ELISA. Also, flow-mediated dilation (FMD) and pulse wave velocity (PWV) were evaluated to determine endothelial function and arterial stiffness, respectively. RESULTS: Levels of 8-isoprostane were markedly higher in RHTN compared to HT patients (22.5±11.2 vs. 17.3±9.8pg/ml, p<0.05, respectively). A significant inverse correlation was observed between FMD and 8-isoprostane (r=-0.35, p=0.001) in RHTN. Finally, multiple logistic regression revealed that 8-isoprostane was a significant predictor of endothelial dysfunction (FMD≤median) in RHTN group. CONCLUSION: RHTN showed markedly higher oxidative stress measured by 8-isoprostane, compared to HT patients. Taken together, our findings suggest the involvement of oxidative stress in endothelial function in RHTN.

Modulation of aldosterone levels by -344 C/T CYP11B2 polymorphism and spironolactone use in resistant hypertension.

Interindividual variability in plasma aldosterone levels comprises environmental and genetic sources. Increased aldosterone levels have been associated with higher risk of hypertension and target-organ damage related to hypertension. Aldosterone excess and intravascular volume expansion are implicated in pathophysiology of resistant hypertension (RH). We sought to investigate whether -344 C/T polymorphism (rs1799998) in aldosterone synthase gene (CYP11B2) is associated with plasma aldosterone levels in patients with resistant hypertension. Sixty-two patients with resistant hypertension were enrolled in this cross-sectional study. Genotypes were obtained by allelic discrimination assay using real time polymerase chain reaction. Multivariable linear regression was used to identify whether TT genotype was a predictor of aldosterone levels. No differences in clinical and laboratorial parameters were found among genotype groups. We found an additive effect of the T allele on plasma aldosterone concentration in RH. Also, there was higher aldosterone levels in TT homozygous under use of spironolactone compared with C carriers and compared with TT subjects who was not under use of spironolactone. TT genotype and the use of spironolactone were significant predictors of aldosterone levels in RH subjects. Plasma aldosterone concentration is significantly associated with -344 C/T CYP11B2 polymorphism and with the treatment with spironolactone in resistant hypertensive subjects.

Fisiopatologia da hipertensão no diabetes e na obesidade / Pathophysiology of hypertension in diabetes and obesity

A hipertensão arterial sistêmica (HAS) é uma síndrome que tem alta prevalência e baixas taxas de controle no mundo ocidental. Atualmente, os mecanismos que contribuem para o desenvolvimento de HAS e suas complicações estão mais elucidados. Por exemplo, hoje se sabe que pacientes hipertensos apresentam mais frequentemente resistência à insulina/hiperinsulinemia, dislipidemia, micro albuminúria e obesidade do que normo tensos. Embora classicamente a associação entre obesidade e hipertensão tenha sido atribuída a alterações hemodinâmicas, sabe-se que inúmeras alterações interferem no desenvolvimento desse quadro, como a disfunção endotelial, o aumento da atividade do sistema renina-angiotensina-aldosterona (SRAA) e do sistema nervoso simpático (SNS)...

Hypertension has a high prevalence and low rates of control worldwide. Nowadays, the mechanisms that contribute to the development of hypertension and its complications are better understood. For example, it is well known that hypertensive patients frequently have insulin resistance/hyper insulinemia, dyslipidemia, obesity and micro albumiria compared to normotensive subjects. Classically, the association between obesity and hypertension has been attributed to hemodynamic alterations but more recently other factors such as endothelial dysfunction, aldosterone--angiotensin-renin system (AARS) and sympathetic nervoussystem activity (SNS)...