An “on-matrix” digestion procedure for AP-MS experiments dissects the interplay between complex-conserved and serotype-specific reactivities in Dengue virus-human plasma interactome.
Ramos Y, Huerta V, Martín D, Palomares S, Yero A, Pupo D, et al. J Proteomics. 2017 Jul 13. pii: S1874-3919(17)30238-5.
The interactions between the four Dengue virus (DENV) serotypes and plasma proteins are crucial in the initial steps of viral infection to humans. Affinity purification combined with quantitative mass spectrometry analysis, has become one of the most powerful tools for the investigation on novel protein-protein interactions. Using this approach, we report here that a significant number of bait-interacting proteins do not dissociate under standard elution conditions, i.e. acid pH and chaotropic agents, and that this problem can be circumvented by using the “on-matrix” digestion procedure described here. This procedure enabled the identification of 16 human plasma proteins interacting with domain III from the envelope protein of DENV serotypes 1, 3 and 4 that would have not been detected otherwise and increased the known DIIIE interactors in human plasma to 59 proteins. Selected Reaction Monitoring analysis evidenced DENV interactome in human plasma is rather conserved although significant differences on the reactivity of viral serotypes with specific proteins do exist. A comparison between the serotype-dependent profile of reactivity and the conservation pattern of amino acid residues suggests an evolutionary selection of highly conserved interactions with the host and other interactions mediated for surface regions of higher variability. Significance False negative results on the identification of interacting proteins in pull-down experiments compromise the subsequent interpretation of results and the formulation of a working hypothesis for the derived future work. In this study we demonstrate the presence of bait-interacting proteins reluctant to dissociate under elution conditions of acid pH and presence of chaotropics. We propose the direct proteolytic digestion of proteins while still bound to the affinity matrix (“on-matrix” digestion) and evaluate the impact of this methodology in the comparative study of the interactome of the four serotypes of Dengue virus mediated by the domain III of the viral envelope glycoprotein. Fifty nine proteins were identified as putative interaction partners of Dengue virus (IPs) either due to direct binding or by co-isolation with interacting proteins. Collectively the IPs identified from the pull-down with the recombinant domain III proteins representing the four viral serotypes, 29% were identified only after “on-matrix” digestion which demonstrate the usefulness of this method of recovering bait-bound proteins. Results highlight a particular importance of “on-matrix” digestion procedure for comparative studies where a stronger interaction with one of the interest baits could prevent a bound protein to elute under standard conditions thus leading to misinterpretation as absent in the interactome of this particular bait. The analysis of the Interaction Network indicates that Dengue virus interactome mediated by the domain III of the envelope protein is rather conserved in the viral complex suggesting a key role of these interactions for viral infection thus making candidates to explore for potential biomarkers of clinical outcome in DENV-caused disease. Interestingly, some particular IPs exhibit significant differences in the strength of the interaction with the viral serotypes representing interactions that involve more variable regions in the surface of the domain III. Since such variable regions are the consequence of the interaction with antibodies generated by human immune response; this result relates the interaction with proteins from human plasma with the interplay of the virus and the human immune system.
Association between endothelial dysfunction, epicardial fat and sub-clinical atherosclerosis during menopause.
Cabrera-Rego JO, Navarro-Despaigne D, Staroushik-Morel L, Díaz-Reyes K, Lima-Martínez MM, Iacobellis G. Clin Investig Arterioscler. 2017 Sep 19. pii: S0214-9168(17)30092-X. [Epub ahead of print]. English, Spanish.
Background Menopausal transition is critical for the development of early, subclinical vascular damage. Multiple factors, such as atherosclerosis, increased epicardial fat, and endothelial dysfunction can play a role. Hence, the objective of this study was the comparison of epicardial adipose tissue and carotid intima media thickness in order to establish the best predictor of carotid stiffness in middle-aged women with endothelial dysfunction. Methods A total of 43 healthy women aged 40-59 years old with endothelial dysfunction previously demonstrated by flow mediated dilation were recruited to have anthropometric, biochemical, hormonal and ultrasound determinations of carotid intima media thickness and epicardial fat thickness. Results Carotid arterial stiffness parameters (local pulse wave velocity [4.7±0.7 vs 4.8±0.5 vs 5.6±0.5m/s, respectively, p<0.001], pressure strain elastic modulus [55.2±13.4 vs 59.2±11.8 vs 81.9±15.6kPa, respectively, p<0.001], arterial stiffness index β [4.4±1.4 vs 5.0±1.1 vs 6.4±1.3, respectively, p<0.001]) and epicardial fat thickness (2.98±1.4 vs 3.28±1.9 vs 4.70±1.0mm, respectively, p=0.007) showed a significant and proportional increase in the group of late post-menopausal women when compared to early post-menopausal and pre-menopausal groups, respectively. Among body fat markers, epicardial fat was the strongest predictor of local pulse wave velocity, independent of age. Conclusions In menopausal women with endothelial dysfunction, menopausal transition is associated with increased carotid arterial stiffness and epicardial fat thickness, independent of age. Ultrasound measured epicardial fat was a better independent predictor of arterial stiffness than carotid intima media thickness in these women.
Biometeorological forecasts for health surveillance and prevention of meteor-tropic effects.
Lecha Estela LB. Int J Biometeorol. 2017 Sep 13. [Epub ahead of print].
An early method of biometeorological forecasts was developed for Cuba during the late 90s. It was based on the relationship between the daily occurrence of massive health crisis and the magnitude of the 24-h differences of partial density of oxygen in the air (PODA index). Ten years later, applying new technological facilities, a new model was developed in order to offer operational biometeorological forecast to Cuban health institutions. After a satisfactory validation process, the official bioforecast service to health institutions in Villa Clara province began on February of 2012. The effectiveness had different success levels: for the bronchial asthma crisis (94%), in the hypertensive crisis (88%), with the cerebrovascular illnesses (85%), as well as migraines (82%) and in case of cardiovascular diseases (75%) were acceptable. Since 2008, the application of the model was extended to other regions of the world, including some national applications. Furthermore, it allowed the beginning of regional monitoring of meteor-tropic effects, following the occurrence and movement of areas with higher weather contrasts, defined according to the normalized scale of PODA index. The paper describes the main regional results already available, with emphasis in the observed meteor-tropic effects increasing in all regions during recent years. It coincides with the general increase of energy imbalance in the whole climate system. Finally, the paper describes the current development of new global biometeorological forecast services.
Cellular uptake pathways of sepiolite nanofibers and DNA transfection improvement.
Castro-Smirnov FA, Ayache J, Bertrand JR, Dardillac E, Le Cam E, Piétrement O, et al. Sci Rep. 2017 Jul 17;7(1):5586.
Sepiolite is a nanofibrous natural silicate that can be used as a nanocarrier because it can be naturally internalized into mammalian cells, due to its nano-size dimension. Therefore, deciphering the mechanisms of sepiolite cell internalization constitutes a question interesting biotechnology, for the use of sepiolite as nanocarrier, as well as environmental and public health concerns. Though it is low, the perfectly stable and natural intrinsic fluorescence of sepiolite nanofibers allows to follow their fate into cells by specifically sensitive technics. By combining fluorescence microscopy (including confocal analysis), time-lapse video microscopy, fluorescence activated cell sorting and transmission electron microscopy, we show that sepiolite can be spontaneously internalized into mammalian cells through both non-endocytic and endocytic pathways, macropinocytosis being one of the main pathways. Interestingly, exposure of the cells to endocytosis inhibitors, such as chloroquine, two-fold increase the efficiency of sepiolite-mediated gene transfer, in addition to the 100-fold increased resulting from sepiolite sonomechanical treatment. As sepiolite is able to bind various biological molecules, this nanoparticulate silicate could be a good candidate as a nanocarrier for simultaneous vectorization of diverse biological molecules.
Characteristics of the specific humoral response in patients with advanced solid tumors after active immunotherapy with a VEGF vaccine, at different antigen doses and using two distinct adjuvants.
Sánchez Ramírez J, Morera Díaz Y, Bequet-Romero M, Hernández-Bernal F, Selman-Housein Bernal KH, de la Torre Santos A, et al. BMC Immunol. 2017 Jul 26;18(1):39.
Background CIGB-247, a VSSP-adjuvanted VEGF-based vaccine, was evaluated in a phase I clinical trial in patients with advanced solid tumors (CENTAURO). Vaccination with the maximum dose of antigen showed an excellent safety profile, exhibited the highest immunogenicity and was the only one showing a reduction on platelet VEGF bioavailability. However, this antigen dose level did not achieve a complete seroconversion rate in vaccinated patients. These clinical results led us to the question whether a “reserve” of untapped immune response potential against VEGF could exist in cancer patients. To address this matter, CENTAURO-2 clinical trial was conducted where antigen and VSSP dose scale up were studied, and also incorporated the exploration of aluminum phosphate as adjuvant. These changes were made with the aim to increase immune response against VEGF. Results The present study reports the characterization of the humoral response elicited by CIGB-247 from the combining of different antigen doses and adjuvants. Cancer patients were immunologically monitored for approximately 1 year. Vaccination with different CIGB-247 formulations exhibited a very positive safety profile. Cancer patients developed IgM, IgG or IgA antibodies specific to VEGF. Elicited polyclonal antibodies had the ability to block the interaction between VEGF and its receptors, VEGFR1 and VEGFR2. The highest humoral response was detected in patients immunized with 800 μg of antigen + 200 μg of VSSP. Off-protocol long-term vaccination did not produce negative changes in humoral response. Conclusions Vaccination with a human VEGF variant molecule as antigen in combination with VSSP or aluminum phosphate is immunogenic. The results of this study could contribute to the investigation of this vaccine therapy in an adequately powered efficacy trial. Trial Registration Trial registration number: RPCEC00000155. Cuban Public Clinical Trial Registry. Date of registration: June 06, 2013. Available from: http://registroclinico.sld.cu/
Characterization of the sensorimotor rhythm in 4-month-old Infants born at term and premature.
Roca-Stappung M, Moguel-González M, Fernández T, Harmony T, Mendoza-Montoya O, Marroquín JL, et al. Appl Psychophysiol Biofeedback. 2017 Jul 22. DOI: 10.1007/s10484-017-9370-4. [Epub ahead of print]
The sensorimotor rhythm (SMR) is an electroencephalographic rhythm associated with motor and cognitive development observed in the central brain regions during wakefulness in the absence of movement, and it reacts contralaterally to generalized and hemibody movements. The purpose of this work was to characterize the SMR of 4-month-old infants, born either healthy at term or prematurely with periventricular leukomalacia (PVL). Two groups of infants were formed: healthy and premature with PVL. Their electroencephalograms (EEGs) were recorded in four conditions: rest, free movement, right-hand grasping and left-hand grasping, in order to explore general reactivity to free movement and contralateral reactivity in hand-grasping conditions. Associations between SMR, and cognitive and motor performance were analyzed. The healthy infants showed a SMR between 5.47 and 7.03 Hz, with clear contralateral reactivity to free movement and right-hand grasping. However, the premature infants with PVL did not show enough electroencephalographic characteristics to evidence the presence of SMR. Poor performance, characteristic of children with PVL, was related to low-frequency SMR, while good performance was associated with a higher frequency rhythm in the left hemisphere. The presence of SMR in the group of healthy infants could be considered a sign of health at this age. Thus, poor SMR evidence in the EEG of infants with PVL is probably a sign of brain immaturity or brain dysfunction. Our results provide data on infant SMR development that is needed to design neurofeedback protocols for infants with PVL.
Detection and identification of Leishmania spp.: application of two hsp70-based PCR-RFLP protocols to clinical samples from the New World.
Montalvo AM, Fraga J, Tirado D, Blandón G, Alba A, Van der Auwera G, et al. Parasitol Res. 2017 Jul;116(7):1843–8.
Leishmaniasis is highly prevalent in New World countries, where several methods are available for detection and identification of Leishmania spp. Two hsp70-based PCR protocols (PCR-N and PCR-F) and their corresponding restriction fragment length polymorphisms (RFLP) were applied for detection and identification of Leishmania spp. in clinical samples recruited in Colombia, Guatemala, and Honduras. A total of 93 cases were studied. The samples were classified into positive or suspected of leishmaniasis according to parasitological criteria. Molecular amplification of two different hsp70 gene fragments and further RFLP analysis for identification of Leishmania species was done. The detection in parasitologically positive samples was higher using PCR-N than PCR-F. In the total of samples studied, the main species identified were Leishmania panamensis, Leishmania braziliensis, and Leishmania infantum (chagasi). Although RFLP-N was more efficient for the identification, RFLP-F is necessary for discrimination between L. panamensis and Leishmania guyanesis, of great importance in Colombia. Unexpectedly, one sample from this country revealed an RFLP pattern corresponding to Leishmania naiffi. Both molecular variants are applicable for the study of clinical samples originated in Colombia, Honduras, and Guatemala. Choosing the better tool for each setting depends on the species circulating. More studies are needed to confirm the presence of L. naiffi in Colombian territory.
Detection of Naturally Occurring Human Antibodies Against Gangliosides by ELISA.
Hernández AM, Rodríguez-Zhurbenko N. Methods Mol Biol. 2017;1643:179–86.
Gangliosides are sialic acid-containing glycolipids that have been considered attractive targets for cancer immunotherapy, based on the qualitative and quantitative changes they suffer during malignant transformation and due to their importance for tumor biology. Natural antibodies against gangliosides have been detected not only in cancer patients but also in healthy donors. The presence of these antibodies can be used as diagnostic or prognostic factor. However, these responses are difficult to detect because anti-ganglioside antibodies are usually of IgM isotype and low affinity. Enzyme Linked Immunosorbent Assay (ELISA) is an immunoassay based on the specific binding of antibodies to antigens bound to a solid phase. These antigens can be glycolipids like gangliosides. An enzyme linked to the last reactant allows the detection of specific binding through the development of color after the addition of a suitable substrate. ELISA combines the specificity of antibodies with the sensitivity of enzyme reactions. The ELISA method described herein can be used to detect antibody responses against gangliosides not only related to cancer but also to autoimmune diseases and infections, both in healthy donors, and patients, untreated or receiving specific immunotherapy.
Development and validation of a bioanalytical method based on LC-MS/MS analysis for the quantitation of CIGB-814 peptide in plasma from Rheumatoid Arthritis patients.
Cabrales-Rico A, Ramos Y, Besada V, Del Carmen Domínguez M, Lorenzo N, García O, et al. J Pharm Biomed Anal. 2017 Jun 1;143:130–40. [Epub ahead of print]
CIGB-814, originally named as E18-3 APL1 or APL1 in preclinical experiments, is a novel therapeutic peptide candidate for Rheumatoid Arthritis (RA). It is an altered peptide ligand containing a novel CD4+ T-cell epitope of human heat shock protein 60 (83-109, MW 2988.38g/mol) with a mutation (D100→L) that increases its affinity for HLA-II type molecules associated to RA. A bioanalytical method, based on LC-MS/MS analysis, in the SRM mode was developed and fully validated to quantify this peptide in human plasma. An internal standard with the same amino acid sequence but labeled with three (13C615N2)-Lys residues was used for quantitation. The method provides a linear range from 1.5 to 48ng/mL (without matrix effect and carry over) and an accuracy and precision good enough for monitoring more than 80% of the AUC of the PK profile in a phase I clinical trial. The peptide was administered subcutaneously in three dose levels (1, 2.5 and 5mg) not normalized to the body weight of patients with RA. The low doses imposed an analytical challenge; however, a LLOQ of 1.5ng/mL enabled the PK analysis. The Cmax, reached at 0.5h, showed a great variability, that was most likely due to the non-normalized doses; the proposed mechanism for this peptide; and the variability between patients. A rapid clearance of this peptide (4-6h) is advantageous for an immunomodulatory drug, because the therapeutic schedule requires repeated dosages to restore peripheral tolerance.
Diabetic Foot Ulcers and Epidermal Growth Factor: Revisiting the Local Delivery Route for a Successful Outcome.
Berlanga-Acosta J, Fernández-Montequín J, Valdés-Pérez C, Savigne-Gutiérrez W, Mendoza-Marí Y, García-Ojalvo A, et al. Biomed Res Int. 2017;2017:2923759. Epub 2017 Aug 21.
Soon after epidermal growth factor (EGF) discovery, some in vivo models appeared demonstrating its property to enhance cutaneous wound healing. EGF was the first growth factor (GF) introduced in the clinical arena as a healing enhancer, exerting its mitogenic effects on epithelial, fibroblastoid, and endothelial cells via a tyrosine kinase membrane receptor. Compelling evidences from the 90s documented that, for EGF, locally prolonged bioavailability and hourly interaction with the receptor were necessary for a successful tissue response. Eventually, the enthusiasm on the clinical use of EGF to steer the healing process was wiped out as the topical route to deliver proteins started to be questioned. The simultaneous in vivo experiments, emphasizing the impact of the parenterally administered EGF on epithelial and nonepithelial organs in terms of mitogenesis and cytoprotection, rendered the theoretical fundamentals for the injectable use of EGF and shaped the hypothesis that locally infiltrating the diabetic ulcers would lead to an effective healing. Although the diabetic chronic wounds microenvironment is hostile for local GFs bioavailability, EGF local infiltration circumvented the limitations of its topical application, thus expanding its therapeutic prospect. Our clinical pharmacovigilance and basic studies attest the significance of the GF local infiltration for chronic wounds healing.
Digital imaging information technology for biospeckle activity assessment relative to bacteria and parasites.
Ramírez-Miquet EE, Cabrera H, Grassi HC, de J Andrades E, Otero I, Rodríguez D. Lasers Med Sci. 2017 Aug;32(6):1375–86.
This paper reports on the biospeckle processing of biological activity using a visualization scheme based upon the digital imaging information technology. Activity relative to bacterial growth in agar plates and to parasites affected by a drug is monitored via the speckle patterns generated by a coherent source incident on the microorganisms. We present experimental results to demonstrate the potential application of this methodology for following the activity in time. The digital imaging information technology is an alternative visualization enabling the study of speckle dynamics, which is correlated to the activity of bacteria and parasites. In this method, the changes in Red-Green-Blue (RGB) color component density are considered as markers of the growth of bacteria and parasites motility in presence of a drug. The RGB data was used to generate a two-dimensional surface plot allowing an analysis of color distribution on the speckle images. The proposed visualization is compared to the outcomes of the generalized differences and the temporal difference. A quantification of the activity is performed using a parameterization of the temporal difference method. The adopted digital image processing technique has been found suitable to monitor motility and morphological changes in the bacterial population over time and to detect and distinguish a short term drug action on parasites.
Electrocardiographic Markers of Appropriate Implantable Cardioverter-Defibrillator Therapy in Young People with Congenital Heart Diseases.
Benítez Ramos DB, Cabrera Ortega M, Castro Hevia J, Dorantes Sánchez M, Alemán Fernández AA, Castañeda Chirino O, et al.
Implantable cardioverter-defibrillators (ICDs) are increasingly utilized in patients with congenital heart disease (CHD). Prediction of the occurrence of shocks is important if improved patient selection is desired. The electrocardiogram (ECG) has been the first-line tool predicting the risk of sudden death, but data in CHD patients are lacking. We aim to evaluate the predictive value of electrocardiographic markers of appropriate therapy of ICD in young people with CHD. We conducted a prospective, longitudinal study, in twenty-six CHD patients (mean age 24.7 ± 5.3 years) who underwent first ICD implantation. Forty-two age- and diagnosis-matched controls were recruited. Twelve-lead ECG and 24 h Holter analysis were performed during a mean follow-up of 38.9 months. Data included heart rate, heart rate variability, QRS duration (QRSd), QTc interval and its dispersion, Tpeak-Tend (Tp-Te) interval and its dispersion, presence of fragmented QRS (fQRS), T wave alternans, atrial arrhythmias, and non-sustained ventricular tachycardia. Implant indication was primary prevention in ten cases (38.5%) and secondary prevention in 16 (61.5%). Overall, 17 subjects (65.3%) received at least one appropriate and effective ICD discharge. fQRS was present in 64.7% of cases with ICD therapy compared with patients without events or controls (p < 0.0001). Tp-e and Tp-e dispersion were significantly prolonged in patients with recurrences (113.5 and 37.2 ms) versus patients without ICD discharge (89.6 and 24.1 ms) or controls (72.4 and 19.3 ms) (p < 0.0001 and p < 0.0001, respectively). On univariate Cox regression analysis QRSd (hazard ratio: 1.19 per ms, p = 0.003), QTc dispersion (hazard ratio: 1.57 per ms, p = 0.002), fQRS (hazard ratio: 3.58 p < 0.0001), Tp-e (hazard ratio: 2.27 per ms, p < 0.0001), and Tp-e dispersion (hazard ratio: 4.15 per ms, p < 0.0001), emerged as strong predictors of outcome. On multivariate Cox analysis fQRS, Tp-e and Tp-e dispersion remained in the model. The presence of fQRS, and both Tp-e and Tp-e dispersion are useful ECG tools in daily clinical practice to identify CHD patients at risk for appropriate ICD therapy.
First record of natural vertical transmission of dengue virus in Aedes aegypti from Cuba.
Gutiérrez-Bugallo G, Rodríguez-Roche R, Díaz G, Vázquez AA, Álvarez M, Rodríguez M, et al. Acta Trop. 2017 Oct;174:146–8.
While horizontal transmission (human-mosquito-human) of dengue viruses largely determines the epidemiology of the disease, vertical transmission (infected female mosquito- infected offspring) has been suggested as a mechanism that ensures maintenance of the virus during adverse conditions for horizontal transmission to occur. The purpose of this study was to analyze the natural infection of larval stages of Aedes aegypti (Diptera: Culicidae) with the dengue virus (DENV) in Cuba. Here, we report vertical transmission of DENV-3 genotype III in natural populations of Ae. aegypti through RT-PCR detection and serotyping plus sequencing. Our report constitutes the first record of vertical transmission of DENV in Ae. aegypti from Cuba with details of its serotype and genotype.
Immunologic Response Elicited in Breast Cancer Patients Receiving a NeuGcGM3-based Vaccine as Adjuvant Therapy.
Valdés-Zayas A, González Z, Mulens V, Vega AM, Pérez K, Lorenzo-Luaces P. J Immunother. 2017 Oct;40(8):289–301.
This study aimed to investigate the immunogenicity of a cancer vaccine consisting of the NeuGcGM3 ganglioside combined with the outer membrane protein complex of Neisseria meningitides to form very small size particles. The vaccine is administered together with Montanide ISA51, as adjuvant treatment for breast cancer patients. After surgical resection and standard first-line chemo/radiotherapy, breast cancer patients in stage II-III were enrolled in a phase III clinical trial and allocated into 2 strata, according to the number of positive lymph nodes [stratum I (0-3); stratum II (≥4)]. Subsequently, patients were randomly assigned to receive the vaccine or placebo. The treatment consisted of 5 vaccine doses (200 μg) every 2 weeks and thereafter monthly reimmunizations to complete 15 doses. The vaccine was well-tolerated and high titers of immunoglobulin M and immunoglobulin G anti-NeuGcGM3 antibodies were similarly detected in each stratum. Hyperimmune sera were able to specifically recognize and kill the NeuGcGM3-expressing L1210 tumor cell line, and these functional capacities were significantly associated with a better clinical outcome in patients of stratum II. Besides, postimmune sera had the capacity to revert in vitro the immunosuppression induced by NeuGcGM3, as measured by the prevention of CD4 downmodulation on human T lymphocytes. Vaccination had no impact on the frequency of regulatory T cells or circulating NK cells. This study demonstrated, for the first time, the immunogenicity of the NeuGcGM3/VSSP/Montanide ISA 51 vaccine in the adjuvant setting and describes the functionality of induced anti-NeuGcGM3 antibodies as potential surrogate biomarkers of clinical benefit.
Importance of tissue sampling, laboratory methods, and patient characteristics for detection of Pneumocystis in autopsied lungs of non-immunosuppressed individuals.
Vargas SL, Ponce C, Bustamante R, Calderón E, Nevez G, De Armas Y, et al. Eur J Clin Microbiol Infect Dis. 2017 Jun 5. Epub ahead of print
To understand the epidemiological significance of Pneumocystis detection in a lung tissue sample of non-immunosuppressed individuals, we examined sampling procedures, laboratory methodology, and patient characteristics of autopsy series reported in the literature. Number of tissue specimens, DNA-extraction procedures, age and underlying diagnosis highly influence yield and are critical to understand yield differences of Pneumocystis among reports of pulmonary colonization in immunocompetent individuals.
Is peanut causing food allergy in Cuba? Preliminary assessment of allergic sensitization and IgE specificity profile to peanut allergens in Cuban allergic patients.
Mateo-Morejón M, Labrada-Rosado A, Torralba-Averoff D, Cruz-Jiménez R, Oliva-Díaz Y, Álvarez-Castelló M, et al. World Allergy Organ J. 2017 Jul 11;10(1):26.
Background Peanut allergy is increasing at an alarming pace in developed countries. Peanut (Arachis hypogaea) is a common food in Cuba. Nevertheless, reported values of sensitization and symptom severity are usually low. As our objective, we carried out an evaluation of allergic sensitivity to perform an assessment of allergic sensitization and IgE specificity profile to peanut allergens in Cuban allergic patients. Methods The Skin Prick Test (SPT) was performed for each patient, using two glycerinated allergenic extracts, prepared from raw or roasted peanuts. Overall, 316 food allergic patients (159 adults and 157 children) attending allergy services at four hospitals in Havana were included, as well as 303 adult non- allergic volunteers. The IgE binding profile of 26 selected SPT positive patients was further analyzed by immunoblotting. Results The prevalence of sensitization to peanut was 4.6% in general adult population, whereas in adult food-allergic patients it was 18.6%. Prevalence rates were even greater in food allergic children achieving 25.8%. Sensitization frequencies were apparently greater for roasted, as compared to raw peanuts, although the difference was not significant (p> 0.05, Mc Nemar’s). IgE binding was shown mostly by the 15 and 17 kDa bands, tentatively identified as the major allergens Ara h 2 and Ara h 6. The IgG4 binding profile was similar to IgE, although with more prominence of the bands at 37 and 28 KDa, corresponding to an Ara h 3 fragment and Peanut Agglutinin. Discussion The study estimated a relatively high prevalence of peanut sensitization in population. Data reported here suggest that IgE sensitization in Cuban patients is focused mostly on MW bands corresponding to the major allergens Ara h 6 and Ara h 2. Conclusions Sensitization to peanut allergen is indeed relatively frequent in Cuba. The IgE profile is congruent to a sensitization pattern by ingestion of roasted peanuts and is directed to well-known major allergens.
KBE009: An antimalarial bestatin-like inhibitor of the Plasmodium falciparum M1 aminopeptidase discovered in an Ugi multicomponent reaction-derived peptidomimetic library.
González-Bacerio J, Maluf SEC, Méndez Y, Pascual I, Florent I, Melo PMS, et al. Bioorg Med Chem. 2017 Sep 1;25(17):4628–36.
Malaria is a global human parasitic disease mainly caused by the protozoon Plasmodium falciparum. Increased parasite resistance to current drugs determines the relevance of finding new treatments against new targets. A novel target is the M1 alanyl-aminopeptidase from P. falciparum (PfA-M1), which is essential for parasite development in human erythrocytes and is inhibited by the pseudo-peptide bestatin. In this work, we used a combinatorial multicomponent approach to produce a library of peptidomimetics and screened it for the inhibition of recombinant PfA-M1 (rPfA-M1) and the in vitro growth of P. falciparum erythrocytic stages (3D7 and FcB1 strains). Dose-response studies with selected compounds allowed identifying the bestatin-based peptidomimetic KBE009 as a submicromolar rPfA-M1 inhibitor (Ki=0.4μM) and an in vitro antimalarial compound as potent as bestatin (IC50=18μM; without promoting erythrocyte lysis). At therapeutic-relevant concentrations, KBE009 is selective for rPfA-M1 over porcine APN (a model of these enzymes from mammals), and is not cytotoxic against HUVEC cells. Docking simulations indicate that this compound binds PfA-M1 without Zn2+ coordination, establishing mainly hydrophobic interactions and showing a remarkable shape complementarity with the active site of the enzyme. Moreover, KBE009 inhibits the M1-type aminopeptidase activity (Ala-7-amido-4-methylcoumarin substrate) in isolated live parasites with a potency similar to that of the antimalarial activity (IC50=82μM), strongly suggesting that the antimalarial effect is directly related to the inhibition of the endogenous PfA-M1. These results support the value of this multicomponent strategy to identify PfA-M1 inhibitors, and make KBE009 a promising hit for drug development against malaria.
Medical ozone promotes Nrf2 phosphorylation reducing oxidative stress and pro-inflammatory cytokines in multiple sclerosis patients.
Delgado-Roche L, Riera-Romo M, Hernández-Matos Y, Barrios JM, Martínez-Sánchez G, et al. Eur J Pharmacol. 2017 Sep 15;811:148–54.
Oxidative stress and inflammation play key roles in the pathogenesis of Multiple sclerosis (MS). Different drugs have been used in the clinical practice, however, there is not a completely effective treatment. Due to its potential therapeutic action, medical ozone represents a promising approach for neurodegenerative disorders. The aim of the present study was to address the role of ozone therapy on the cellular redox state in MS patients. Ozone (20μg/ml) was administered three times per week during a month by rectal insufflation. The effect of ozone therapy on biomarkers of oxidative stress and inflammation was addressed by spectrophotometric and immunoenzymatic assays. Furthermore, we investigated the action of ozone on CK2 expression and Nrf2 phosphorylation by western blotting analysis. Medical ozone significantly improved (P < 0.05) the activity of antioxidant enzymes and increased the levels of cellular reduced glutathione. In accordance, a significant reduction (P < 0.05) of oxidative damage on lipids and proteins was observed in ozone-treated patients. As well, the levels of pro-inflammatory cytokines TNFα and IL-1β were lower after ozone treatment. Ozone therapy incremented the CK2 expression together with Nrf2 phosphorylation in mononuclear cells of MS patients. These findings suggest that ozone´s antioxidant and anti-inflammatory effects might be partially associated with an induction of Nrf2 phosphorylation and activation. These results provide new insights on the molecular events modulated by ozone, and pointed out ozone therapy as a potential therapeutic alternative for MS patients.
Nasal administration of the neuroprotective candidate NeuroEPO to healthy volunteers: a randomized, parallel, open-label safety study. Santos-Morales O, Díaz-Machado A, Jiménez-Rodríguez D, Pomares-Iturralde Y, Festary-Casanovas T, González-Delgado CA, et al. BMC Neurol. 2017 Jul 4;17(1):129.
Background Delivery of therapeutic agents as erythropoietin (EPO) into Central Nervous System through intranasal route could benefit patients with neurological disorders. A new nasal formulation containing a non-hematopoietic recombinant EPO (NeuroEPO) has shown neuroprotective actions in preclinical models. In the current study, the safety of NeuroEPO was evaluated for the first time in humans. Methods A phase I, randomized, parallel, open-label study was carried out in healthy volunteers. They received, intranasally, 1 mg of NeuroEPO every 8 h during 4 days (Group A) or 0.5 mg of NeuroEPO (Group B) with the same schedule. The working hypothesis was that intranasal NeuroEPO produce <10% of severe adverse reactions in the evaluated groups. Therefore, a rigorous assessment of possible adverse events was carried out, which included tolerance of the nasal mucosa and the effect on hematopoietic activity. Clinical safety evaluation was daily during treatment and laboratory tests were done before and on days 5 and 14 after starting treatment. Results Twenty-five volunteers, 56% women, with a mean age of 27 yrs. were included. Twelve of them received the highest NeuroEPO dose. Twenty types of adverse events occurred, with headache (20%) and increase of hepatic enzymes (20%) as the most reported ones. Nasopharyngeal itching was the most common local event but only observed in four patients (16%), all of them from the lowest dose group. About half of the events were very probably or probably caused by the studied product. Most of the events were mild (95.5%), did not require treatment (88.6%) and were completely resolved (81.8%). No severe adverse events were reported. During the study the hematopoietic variables were kept within reference values. Conclusions NeuroEPO was a safe product, well tolerated at the nasal mucosa level and did not stimulate erythropoiesis in healthy volunteers. Trial Registration Cuban Public Registry of Clinical Trials RPCEC00000157 , June 10, 2013.
Nimotuzumab Induces NK Cell Activation, Cytotoxicity, Dendritic Cell Maturation and Expansion of EGFR-Specific T Cells in Head and Neck Cancer Patients.
Mazorra Z, Lavastida A, Concha-Benavente F, Valdés A, Srivastava RM, García-Bates TM, et al. Front Pharmacol. 2017 Jun 19;8:382.
Survival benefit and long-term duration of clinical response have been seen using the epidermal growth factor receptor (EGFR)-targeted monoclonal antibody (mAb) nimotuzumab. Blocking EGFR signaling may not be the only mechanism of action underlying its efficacy. As an IgG1 isotype mAb, nimotuzumab’s capacity of killing tumor cells by antibody dependent cellular cytotoxicity (ADCC) and to induce an immune response in cancer patients have not been studied. ADCC-induced by nimotuzumab was determined using a 51Cr release assay. The in vitro effect of nimotuzumab on natural killer (NK) cell activation and dendritic cell (DC) maturation and the in vivo frequency of circulating regulatory T cells (Tregs) and NK cells were assessed by flow cytometry. Cytokine levels in supernatants were determined by ELISA. ELISpot was carried out to quantify EGFR-specific T cells in nimotuzumab-treated head and neck cancer (HNSCC) patients. Nimotuzumab was able to kill EGFR+ tumor cells by NK cell-mediated ADCC. Nimotuzumab-activated NK cells promoted DC maturation and EGFR-specific CD8+ T cell priming. Interestingly, nimotuzumab led to upregulation of some immune checkpoint molecules on NK cells (TIM-3) and DC (PD-L1), to a lower extent than another EGFR mAb, cetuximab. Furthermore, circulating EGFR-specific T cells were identified in nimotuzumab-treated HNSCC patients. Notably, nimotuzumab combined with cisplatin-based chemotherapy and radiation increased the frequency of peripheral CD4+CD39+FOXP3+Tregs which otherwise were decreased to baseline values when nimotuzumab was used as monotherapy. The frequency of circulating NK cells remained constant during treatment. Nimotuzumab-induced, NK cell-mediated DC priming led to induction of anti-EGFR specific T cells in HNSCC patients. The association between EGFR-specific T cells and patient clinical benefit with nimotuzumab treatment should be investigated.
Non-Invasive Brain Stimulation for Children with Autism Spectrum Disorders: A Short-Term Outcome Study.
Gómez L, Vidal B, Maragoto C, Morales LM, Berrillo S, Vera Cuesta H, et al. Behav Sci (Basel). 2017 Sep 17;7(3).
Non-Invasive Brain Stimulation (NIBS) is a relatively new therapeutic approach that has shown beneficial effects in Autism Spectrum Disorder (ASD). One question to be answered is how enduring its neuromodulatory effect could be. Twenty-four patients with ASD (mean age: 12.2 years) received 20 sessions of NIBS over the left dorsolateral prefrontal cortex (L-DLPFC). They were randomized into two groups with two (G1) or three (G2) clinical evaluations before NIBS. Both groups had a complete follow-up at six months after the intervention, with the aim of determining the short-term outcome using the total score on the Autism Behavior Checklist, Autism Treatment Evaluation Checklist, and the Autism Diagnostic Interview. Transcranial Direct Current Stimulation (tDCS) was used in ASD patients aged <11 years, and repetitive Transcranial Magnetic Stimulation (rTMS) for 11-13-year-olds. Observation points were at one, three, and six months after completing all the sessions of NIBS. A significant reduction in the total score on the three clinical scales was observed and maintained during the first six months after treatment, with a slight and non-significant tendency to increase the scores in the last evaluation. Twenty sessions of NIBS over the L-DLPFC improves autistic symptoms in ASD children, with a lasting effect of six months.
Prevalence of Giardiaduodenalis among children from a central region of Cuba: molecular characterization and associated risk factors.
Puebla LJ, Núñez FA, García AB, Rivero LR, Millán IA, Prado RC. J Parasit Dis. 2017 Jun;41(2):405–13.
Giardia duodenalis is one of the most frequent intestinal parasitic infections in children worldwide. To date, eight main assemblages of G. duodenalis have been described, but only A and B genetic groups are known to infect humans. A cross-sectional study was conducted to determine the prevalence and risk factors of Giardia duodenalis infection in 417 preschool children from the Fomento municipality in the central region of Cuba between January and June 2013. The overall prevalence of Giardia infection was 10.79 %. Assemblage identification was carried out by the amplification of a fragment of the triose phosphate isomerase (tpi) gene. DNA from 36 of 45 (80 %) samples was successfully amplified by PCR-tpi. Assemblage B and mixed assemblages A + B represented 52.78 and 36.11 % respectively, of genotyped samples. Assemblage A accounts for only 11.11 %. Children who were cared for at home were associated with diarrhea caused by assemblage B. No associations were found between other clinical variables with infecting assemblage of Giardia. Univariate analysis identified the use of unsafe water resources (OR 2.9; CI 1.2-6.8) and-even more interestingly-keeping dogs indoor (OR 2.5; CI 1.2-5.4) were significant risk factors associated with Giardia infection among children. Multivariate analysis using introduction test logistic regression ratified the association of these two risk factors: kept dogs indoor (OR 2.8, CI 1.1-5.3), and untreated water (OR 1.4, CI 1.4-4.9) with Giardia infection. This information may be useful for an effective prevention and control programme of giardiasis in this population.
Real-time parameter estimation of Zika outbreaks using model averaging.
Sebrango-Rodríguez CR, Martínez-Bello DA, Sánchez-Valdés L, Thilakarathne PJ, Del Fava E, Van der Stuyft P, et al. Epidemiol Infect. 2017 Aug;145(11):2313–23.
Early prediction of the final size of any epidemic and in particular for Zika disease outbreaks can be useful for health authorities in order to plan the response to the outbreak. The Richards model is often been used to estimate epidemiological parameters for arboviral diseases based on the reported cumulative cases in single- and multi-wave outbreaks. However, other non-linear models can also fit the data as well. Typically, one follows the so called post selection estimation procedure, i.e., selects the best fitting model out of the set of candidate models and ignores the model uncertainty in both estimation and inference since these procedures are based on a single model. In this paper we focus on the estimation of the final size and the turning point of the epidemic and conduct a real-time prediction for the final size of the outbreak using several non-linear models in which these parameters are estimated via model averaging. The proposed method is applied to Zika outbreak data in four cities from Colombia, during the outbreak occurred in 2015-2016.
Remission and Low Disease Activity Status (LDAS) protect lupus patients from damage occurrence: data from a multiethnic, multinational Latin American Lupus Cohort (GLADEL).
Ugarte-Gil MF, Wojdyla D, Pons-Estel GJ, Catoggio LJ, Drenkard C, Sarano J, et al. Ann Rheum Dis. 2017 Sep 22. pii: annrheumdis-2017-211814.
Objective To evaluate disease activity statuses’ (DAS’) impact on systemic lupus erythematosus (SLE) outcomes. Materials and methods Four DAS were defined: remission off-therapy: SLE Disease Activity Index (SLEDAI)=0, no prednisone or immunosuppressive drugs (IS); remission on-therapy: SLEDAI=0, prednisone ≤5 mg/day and/or IS (maintenance); low (L) DAS: SLEDAI ≤4, prednisone ≤7.5 mg/day and/or IS (maintenance); non-optimally controlled: SLEDAI >4 and/or prednisone >7.5 mg/day and/or IS (induction). Antimalarials were allowed in all. Predefined outcomes were mortality, new damage (increase of at least one Systemic Lupus International Collaborating Clinics/American College of Rheumatology (SLICC/ACR) damage index (SDI) point) and severe new damage (increase of at least 3 SDI points). Univariable and multivariable Cox regression models were performed to define the impact of DAS, as time-dependent variable, on these outcomes. Results 1350 patients were included, 79 died during follow-up, 606 presented new and 177 severe new damage. In multivariable analyses, remission (on/off-therapy) was associated with a lower risk of new (HR 0.60; 95% CI 0.43 to 0.85), and of severe new damage (HR 0.32; 95% CI 0.15 to 0.68); low disease activity status (LDAS) was associated with a lower risk of new damage (HR 0.66; 95% CI 0.48 to 0.93) compared with non-optimally controlled. No significant effect on mortality was observed. Conclusions Remission was associated with a lower risk of new and severe new damage; LDAS with a lower risk of new damage after adjusting for other damage confounders.
Rey- Osterrieth Complex Figure – copy and immediate recall (3 minutes): Normative data for Spanish-speaking pediatric populations.
Arango-Lasprilla JC, Rivera D, Erlt MM, Muñoz Mancilla JM, García-Guerrero CE, Rodriguez-Irizarry W, et al. NeuroRehabilitation. 2017 Sep 5. [Epub ahead of print].
Objective To generate normative data for the Rey- Osterrieth Complex Figure (ROCF) in Spanish-speaking pediatric populations. Method The sample consisted of 4,373 healthy children from nine countries in Latin America (Chile, Cuba, Ecuador, Guatemala, Honduras, Mexico, Paraguay, Peru, and Puerto Rico) and Spain. Each participant was administered the ROCF as part of a larger neuropsychological battery. The ROCF copy and immediate recall (3 minutes) scores were normed using multiple linear regressions and standard deviations of residual values. Age, age2, sex, and mean level of parental education (MLPE) were included as predictors in the analyses. Results The final multiple linear regression models showed main effect for age on copy and immediate recall scores, such that scores increased linearly as a function of age. Age2 affected ROCF copy scores for all countries, except Puerto Rico; and ROCF immediate recall scores for all countries, except Chile, Guatemala, Honduras, Paraguay, and Puerto Rico. Models indicated that children whose parents had a MLPE >12 years of education obtained higher scores compared to children whose parents had a MLPE≤12 years for Chile, Puerto Rico, and Spain in the ROCF copy, and Paraguay and Spain for the ROCF immediate recall. Sex affected ROCF copy and immediate recall scores for Chile and Puerto Rico with girls scoring higher than boys. Conclusions This is the largest Spanish speaking pediatric normative study in the world, and it will allow neuropsychologists from these countries to have a more accurate approach to interpret the ROCF Test in pediatric populations.
The Cuba-United States Thaw: Building Bridges Through Science and Global Health.
Bausch DG, Kourí V, Resik S, Acosta B, Guillén G, Goraleski K, et al. Am J Trop Med Hyg. 2017 Jun;96(6):1267–9.
Beginning in 2014, there has been significant progress in normalization of relations between Cuba and the United States. Herein, we discuss the history and recent progress in scientific collaboration between the two countries as well as the continued challenges. Science and global health diplomacy can be key tools in reestablishing a trusting and productive relationship of mutual and global benefit, bringing about better and healthier lives for people in both Cuba and the United States.
The influence of physical activity in water on sleep quality in pregnant women: A randomised trial. Rodríguez-Blanque R, Sánchez-García JC, Sánchez-López AM, Mur-Villar N, Aguilar-Cordero MJ. Women Birth. 2017 Jul 7. pii: S1871-5192(16)30267-0.
Introduction Sleep is a physiological state of self-regulation. The international classification of sleep disorders now includes as a new category those occurring during pregnancy. Regular physical activity is known to improve the quality of life, one aspect of which is sleep quality. During pregnancy, physical activity is decreased but should not be eliminated, as studies have reported a high correlation between sleep disorders and the absence of physical activity. Regular physical exercise during pregnancy, whether performed in water or out of it, provides greater control of gestational weight gain. Furthermore, the reduced weight gain during pregnancy, as a result of physical exercise, is associated with greater physical resistance to the demands of childbirth, combats the fatigue caused by pregnancy and reduces back pain. All of these outcomes tend to enhance sleep quality, among other beneficial effects. Objective To determine whether, in pregnant women, there is an association between moderate-intensity physical activity in an aquatic environment and sleep quality. Materials and Methods A randomised clinical trial was conducted with a sample of 140 pregnant women aged 21-43 years, divided into two groups; Intervention Group and Control Group. The women were recruited in the twelfth week of gestation and took part in the [Study of] Water Exercise in Pregnancy programme from week 20 to week 37. Sleep quality was evaluated in the first and third trimesters of pregnancy, using the Pittsburgh Sleep Quality Index questionnaire. Results The Mann-Whitney U test showed that the results obtained were statistically significant (p<0.05). In the Intervention Group, 44 of the women (65.67%) were classified as “poor sleepers” versus 62 women (92.54%) in the Control Group. Conclusions The [Study of] Water Exercise in Pregnancy method improves the quality of sleep in pregnant women, both subjectively and in terms of latency, duration and efficiency.
The role of methionine on metabolism, oxidative stress, and diseases.
Martínez Y, Li X, Liu G, Bin P, Yan W, Más D, et al. Amino Acids. 2017 Sep 19. [Epub ahead of print]
Methionine is an aliphatic, sulfur-containing, essential amino acid, and a precursor of succinyl-CoA, homocysteine, cysteine, creatine, and carnitine. Recent research has demonstrated that methionine can regulate metabolic processes, the innate immune system, and digestive functioning in mammals. It also intervenes in lipid metabolism, activation of endogenous antioxidant enzymes such as methionine sulfoxide reductase A, and the biosynthesis of glutathione to counteract oxidative stress. In addition, methionine restriction prevents altered methionine/transmethylation metabolism, thereby decreasing DNA damage and carcinogenic processes and possibly preventing arterial, neuropsychiatric, and neurodegenerative diseases. This review focuses on the role of methionine in metabolism, oxidative stress, and related diseases.
The tetravalent formulation of domain III-capsid proteins recalls memory B- and T-cell responses induced in monkeys by an experimental dengue virus infection.
Gil L, Lazo, Valdés I, Suzarte E, Yen P, Remírez R, et al. Clin Trans Immunology. 2017 Jun 23;6(6):e148.
Tetra DIIIC is a vaccine candidate against dengue virus (DENV) composed by four chimeric proteins that fuse the domain III of the envelope protein of each virus to the corresponding capsid protein. Containing B- and T-cell epitopes, these proteins form aggregates after the incubation with an immunostimulatory oligodeoxynucleotide, and their tetravalent formulation induces neutralizing antibodies and cellular immune response in mice and monkeys. Also, Tetra DIIIC protects mice after challenge with each DENV, and the monovalent formulation obtained from DENV-2 protects monkeys upon homologous viral challenge. However, in the last years, new evidences have arisen regarding domain III of DENV envelope protein as irrelevant target for neutralizing antibodies in humans. Nevertheless, vaccination with domain III induces a neutralizing antibody response that confers protection against re-infection. In addition, it has been demonstrated that the induction of a cellular immune response is essential to protect during the infection. This response can also avoid severe manifestations of dengue disease, associated to the antibody-dependent enhancement of the infection. In this study, we observed that Tetra DIIIC was able to boost the antiviral and neutralizing antibody responses previously generated in monkeys during an experimental DENV infection, demonstrating that domain III is targeted by B cells during the viral infection. Additionally, Tetra DIIIC successfully boosted the cellular immune response generated by the viruses, probably against T-cells epitopes in the capsid proteins. These results highlight the functionality of Tetra DIIIC as a vaccine candidate against DENV.
Why Did Zika Not Explode in Cuba? The Role of Active Community Participation to Sustain Control of Vector-Borne Diseases.
Castro M, Pérez D, Guzmán MG, Barrington C. Am J Trop Med Hyg. 2017 Aug;97(2):311–2.
As the global public health community develops strategies for sustainable Zika prevention and control, assessment of the Cuban response to Zika provides critical lessons learned. Cuba’s early and successful response to Zika, grounded in the country’s long-standing dengue prevention and control program, serves as a model of rapid mobilization of intersectoral efforts. Sustaining this response requires applying the evidence generated within the Cuban dengue program that active community participation improves outcomes and is sustainable and cost-effective. There is also a need for implementation science efforts to assess the transferability of lessons learned from Zika prevention and control to other pathogens and from one context to another in addition to how to take these efforts to scale.
