A multicomponent macrocyclization strategy to natural product-like cyclic lipopeptides: synthesis and anticancer evaluation of surfactin and mycosubtilin analogues. Morejón MC, Laub A, Kaluđerović GN, Puentes AR, Hmedat AN, Otero-González AJ. Org Biomol Chem. 2017 Apr 13. DOI: 10.1039/c7ob00459a. [Epub ahead of print]

A multicomponent macrocyclization strategy towards cyclic lipopeptides is described. The approach relies on the utilization of the Ugi and Passerini multicomponent reactions for the cyclization of peptides and oxo-peptides, and here it is employed for the construction of a small library of analogues of the natural products mycosubtilin and surfactin A. A key feature of this method is the simultaneous incorporation of either one or two exocyclic lipid tails along with the macrocyclic ring closure, which is only possible due to the multicomponent nature of the macrocyclization step. The evaluation of the anticancer activity of the lipopeptide library showed that the installation of a second lipid moiety in the surfactin scaffold leads to a more potent cytotoxicity in cancer cells. This is a new example of the multicomponent reaction potential in rapidly producing natural product analogues for biological screening.

An improved method for purification and refolding of recombinant HIV Vif expressed in E. coli. Duarte CA, Palomino M. Biotechnol Appl Biochem. 2017 Feb 8. DOI: 10.1002/bab.1557. [Epub ahead of print]

Virion infectivity factor (Vif) is a 23 KDa protein which protects HIV-1 from deamination of its proviral DNA by APOBEC3G. The active form of Vif is a multimer that interacts simultaneously with CBF-beta, the Elongin B and C subunits, Cullin 5 and APOBEC3G to form a ubiquitin ligase complex targeting the latter for degradation. Vif clearly represents an attractive target for developing novel antiviral drugs for the therapy of HIV/AIDS, and this goal requires a source of well folded, readily available protein. For that purpose, we have cloned Vif in the pET28a expression vector, expressing the resulting His-tagged recombinant protein in the BL21 (DE3) E. coli strain. After lysis, Vif was solubilized from the insoluble fraction with 6 M guanidinium chloride and purified by denaturing immobilized-metal affinity chromatography, refolding the protein afterwards by dialysis. The use of MES buffer at pH 6.2 and the presence of EDTA improved Vif refolding yields by reducing the formation of insoluble aggregates. The purified protein was bound by two monoclonal antibodies against sequential and conformational epitopes located at the C and N terminus, respectively.

A simple, fast and inexpensive method for mutation scanning of CFTR. Figueredo Lago JE, Armas Cayarga A, González González YJ, Collazo Mesa T. BMC Med Genet. 2017 May 25;18(1):58. DOI: 10.1186/s12881-017-0420-9.

Background Mutation scanning methods in Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene may not distinguish between a Cystic Fibrosis (CF) causing mutation and a benign variant. We have developed a simple and fast method for scanning 14 selected CF-causing mutations which have high frequency in Latin America. Methods In a group of 35 samples coming from CF patients previously characterized and using two allele-specific real-time multiplex PCRs targeting wild-type and mutant alleles respectively, we detect the presence of mutations by analyzing the Ct variation. Twenty-five samples without mutations considered non-carrier samples, were also included in this study. High Resolution Melting Analysis (HRMA) was performed to confirm the result of the scanning method and in most cases allowed the genotype determination. Results The results validate this method for CF diagnosis. A least one CFTR gene mutation was detected in the samples of CF patients, as predicted by their ΔCt values. The ΔCt value also indicated the zygosity of the sample according to the distribution of CFTR gene mutations. In most cases, HRMA allowed the identification of the mutation(s), thereby confirming the efficiency of this scanning strategy. Conclusions This strategy simplifies the detection of CF, reducing the analysis of 14 CF-causing mutations to two parallel reactions and making the procedure compatible with the analysis of a large number of samples. As the method is fast, inexpensive and highly reliable, it is advisable for scanning CFTR gene mutations in newborns, patients with a clinical suspicion of CF as well as in the preconception carrier screening.

A Simple Method to Predict Blood-Brain Barrier Permeability of Drug-Like Compounds Using Classification Trees. Castillo-Garit JA, Casañola-Martin GM, Le-Thi-Thu H, Pham-The H, Barigye SJ. Med Chem. 2017 Feb 9. DOI: 10.2174/1573406413666170209124302. [Epub ahead of print]

Background To know the ability of a compound to penetrate the blood-brain barrier (BBB) is a challenging task; despite the numerous efforts realized to predict/measure BBB passage, they still have several drawbacks. Methods The prediction of the permeability through the BBB is carried out using classification trees. A large data set of 497 compounds (recently published) is selected to develop the tree model. Results The best model shows an accuracy higher than 87.6% for training set; the model was also validated using 10-fold cross-validation procedure and through a test set achieving accuracy values of 86.1% and 87.9%, correspondingly. We give a brief explanation, in structural terms, of how our model describes the passage of molecules through the BBB. Additionally, the obtained model is compared with other approaches previously published in the international literature showing better results. Conclusions Finally, we can say that, the present model would be a valuable tool in the early stages of drug discovery process of neuropharmaceuticals.

Assessment of IL-28: rs12979860 and rs8099917 Polymorphisms in a Cohort of Cuban Chronic HCV Genotype 1b Patients. Palenzuela Gardón D, Guillén IA, Fernández JR, Camacho H, Estévez ZC, Dueñas S, et al. J Biomol Tech. 2016 Dec 16. pii: jbt. 17-2801-001. DOI: 10.7171/jbt.17-2801-001. [Epub ahead of print]

Hepatitis C virus (HCV) is a significant global public health problem with >185 million infections worldwide. A series of genome-wide association studies (GWAS) has identified IL-28B polymorphisms as a predictor of sustained virologic response (SVR), as well as spontaneous clearance in chronic HCV genotype 1 patients. The objective of this work was to evaluate the prevalence of IL-28B rs12979860 and rs8099917 polymorphisms in Cuban chronic HCV patients. The study cohort included 73 chronic HCV patients treated with concomitant administration of CIGB-230 and nonpegylated IFN-α plus ribavirin (non-pegIFN-α/R) antiviral therapy. The genotype distribution of IL-28B rs12979860CC, -CT, and -TT was 29, 41, and 30%, respectively, and the distribution for rs8099917TT, -TG, and -GG was 63, 31, and 5%, respectively. The allele frequencies for rs12979860C and -T alleles were 51 and 49%, respectively, and for rs8099917G and -T alleles, the values were 21 and 79%, respectively. SVR rates were 55, 42, and 35% for rs12979860CC, -CT, and -TT, respectively, and 52, 30, and 25% for rs8099917TT, -GT, and -GG, respectively. The combined assessment of both single nucleotide polymorphisms (SNPs) resulted in 3 major genotypes (rs12979860CC/rs8099917TT, rs12979860CT/rs8099917TT, and rs12979860CT/rs8099917GG) with a frequency of 30.1, 21.9, and 20.5%, respectively. In patients with heterozygous variant rs12979860CT, the additional genotyping of rs8099917 contributed to increase the SVR rate. It is concluded that in Cuban HCV-infected patients, the responder homogeneous variant rs8099917TT is the most frequent genotype. The simultaneous genotyping of 2 IL-28B SNPs could improve the prediction of SVR contributing to better therapeutic decisions and treatment management.

Boosting Immune Responses Following Fractional-Dose Inactivated Poliovirus Vaccine: A Randomized, Controlled Trial. Resik S, Tejeda A, Díaz M, Okayasu H, Sein C, Molodecky NA, et al. J Infect Dis. 2017 Jan 9. pii: jiw492. DOI: 10.1093/infdis/jiw492. [Epub ahead of print]

Background Fractional-dose administration of inactivated poliovirus vaccine (fIPV) could increase IPV affordability and stretch limited supplies. We assessed immune responses following fIPV administered intradermally, compared with full-dose IPV administered intramuscularly, among adults with a history of oral poliovirus vaccine (OPV) receipt. Methods We conducted a randomized, controlled noninferiority trial in Cuba. fIPV or IPV were administered on days 0 and 28; serum was collected on days 0, 7, 28, and 56 for analysis by a neutralization assay. The primary end point was seroconversion or a ≥4-fold rise in antibody titer. The noninferiority limit was 10%. The secondary end point was safety, assessed by the number and intensity of adverse reactions. Results A total of 503 of 534 enrolled participants (94.2%) completed all study requirements. Twenty-eight days after the first dose, 94.8%, 98.0%, and 98.0% of fIPV recipients had an immune response to poliovirus types 1, 2, and 3, respectively, compared with 98.1% (P = .06), 98.0% (P = 1.00), and 99.2% (P = .45) in the IPV arm. Noninferiority was achieved on days 7, 28, and 56 for all serotypes. No serious adverse events were reported. Conclusion fIPV induced similar boosting immune responses, compared with full-dose IPV. This suggests that fIPV would be an effective strategy to boost population immunity in an outbreak situation.

Clinical Perspective on Antihypertensive Drug Treatment in Adults With Grade 1 Hypertension and Low-to-Moderate Cardiovascular Risk: An International Expert Consultation. Morales Salinas A, Coca A, Olsen MH, Sánchez RA, Sebba-Barroso WK, Kones R, et al. Curr Probl Cardiol. 2017 Jul;42(7):198–225.

Hypertension is a leading risk factor for disease burden globally. An unresolved question is whether grade 1 hypertension (140-159/90-99mmHg) with low (cardiovascular mortality <1% at 10 years) to moderate (cardiovascular mortality ≥1% and <5% at 10 years) absolute total cardiovascular risk (CVR) should be treated with antihypertensive agents. A virtual international consultation process was undertaken to summarize the opinions of select experts. After holistic analysis of all epidemiological, clinical, psychosocial, and public health elements, this consultation process reached the following consensus in hypertensive adults aged <80 years: (1) The question of whether drug treatment in grade 1 should be preceded by a period of some weeks or months during which only lifestyle measures are recommended cannot be evidence based, but the consensus opinion is to have a period of lifestyle alone reserved only to patients with grade 1 “isolated” hypertension (grade 1 uncomplicated hypertension with low absolute total CVR, and without other major CVR factors and risk modifiers). (2) The initiation of antihypertensive drug therapy in grade 1 hypertension with moderate absolute total CVR should not be delayed. (3) Men ≥55 years and women ≥60 years with uncomplicated grade 1 hypertension should automatically be classified within the moderate absolute total CVR category, even in the absence of other major CVR factors and risk modifiers. (4) Statins should be considered along with blood-pressure lowering therapy, irrespective of cholesterol levels, in patients with grade 1 hypertensive with moderate CVR.

Data Mining Techniques Applied to Hydrogen Lactose Breath Test. Rubio-Escudero C, Valverde-Fernández J, Nepomuceno Chamorro I, Pontes-Balanza B, Hernández-Mendoza Y, Rodríguez-Herrera A. PLoS One. 2017 Jan 26;12(1): e0170385. DOI: 10.1371/journal.pone.0170385.

Objectives Analyze a set of data of hydrogen breath tests by use of data mining tools. Identify new patterns of H2 production. Methods Hydrogen breath tests data sets as well as k-means clustering as the data mining technique to a dataset of 2571 patients. Results Six different patterns have been extracted upon analysis of the hydrogen breath test data. We have also shown the relevance of each of the samples taken throughout the test. Conclusions Analysis of the hydrogen breath test data sets using data mining techniques has identified new patterns of hydrogen generation upon lactose absorption. We can see the potential of application of data mining techniques to clinical data sets. These results offer promising data for future research on the relations between gut microbiota produced hydrogen and its link to clinical symptoms.

Digital devide, biometeorological data infrastructures and human vulnerability definition. Fdez-Arroyabe P, Lecha Estela L, Schimt F. Int J Biometeorol. 2017 Jun 28. DOI: 10.1007/s00484-017-1398-x. [Epub ahead of print]

The design and implementation of any climate-related health service, nowadays, imply avoiding the digital divide as it means having access and being able to use complex technological devices, massive meteorological data, user’s geographic location and biophysical information. This article presents the co-creation, in detail, of a biometeorological data infrastructure, which is a complex platform formed by multiple components: a mainframe, a biometeorological model called Pronbiomet, a relational database management system, data procedures, communication protocols, different software packages, users, datasets and a mobile application. The system produces four daily world maps of the partial density of the atmospheric oxygen and collects user feedback on their health condition. The infrastructure is shown to be a useful tool to delineate individual vulnerability to meteorological changes as one key factor in the definition of any biometeorological risk. This technological approach to study weather-related health impacts is the initial seed for the definition of biometeorological profiles of persons, and for the future development of customized climate services for users in the near future.

Evaluation in Mice of the Immunogenicity of a Tetravalent Subunit Vaccine Candidate Against Dengue Virus Using Mucosal and Parenteral Immunization Routes. Lazo Vázquez L, Gil González L, Marcos López E, Pérez Fuentes Y, Cervetto de Armas L, Brown Richards E, et al. Viral Immunol. 2017 Apr 18. DOI: 10.1089/vim.2016.0150. [Epub ahead of print]

Our group has developed a subunit vaccine candidate against Dengue virus (DENV) based on two different viral regions, the domain III of the envelope protein and the capsid protein. The chimeric proteins for each serotype (DIIIC1-4), aggregated with the oligodeoxynucleotide 39 M, form the tetravalent formulation named Tetra DIIIC. Tetra DIIIC induces a protective immune response in mice when it is inoculated by intraperitoneal route. However, if children are the main targets for a DENV vaccine, then a needle-free route of administration should be attractive and advantageous. In this study, we evaluated for the first time, in vivo, a vaccine candidate against DENV based on recombinant proteins using the intranasal route. After three doses of Tetra DIIIC in mice, we measured the humoral immune response against the four DENV serotypes and the corresponding recombinant proteins. Moreover, the functionality of these antibodies was evaluated through a plaque reduction neutralization test. Finally, to assess the cellular immune response induced, we measured the IFN-γ-levels secreted by spleen cells after in vitro stimulation with DENV. The results presented in this study indicate that the intranasal immunization with Tetra DIIIC favors the generation of DENV-specific cell-mediated immunity. On the other hand, the immunization using intraperitoneal and intranasal routes, simultaneously, generate functional antibodies (anti-DIIIC and anti-DENV) and an in vitro response of IFN-γ secretion.

From individual to herd protection with pneumococcal vaccines: the contribution of the Cuban pneumococcal conjugate vaccine implementation strategy. Linares-Pérez N, Toledo-Romaní ME, Santana-Mederos D, García-Fariñas A, García-Rivera D, Valdéz Balbín Y, et al. Int J Infect Dis. 2017 Jul;60:98–102.

A new pneumococcal conjugate vaccine is currently undergoing advanced clinical evaluation prior to its planned introduction in Cuba. The implementation of the pneumococcal vaccination strategy has been designed with consideration of the need to maximize both its direct and indirect effects. A novel approach is suggested, which addresses preschool children as the first-line target group to generate herd immunity in infants and to have an impact on transmission at the community level. The clinical evaluation pipeline is described herein, including evaluations of effectiveness, cost-effectiveness, and impact. The scientific contribution of the Cuban strategy could support a paradigm shift from individual protection to a population effect based on a rigorous body of scientific evidence.

Genetic analysis of age at onset variation in spinocerebellar ataxia type 2. Figueroa KP, Coon H, Santos N, Velázquez L, Mederos LA, Pulst SM. Neurol Genet. 2017 May 15;3(3):e155. DOI: 10.1212/NXG.0000000000000155.

Objective To examine heritability of the residual variability of spinocerebellar ataxia type 2 (SCA2) age at onset (AO) after controlling for CAG repeat length. Methods From 1955 to 2001, dates of birth, CAG repeat lengths, AO, sex, familial inheritances, and clinical manifestations were collected for a large Cuban SCA2 cohort of 382 affected individuals, including 129 parent-child pairs and 69 sibships. Analyses were performed with log-transformed AO in the GENMOD procedure to predict AO using repeat length, taking into account family structure. Because all relationships were first degree, the model was implemented with an exchangeable correlation matrix. Familial correlations were estimated using the Pedigree Analysis Package to control for similarity due to genetic relatedness. Results For the entire sample, the mutant CAG repeat allele explained 69% of AO variance. When adjusted for pedigree structure, this decreased to 50%. Evidence for imprinting or sex-specific effects of the CAG repeat on AO was not found. For the entire sample, we determined an upper bound for heritability of the residual variance of 33% (p = 0.008). Heritability was higher in sib-sib pairs, especially in female sib-sib pairs, than in parent-child pairs. Conclusions We established that a large proportion of AO variance in SCA2 was determined by genetic modifiers in addition to CAG repeat length. The genetic structure of heritability of the residual AO variance was surprisingly similar to Huntington disease, suggesting the presence of recessive modifying alleles and possibly X-chromosome-linked modifiers.

Hemodynamic Impact of the C-Pulse Cardiac Support Device: A One-Dimensional Arterial Model Study. Campos Arias D, Londono F, Rodríguez Moliner T, Georgakopoulos D, Stergiopulos N, Segers P. Artif Organs. 2017 May 26. DOI: 10.1111/aor.12922. [Epub ahead of print]

The C-Pulse is a novel extra-aortic counter-pulsation device to unload the heart in patients with heart failure. Its impact on overall hemodynamics, however, is not fully understood. In this study, the function of the C-Pulse heart assist system is implemented in a one-dimensional (1-D) model of the arterial tree, and central and peripheral pressure and flow waveforms with the C-Pulse turned on and off were simulated. The results were studied using wave intensity analysis and compared with in vivo data measured non-invasively in three patients with heart failure and with invasive data measured in a large animal (pig). In all cases the activation of the C-Pulse was discernible by the presence of a diastolic augmentation in the pressure and flow waveforms. Activation of the device initiates a forward traveling compression wave, whereas a forward traveling expansion wave is associated to the device relaxation, with waves exerting an action in the coronary and the carotid vascular beds. We also found that the stiffness of the arterial tree is an important determinant of action of the device. In settings with reduced arterial compliance, the same level of aortic compression demands higher values of external pressure, leading to stronger hemodynamic effects and enhanced perfusion. We conclude that the 1-D model may be used as an efficient tool for predicting the hemodynamic impact of the C-Pulse system in the entire arterial tree, complementing in vivo observations.

Hepatitis B virus infection assessed 3 to 18 years after vaccination in Cuban children and adolescents born to HBsAg-positive mothers. Rodríguez Lay LLA, Bello Corredor M, Montalvo Villalba MC, Chibás Ojeda AG, Sariego Frómeta S, Díaz González M, et al. Arch Virol. 2017 Apr 24. DOI : 10.1007/s00705-017-3365-6. [Epub ahead of print]

Thirty-two participants, aged between 3-18 years, born to hepatitis B surface antigen (HBsAg)-positive mothers and vaccinated at birth were analyzed for hepatitis B virus (HBV) infection. Overall, 56% had anti-HB titers ≥10 IU/L; five were positive for antibodies to the core antigen (anti-HBc), and two of these were also positive for HBsAg/DNA. One of the HBsAg/anti-HBc double-negative children presented with an unusual occult infection (HBV DNA-positive). No known vaccine escape mutations were detectable. Our data suggest that the vaccine protected 93.8% of children in this high-risk group against chronic HBV infection. Occult infections should be considered even in countries with low endemicity and high vaccination coverage.

Human papillomavirus infection in anal intraepithelial lesions from HIV infected Cuban men. Limia CM, Soto Y, García Y, Blanco O, Kourí V, López MV. Infect Agent Cancer. 2017 Jan;12:5 . DOI: 10.1186/s13027-017-0118-9.

Background An association between HPV infection and progression to anal squamous intraepithelial lesions (ASIL) has been established, specifically in high-risk populations such as HIV-infected men. In this population, anal cancer is one of the most common non-AIDS-defining malignancies. Methods A cross-sectional study to detect anal lesions and HPV infection was performed. Anal mucosa samples were collected from 56 HIV-infected men from Cuba. The cytological diagnosis was done according to Bethesda 2001 System. HPV DNA detection was determined by qPCR for six high-risk HPV types and end point PCR for low-risk HPV types (6 and 11). The end point PCR with nucleotide sequencing technique was achieved to detect other genotypes of HPV not included in the qPCR in those samples negative for HPV- 6 and 11 or negative for the six genotypes identified in the qPCR. Results Cytological diagnosis identified 53 of 56 (95%) men with abnormal anal cytology. Among those, 26% (14/53) had atypical squamous cells of undetermined significance (ASC-US), 4% (2/53) had atypical squamous cells of undetermined significance cannot exclude high-grade lesions (ASC-H), 64% (34/53) had low-grade squamous intraepithelial lesions (LSIL), and 6% (3/53) had high-grade squamous intraepithelial lesions (HSIL). HPV DNA was detected in 89% (50/56) of men and 79% had at least one of the high-risk HPV types. HPV- 16 was the most common genotype (52%), while HPV-18 was the most frequently detected genotype in men with HSIL. We found statistically significant differences in the HPV viral loads with respect to the cytology results (p = 0.0006) and that the practice of receptive anal sex was a risk factor for anal HPV infection (p = 0.032). Conclusion This study shows a high prevalence of ASIL and high-risk HPV infections in the study group and is the first study showing the distribution of HPV genotypes in HIV infected Cuban men with abnormal anal cytology. This information may be of importance for local decision makers to improve prevention strategies, including the introduction of HPV vaccine in Cuba.

Identification of Inhibitors of CD36-Amyloid Beta Binding as Potential Agents for Alzheimer’s Disease. Doens D, Valiente PA, Mfuh AM, X T Vo A, Tristan A, Carreño L, et al. ASC Chem Neurosci. 2017 Jun 21;8(6):1232–41. DOI: 10.1021/acschemneuro.6b00386. Epub 2017 Feb 15.

Neuroinflammation is one of the hallmarks of Alzheimer’s disease pathology. Amyloid β has a central role in microglia activation and the subsequent secretion of inflammatory mediators that are associated with neuronal toxicity. The recognition of amyloid β by microglia depends on the expression of several receptors implicated in the clearance of amyloid and in cell activation. CD36 receptor expressed on microglia interacts with fibrils of amyloid inducing the release of proinflammatory cytokines and amyloid internalization. The interruption of the interaction CD36-amyloid β compromises the activation of microglia cells. We have developed and validated a new colorimetric assay to identify potential inhibitors of the binding of amyloid β to CD36. We have found seven molecules, structural analogues of the Trichodermamide family of natural products that interfere with the interaction CD36-amyloid β. By combining molecular docking and dynamics simulations, we suggested the second fatty acids binding site within the large luminal hydrophobic tunnel, present in the extracellular domain of CD36, as the binding pocket of these compounds. Free energy calculations predicted the nonpolar component as the driving force for the binding of these inhibitors. These molecules also inhibited the production of TNF-α, IL-6, and IL-1β by peritoneal macrophages stimulated with fibrils of amyloid β. This work serves as a platform for the identification of new potential anti-inflammatory agents for the treatment of Alzheimer’s disease.

Identification of Tight-Binding Plasmepsin II and Falcipain 2 Inhibitors in Aqueous Extracts of Marine Invertebrates by the Combination of Enzymatic and Interaction-Based Assays. Salas-Sarduy E, Guerra Y, Covaleda Cortés G, Avilés FX, Chávez Planes MA. Mar Drugs. 2017 Apr 21;15(4). pii: E123. DOI: 10.3390/md15040123.

Natural products from marine origin constitute a very promising and underexplored source of interesting compounds for modern biotechnological and pharmaceutical industries. However, their evaluation is quite challenging and requires specifically designed assays to reliably identify the compounds of interest in a highly heterogeneous and interfering context. In the present study, we describe a general strategy for the confident identification of tight-binding protease inhibitors in the aqueous extracts of 62 Cuban marine invertebrates, using Plasmodium falciparum hemoglobinases Plasmepsin II and Falcipain 2 as model enzymes. To this end, we first developed a screening strategy that combined enzymatic with interaction-based assays and then validated screening conditions using five reference extracts. Interferences were evaluated and minimized. The results from the massive screening of such extracts, the validation of several hits by a variety of interaction-based assays and the purification and functional characterization of PhPI, a multifunctional and reversible tight-binding inhibitor for Plasmepsin II and Falcipain 2 from the gorgonian Plexaura homomalla, are presented.

LALF32-51-E7 therapeutic vaccine induces antitumor immunity against human papillomavirus type 16 E7-expressing murine tumor metastases in the lungs. Granadillo M, Batte A, Blanco A, Alfonso AB, Suárez J, Merino N, et al. Clin Exp Metastasis. 2017 Apr;34(3–4):241–9.

One important goal of cancer immunotherapy is to prevent and treat tumor metastasis. We have previously reported the significant antitumor effect induced by the immunization with our human papillomavirus therapeutic protein-based vaccine (LALF32-51-E7) without adjuvant and admixed with clinically relevant adjuvants in the subcutaneous TC-1 tumor challenge model. In the present study, we evaluated the efficacy of the above mentioned vaccine formulations in controlling the hematogenous spread of TC-1 tumor cells using a more tumourigenic clone named TC-1* and other intravenous injection site less stressful than the tail vein. We generated a lung metastasis model by injecting TC-1* cells into the retro-orbital venous sinus and this is the first study describing it. Also, this is the first study that demonstrates the efficacy of the immunization with LALF32-51-E7 without adjuvant and admixed with VSSP or Al(OH)3 in controlling metastatic tumors increasing the survival of the mice. Our TC-1 lung metastasis model can be used to test the efficacy of other immunotherapeutic strategies based on E6/E7 antigens.

Outer membrane vesicles extracted from Neisseria meningitidis serogroup X for prevention of meningococcal disease in Africa. Acevedo R, Zayas C, Norheim G, Fernández S, Cedré B, Aranguren Y, et al. Pharmacol Res. 2017 Jul;121:194–201.

Meningococcal disease is caused mainly by serogroups A, B, C, Y, W of N. meningitidis. However, numerous cases of meningitis caused by serogroup X N. meningitidis (MenX) have recently been reported in several African countries. Currently, there are no licensed vaccines against this pathogen and most of the MenX cases have been caused by meningococci from clonal complex (c.c) 181. Detergent extracted meningococcal outer membrane vesicle (dOMV) vaccines have previously shown to be safe and effective against epidemics of serogroup B meningococcal disease in all age groups. The aim of this work is therefore to obtain, characterize and evaluate the vaccine potential of dOMVs derived from a MenX strain (OMVx). Three experimental lots of OMVx were prepared by deoxycholate extraction from the MenX strain BF 2/97. Size and morphology of the vesicles was determined by Dynamic Light Scattering and electron microscopy, whereas the antigenic composition was characterized by gel electrophoresis and immunoblotting. OMVx were thereafter adsorbed to aluminium hydroxide (OMVx/AL) and two doses of OMVx were administered s.c. to groups of Balb/c mice three weeks apart. The immunogenicity and functional antibody activities in sera were evaluated by ELISA (anti-OMVx specific IgG responses) and serum bactericidal activity (SBA) assay. The size range of OMVx was shown to be between 90 and 120nm, whereas some of the antigens detected were the outer membrane proteins PorA, OpcA and RmpM. The OMVx/AL elicited high anti-OMVx antibody responses with bactericidal activity and no bactericidal activity was observed in the control group of no immunised mice. The results demonstrate that OMVx are immunogenic and could form part of a future vaccine to prevent the majority of meningococcal disease in the African meningitis belt.

Permutation entropy analysis of heart rate variability for the assessment of cardiovascular autonomic neuropathy in type 1 diabetes mellitus. Carricarte Naranjo C, Sánchez-Rodríguez LM, Brown Martínez M, Estévez Báez M, Machado García A. Comput Biol Med. 2017 Jul 1;86:90–7.

Heart rate variability (HRV) analysis is a relevant tool for the diagnosis of cardiovascular autonomic neuropathy (CAN). To our knowledge, no previous investigation on CAN has assessed the complexity of HRV from an ordinal perspective. Therefore, the aim of this work is to explore the potential of permutation entropy (PE) analysis of HRV complexity for the assessment of CAN. For this purpose, we performed a short-term PE analysis of HRV in healthy subjects and type 1 diabetes mellitus patients, including patients with CAN. Standard HRV indicators were also calculated in the control group. A discriminant analysis was used to select the variables combination with best discriminative power between control and CAN patients groups, as well as for classifying cases. We found that for some specific temporal scales, PE indicators were significantly lower in CAN patients than those calculated for controls. In such cases, there were ordinal patterns with high probabilities of occurrence, while others were hardly found. We posit this behavior occurs due to a decrease of HRV complexity in the diseased system. Discriminant functions based on PE measures or probabilities of occurrence of ordinal patterns provided an average of 75% and 96% classification accuracy. Correlations of PE and HRV measures showed to depend only on temporal scale, regardless of pattern length. PE analysis at some specific temporal scales, seem to provide additional information to that obtained with traditional HRV methods. We concluded that PE analysis of HRV is a promising method for the assessment of CAN.

Pharmacokinetic and pharmacodynamic characterization of a novel formulation containing co-formulated interferons alpha-2b and gamma in healthy male volunteers. García-García I, Hernández-González I, Díaz-Machado A, González-Delgado CA, Pérez-Rodríguez S, García-Vega Y, et al. BMC Pharmacol Toxicol. 2016 Dec 7;17(1):58.
DOI: 10.1186/s40360-016-0103-8

Background More potent antitumor activity is desired in Interferon (IFN)-treated cancer patients. This could be achieved by combining IFN alpha and IFN gamma. The aim of this work was to characterize the pharmacokinetics and pharmacodynamics of a novel formulation containing a co-formulated combination of IFNs alpha-2b and gamma (CIGB-128-A). Methods A group of nine healthy male subjects received intramuscularly 24.5 × 106 IU of CIGB-128-A. IFN concentrations were evaluated for 48 h. Serum neopterin, beta2-microglobulin (β2M) and 2′–5′ oligoadenylate synthetase (2′–5′ OAS), classical IFN-inducible serum markers, were measured during 192 h by enzyme immunoassay and body temperature was used as pharmacodynamic variable as well. Results Concerning pharmacokinetics, serum IFNs’ profiles were better fitted to a mono-compartmental model with consecutive zero order and first order absorption, one bioavailability value. No interferences by simultaneous administered IFNs were observed in their typical similar systemic profiles. Neopterin and β2M time profiles showed a delay that was efficiently linked to pharmacokinetics by means of a zero order absorption rate constant. Neopterin level was nine-fold higher than initial values, 48 h post-administration, an increment not described before. At this time, mean serum β2M peaked around the double from baseline. Serum concentrations of the enzyme 2′–5′ OAS was still elevated on the 8 day post-injection. The formulation was well tolerated. Most frequent adverse reactions were fever, headache, arthralgia and lymphopenia, mostly mild. Conclusions The administration of co-formulated IFN alpha-2b and IFN gamma likely provides improved pharmacodynamic properties that may be beneficial to treat several malignancies.

Physical interactions between DNA and sepiolite nanofibers, and potential application for DNA transfer into mammalian cells. Castro-Smirnov FA, Piétrement O, Aranda P, Bertrand JR, Ayache J, Le Cam E. Sci Rep. 2016 Nov 3;6:6341. DOI: 10.1038/srep36341.

Nanofibers of sepiolite, a natural silicate belonging to the clay minerals family, might constitute a potential promising nanocarrier for the non-viral transfer of bio-molecules. We show here that sepiolite nanofibers efficiently bind different types of DNA molecules through electrostatic interactions, hydrogen bonding, cation bridges, and van der Waals forces. Moreover, Fourier-transform infrared spectroscopy identified the external silanol groups as the main sites of interaction with the DNA. Furthermore, as a proof of concept, we show that sepiolite is able to stably transfer plasmid DNA into mammalian cells and that the efficiency can be optimized. Indeed, sonication of sepiolite 100-fold stimulated DNA transfection efficiency. These results open the way to the use of sepiolite-based biohybrids as a novel class of nanoplatform for gene transfer with potential clinical applications.

Procedure to describe clavicular motion. Gutiérrez Delgado G, De Beule M, Ortega Cardentey DR, Segers P, Iznaga Benítez AM, Rodrígues Moliner T, et al. J Shoulder Elbow Surg. 2017 Mar;26(3):490–6.

Background For many years, researchers have attempted to describe shoulder motions by using different mathematical methods. The aim of this study was to describe a procedure to quantify clavicular motion. Methods The procedure proposed for the kinematic analysis consists of 4 main processes: 3 transcortical pins in the clavicle, motion capture, obtaining 3-dimensional bone models, and data processing. Results Clavicular motion by abduction (30° to 150°) and flexion (55° to 165°) were characterized by an increment of retraction of 27° to 33°, elevation of 25° to 28°, and posterior rotation of 14° to 15°, respectively. In circumduction, clavicular movement described an ellipse, which was reflected by retraction and elevation. Kinematic analysis shows that the articular surfaces move by simultaneously rolling and sliding on the convex surface of the sternum for the 3 movements of abduction, flexion, and circumduction. Conclusion The use of 3 body landmarks in the clavicle and the direct measurement of bone allowed description of the osteokinematic and arthrokinematic movement of the clavicle.

Prognostic indicators in pediatric clinically isolated syndrome. Iaffaldano P, Simone M, Lucisano G, Ghezzi A, Coniglio G, Brescia Morra V, et al. Ann Neurol. 2017 May;81(5):729–39.

Objective To assess prognostic factors for a second clinical attack and a first disability worsening event in pediatric clinically isolated syndrome (pCIS) suggestive of Multiple Sclerosis (MS) patients. Methods A cohort of 770 pCIS patients was followed-up for at least 10 years. Cox proportional hazard models and RECursive Partitioning and AMalgamation (RECPAM) tree-regression were used to analyze data. Results In pCIS, female sex and a multifocal onset were risk factors for a second clinical attack (HR, 95% CI: 1.28, 1.06-1.55; 1.42, 1.10-1.84, respectively), whereas disease modifying drugs (DMDs) exposure reduced this risk (HR, 95% CI: 0.75, 0.60-0.95). After pediatric onset MS (POMS) diagnosis, age at onset younger than 15 years and DMDs exposure decreased the risk of a first EDSS worsening event (HR, 95% CI: 0.59, 0.42-0.83; 0.75, 0.71-0.80, respectively), whereas the occurrence of relapse/s increased this risk (HR, 95% CI: 5.08, 3.46-7.46). An exploratory RECPAM analysis highlighted a significant higher incidence of a first EDSS worsening event in patients with multifocal or isolated spinal-cord or optic neuritis involvement at onset in comparison to those with an isolated supratentorial or brainstem syndrome. A Cox regression model including RECPAM classes confirmed DMDs exposure as the most protective factor against EDSS worsening events and relapses as the most important risk factor for attaining EDSS worsening. Interpretation This work represents an important step forward in identifying predictors of unfavorable course in pCIS and POMS and supports a protective effect of early DMDs treatment in preventing MS development and disability accumulation in this population.

Safety and Immunogenicity of a Human Epidermal Growth Factor Receptor 1 (HER1)-Based Vaccine in Prostate Castration-Resistant Carcinoma Patients: A Dose-Escalation Phase I Study Trial. Caballero I, Aira LE, Lavastida A, Popa X, Rivero J, González J, et al. Front Pharmacol. 2017 May 10;8:263. DOI: 10.3389/fphar.2017.00263.

Metastatic castration-resistant prostate cancer (CRPC) remains incurable due to the lack of effective therapies. Activation of the human epidermal growth factor receptor 1 (HER1) in prostate cancer contributes to metastatic progression as well as to disease relapse. Here, we determined the toxicity and immunogenicity of a HER1-based cancer vaccine in CRPC patients included in a phase I clinical trial. CRPC patients (n = 24) were intramuscularly vaccinated with HER1 vaccine consisting of the extracellular domain of HER1 molecule (ECD) and very small size proteoliposome from Neisseria meningitidis (VSSP) and Montanide ISA-51 VG as adjuvants. Patients were included in five groups according to the vaccine dose (100, 200, 400, 600, and 800 μg). The primary endpoints were safety and immunogenicity. The anti-HER1 antibodies were measured by an ELISA, the recognition of an HER1 positive tumor cell line and the inhibition of HER1 phosphorylation by sera were determined by flow cytometry and western blot analysis, respectively. The HER1-specific T cell response was assessed by determination of IFN-γ-producing T cells using ELISpot assay. The vaccine was well tolerated. No grade III or IV adverse events were reported. High titers of anti-HER1 antibodies were observed in most of the evaluated patients. There were no significant differences regarding the geometric means of the anti-HER1 titers among the dose groups except the group of 100 μg in which antibody titers were significantly lower. A Th1-type IgG subclasses pattern was predominant in most patients. Only patients receiving the higher doses of vaccine showed significant tumor cell recognition and HER1 phosphorylation inhibition by hyperimmune sera. Forty two percent of the patients showed a specific T cell response against HER1 peptides pool in post-treatment samples. There was a trend toward survival benefit in those patients showing high anti-HER1 specific antibody titers and a significant association between cellular immune response and clinical outcome.

Screening for immune response against Dengue virus in Vietnamese non-human primates: implications for vaccine developers. Lazo Vázquez L, Suzarte Portal E, Castro Velazco J, Yen P, Dung LT,  Gil González L, et al. Clin Transl Immunology. 2017 Mar 24;6(3):e135.
DOI: 10.1038/cti.2016.79.

One of the major problems faced for the development of a vaccine against Dengue virus is the lack of a suitable animal model. Although non-human primates do not show overt signs of disease, these animals develop viremia after the infection and are the best model to evaluate vaccine candidates against this pathogen. However, for that purpose, the screening of all animals is mandatory to discard those with previous natural immunity. The most common technique used in the screening is the plaque reduction neutralization test (PRNT). However, most recent studies points to the cell-mediated immunity (CMI) as an important player in the process of controlling Dengue virus (DENV) infections. Here we presented the results from the screening of 55 rhesus monkeys housed in an animal breeding facility at Quang Ninh province, Vietnam. We evaluated the neutralizing antibody response by PRNT and determined the levels of interferon γ (IFNγ)-secretion after the viral stimulation of monkey-peripheral blood mononuclear cells, by enzyme-linked immunosorbent assay (ELISA). We found no correspondence between PRNT and IFNγ-ELISA. In fact, 19 animals were positive only by IFNγ-ELISA. Moreover, to study the protective capacity of the CMI detected, three animals with positive response by IFNγ-ELISA and negative by PRNT were inoculated with an infective preparation of DENV-3 and, as a result, no viremia was detected during 10 days after the challenge. This fact points to the importance of screening non-human primates through a CMI assay together with PRNT. This procedure should discard those false-negative cases which would be protected after the viral challenge in the immunization schedule.

Self-assembled particulate PsaA as vaccine against Streptococcus pneumoniae infection. González-Mir M, Rodríguez-Noda L, Fariñas-Medina M, García-Rivera D, Vérez-Bencomo V, Rehm BHA. Heliyon. 2017 Apr 11;3(4):e00291. DOI: 10.1016/j.heliyon.2017.e00291.

Streptococcus pneumoniae is a human pathogen responsible for the majority of childhood pneumonia and media otitis cases worldwide. The diversity of its capsular polysaccharides (CPS) results in more than 91 serotypes of which at least 23 are virulent. Various CPS conjugated to immunogenic carrier proteins are currently licensed and provide protection against the infection caused by the respective serotypes but not against new and emerging virulent serotypes. In this study, we considered the conserved protein antigen PsaA, the pneumococcal surface adhesin A, in order to overcome the limitations of CPS antigens. The PsaA was translationally fused to a polyhydroxybutyrate (PHB) synthase which mediated production of PsaA displayed on PHB inclusions in recombinant Escherichia coli. This suggested that the PsaA fusion to the PHB synthase did not interfere with PHB synthase activity and its ability to mediate formation of nano-sized inclusions composed of a PHB core surrounded by the PHB synthase fused to PsaA. Isolated PHB beads showed a negative surface charge. Transmission electron microscopy analysis suggested that the PsaA fusion to the PHB synthase reduced the size of PHB beads from about 500 nm to 100 nm. The integrity and antigenicity of the fusion protein attached to isolated PHB beads was confirmed by SDS-PAGE, tryptic peptide fingerprinting analysis using MALDI-TOF-MS/MS and immunoblotting using a monoclonal anti-PsaA antibody. Mice immunized with PsaA displaying PHB beads produced high and specific IgG levels dominated by IgG1 isotype. While IgG1 titer were similar between soluble and insoluble PsaA, the IgG2 titers were strongly increased upon vaccination with insoluble PsaA i.e. PsaA displayed on PHB beads. Particulate PsaA-PHB beads elicited IgG antibodies recognizing PsaA in whole cell lysates of seven different serotypes of S. pneumoniae. This study suggested that PHB beads are suitable carriers for PsaA in order to induce a significant and specific Th-2-type immune response.

Specific humoral and cellular immune responses in cancer patients undergoing chronic immunization with a VEGF-based therapeutic vaccine. Moreara Y, Sánchez J, Bequet-Romero M, Selman-Housein KH, de la Torre A, Hernández-Bernal F, et al. Vaccine. 2017 Jun 16;35(28):3582–90.

CIGB-247 is a cancer therapeutic vaccine, based on recombinant modified human vascular endothelial growth factor (VEGF) as antigen, in combination with the adjuvant VSSP, a bacterially-derived adjuvant. The vaccine have demonstrated efficacy in several murine malignancy models. These studies supported the rationale for a phase I clinical trial where safety, tolerance, and immunogenicity of CIGB-247 was studied in patients with advanced solid tumors at three antigen dose level. Surviving individuals of this clinical trial were eligible to receive off-trial voluntary re-immunizations. The present work is focus in the immunological follow up of these patients after approximately three years of immunizations, without additional oncological treatments. Long term vaccination was feasible and safe. Our results indicated that after sustained vaccination most of the patients conserved their seroconversion status. The specific anti-VEGF IgG titer diminished, but in all the cases keeps values up from the pre-vaccination levels. Continued vaccination was also important to produce a gradual shift in the anti-VEGF IgG response from IgG1 to Ig4. Outstanding, our results indicated that long-term off-trial vaccination could be associated with the maintaining of one reserve of antibodies able to interfere with the VEGF/Receptor interaction and the production of IFNγ secretion in CD8+ cells. The results derived from the study of this series of patients suggest that long term therapeutic vaccination is a feasible strategy, and highlight the importance of continuing the clinical development program of this novel cancer therapeutic vaccine candidate. We also highlight the future clinical applications of CIGB-247 in cancer and explain knowledge gaps that future studies may address. Registration number and name of trial registry: RPCEC00000102. Cuban Public Clinical Trial Registry (WHO accepted Primary Registry). Available from: http://registroclinico.sld.cu/.

Subtle alterations in cerebrovascular reactivity in mild cognitive impairment detected by graph theoretical analysis and not by the standard approach. Sánchez-Catasús CA, Sanabria-DíazG, Willemsen A, Martínez-Montes E, Samper-Noa J, Águila-Ruiz A, et al. Neuroimage Clin. 2017 Apr 25;15:151–60.

There is growing support that cerebrovascular reactivity (CVR) in response to a vasodilatory challenge, also defined as the cerebrovascular reserve, is reduced in Alzheimer’s disease dementia. However, this is less clear in patients with mild cognitive impairment (MCI). The current standard analysis may not reflect subtle abnormalities in CVR. In this study, we aimed to investigate vasodilatory-induced changes in the topology of the cerebral blood flow correlation (CBFcorr) network to study possible network-related CVR abnormalities in MCI. For this purpose, four CBFcorr networks were constructed: two using CBF SPECT data at baseline and under the vasodilatory challenge of acetazolamide (ACZ), obtained from a group of 26 MCI patients; and two equivalent networks from a group of 26 matched cognitively normal controls. The mean strength of association (SA) and clustering coefficient (C) were used to evaluate ACZ-induced changes on the topology of CBFcorr networks. We found that cognitively normal adults and MCI patients show different patterns of C and SA changes. The observed differences included the medial prefrontal cortices and inferior parietal lobe, which represent areas involved in MCI’s cognitive dysfunction. In contrast, no substantial differences were detected by standard CVR analysis. These results suggest that graph theoretical analysis of ACZ-induced changes in the topology of the CBFcorr networks allows the identification of subtle network-related CVR alterations in MCI, which couldn’t be detected by the standard approach.

Wasp venomic: Unravelling the toxins arsenal of Polybia paulista venom and its potential pharmaceutical applications. Pérez-Riverol A, Dos Santos-Pinto JRA, Lasa AM, Palma MS, Brochetto-Braga MR. J Proteomics. 2017 May 24;161:88–103.
DOI: 10.1016/j.jprot.2017.04.016. Epub 2017 Apr 19.

Polybia paulista (Hymenoptera: Vespidae) is a neotropical social wasp from southeast Brazil. As most social Hymenoptera, venom from P. paulista comprises a complex mixture of bioactive toxins ranging from low molecular weight compounds to peptides and proteins. Several efforts have been made to elucidate the molecular composition of the P. paulista venom. Data derived from proteomic, peptidomic and allergomic analyses has enhanced our understanding of the whole envenoming process caused by the insect sting. The combined use of bioinformatics, -omics- and molecular biology tools have allowed the identification, characterization, in vitro synthesis and recombinant expression of several wasp venom toxins. Some of these P. paulista – derived bioactive compounds have been evaluated for the rational design of antivenoms and the improvement of allergy specific diagnosis and immunotherapy. Molecular characterization of crude venom extract has enabled the description and isolation of novel toxins with potential biotechnological applications. Here, we review the different approaches that have been used to unravel the venom composition of P. paulista. We also describe the main groups of P. paulista – venom toxins currently identified and analyze their potential in the development of component-resolved diagnosis of allergy, and in the rational design of antivenoms and novel bioactive drugs.