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Curtidor H.,Fundacion Institute Inmunologia Of Colombia | Curtidor H.,El Rosario University | Vanegas M.,Fundacion Institute Inmunologia Of Colombia | Vanegas M.,El Rosario University | And 4 more authors.
Current Medicinal Chemistry | Year: 2011

Our ongoing search for a fully-effective vaccine against the Plasmodium falciparum parasite (causing the most lethal form ofhuman malaria) has been focused on identifying and characterising proteins' amino acid sequences (high activity binding peptides orHABPs) involved in parasite invasion of red blood cells (RBC) by the merozoite and hepatocytes by the sporozoite. Many such merozoiteHABPs have been recognised and molecularly and structurally characterised; however, native HABPs are immunologically silentsince they do not induce any immune response or protection against P. falciparum malaria infection and they have to be structurallymodified to allow them to fit perfectly into immune system molecules.A deeply structural analysis of these conserved merozoite HABPs and their modified analogues has led to rules or principles becomingrecognised for constructing a logical and rational methodology for a minimal subunit-based, multi-epitope, multi-stage, chemicallysynthesisedvaccine. The same in-depth analysis of the most relevant sporozoite proteins involved in sporozoite cell-traversal and hepatocyteinvasion as well as the hepatic stage is shown here.Specifically modifying these HABPs has resulted in a new set of potential pre-erythrocyte targets which are able to induce high, longlastingantibody titres in Aotus monkeys, against their corresponding recombinant proteins and the complete parasite native molecules.This review shows how these rules may be applied against the first stage of parasite invasion (i.e. the sporozoite) to mount the first line ofdefence against the malarial parasite, which may indeed be the most effective one. Our results strongly support including some of thesemodified sporozoite HABPs in combination with the previously-described modified merozoite HABPs for obtaining the aforementionedfully-protective, multiepitope, multi-stage, minimal subunit-based, chemically-synthesized, antimalarial vaccine. © 2011 Bentham Science Publishers.

Bohorquez H.J.,Fundacion Institute Inmunologia Of Colombia | Reyes A.,National University of Colombia
Molecular Physics | Year: 2014

We investigate the Pauli energy in atoms and molecules as a measure of electron localisation. Our results indicate that the Pauli energy has an exponential dependence on the number of localised electrons. This relationship yields to a kinetic energy density expression that depends on the electron density ρ(r) and the pair density ρ2(r, r′). The proposed equation shows certain advantages over a similar orbital-free kinetic energy functional recently proposed by Delle Site and co-workers. The methodology introduced here is a novel approach for exploring electronic quantities with a partition scheme that might be useful for research in density functional theory. © 2013 Taylor & Francis.

Patarroyo M.A.,Fundacion Institute Inmunologia Of Colombia | Patarroyo M.A.,El Rosario University | Bermudez A.,Fundacion Institute Inmunologia Of Colombia | Bermudez A.,El Rosario University | And 5 more authors.
PLoS ONE | Year: 2010

T-cell receptor gene rearrangements were studied in Aotus monkeys developing high antibody titers and sterilizing immunity against the Plasmodium falciparum malaria parasite upon vaccination with the modified synthetic peptide 24112, which was identified in the Merozoite Surface Protein 2 (MSP-2) and is known to bind to HLA-DRβ1*0403 molecules with high capacity. Spectratyping analysis showed a preferential usage of Vβ12 and Vβ6 TCR gene families in 67% of HLADRβ1* 0403-like genotyped monkeys. Docking of peptide 24112 into the HLA-DRβ1*0401-HA peptide-HA1.7TCR complex containing the VDJ rearrangements identified in fully protected monkeys showed a different structural signature compared to nonprotected monkeys. These striking results show the exquisite specificity of the TCR/pMHCII complex formation needed for inducing sterilizing immunity and provide important hints for a logical and rational methodology to develop multiepitopic, minimal subunit-based synthetic vaccines against infectious diseases, among them malaria. © 2010 Patarroyo et al.

Patarroyo M.E.,Fundacion Institute Inmunologia Of Colombia | Patarroyo M.E.,National University of Colombia | Alba M.P.,Fundacion Institute Inmunologia Of Colombia | Alba M.P.,El Rosario University | And 2 more authors.
Peptides | Year: 2011

The sporozoite microneme proteins essential for cell traversal, SPECT-1 and SPECT-2, are considered attractive pre-erythrocytic immune targets due to the key role they play in crossing of the malaria parasite across the dermis and the liver sinusoidal wall, prior to invasion of hepatocytes. In this study, the sequences of SPECT-1 and SPECT-2 were mapped using 20 mer-long synthetic peptides to identify high-activity binding peptides (HABPs) to HeLa cells. 17 HABPs with enzyme sensitive bindings to HeLa cells were identified: 3 predominantly α-helical in SPECT-1, and 10 α-helical and 4 β-turns/random coils in SPECT-2. Immunofluorescence assays (IFA) with antibodies raised in rabbits against chemically synthesized B-cell epitopes suggests the presence of these two proteins in the micronemes and in sporozoite membrane. 1H NMR studies showed that HABPs located in the membrane-attack complex/perforin (MACPF) domain of SPECT-2 share high similarity with the 3D structure of C8α. Altogether, the results highlight the potential of including HABPs from SPECT-1 and SPECT-2 as components of a fully effective multistage, multiepitopic, minimal subunit-based synthetic vaccine against Plasmodium falciparum malaria. © 2010 Elsevier Inc. All rights reserved.

Diaz-Roa A.,El Rosario University | Gaona M.A.,El Rosario University | Segura N.A.,El Rosario University | Ramirez-Hernandez A.,National University of Colombia | And 5 more authors.
Acta Tropica | Year: 2016

Larval therapy is used as alternative treatment for hard-to-heal chronic and infected wounds. Lucilia sericata is the most used blowfly species. However, it has been shown recently that Sarconesiopsis magellanica larval excretions and secretions have potent antibacterial activity; this blowfly belongs to the Calliphoridae family.The present work has dealt with evaluating larval therapy using S. magellanica on wounds induced in diabetic rabbits and its action was compared to the effect induced by L. sericata. Twelve New Zealand White rabbits (. Oryctolagus cuniculus) were used; they were divided into 4 groups, the first two being treated with larval therapy derived from both aforementioned necrophagous blowflies, an antibiotic was used in the third and the fourth was used as control. All the animals were wounded on the back and infected with Pseudomonas aeruginosa and Staphylococcus aureus.Samples of the secretion from each animal's infected wound were taken and sown on blood agar. The colony forming units were then counted. The PUSH scale was used for the macroscopic evaluation of the wounds.Bacterial control was encountered 48. h post-treatment in the treatments involving larval therapy and to a lesser extent with the antibiotic. Likewise, wound debridement was quicker and more efficient with larval therapy compared to the antibiotic group; however, wound closing time was 23 days in all treatments. The group treated with S. magellanica larvae had relatively quicker evolution until the proliferation phase and the start of maturation, even though there were no significant differences between both blowfly species evaluated here regarding treatments by the end of the treatment period.The present study has validated the diabetic rabbit model for inducing chronic wounds regarding larval therapy and has likewise confirmed the effectiveness of S. magellanica-derived larval therapy as an alternative for curing and healing wounds. © 2015 Elsevier B.V.

Galvis-Jimenez J.M.,El Rosario University | Curtidor H.,El Rosario University | Curtidor H.,Fundacion Institute Inmunologia Of Colombia | Patarroyo M.A.,El Rosario University | And 3 more authors.
Cancer Biology and Therapy | Year: 2013

Among the different types of tests used for cancer diagnosis, molecular tests have been increrasingly incorporated because of their ability to detect either expression or functional changes in the molecules associated with the disease. Mammaglobin is a protein found in mammary tissue and can be detected in serum. This protein has been proposed as a biomarker to diagnose breast cancer, given that patients exhibit an increased amount of the protein in serum and tumor tissue, in comparison to healthy individuals. The ELISA test was used in the present study to detect mammaglobin in blood samples from 51 breast cancer patients and 51 control individuals. Antibodies against mamaglobin were generated in rabbits by using the following synthetic peptides: A (amino acids 13 to 21), B (amino acids 31 to 39), C (amino acids 56 to 64) and a D peptide, corresponding to the protein isoform without three amino acids (59, 60 and 61 amino acids) from peptide C. All peptides were immunogenic and allowed generation of antibodies that were able to discriminate patients from controls. The best results were obtained for antiserum B, achieving the best sensitivity (86.3%) and specificity (96%). © 2012 Landes Bioscience.

Pinilla Y.T.,El Rosario University | Patarroyo M.A.,El Rosario University | Patarroyo M.A.,Fundacion Institute Inmunologia Of Colombia | Bello F.J.,El Rosario University
Forensic Science International | Year: 2013

Sarconesiopsis magellanica is a forensically relevant necrophagous blowfly that can aid in determining the post-mortem interval (PMI) as it is the first to colonise decomposing corpses. The blowfly has been reported in several South-American countries including Colombia, in high-altitude regions ranging from 1200 to 3100m above sea level. The present study reports this blowfly's life cycle and an analysis of its reproductive and population parameters under laboratory conditions for the first time. Six successive generations of flies were produced with an average of 65.38% adults emerging with respect to the total number of puparia. The shortest life cycle from egg to adult emergence was found in individuals fed on a lyophilised liver (LL) diet, while the longest one was found in individuals fed with an egg-powdered milk (E-PM) diet; intermediate values were found when the pig liver (PL) diet was tested. The greatest adult longevity was achieved when the PL diet was used, the LL diet giving the shortest. The population parameters based on the horizontal life table were: net reproductive rate (Ro)=447.752±9.9, mean generational time (Tc)=18.18±0.38, natural population increase rate (rm)=0.145 and finite population increase rate (λ)=1.398. This blowfly colony represents a valuable asset for both basic and applied studies. Members of the S. magellanica colony so established were used for analysing the life-cycle, reproductive and population parameters, and further medical and forensic application studies are currently underway. © 2013 Elsevier Ireland Ltd.

Diaz-Roa A.,El Rosario University | Gaona M.A.,El Rosario University | Segura N.A.,El Rosario University | Suarez D.,Fundacion Institute Inmunologia Of Colombia | And 3 more authors.
Acta Tropica | Year: 2014

The most important mechanism for combating infection using larval therapy depends on larval excretions and secretions (ES). The present work was aimed at evaluating Sarconesiopsis magellanica (Diptera: Calliphoridae) ES antibacterial activity in six bacterial strains (three Gram-positive and three Gram-negative) and comparing this to the effect of Lucilia sericata-derived ES. Antibacterial activity at 50. μg/mL minimum inhibitory concentration (MIC) was observed for Staphylococcus epidermidis ATCC-12228 and Staphylococcus aureus ATCC-29213 strains, when the turbidimetry test involving S. magellanica ES was used; the rest of the bacterial strains (Staphylococcus aureus ATCC-6538, Pseudomonas aeruginosa ATCC-10145, Pseudomonas aeruginosa ATCC-9027 and Pseudomonas aeruginosa ATCC-27853) were inhibited at a 100. μg/mL MIC. Twice the amount was required to inhibit the aforementioned bacteria with L. sericata-derived ES using this same technique; a similar trend was observed when the agar diffusion method was used instead. Furthermore, when the previously established MIC for each bacterial strain was used, their colonies became reduced following 1-6. h incubation with S. magellanica derived ES, whilst the reduction occurred from 2 to 6. hours with those from L. sericata. Although the MIC for each strain obtained with ciprofloxacin was lower than those established when using either blowfly derived-ES, the gradual reduction of the colonies occurred at a longer incubation time (6. h or more). The results showed that S. magellanica ES antibacterial activity was more potent and effective, compared to that of L. sericata-derived ES. © 2014 Elsevier B.V.

Pinilla Y.T.,El Rosario University | Moreno-Perez D.A.,El Rosario University | Moreno-Perez D.A.,Fundacion Institute Inmunologia Of Colombia | Patarroyo M.A.,El Rosario University | And 2 more authors.
Acta Tropica | Year: 2013

Sarconesiopsis magellanica (Diptera: Calliphoridae) is a medically important necrophagous fly which is used for establishing the post-mortem interval. Diptera maggots release proteolytic enzymes contained in larval excretion and secretion (ES) products playing a key role in digestion. Special interest in proteolytic enzymes has also been aroused regarding understanding their role in wound healing since they degrade necrotic tissue during larval therapy. This study was thus aimed at identifying and characterising S. magellanica proteolytic enzyme ES products for the first time. These products were obtained from first-, second- and third-instar larvae taken from a previously-established colony. ES proteins were separated by SDS-PAGE and their proteolytic activity was characterised by zymograms and inhibition assays involving BAPNA (Nα-benzoyl- dl-Arg- p-nitroanilide) and SAPNA substrates, using synthetic inhibitors. The protein profile ranged from ~69. kDa to ~23. kDa; several of them coincided with the Lucilia sericata ES protein profile. Serine-protease hydrolysis activity (measured by zymogram) was confirmed when a ~25. kDa band disappeared upon ES incubation with PMSF inhibitor at pH 7.8. Analysis of larval ES proteolytic activity on BAPNA and SAPNA substrates (determined by using TLCK and TPCK specific inhibitors) suggested a greater amount of trypsin-like protease. These results support the need for further experiments aimed at validating S. magellanica use in larval therapy. © 2013 Elsevier B.V.

PubMed | Fundacion Institute Inmunologia Of Colombia
Type: Journal Article | Journal: AMB Express | Year: 2017

DNA cloning is an essential tool regarding DNA recombinant technology as it allows the replication of foreign DNA fragments within a cell. pELMO was here constructed as an in-house cloning vector for rapid and low-cost PCR product propagation; it is an optimally designed vector containing the ccdB killer gene from the pDONR 221 plasmid, cloned into the pUC18 vectors multiple cloning site (Thermo Scientific). The ccdB killer gene has a cleavage site (CCC/GGG) for the SmaI restriction enzyme which is used for vector linearisation and cloning blunt-ended products. pELMO transformation efficiency was evaluated with different sized inserts and its cloning efficiency was compared to that of the pGEM-T Easy commercial vector. The highest pELMO transformation efficiency was observed for ~500bp DNA fragments; pELMO vector had higher cloning efficiency for all insert sizes tested. In-house and commercial vector cloned insert reads after sequencing were similar thus highlighting that sequencing primers were designed and localised appropriately. pELMO is thus proposed as a practical alternative for in-house cloning of PCR products in molecular biology laboratories.

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