Expression of substance P, calcitonin gene-related peptide and vascular endothelial growth factor in human dental pulp under different clinical stimuli.

BACKGROUND: The aim of this study was to measure the dental pulp inflammatory response through neuropeptides (SP and CGRP) as a response to occlusal trauma, orthodontic movements and a combination of both, as well as the angiogenic defense mechanism through VEGF expression, which could be the initial step to mineralized tissue formation. METHODS: Forty human dental pulp samples were collected from healthy first premolars with extraction indicated due to orthodontic reasons from a sample of 20 patients. Patients were divided into four groups with 10 premolars each (1 mandibular and 1 maxillary premolar from each patient): healthy pulp control group, occlusal trauma group, moderate orthodontic forces group; and occlusal trauma plus moderate orthodontic forces group. Stimuli were applied for 24 h before tooth extraction in all experimental groups. All samples were processed, and SP, CGRP, and VEGF were measured by radioimmunoassay. The Kruskal-Wallis test was performed to assess significant differences among groups and Mann-Whitney's U post hoc pairwise comparisons were also performed. RESULTS: The highest increase in SP, CGRP, and VEGF expressions was found in the occlusal trauma plus orthodontic forces group, followed by the moderate orthodontic forces, the occlusal trauma and the control groups, with statistically significant differences between all groups for each of the 3 peptides analyzed (Kruskal-Wallis p < 0.001). All possible pairwise post-hoc comparisons were also significant for each peptide analyzed (Mann-Whitney's U p < 0.001). CONCLUSION: SP, CGRP, and VEGF expressions significantly increase in human dental pulps when stimulated by occlusal trauma combined with moderate orthodontic forces, as compared with these two stimuli applied independently. Name of the registry: Importance of Neurogenic Inflammation in the Angiogenic Response of the Dental Pulp as a Defensive Response. TRIAL REGISTRATION NUMBER: NCT03804034. Date of registration: 01/15/2019 Retrospectively registered. URL of trial registry record: https://clinicaltrials.gov/ct2/show/NCT03804034?term=NCT03804034&draw=2&rank=1 .

Sexual forms obtained in a continuous in vitro cultured Colombian strain of Plasmodium falciparum (FCB2).

BACKGROUND: The epidemiological control of malaria has been hampered by the appearance of parasite resistance to anti-malarial drugs and by the resistance of mosquito vectors to control measures. This has also been associated with weak transmission control, mostly due to poor control of asymptomatic patients associated with host-vector transmission. This highlights the importance of studying the parasite's sexual forms (gametocytes) which are involved in this phase of the parasite's life-cycle. Some African and Asian strains of Plasmodium falciparum have been fully characterized regarding sexual forms' production; however, few Latin-American strains have been so characterized. This study was aimed at characterizing the Colombian FCB2 strain as a gametocyte producer able to infect mosquitoes. METHODS: Gametocyte production was induced in in vitro cultured P. falciparum FCB2 and 3D7 strains. Pfap2g and Pfs25 gene expression was detected in FCB2 strain gametocyte culture by RT-PCR. Comparative analysis of gametocytes obtained from both strains was made (counts and morphological changes). In vitro zygote formation from FCB2 gametocytes was induced by incubating a gametocyte culture sample at 27 °C for 20 min. A controlled Anopheles albimanus infection was made using an artificial feed system with cultured FCB2 gametocytes (14-15 days old). Mosquito midgut dissection was then carried out for analyzing oocysts. RESULTS: The FCB2 strain expressed Pfap2g, Pfs16, Pfg27/25 and Pfs25 sexual differentiation-related genes after in vitro sexual differentiation induction, producing gametocytes that conserved the expected morphological features. The amount of FCB2 gametocytes produced was similar to that from the 3D7 strain. FCB2 gametocytes were differentiated into zygotes and ookinetes after an in vitro low-temperature stimulus and infected An. albimanus mosquitoes, developing to oocyst stage. CONCLUSIONS: Even with the history of long-term FCB2 strain in vitro culture maintenance, it has retained its sexual differentiation ability. The gametocytes produced here preserved these parasite forms' usual characteristics and An. albimanus infection capability, thus enabling its use as a tool for studying sexual form biology, An. albimanus infection comparative analysis and anti-malarial drug and vaccine development.

Designing and optimizing new antimicrobial peptides: all targets are not the same.

Because the resistance of microorganisms to the available antibiotics is a growing healthcare problem worldwide, the search for new antimicrobial peptides (AMPs) that provide useful therapeutic options has been increasing in importance. Many initial candidates have had to be discarded after having advanced to the preclinical and clinical stages. This has led to substantial losses in terms of time and money. For that reason, the essential characteristics of AMPs (i.e. their activity, selectivity, stability in physiological conditions and low production cost) must be considered in their design. In addition, peptides could be active against several kinds of cells with activity and selectivity resulting from interaction with multiple target cell components, which sometimes are present in mammalian cells as well. Thus, the cellular composition is important in the AMP-target cell interaction and must be considered in the design of AMPs, too. This review describes general aspects of AMP design, limitations concerning their therapeutic application, and optimization strategies for overcoming such limitations.

Receptor-ligand and parasite protein-protein interactions in Plasmodium vivax: Analysing rhoptry neck proteins 2 and 4.

Elucidating receptor-ligand and protein-protein interactions represents an attractive alternative for designing effective Plasmodium vivax control methods. This article describes the ability of P. vivax rhoptry neck proteins 2 and 4 (RON2 and RON4) to bind to human reticulocytes. Biochemical and cellular studies have shown that two PvRON2- and PvRON4-derived conserved regions specifically interact with protein receptors on reticulocytes marked by the CD71 surface transferrin receptor. Mapping each protein fragment's binding region led to defining the specific participation of two 20 amino acid-long regions selectively competing for PvRON2 and PvRON4 binding to reticulocytes. Binary interactions between PvRON2 (ligand) and other parasite proteins, such as PvRON4, PvRON5, and apical membrane antigen 1 (AMA1), were evaluated and characterised by surface plasmon resonance. The results revealed that both PvRON2 cysteine-rich regions strongly interact with PvAMA1 Domains II and III (equilibrium constants in the nanomolar range) and at a lower extent with the complete PvAMA1 ectodomain and Domains I and II. These results strongly support that these proteins participate in P. vivax's complex invasion process, thus providing new pertinent targets for blocking P. vivax merozoites' specific entry to their target cells.

Plasmodium vivax in vitro continuous culture: the spoke in the wheel.

Understanding the life cycle of Plasmodium vivax is fundamental for developing strategies aimed at controlling and eliminating this parasitic species. Although advances in omic sciences and high-throughput techniques in recent years have enabled the identification and characterization of proteins which might be participating in P. vivax invasion of target cells, exclusive parasite tropism for invading reticulocytes has become the main obstacle in maintaining a continuous culture for this species. Such advance that would help in defining each parasite protein's function in the complex process of P. vivax invasion, in addition to evaluating new therapeutic agents, is still a dream. Advances related to maintenance, culture medium supplements and the use of different sources of reticulocytes and parasites (strains and isolates) have been made regarding the development of an in vitro culture for P. vivax; however, only some cultures having few replication cycles have been obtained to date, meaning that this parasite's maintenance goes beyond the technical components involved. Although it is still not yet clear which molecular mechanisms P. vivax prefers for invading young CD71+ reticulocytes [early maturation stages (I-II-III)], changes related to membrane proteins remodelling of such cells could form part of the explanation. The most relevant aspects regarding P. vivax in vitro culture and host cell characteristics have been analysed in this review to explain possible reasons why the species' continuous in vitro culture is so difficult to standardize. Some alternatives for P. vivax in vitro culture have also been described.

Self-assembling functional programmable protein array for studying protein-protein interactions in malaria parasites.

BACKGROUND: Plasmodium vivax is the most widespread malarial species, causing significant morbidity worldwide. Knowledge is limited regarding the molecular mechanism of invasion due to the lack of a continuous in vitro culture system for these species. Since protein-protein and host-cell interactions play an essential role in the microorganism's invasion and replication, elucidating protein function during invasion is critical when developing more effective control methods. Nucleic acid programmable protein array (NAPPA) has thus become a suitable technology for studying protein-protein and host-protein interactions since producing proteins through the in vitro transcription/translation (IVTT) method overcomes most of the drawbacks encountered to date, such as heterologous protein production, stability and purification. RESULTS: Twenty P. vivax proteins on merozoite surface or in secretory organelles were selected and successfully cloned using gateway technology. Most constructs were displayed in the array expressed in situ, using the IVTT method. The Pv12 protein was used as bait for evaluating array functionality and co-expressed with P. vivax cDNA display in the array. It was found that Pv12 interacted with Pv41 (as previously described), as well as PvMSP142kDa, PvRBP1a, PvMSP8 and PvRAP1. CONCLUSIONS: NAPPA is a high-performance technique enabling co-expression of bait and query in situ, thereby enabling interactions to be analysed rapidly and reproducibly. It offers a fresh alternative for studying protein-protein and ligand-receptor interactions regarding a parasite which is difficult to cultivate (i.e. P. vivax).

Towards designing a synthetic antituberculosis vaccine: The Rv3587c peptide inhibits mycobacterial entry to host cells.

Mycobacterium tuberculosis is considered one of the most successful pathogens in the history of mankind, having caused 1.7 million deaths in 2016. The amount of resistant and extensively resistant strains has increased; BCG has been the only vaccine to be produced in more than 100 years though it is still unable to prevent the disease's most disseminated form in adults; pulmonary tuberculosis. The search is thus still on-going for candidate antigens for an antituberculosis vaccine. This paper reports the use of a logical and rational methodology for finding such antigens, this time as peptides derived from the Rv3587c membrane protein. Bioinformatics tools were used for predicting mycobacterial surface location and Rv3587c protein structure whilst circular dichroism was used for determining its peptides' secondary structure. Receptor-ligand assays identified 4 high activity binding peptides (HABPs) binding specifically to A549 alveolar epithelial cells and U937 monocyte-derived macrophages, covering the region between amino acids 116 and 193. Their capability for inhibiting Mtb H37Rv invasion was evaluated. The recognition of antibodies from individuals suffering active and latent tuberculosis and from healthy individuals was observed in HABPs capable of avoiding mycobacterial entry to host cells. The results showed that 8 HABPs inhibited such invasion, two of them being common for both cell lines: 39265 (155VLAAYVYSLDNKRLWSNLDT173) and 39266 (174APSNETLVKTFSPGEQVTTY192). Peptide 39265 was the least recognised by antibodies from the individuals' sera evaluated in each group. According to the model proposed by FIDIC regarding synthetic vaccine development, peptide 39265 has become a candidate antigen for an antituberculosis vaccine.

Identifying and characterising PPE7 (Rv0354c) high activity binding peptides and their role in inhibiting cell invasion.

This study was aimed at characterising the PPE7 protein from the PE/PPE protein family. The presence and transcription of the rv0354c gene in the Mycobacterium tuberculosis complex was determined and the subcellular localisation of the PPE7 protein on mycobacterial membrane was confirmed by immunoelectron microscope. Two peptides were identified as having high binding activity (HABPs) and were tested in vitro regarding the invasion of Mycobacterium tuberculosis H37Rv. HABP 39224 inhibited invasion in A549 epithelial cells and U937 macrophages by more than 50%, whilst HABP 39225 inhibited invasion by 40% in U937 cells. HABP 39224, located in the protein's C-terminal region, has a completely conserved amino acid sequence in M. tuberculosis complex species and could be selected as a base peptide when designing a subunit-based, anti-tuberculosis vaccine.

Conserved Binding Regions Provide the Clue for Peptide-Based Vaccine Development: A Chemical Perspective.

Synthetic peptides have become invaluable biomedical research and medicinal chemistry tools for studying functional roles, i.e., binding or proteolytic activity, naturally-occurring regions' immunogenicity in proteins and developing therapeutic agents and vaccines. Synthetic peptides can mimic protein sites; their structure and function can be easily modulated by specific amino acid replacement. They have major advantages, i.e., they are cheap, easily-produced and chemically stable, lack infectious and secondary adverse reactions and can induce immune responses via T- and B-cell epitopes. Our group has previously shown that using synthetic peptides and adopting a functional approach has led to identifying Plasmodium falciparumconserved regions binding to host cells. Conserved high activity binding peptides' (cHABPs) physicochemical, structural and immunological characteristics have been taken into account for properly modifying and converting them into highly immunogenic, protection-inducing peptides (mHABPs) in the experimental Aotus monkey model. This article describes stereo-electron and topochemical characteristics regarding major histocompatibility complex (MHC)-mHABP-T-cell receptor (TCR) complex formation. Some mHABPs in this complex inducing long-lasting, protective immunity have been named immune protection-inducing protein structures (IMPIPS), forming the subunit components in chemically synthesized vaccines. This manuscript summarizes this particular field and adds our recent findings concerning intramolecular interactions (H-bonds or π-interactions) enabling proper IMPIPS structure as well as the peripheral flanking residues (PFR) to stabilize the MHCII-IMPIPS-TCR interaction, aimed at inducing long-lasting, protective immunological memory.

The Plasmodium vivax rhoptry neck protein 5 is expressed in the apical pole of Plasmodium vivax VCG-1 strain schizonts and binds to human reticulocytes.

BACKGROUND: Different proteins derived from the membrane or the apical organelles become involved in malarial parasite invasion of host cells. Among these, the rhoptry neck proteins (RONs) interact with a protein component of the micronemes to enable the formation of a strong bond which is crucial for the parasite's successful invasion. The present study was aimed at identifying and characterizing the RON5 protein in Plasmodium vivax and evaluating its ability to bind to reticulocytes. METHODS: Taking the Plasmodium falciparum and Plasmodium knowlesi RON5 amino acid sequences as template, an in-silico search was made in the P. vivax genome for identifying the orthologous gene. Different molecular tools were used for experimentally ascertaining pvron5 gene presence and transcription in P. vivax VCG-1 strain schizonts. Polyclonal antibodies against PvRON5 peptides were used for evaluating protein expression (by Western blot) and sub-cellular localization (by immunofluorescence). A 33 kDa PvRON5 fragment was expressed in Escherichia coli and used for evaluating the reactivity of sera from patients infected by P. vivax. Two assays were made for determining the RON5 recombinant fragment's ability to bind to reticulocyte-enriched human umbilical cord samples. RESULTS: The pvron5 gene (3,477 bp) was transcribed in VCG-1 strain schizonts and encoded a ~133 kDa protein which was expressed in the rhoptry neck of VCG-1 strain late schizonts, together with PvRON2 and PvRON4. Polyclonal sera against PvRON5 peptides specifically detected ~85 and ~30 kDa fragments in parasite lysate, thereby suggesting proteolytic processing in this protein. Comparative analysis of VCG-1 strain PvRON5 with other P. vivax strains having different geographic localizations suggested its low polymorphism regarding other malarial antigens. A recombinant fragment of the PvRON5 protein (rPvRON5) was recognized by sera from P. vivax-infected patients and bound to red blood cells, having a marked preference for human reticulocytes. CONCLUSIONS: The pvron5 gene is transcribed in the VCG-1 strain, the encoded protein is expressed at the parasite's apical pole and might be participating in merozoite invasion of host cells, taking into account its marked binding preference for human reticulocytes.

Protecting capacity against malaria of chemically defined tetramer forms based on the Plasmodium falciparum apical sushi protein as potential vaccine components.

Developing novel generations of subunit-based antimalarial vaccines in the form of chemically-defined macromolecule systems for multiple antigen presentation represents a classical problem in the field of vaccine development. Many efforts involving synthesis strategies leading to macromolecule constructs have been based on dendrimer-like systems, the condensation of large building blocks and conventional asymmetric double dimer constructs, all based on lysine cores. This work describes novel symmetric double dimer and condensed linear constructs for presenting selected peptide multi-copies from the apical sushi protein expressed in Plasmodium falciparum. These molecules have been proved to be safe and innocuous, highly antigenic and have shown strong protective efficacy in rodents challenged with two Plasmodium species. Insights into systematic design, synthesis and characterisation have led to such novel antigen systems being used as potential platforms for developing new anti-malarial vaccine candidates.

Characterisation of the erythrocyte invasion phenotype of FCB-2: A South American P. falciparum reference strain.

The extent of parasite adaptive capability involved in erythrocyte invasion represents a significant challenge for the development of a Plasmodium falciparum vaccine. The parasite's geographical and populational origin may influence such adaptive behaviour; in vitro culture-adapted parasite strains are typically used for such studies. Previous studies have reported invasion phenotypes in strains from Africa and Asia and, to a lesser extent, from Latin America. This study was aimed at expanding the pool of characterised parasite strains from Latin America by describing the invasion phenotype of the P. falciparum Colombia Bogotá 2 (FCB2) strain. The FCB2 genome was sequenced and erythrocyte invasion ligand sequences were analysed and compared to other previously reported ones. RT-PCR was used for assessing Pfeba family erythrocyte invasion ligands and reticulocyte binding homologue (Pfrh) gene transcription. A flow cytometry-based erythrocyte invasion assay (using enzymatically-treated erythrocytes) was used for determining the FCB2 strain's invasion phenotype. The P. falciparum FCB2 genome sequence was analysed, bearing in mind that prolonged in vitro parasite culture may affect its genome sequence and, in some cases, lead to the deletion of certain genes; it was demonstrated that all erythrocyte invasion ligand gene sequences studied here were preserved. Comparative analysis showed that the target genome sequences were conserved whereas transcriptional analysis highlighted Pfebas and Pfrhs gene expression. Erythrocyte invasion analysis demonstrated that the FCB2 strain has a sialic acid-resistant invasion phenotype.

Annotation and characterization of the Plasmodium vivax rhoptry neck protein 4 (PvRON4).

BACKGROUND: The tight junction (TJ) is one of the most important structures established during merozoite invasion of host cells and a large amount of proteins stored in Toxoplasma and Plasmodium parasites' apical organelles are involved in forming the TJ. Plasmodium falciparum and Toxoplasma gondii apical membrane antigen 1 (AMA-1) and rhoptry neck proteins (RONs) are the two main TJ components. It has been shown that RON4 plays an essential role during merozoite and sporozoite invasion to target cells. This study has focused on characterizing a novel Plasmodium vivax rhoptry protein, RON4, which is homologous to PfRON4 and PkRON4. METHODS: The ron4 gene was re-annotated in the P. vivax genome using various bioinformatics tools and taking PfRON4 and PkRON4 amino acid sequences as templates. Gene synteny, as well as identity and similarity values between open reading frames (ORFs) belonging to the three species were assessed. The gene transcription of pvron4, and the expression and localization of the encoded protein were also determined in the VCG-1 strain by molecular and immunological studies. Nucleotide and amino acid sequences obtained for pvron4 in VCG-1 were compared to those from strains coming from different geographical areas. RESULTS: PvRON4 is a 733 amino acid long protein, which is encoded by three exons, having similar transcription and translation patterns to those reported for its homologue, PfRON4. Sequencing PvRON4 from the VCG-1 strain and comparing it to P. vivax strains from different geographical locations has shown two conserved regions separated by a low complexity variable region, possibly acting as a "smokescreen". PvRON4 contains a predicted signal sequence, a coiled-coil α-helical motif, two tandem repeats and six conserved cysteines towards the carboxy-terminus and is a soluble protein lacking predicted transmembranal domains or a GPI anchor. Indirect immunofluorescence assays have shown that PvRON4 is expressed at the apical end of schizonts and co-localizes at the rhoptry neck with PvRON2. CONCLUSIONS: Genomic, transcriptional and expression data reported for PvRON4, as well as its primary structure characteristics suggest that this protein participates in reticulocyte invasion, as has been shown for its homologue PfRON4.

Binding activity, structure, and immunogenicity of synthetic peptides derived from Plasmodium falciparum CelTOS and TRSP proteins.

Several sporozoite proteins have been associated with Plasmodium falciparum cell traversal and hepatocyte invasion, including the cell-traversal protein for ookinetes and sporozoites (CelTOS), and thrombospondin-related sporozoite protein (TRSP). CelTOS and TRSP amino acid sequences have been finely mapped to identify regions specifically binding to HeLa and HepG2 cells, respectively. Three high-activity binding peptides (HABPs) were found in CelTOS and one HABP was found in TRSP, all of them having high α-helical structure content. These HABPs' specific binding was sensitive to HeLa and HepG2 cells' pre-treatment with heparinase I and chondroitinase ABC. Despite their similarity at three-dimensional (3D) structural level, TRSP and TRAP HABPs located in the TSR domain did not compete for the same binding sites. CelTOS and TRSP HABPs were used as a template for designing modified sequences to then be assessed in the Aotus monkey experimental model. Antibodies directed against these modified HABPs were able to recognize both the native parasite protein by immunofluorescence assay and the recombinant protein (expressed in Escherichia coli) by Western blot and ELISA assays. The results suggested that these modified HABPs could be promising targets in designing a fully effective, antimalarial vaccine.

PvRON2, a new Plasmodium vivax rhoptry neck antigen.

BACKGROUND: Rhoptries are specialized organelles from parasites belonging to the phylum Apicomplexa; they secrete their protein content during invasion of host target cells and are sorted into discrete subcompartments within rhoptry neck or bulb. This distribution is associated with these proteins' role in tight junction (TJ) and parasitophorous vacuole (PV) formation, respectively. METHODS: Plasmodium falciparum RON2 amino acid sequence was used as bait for screening the codifying gene for the homologous protein in the Plasmodium vivax genome. Gene synteny, as well as identity and similarity values, were determined for ron2 and its flanking genes among P. falciparum, P. vivax and other malarial parasite genomes available at PlasmoDB and Sanger Institute databases. Pvron2 gene transcription was determined by RT-PCR of cDNA obtained from the P. vivax VCG-1 strain. Protein expression and localization were assessed by Western blot and immunofluorescence using polyclonal anti-PvRON2 antibodies. Co-localization was confirmed using antibodies directed towards specific microneme and rhoptry neck proteins. RESULTS AND DISCUSSION: The first P. vivax rhoptry neck protein (named here PvRON2) has been identified in this study. PvRON2 is a 2,204 residue-long protein encoded by a single 6,615 bp exon containing a hydrophobic signal sequence towards the amino-terminus, a transmembrane domain towards the carboxy-terminus and two coiled coil α-helical motifs; these are characteristic features of several previously described vaccine candidates against malaria. This protein also contains two tandem repeats within the interspecies variable sequence possibly involved in evading a host's immune system. PvRON2 is expressed in late schizonts and localized in rhoptry necks similar to what has been reported for PfRON2, which suggests its participation during target cell invasion. CONCLUSIONS: The identification and partial characterization of the first P. vivax rhoptry neck protein are described in the present study. This protein is homologous to PfRON2 which has previously been shown to be associated with PfAMA-1, suggesting a similar role for PvRON2.

Conserved regions from Plasmodium falciparum MSP11 specifically interact with host cells and have a potential role during merozoite invasion of red blood cells.

Despite significant global efforts, a completely effective vaccine against Plasmodium falciparum, the species responsible for the most serious form of malaria, has not been yet obtained. One of the most promising approaches consists in combining chemically synthesized minimal subunits of parasite proteins involved in host cell invasion, which has led to the identification of peptides with high binding activity (named HABPs) to hepatocyte and red blood cell (RBC) surface receptors in a large number of sporozoite and merozoite proteins, respectively. Among these proteins is the merozoite surface protein 11 (MSP11), which shares important structural and immunological features with the antimalarial vaccine candidates MSP1, MSP3, and MSP6. In this study, 20-mer-long synthetic peptides spanning the complete sequence of MSP11 were assessed for their ability to bind specifically to RBCs. Two HABPs with high ability to inhibit invasion of RBCs in vitro were identified (namely HABPs 33595 and 33606). HABP-RBC bindings were characterized by means of saturation assays and Hill analysis, finding cooperative interactions of high affinity for both HABPs (n(H) of 1.5 and 1.2, K(d) of 800 and 600 nM for HABPs 33595 and 33606, respectively). The nature of the possible RBC receptors for MSP11 HABPs was studied in binding assays to enzyme-treated RBCs and cross-linking assays, finding that both HABPs use mainly a sialic acid-dependent receptor. An analysis of the immunological, structural and polymorphic characteristics of MSP11 HABPs supports including these peptides in further studies with the aim of designing a fully effective protection-inducing vaccine against malaria.

Peptides from the Mycobacterium tuberculosis Rv1980c protein involved in human cell infection: insights into new synthetic subunit vaccine candidates.

Mycobacterium tuberculosis infection continues to be a major cause of morbidity and mortality throughout the world. The vast complexity of the intracellular pathogen M. tuberculosis and the diverse mechanisms by which it can invade host cells highlight the importance of developing a fully protective vaccine. Our vaccine development strategy consists of including fragments from multiple mycobacterial proteins involved in cell invasion. The aim of this study was to identify high activity binding peptides (HABPs) in the immunogenic protein Rv1980c from M. tuberculosis H37Rv with the ability to inhibit mycobacterial invasion into U937 monocyte-derived macrophages and A549 cells. The presence and transcription of the Rv1980c gene was assessed in members belonging to the M. tuberculosis complex and other nontuberculous mycobacteria by PCR and RT-PCR, respectively. Cell surface localization was confirmed by immuno-electron microscopy. Three peptides binding with high activity to U937 cells and one to A549 cells were identified. HABPs 31100, 31101, and 31107 inhibited invasion of M. tuberculosis into A549 and U937 cells and therefore could be promising candidates for the design of a subunit-based antituberculous vaccine.

Mycobacterium tuberculosis Rv0679c protein sequences involved in host-cell infection: potential TB vaccine candidate antigen.

BACKGROUND: To date, the function of many hypothetical membrane proteins of Mycobacterium tuberculosis is still unknown and their involvement in pathogen-host interactions has not been yet clearly defined. In this study, the biological activity of peptides derived from the hypothetical membrane protein Rv0679c of M. tuberculosis and their involvement in pathogen-host interactions was assessed. Transcription of the Rv0679c gene was studied in 26 Mycobacterium spp. Strains. Antibodies raised against putative B-cell epitopes of Rv0679c were used in Western blot and immunoelectron microscopy assays. Synthetic peptides spanning the entire length of the protein were tested for their ability to bind to A549 and U937 cells. High-activity binding peptides (HABPs) identified in Rv0679c were tested for their ability to inhibit mycobacterial invasion into cells. RESULTS: The gene encoding Rv0679c was detected in all strains of the M. tuberculosis complex (MTC), but was only transcribed in M. tuberculosis H37Rv, M. tuberculosis H37Ra and M. africanum. Anti-Rv0679c antibodies specifically recognized the protein in M. tuberculosis H37Rv sonicate and showed its localization on mycobacterial surface. Four HABPs inhibited invasion of M. tuberculosis to target cells by up to 75%. CONCLUSIONS: The results indicate that Rv0679c HABPs and in particular HABP 30979 could be playing an important role during M. tuberculosis invasion of host cells, and therefore could be interesting research targets for studies aimed at developing strategies to control tuberculosis.

Shorter Antibacterial Peptide Having High Selectivity for E. coli Membranes and Low Potential for Inducing Resistance.

Antimicrobial peptides (AMPs) have been recognised as a significant therapeutic option for mitigating resistant microbial infections. It has been found recently that Plasmodium falciparum-derived, 20 residue long, peptide 35409 had antibacterial and haemolytic activity, making it an AMP having reduced selectivity, and suggesting that it should be studied more extensively for obtaining new AMPs having activity solely targeting the bacterial membrane. Peptide 35409 was thus used as template for producing short synthetic peptides (<20 residues long) and evaluating their biological activity and relevant physicochemical characteristics for therapeutic use. Four of the sixteen short peptides evaluated here had activity against E. coli without any associated haemolytic effects. The 35409-1 derivative (17 residues long) had the best therapeutic characteristics as it had high selectivity for bacterial cells, stability in the presence of human sera, activity against E. coli multiresistant clinical isolates and was shorter than the original sequence. It had a powerful membranolytic effect and low potential for inducing resistance in bacteria. This peptide's characteristics highlighted its potential as an alternative for combating infection caused by E. coli multiresistant bacteria and/or for designing new AMPs.

Evaluating the immunogenicity of chemically-synthesised peptides derived from foot-and-mouth disease VP1, VP2 and VP3 proteins as vaccine candidates.

Foot-and-mouth disease (FMD) is one of the most contagious veterinary viral diseases known, having economic, social and potentially devastating environmental impacts. The vaccines currently being marketed/sold around the world for disease control and prevention in bovines do not stimulate the production of antibodies having crossed reactions to different serotypes. This means that if an animal becomes infected by a serotype which has not been included in a vaccine then it will develop the disease. Synthetic peptide vaccines represent a safer option and (depending on the design) can stimulate antibodies protecting against different variants. Based on the forgoing, this work was aimed at evaluating FMDV VP1, VP2 and VP3 protein-derived, modified and chemically-synthesised peptides' ability to induce an immune response for developing a vaccine contributing towards controlling the disease. VP1, VP2 and VP3 proteins' conserved regions were selected for this. Peptides from these regions were chemically synthesised; binding assays were then carried out for ascertaining whether they were involved in BHK-21 cell binding. Selected peptides' structure and location were studied. Peptides which did bind were modified and formulated with Montanide ISA 70 adjuvant; 17 animals were immunised twice with the formulation. The animals were genotyped by amplifying the BoLA-DRB3.2 gene. Blood samples were taken from 17 cattle on day 43 post-first immunisation for studying the formulation's immunogenicity. The sera were used in ELISA, immunofluorescence, flow cytometry, immunoadsorption and seroneutralisation assays. The A24 Cruzeiro and O1 Campos virus serotypes were used for these assays. The results revealed that even though protein exposure and 3D structure might be different amongst serotypes, the antibodies so produced could inhibit virus entry to cells, thereby showing the selected peptides' in vitro protection-inducing ability.