Memantine Improves Memory and Neurochemical Damage in a Model of Maple Syrup Urine Disease.

Maple Syrup Urine Disease (MSUD) is a metabolic disease characterized by the accumulation of branched-chain amino acids (BCAA) in different tissues due to a deficit in the branched-chain alpha-ketoacid dehydrogenase complex. The most common symptoms are poor feeding, psychomotor delay, and neurological damage. However, dietary therapy is not effective. Studies have demonstrated that memantine improves neurological damage in neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases. Therefore, we hypothesize that memantine, an NMDA receptor antagonist can ameliorate the effects elicited by BCAA in an MSUD animal model. For this, we organized the rats into four groups: control group (1), MSUD group (2), memantine group (3), and MSUD + memantine group (4). Animals were exposed to the MSUD model by the administration of BCAA (15.8 µL/g) (groups 2 and 4) or saline solution (0.9%) (groups 1 and 3) and treated with water or memantine (5 mg/kg) (groups 3 and 4). Our results showed that BCAA administration induced memory alterations, and changes in the levels of acetylcholine in the cerebral cortex. Furthermore, induction of oxidative damage and alterations in antioxidant enzyme activities along with an increase in pro-inflammatory cytokines were verified in the cerebral cortex. Thus, memantine treatment prevented the alterations in memory, acetylcholinesterase activity, 2',7'-Dichlorofluorescein oxidation, thiobarbituric acid reactive substances levels, sulfhydryl content, and inflammation. These findings suggest that memantine can improve the pathomechanisms observed in the MSUD model, and may improve oxidative stress, inflammation, and behavior alterations.

Development of an antibody against EtpA from enterotoxigenic Escherichia coli and evaluation of its use for bacterial isolation using magnetic beads.

The enterotoxigenic Escherichia coli (ETEC) strain is one of the most frequent causative agents of childhood diarrhea and travelers' diarrhea in low-and middle-income countries. Among the virulence factors secreted by ETEC, the exoprotein EtpA has been described as an important. In the present study, a new detection tool for enterotoxigenic E. coli bacteria using the EtpA protein was developed. Initially, antigenic sequences of the EtpA protein were selected via in silico prediction. A chimeric recombinant protein, corresponding to the selected regions, was expressed in an E. coli host, purified and used for the immunization of mice. The specific recognition of anti-EtpA IgG antibodies generated was evaluated using flow cytometry. The tests demonstrated that the antibodiesdeveloped were able to recognize the native EtpA protein. By coupling these antibodies to magnetic beads for the capture and detection of ETEC isolates, cytometric analyses showed an increase in sensitivity, specificity and the effectiveness of the method of separation and detection of these pathogens. This is the first report of the use of this methodology for ETEC separation. Future trials may indicate their potential use for isolating these and other pathogens in clinical samples, thus accelerating the diagnosis and treatment of diseases.

Mesoporous silica nanoparticles adsorb aflatoxin B1 and reduce mycotoxin-induced cell damage.

The present study examined the effects of mesoporous silica nanoparticles (MSNs) on its adsorption capacity of aflatoxin B1 (AFB1). Moreover, the study evaluated the toxicity of MSNs with AFB1 using NIH3T3 cells and hemolysis test. The obtained MSNs were spherical, irregular-like in shape, having a mean size of 39.97 ± 7.85 nm and a BET surface area of 1195 m2/g. At 0.1 mg mL-1 concentration of MSN, the AFB1 adsorption capacity was 30%, which reached 70% when the MSN concentration increased to 2.0 mg mL-1. Our findings showed that AFB1 was adsorbed (∼67%) in the first few minutes on being in contact with MSNs, reaching an adsorption capacity of ∼70% after 15 min. Thereafter, the adsorption capacity remained constant in solution, demonstrating that the MSNs adsorbed toxins even beyond overnight. MSN treatment (0.5-2.0 mg mL-1) using NIH3T3 cells did not result in any reduction in cell viability. In addition, MSN treatment completely reversed the cytotoxic effect of AFB1 at all concentrations. Hemolysis test also revealed no hemolysis in MSNs evaluated alone and in those combined with AFB1. To the best of our knowledge, this study is the first to demonstrate that MSN can reduce cell toxicity produced by AFB1 due to its potential to adsorb mycotoxins.

Evaluation from a B-cell epitope-based chimeric protein for the serodiagnosis of tegumentary and visceral leishmaniasis.

The diagnosis of leishmaniasis presents problems due to the variable sensitivity and/or specificity of tests. In addition, high levels of anti-parasite antibodies can remain after treatment, making it difficult to conduct a prognostic follow-up of patients. In this context, it is necessary to identify new candidates to be examined for the sensitive and specific diagnosis of the disease. In the present study, four Leishmania proteins, previously shown as antigenic for tegumentary leishmaniasis (TL), were evaluated, and their linear specific B-cell epitopes were predicted and used to generate a new gene codifying chimeric protein called ChimB, which was cloned, and the recombinant version was expressed, purified, and evaluated in ELISA (Enzyme-Linked Immunosorbent Assay) to diagnose TL and visceral leishmaniasis (VL). A total of 220 human serum samples were used, and, when ChimB was used, results showed sensitivity, specificity, and positive and negative predictive values of 100% for the diagnosis of both diseases; however, when using peptides, the sensitivity values reached from 28.0% to 57.3% and specificity varied from 16.3% to 83.7%. A soluble Leishmania extract (SLA) showed sensitivity and specificity values of 30.7% and 45.9%, respectively. The area under the curve (AUC) value for ChimB was 1.0, while for synthetic peptides, this value reached between 0.502 and 0.635, whereas for SLA, the value was of 0.589. Serological assays using sera samples collected before and after treatment showed significant reductions in the anti-ChimB antibody levels after therapy, suggesting a prognostic role of this recombinant antigen. In conclusion, preliminary data suggest the use from ChimB as a potential candidate for the diagnosis and prognosis of leishmaniasis.

Sensitive and specific serodiagnosis of tegumentary leishmaniasis using a new chimeric protein based on specific B-cell epitopes of Leishmania antigenic proteins.

Serological tests used for the diagnosis of tegumentary leishmaniasis (TL) presents problems, mainly related to their variable sensitivity and/or specificity, which can be caused by low levels of antileishmanial antibodies or by presence of cross-reactive diseases, respectively. In this context, the search for new antigenic candidates presenting higher sensitivity and specificity is urgently required. In the present study, the amino acid sequences of the LiHyT, LiHyD, LiHyV, and LiHyP proteins, which were previously showed to be antigenic in the visceral leishmaniasis (VL), were evaluated and eight B-cell epitopes were predicted and used for construction of gene codifying a chimeric protein called ChimLeish. The protein was expressed, purified and evaluated as a recombinant antigen in ELISA (Enzyme-Linked Immunosorbent Assay) for the diagnosis of TL. The own B cell epitopes used to construct the chimera were synthetized and also evaluated as antigens, as well as a soluble Leishmania braziliensis antigenic extract (SLA). Results showed that ChimLeish presented 100% sensitivity and specificity to diagnose TL, while synthetic peptides showed sensitivity varying from 9.1% to 90.9%, while specificity reached from 98.3% to 99.1%. SLA showed sensitivity and specificity of 18.2% and 98.3%, respectively. A preliminary prognostic evaluation showed that anti-ChimLeish IgG antibodies declined in significant levels, when serological reactivity was compared before and six months after treatment, suggesting also a possible prognostic role of this antigen for TL.

Gallic acid modulates purine metabolism and oxidative stress induced by ethanol exposure in zebrafish brain.

Gallic acid (GA) is a secondary metabolite found in plants. It has the ability to cross the blood-brain barrier and, through scavenging properties, has a protective effect in a brain insult model. Alcohol metabolism generates reactive oxygen species (ROS); thus, alcohol abuse has a deleterious effect on the brain. The zebrafish is a vertebrate often used for screening toxic substances and in acute ethanol exposure models. The aim of this study was to evaluate whether GA pretreatment (24 h) prevents the changes induced by acute ethanol exposure (1 h) in the purinergic signaling pathway in the zebrafish brain via degradation of extracellular nucleotides and oxidative stress. The nucleotide cascade promoted by the nucleoside triphosphate diphosphohydrolase (NTPDase) and 5'-nucleotidase was assessed by quantifying nucleotide metabolism. The effect of GA alone at 5 and 10 mg L-1 did not change the nucleotide levels. Pretreatment with 10 mg L-1 GA prevented an ethanol-induced increase in ATP and ADP levels. No significant difference was found between the AMP levels of the two pretreatment groups. Pretreatment with 10 mg L-1 GA prevented ethanol-enhanced lipid peroxidation and dichlorodihydrofluorescein (DCFH) levels. The higher GA concentration was also shown to positively modulate against ethanol-induced effects on superoxide dismutase (SOD), but not on catalase (CAT). This study demonstrated that GA prevents the inhibitory effect of ethanol on NTPDase activity and oxidative stress parameters, thus consequently modulating nucleotide levels that may contribute to the possible protective effects induced by alcohol and purinergic signaling.

Mesoporous silica nanoparticles incorporated with zinc oxide as a novel antifungal agent against toxigenic fungi strains.

Developing environmentally friendly alternative strategies to reduce the damage caused by fungi in agriculture has been widely investigated. In this study, we evaluated using mesoporous silica nanoparticles (MSNs) incorporated with zinc oxide (MSNs-ZnO) as a potential antifungal agent against Fusarium graminearum and Aspergillus flavus strains, as well as their antimycotoxin properties. The MSNs that synthesized and characterized could release abundant ZnO in the first 24 h. Subsequently, the ZnO release became slower, providing greater durability of the antifungal effect. Significant (P < 0.001) growth reductions in F. graminearum (81%) and A. flavus (65%) compared to the control were obtained at a high concentration of the MSNs-ZnO (1.0 mg mL-1). Moreover, the MSNs-ZnO treatment at a high concentration (1.0 mg mL-1) caused morphology alteration in both fungi, showing ruptures and deformations in the fungal hyphae, affecting their growth and toxin production. A significant reduction (P < 0.001) in the productions of deoxynivalenol (89%) and aflatoxin B1 (58%) by F. graminearum and A. flavus were also observed. These findings imply that using MSNs as the carriers of zinc compounds, such as ZnO, could be investigated as a safe alternative for effectively controlling toxigenic fungi in agriculture.

ChimLeish, a new recombinant chimeric protein evaluated as a diagnostic and prognostic marker for visceral leishmaniasis and human immunodeficiency virus coinfection.

Visceral leishmaniasis (VL) is a neglected tropical disease of global importance caused by parasites of the genus Leishmania, and coinfection with human immunodeficiency virus (HIV) is common in countries where both diseases are endemic. In particular, widely used immunological tests for VL diagnosis have impaired sensitivity (Se) and specificity (Sp) in VL/HIV coinfected patients and there is also cross-reactivity with other endemic diseases, e.g., Chagas disease, malaria, and tuberculosis. To develop new antigens to improve the diagnosis of VL and VL/HIV coinfection, we predicted eight specific B-cell epitopes of four Leishmania infantum antigens and constructed a recombinant polypeptide chimera antigen called ChimLeish. A serological panel of 195 serum samples was used to compare the diagnostic capabilities of ChimLeish alongside the individual synthetic peptides. ChimLeish reacted with sera from all VL and VL/HIV coinfected patients [Se = 100%; Sp = 100%; area under the curve (AUC) = 1.0]. Peptides showed lower reactivities (Se = 76.8 to 99.2%; Sp = 67.1 to 95.7%; AUC between 0.87 and 0.98) as did a L. infantum antigenic preparation used as an antigen control (Se = 56.8%; Sp = 69.5%: AUC = 0.45). Notably, ChimLeish demonstrated a significant reduction (p < 0.05) of anti-ChimLeish antibodies after treatment and cure of a small number of patients. Although only a limited serological panel was tested, preliminary data suggest that ChimLeish should be evaluated in larger sample studies for the diagnosis of VL and VL/HIV coinfection.

Leishmania infantum amastin protein incorporated in distinct adjuvant systems induces protection against visceral leishmaniasis.

The control measures against visceral leishmaniasis (VL) include a precise diagnosis of disease, the treatment of human cases, and reservoir and vector controls. However, these are insufficient to avoid the spread of the disease in specific countries worldwide. As a consequence, prophylactic vaccination could be interesting, although no effective candidate against human disease is available. In the present study, the Leishmania infantum amastin protein was evaluated regarding its immunogenicity and protective efficacy against experimental VL. BALB/c mice immunized with subcutaneous injections of the recombinant protein with or without liposome/saponin (Lip/Sap) as an adjuvant. After immunization, half of the animals per group were euthanized and immunological evaluations were performed, while the others were challenged with L. infantum promastigotes. Forty-five days after infection, the animals were euthanized and parasitological and immunological evaluations were performed. Results showed the development of a Th1-type immune response in rAmastin-Lip and rAmastin-Sap/vaccinated mice, before and after infection, which was based on the production of protein and parasite-specific IFN-γ, IL-12, GM-CSF, and nitrite, as well as the IgG2a isotype antibody. CD4+ T cells were mainly responsible for IFN-γ production in vaccinated mice, which also presented significant reductions in parasitism in their liver, spleen, draining lymph nodes, and bone marrow. In addition, PBMC cultures of treated VL patients and healthy subjects stimulated with rAmastin showed lymphoproliferation and higher IFN-γ production. In conclusion, the present study shows the first case of an L. infantum amastin protein associated with distinct delivery systems inducing protection against L. infantum infection and demonstrates an immunogenic effect of this protein in human cells.

Biotechnological applications from a Leishmania amastigote-specific hypothetical protein in the canine and human visceral leishmaniasis.

The treatment against visceral leishmaniasis (VL) presents problems, mainly related to the toxicity and/or high cost of the drugs. In this context, a rapid and precise diagnosis of the disease should be performed, mainly to treat patients as soon as possible, aiming to reduce the treatment time and the toxicity of the therapeutics. In the present study, the diagnostic role of an amastigote-specific Leishmania protein was evaluated in the canine and human VL. Results showed that the recombinant protein (called rLiHyJ) and one specific B cell epitope (called PeptJ) predicted from protein sequence presented high sensitivity and specificity values to diagnose canine and human disease, showing also a low reactivity against cross-reactive samples. The rA2 protein and a parasite antigenic extract showed variable sensitivity and/or specificity values in the ELISA experiments. A prognostic evaluation of protein and peptide in the human VL indicated that specific IgG antibodies significantly decreased after treatment, when compared to be values obtained before therapy. The in vitro immunogenicity using rLiHyJ in peripheral blood mononuclear cell (PBMC) cultures collected of such patients and healthy subjects suggested that the protein induced lymphoproliferation and high IFN-γ production in the stimulated cells. In conclusion, although preliminary, results suggest that rLiHyJ and PeptJ could present distinct biotechnological applications in the canine and human VL.

Targeting epimastigotes of Trypanosoma cruzi with a peptide isolated from a phage display random library.

Chagas disease, also known as American trypanosomiasis, is a potentially life-threatening illness caused by the protozoan parasite Trypanosoma cruzi, which is transmitted by insects of the family Reduviidae. Since conventional treatments with nitroheterocyclic drugs show serious adverse reactions and have questionable efficiency, different research groups have investigated polypeptide-based approaches to interfere with the parasite cell cycle in other Trypanosomatids. These strategies are supported by the fact that surface players are candidates to develop surface ligands that impair function since they may act as virulence factors. In this study, we used a phage display approach to identify peptides from one library-LX8CX8 (17 aa) (where X corresponds to any amino acid). After testing different biopanning conditions using live or fixed epimastigotes, 10 clones were sequenced that encoded the same peptide, named here as EPI18. The bacteriophage expressing EPI18 binds to epimastigotes from distinct strains of T. cruzi. To confirm these results, this peptide was synthetized, biotinylated, and assayed using flow cytometry and confocal microscopy analyses. These assays confirmed the specificity of the binding capacity of EPI18 toward epimastigote surfaces. Our findings suggest that EPI18 may have potential biotechnological applications that include peptide-based strategies to control parasite transmission.

A new Leishmania hypothetical protein can be used for accurate serodiagnosis of canine and human visceral leishmaniasis and as a potential prognostic marker for human disease.

Distinct antigens have been evaluated with diagnostic purpose for canine and human visceral leishmaniasis (VL), and variable sensitivity and specificity values have been obtained in the assays. In the present study, a Leishmania infantum hypothetical protein called LiHyG, which was identified in an immunoproteomics study in Leishmania infantum amastigote extracts by antibodies in VL dogs sera; was cloned, expressed, purified and evaluated as a recombinant protein (rLiHyG) for the diagnosis of canine and human disease. The recombinant amastigote-specific A2 protein (rA2) and a soluble L. infantum protein extract (SLA) were used as controls. For canine VL, the sensitivity values were of 100%, 57.29% and 48.57%, when rLiHyG, rA2 and SLA were used, respectively, while the specificity values were of 100%, 81.43% and 88.57%, respectively. In addition, AUC values were of 1.00, 0.72 and 0.65, when rLiHyG, rA2 and SLA were used, respectively, while accuracy was of 100%, 72.38% and 75.24%, respectively. For human VL, the sensitivity values were of 100%, 84.00% and 88.00%, when rLiHyG, rA2 and SLA were used, respectively, while the specificity values were of 100%, 58.75% and 73.75%, respectively. In addition, AUC values were of 1.00, 0.76 and 0.83, when rLiHyG, rA2 and SLA were used, respectively, while accuracy was of 100%, 64.8% and 66.6%, respectively. The prognostic role of rLiHyG in the human VL was also evaluated, by means of post-therapeutic serological follow-up with sera samples collected before and six months after treatment. Results showed that treated patients presented significant reductions in the anti-rLiHyG IgG, IgG1, and IgG2 antibody levels, with results being similar to those found in healthy subjects. Testing the rA2 protein and SLA as antigens, lower IgG, IgG1, and IgG2 levels were also found, although they were higher after treatment than those obtained for rLiHyG. In conclusion, results suggested that rLiHyG could be considered for future studies as a diagnostic and/or prognostic marker for canine and human VL.

Evaluation of the protective efficacy of a Leishmania protein associated with distinct adjuvants against visceral leishmaniasis and in vitro immunogenicity in human cells.

The treatment against visceral leishmaniasis (VL) presents problems, mainly related to the toxicity and/or high cost of the drugs. In this context, a prophylactic vaccination is urgently required. In the present study, a Leishmania protein called LiHyE, which was suggested recently as an antigenic marker for canine and human VL, was evaluated regarding its immunogenicity and protective efficacy in BALB/c mice against Leishmania infantum infection. In addition, the protein was used to stimulate peripheral blood mononuclear cells (PBMCs) from VL patients before and after treatment, as well as from healthy subjects. Vaccination results showed that the recombinant (rLiHyE) protein associated with liposome or saponin induced effective protection in the mice, since significant reductions in the parasite load in spleen, liver, draining lymph nodes, and bone marrow were found. The parasitological protection was associated with Th1-type cell response, since high IFN-γ, IL-12, and GM-CSF levels, in addition to low IL-4 and IL-10 production, were found. Liposome induced a better parasitological and immunological protection than did saponin. Experiments using PBMCs showed rLiHyE-stimulated lymphoproliferation in treated patients' and healthy subjects' cells, as well as high IFN-γ levels in the cell supernatant. In conclusion, rLiHyE could be considered for future studies as a vaccine candidate against VL.

PEPOP 2.0: new approaches to mimic non-continuous epitopes.

BACKGROUND: Bioinformatics methods are helpful to identify new molecules for diagnostic or therapeutic applications. For example, the use of peptides capable of mimicking binding sites has several benefits in replacing a protein which is difficult to produce, or toxic. Using peptides is less expensive. Peptides are easier to manipulate, and can be used as drugs. Continuous epitopes predicted by bioinformatics tools are commonly used and these sequential epitopes are used as is in further experiments. Numerous discontinuous epitope predictors have been developed but only two bioinformatics tools have been proposed so far to predict peptide sequences: Superficial and PEPOP 2.0. PEPOP 2.0 can generate series of peptide sequences that can replace continuous or discontinuous epitopes in their interaction with their cognate antibody. RESULTS: We have developed an improved version of PEPOP (PEPOP 2.0) dedicated to answer to experimentalists' need for a tool able to handle proteins and to turn them into peptides. The PEPOP 2.0 web site has been reorganized by peptide prediction category and is therefore better formulated to experimental designs. Since the first version of PEPOP, 32 new methods of peptide design were developed. In total, PEPOP 2.0 proposes 35 methods in which 34 deal specifically with discontinuous epitopes, the most represented epitope type in nature. CONCLUSION: Through the presentation of its user-friendly, well-structured new web site conceived in close proximity to experimentalists, we report original methods that show how PEPOP 2.0 can assist biologists in dealing with discontinuous epitopes.

Evaluation of a Leishmania hypothetical protein administered as DNA vaccine or recombinant protein against Leishmania infantum infection and its immunogenicity in humans.

Visceral leishmaniasis (VL) is a fatal disease when acute and untreated. The treatment against this disease is long and presents toxicity and/or high costs. Moreover, parasite resistance has been increasing. Therefore, alternative control measures to avoid the spread of disease should be considered. It is accepted that the development of the T helper (Th)1 immune response, based on the production of pro-inflammatory cytokines, is required for the control of parasites. Although recombinant protein-based vaccines have been tested against VL, they require supplementation with immune adjuvants. In addition, there is a scarcity of studies that comparatively evaluate the efficacy of the immunogens when administered by different delivery systems in mammalian hosts. In the present study, a Leishmania hypothetical protein, LiHyR, was cloned and evaluated by immunization as a plasmid deoxyribonucleic acid (DNA) vaccine or in a recombinant format plus saponin against Leishmania infantum infection. Results showed that both vaccination regimens induced a Th1 cell-based immunity, since high levels of interferon-gamma (IFN-γ), interleukin (IL)-2, IL-12, granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor alpha (TNF-α) were found, and were associated with the low production of IL-4, IL-10, and anti-parasite immunoglobulin (IgG)1 isotype. In addition, significant reductions in the parasite load were found in the evaluated organs of the DNA LiHyR or rLiHyR/saponin-vaccinated animals. No significant difference was achieved between groups vaccinated with DNA or the recombinant protein. The antigen proved to be also immunogenic in human peripheral blood mononuclear cells (PBMCs) collected from healthy subjects and from untreated and treated VL patients. A higher IgG2 isotype was also found in sera samples of these subjects, thus demonstrating its possible use as a human vaccine. This study demonstrates the protective efficacy of a new Leishmania protein against VL, when it is administered as a DNA vaccine or a recombinant protein plus saponin, and points out its use as a human vaccine against disease.

Diagnostic application of recombinant Leishmania proteins and evaluation of their in vitro immunogenicity after stimulation of immune cells collected from tegumentary leishmaniasis patients and healthy individuals.

The present study evaluated the cytokine profile in PBMC supernatants and the humoral response in mucosal leishmaniasis (ML) patients and in healthy subjects living in an endemic area. Four proteins, which had previously proven to be antigenic in the human disease, were tested: LiHyM, enolase, eukaryotic initiation factor 5a, and Beta-tubulin. Results showed that all of the proteins stimulated human cells with higher IFN-γ and lower IL-4 and IL-10 levels. The analysis of antibody isotypes correlated with cell response, since the IgG2 production was higher than IgG1 in both groups. By contrast, a Th2 response was found when an antigenic Leishmania extract was used. Serological analyses revealed high sensitivity and specificity values for the serodiagnosis of the disease, when compared to the data obtained using the antigenic preparation. In conclusion, this study presents new candidates to be evaluated as biomarkers in tegumentary leishmaniasis.

Recombinant prohibitin protein of Leishmania infantum acts as a vaccine candidate and diagnostic marker against visceral leishmaniasis.

Visceral leishmaniasis (VL) represents a serious public health problem, as Leishmania infantum is one of main disease causative agents in the Americas. In a previous immunoproteomic study, the prohibitin (PHB) protein was identified in L. infantum promastigote and amastigote extracts by antibodies in asymptomatic and symptomatic VL dog sera. This protein was found to be highly conserved between different Leishmania spp., but it presented a low identity with amino acid sequences of other organisms. The aim of the present study was to evaluate the cellular response induced by the recombinant PHB (rPHB) protein in BALB/c mice, as well as in PBMCs purified from untreated and treated VL patients, as well as to evaluate its protective efficacy against an infection by L. infantum promastigotes. Our data showed that there was a Th1 cellular response to rPHB, based on high levels of IFN-γ, IL-12, and GM-CSF in the immunized animals, as well as a proliferative response specific to the protein and higher IFN-γ levels induced in PBMCs from individuals who had recovered from the disease. The protection was represented by significant reductions in the parasite load in the animals' spleen, liver, bone marrow, and draining lymph nodes, as compared to results found in the control groups. In addition, an anti-rPHB serology, using a canine and human serological panel, showed a high performance of this protein when diagnosing VL based on high sensitivity and specificity values, as compared to results found for the rA2 antigen and the soluble Leishmania antigenic extract. Our data suggest that PHB has a potential application for the diagnosis of canine and human VL through antibody detection, as well as an application as a vaccine candidate to protect against disease.

Small Myristoylated Protein-3, Identified as a Potential Virulence Factor in Leishmania amazonensis, Proves to be a Protective Antigen against Visceral Leishmaniasis.

In a proteomics approach conducted with Leishmania amazonensis, parasite proteins showed either an increase or a decrease in their expression content during extensive in vitro cultivation, and were related to the survival and the infectivity of the parasites, respectively. In the current study, a computational screening was performed to predict virulence factors among these molecules. Three proteins were selected, one of which presented no homology to human proteins. This candidate, namely small myristoylated protein-3 (SMP-3), was cloned, and its recombinant version (rSMP-3) was used to stimulate peripheral blood mononuclear cells (PBMCs) from healthy subjects living in an endemic area of leishmaniasis and from visceral leishmaniasis patients. Results showed high interferon-γ (IFN-γ) production and low levels of interleukin 10 (IL-10) in the cell supernatants. An in vivo experiment was then conducted on BALB/c mice, which were immunized with rSMP-3/saponin and later challenged with Leishmania infantum promastigotes. The rSMP-3/saponin combination induced high production of protein-specific IFN-γ, IL-12, and granulocyte-macrophage colony-stimulating factor (GM-CSF) by the spleen cells of the immunized mice. This pattern was associated with protection, which was characterized by a significant reduction in the parasite load in distinct organs of the animals. Altogether, these results have revealed that this new virulence factor is immunogenic in both mice and humans, and have proven its protective efficacy against visceral leishmaniasis in a murine model.

Antifungal activities against toxigenic Fusarium specie and deoxynivalenol adsorption capacity of ion-exchanged zeolites.

Zeolites are often used as adsorbents materials and their loaded cations can be exchanged with metal ions in order to add antimicrobial properties. The aim of this study was to use the 4A zeolite and its derived ion-exchanged forms with Zn2+, Li+, Cu2+ and Co2+ in order to evaluate their antifungal properties against Fusarium graminearum, including their capacity in terms of metal ions release, conidia germination and the deoxynivalenol (DON) adsorption. The zeolites ion-exchanged with Li+, Cu2+, and Co2+ showed an excellent antifungal activity against F. graminearum, using an agar diffusion method, with a zone of inhibition observed around the samples of 45.3 ± 0.6 mm, 25.7 ± 1.5 mm, and 24.7 ± 0.6 mm, respectively. Similar results using agar dilution method were found showing significant growth inhibition of F. graminearum for ion-exchanged zeolites with Zn2+, Li+, Cu2+, and Co2+. The fungi growth inhibition decreased as zeolite-Cu2+>zeolite-Li+>zeolite-Co2+>zeolite-Zn2+. In addition, the conidia germination was strongly affected by ion-exchanged zeolites. With regard to adsorption capacity, results indicate that only zeolite-Li+ were capable of DON adsorption significantly (P < 0.001) with 37% at 2 mg mL-1 concentration. The antifungal effects of the ion-exchanged zeolites can be ascribed to the interactions of the metal ions released from the zeolite structure, especially for zeolite-Li+, which showed to be a promising agent against F. graminearum and its toxin.

Antigenicity of phage clones and their synthetic peptides for the serodiagnosis of canine and human visceral leishmaniasis.

In the Americas, Brazil is responsible by 90% of the cases registered of visceral leishmaniasis (VL), and Leishmania infantum is the most common parasite species responsible by disease in Brazilian dogs and humans. A precise diagnosis may allow to a faster and more effective treatment against the disease, which increases the possibility of cure, as well as to induce less toxic effects, due to a lower time exposition for the chemotherapeutics. In a previous study, two L. infantum mimotopes, B10 and C01 clones, were recognized by antibodies in VL dogs sera by a phage display technology, and were well-successfully evaluated as vaccine candidates against visceral and tegumentary leishmaniasis. In the present work, the diagnostic efficacy of these clones, as well as of their exogenous peptides (B10: LSFPFPG and C01: FTSFSPY), was evaluated to diagnose canine and human VL. ELISA assays were performed with the four antigens, and results showed that both clones, as well as their synthetic peptides; showed high sensitivity and specificity values to identify VL samples, presenting an excellent performance to serologically diagnose VL-developing humans and dogs. On the other hand, a wild-type phage, a random non-specific clone and a L. infantum antigenic preparation were used as controls, and showed worst sensitivity and specificity results. In conclusion, besides their biological action as vaccine, B10 and C01 phages and their synthetic peptides could be considered as new markers for the serodiagnosis of canine and human VL.