RESUMEN

The aim of this clinical, prospective, randomized, and parallel study was to evaluate different in-office treatments for dentin hypersensitivity (DH). One hundred ninety-two teeth with non-cavitated root exposures were treated using different desensitizers: fluoride varnish (Duraphat - FLU); bioactive ceramic solution (Biosilicate - BIOS); universal self-etching adhesive (Single Bond Universal - SBU); bioactive photoactivated varnish (PRG filler - SPRG). The degree of DH was analyzed using a visual analog scale (VAS) and computerized visual scale (CoVAS), before treatments and after 7, 15, and 30 days from the first session. Comparisons among desensitizers were performed using the Kruskal-Wallis and Dunn's tests. Friedman test was used to compare between times (p ≤ 0.05). Comparing desensitizers FLU presented a higher value of DH than BIOS using VAS at 7 days, however, no differences were found using CoVAS analysis. Comparing times, BIOS and SBU showed a reduction in DH after 7 days and SBU showed a reduction at 30 days compared to 7 days using VAS. FLU and SPRG groups reduced DH from 15 days to 30 days using VAS. There was a reduction in DH for FLU, BIOS, and SBU after 7 days and for BIOS this reduction also occurred at 30 days when compared to 15 days using CoVAS. SPRG group showed a reduction from 15 to 30 days. All desensitizers tested were able to reduce the initial sensitivity. The bioactive ceramic solution reduced the DH gradually after 30 days using computerized analysis.

Asunto(s)

Desensibilizantes Dentinarios , Sensibilidad de la Dentina , Humanos , Sensibilidad de la Dentina/tratamiento farmacológico , Desensibilizantes Dentinarios/uso terapéutico , Masculino , Femenino , Estudios Prospectivos , Adulto , Resultado del Tratamiento , Persona de Mediana Edad , Fluoruros Tópicos

RESUMEN

The treatment for visceral leishmaniasis (VL) causes toxicity in patients, entails high cost and/or leads to the emergence of resistant strains. No human vaccine exists, and diagnosis presents problems related to the sensitivity or specificity of the tests. Here, we tested two phage clones, B1 and D11, which were shown to be protective against Leishmania infantum infection in a murine model as immunotherapeutics to treat mice infected with this parasite species. The phages were used alone or with amphotericin B (AmpB), while other mice received saline, AmpB, a wild-type phage (WTP) or WTP/AmpB. Results showed that the B1/AmpB and D11/AmpB combinations induced polarised Th1-type cellular and humoral responses, which were primed by high levels of parasite-specific IFN-γ, IL-12, TNF-α, nitrite and IgG2a antibodies, which reflected in significant reductions in the parasite load in distinct organs of the animals when analyses were performed 1 and 30 days after the treatments. Reduced organic toxicity was also found in these animals, as compared with the controls. In conclusion, preliminary data suggest the potential of the B1/AmpB and D11/AmpB combinations as immunotherapeutics against L. infantum infection.

Asunto(s)

Anfotericina B , Anticuerpos Antiprotozoarios , Inmunoterapia , Leishmania infantum , Leishmaniasis Visceral , Ratones Endogámicos BALB C , Leishmaniasis Visceral/inmunología , Leishmaniasis Visceral/tratamiento farmacológico , Animales , Anfotericina B/uso terapéutico , Anfotericina B/administración & dosificación , Anticuerpos Antiprotozoarios/sangre , Leishmania infantum/inmunología , Leishmania infantum/efectos de los fármacos , Ratones , Inmunoterapia/métodos , Femenino , Antiprotozoarios/uso terapéutico , Antiprotozoarios/administración & dosificación , Inmunoglobulina G/sangre , Carga de Parásitos , Modelos Animales de Enfermedad , Técnicas de Visualización de Superficie Celular , Citocinas/metabolismo , Células TH1/inmunología

RESUMEN

Serodiagnosis methods have been used as platforms for diagnostic tests for many diseases. Due to magnetic nanoparticles' properties to quickly detach from an external magnetic field and particle size effects, these nanomaterials' functionalization allows the specific isolation of target analytes, enhancing accuracy parameters and reducing serodiagnosis time. Superparamagnetic iron oxide nanoparticles (MNPs) were synthesized and functionalized with polyethylene glycol (PEG) and then associated with the synthetic Leishmaniosis epitope. This nano-peptide antigen showed promising results. Regarding Tegumentary leishmaniasis diagnostic accuracy, the AUC was 0.8398 with sensibility 75% (95CI% 50.50 - 89.82) and specificity 87.50% (95CI% 71.93 - 95.03), and Visceral leishmaniasis accuracy study also present high performance, the AUC was 0.9258 with sensibility 87.50% (95CI% 63.98 - 97.78) and specificity 87.50% (95CI% 71.93 - 95.03). Our results demonstrate that the association of the antigen with MNPs accelerates and improves the diagnosis process. MNPs could be an important tool for enhancing serodiagnosis.

Asunto(s)

Ensayo de Inmunoadsorción Enzimática , Polietilenglicoles , Sensibilidad y Especificidad , Humanos , Ensayo de Inmunoadsorción Enzimática/métodos , Polietilenglicoles/química , Antígenos de Protozoos/inmunología , Leishmaniasis/diagnóstico , Nanopartículas Magnéticas de Óxido de Hierro/química , Anticuerpos Antiprotozoarios/sangre

RESUMEN

BACKGROUND: This study aimed to evaluate dentin wear and biological performance of desensitizing materials. METHODS: Seventy bovine root dentin blocks were sectioned. Half of the surface of each specimen was untreated (control) and the other half was immersed in EDTA and treated with the following desensitizing materials: placebo varnish (PLA), fluoride varnish (FLU), sodium fluoride (NaF) varnish + sodium trimetaphosphate (TMP), universal adhesive (SBU), S-PRG varnish (SPRG), biosilicate (BIOS), and amelotin solution (AMTN). After application, the specimens were submitted to an erosive-abrasive challenge and the wear analyzed by optical profilometer. Serial dilutions of extracts obtained from the culture medium containing discs impregnated with those desensitizers were applied on fibroblasts and odontoblasts-like cells cultures. Cytotoxicity and production of total protein (TP) by colorimetric assays were determined after 24 h. Data were statistically analyzed using Kruskal-Wallis, Dunn's, One-way ANOVA and Tukey tests (p ≤ 0.05). RESULTS: No dentin wear was observed only for SBU. The lowest dentin wear was observed for AMTN and TMP. Cell viability was significantly reduced after treatment with undiluted extracts of PLA, FLU, TMP and SBU in fibroblasts and TMP and SBU in odontoblast-like cells. SPRG, BIOS and AMTN were cytocompatible at all dilutions tested. Considering TP results, no statistical difference was observed among the groups and high levels for TP were observed after TMP and FLU treatments. CONCLUSIONS: Universal adhesive system may protect dentin with opened tubules from wear after challenge. Extracts of adhesive and fluoride varnishes presented cytotoxic mainly on fibroblasts. The enamel protein may be a future alternative to treat dentin with opened tubules because it may cause low wear under erosive-abrasive challenge with low cytotoxic effects.

Asunto(s)

Desensibilizantes Dentinarios , Dentina , Fluoruro de Sodio , Animales , Bovinos , Desensibilizantes Dentinarios/farmacología , Fluoruro de Sodio/farmacología , Dentina/efectos de los fármacos , Fluoruros Tópicos/farmacología , Fibroblastos/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Desgaste de los Dientes , Ensayo de Materiales , Polifosfatos/farmacología

RESUMEN

Treatment against leishmaniasis presents problems, mainly due to the toxicity of the drugs, high cost, and the emergence of resistant strains. A previous study showed that two vanillin-derived synthetic molecules, 3s [4-(2-hydroxy-3-(4-octyl-1H-1,2,3-triazol-1-yl)propoxy)-3-methoxybenzaldehyde] and 3t [4-(3-(4-decyl-1H-1,2,3-triazol-1-yl)-2-hydroxypropoxy)-3-methoxybenzaldehyde], presented antileishmanial activity against Leishmania infantum, L. amazonensis, and L. braziliensis species. In the present work, 3s and 3t were evaluated to treat L. amazonensis-infected mice. Molecules were used pure or incorporated into Poloxamer 407-based micelles. In addition, amphotericin B (AmpB) and its liposomal formulation, Ambisome®, were used as control. Animals received the treatment and, one and 30 days after, they were euthanized to evaluate immunological, parasitological, and biochemical parameters. Results showed that the micellar compositions (3s/Mic and 3t/Mic) induced significant reductions in the lesion mean diameter and parasite load in the infected tissue and distinct organs, as well as a specific and significant antileishmanial Th1-type immune response, which was based on significantly higher levels of IFN-γ, IL-12, nitrite, and IgG2a isotype antibodies. Drug controls showed also antileishmanial action; although 3s/Mic and 3t/Mic have presented better and more significant parasitological and immunological data, which were based on significantly higher IFN-γ production and lower parasite burden in treated animals. In addition, significantly lower levels of urea, creatinine, alanine transaminase, and aspartate transaminase were found in mice treated with 3s/Mic and 3t/Mic, when compared to the others. In conclusion, results suggest that 3s/Mic and 3t/Mic could be considered as therapeutic candidates to treat against L. amazonensis infection.

Asunto(s)

Antiprotozoarios , Benzaldehídos , Leishmania mexicana , Ratones Endogámicos BALB C , Micelas , Animales , Ratones , Benzaldehídos/farmacología , Benzaldehídos/química , Leishmania mexicana/efectos de los fármacos , Antiprotozoarios/farmacología , Antiprotozoarios/uso terapéutico , Antiprotozoarios/química , Leishmaniasis Cutánea/tratamiento farmacológico , Femenino , Anfotericina B/farmacología , Anfotericina B/uso terapéutico , Poloxámero/química , Poloxámero/farmacología , Masculino , Bazo/parasitología

RESUMEN

OBJECTIVES: This laboratory study evaluated the influence of the fluorescence intensity of composite resins on additional tooth wear and the presence of restorative material in different dental thirds during the retreatment of direct veneers. MATERIALS AND METHODS: The crown dimensions of 60 bovine incisors were reduced to 10 × 8 mm. The teeth were classified according to the fluorescence intensity of the composites: low (LOW) (TPH Spectra), medium (MED) (Opallis), and high (HIGH) (Essentia) groups. The teeth were divided according to the removal methods: conventional (CON) and fluorescence-aided identification technique (FIT). The specimens were scanned (T0), received veneer preparation, and scanned again (T1). After restorations, the composites were removed and the teeth were scanned (T2). Measurement assessments between T1 and T2 were performed to determine additional wear, presence of residual areas, and the average between additional wear and the presence of residual areas. Kruskal Wallis, Mann-Whitney, Friedman, 2-way ANOVA, and post-Tukey tests were performed (α < 0.05). RESULTS: The comparison of composite resins indicated a smaller area of additional wear and greater residue presence in the HIGH group than the LOW group for both techniques in the cervical third. Regarding removal methods, the FIT produced greater additional wear than the CON method for the LOW and MED groups in the middle and cervical thirds. The incisal third exhibited greater additional wear than the other thirds. CONCLUSIONS: Composite resins with high fluorescence intensity removed using FIT had less tooth wear. The incisal third was the most affected area for direct veneer removal procedures. CLINICAL SIGNIFICANCE: A FIT has been proposed for composite resin removal; however, the different fluorescence intensities of composite resins can influence tooth wear caused during this procedure.

Asunto(s)

Resinas Compuestas , Coronas con Frente Estético , Resinas Compuestas/química , Bovinos , Fluorescencia , Animales

RESUMEN

This study aimed to evaluate the interface of universal adhesive (UA) with bulk-fill resin (BF) and dentin, after endodontic irrigation. The pulp chamber of 96 molars was irrigated with chlorhexidine 2% (CHX) or sodium hypochlorite 5.25% (NaOCl) and restored according to three restorative protocols. Microtensile bond strength (µTBS) and nanoleakage tests were performed after 24 h of thermocycling. Kruskal Wallis, Mann-Whitney and Wilcoxon were performed (p < 0.05). Comparing restorative systems, a difference was found only when irrigated with CHX after 24 h. CHX provided higher µTBS than NaOCl after 24 h for two restorative protocols; after thermocycling, NaOCl provided the greatest µTBS than CHX for one group. Decreased µTBS was observed for all restorative systems when CHX was used after thermocycling. NaOCl demonstrated the highest nanoleakage. The composition of UA influenced the immediate adhesion and sealing to BF when CHX was used; ageing has a deteriorating effect on adhesion and sealing for all restorative systems.

Asunto(s)

Recubrimiento Dental Adhesivo , Humanos , Técnicas In Vitro , Recubrimiento Dental Adhesivo/métodos , Irrigantes del Conducto Radicular/química , Hipoclorito de Sodio/química , Resistencia a la Tracción , Filtración Dental , Resinas Compuestas/química , Clorhexidina/farmacología , Clorhexidina/química , Cementos de Resina/química , Ensayo de Materiales , Cementos Dentales/química

RESUMEN

Treatment against visceral leishmaniasis (VL) presents problems, mainly related to drug toxicity, high cost and/or by emergence of resistant strains. In the present study, two vanillin synthetic derivatives, 3 s [4-(2-hydroxy-3-(4-octyl-1H-1,2,3-triazol-1-yl)propoxy)-3-methoxybenzaldehyde] and 3 t [4-(3-(4-decyl-1H-1,2,3-triazol-1-yl)-2-hydroxypropoxy)-3-methoxybenzaldehyde], were evaluated as therapeutic candidates in a murine model against Leishmania infantum infection. Molecules were used pure (3 s and 3 t) or incorporated into Poloxamer 407-based micelles (3 s/M and 3 t/M) in the infected animals, which also received amphotericin B (AmpB) or Ambisome® as control. Results showed that 3 s/M and 3 t/M compositions induced a Th1-type immune response in treated animals, with higher levels of IFN-γ, IL-2, TNF-α, IL-12, nitrite, and IgG2a antibodies. Animals presented also low toxicity and significant reductions in the parasite load in their spleens, livers, bone marrows and draining lymph nodes, as compared as control groups mice, with the evaluations performed one and 30 days after the application of the therapeutics. In conclusion, preliminary data suggest that 3 s/M and 3 t/M could be considered for future studies as therapeutic agents against VL.

Asunto(s)

Benzaldehídos , Leishmaniasis Visceral , Leishmaniasis , Ratones , Animales , Micelas , Interleucina-12 , Ratones Endogámicos BALB C

RESUMEN

The problems with current strategies to control canine visceral Leishmaniasis (CVL), which include the euthanasia ofinfected animals, and also the toxicity of the drugs currently used in human treatments for CVL, add urgency to the search for newtherapeutic agents. This study aimed to evaluate the activity against Leishmania (L.) infantum of 12 amides that are chemicallyinspired by gibbilimbol B, a bioactive natural product that was initially obtained from Piper malacophyllum. Three of thesecompounds N-(2-ethylhexyl)-4-chlorobenzamide (9), N-(2-ethylhexyl)-4-nitrobenzamide (10), and N-(2-ethylhexyl)-4-(tert-butyl)benzamide (12) demonstrated activity against the intracellular amastigotes without toxicity to mammalian host cells (CC50> 200 μM); compounds 9, 10, and 12 resulted in EC50 values of 12.7, 12.2, and 5.1 μM, respectively. In silico drug-likeness studiespredicted that these compounds would show high levels of gastrointestinal absorption, would be able to penetrate the blood-brainbarrier, would show moderate solubility, and would not show unwanted molecular interactions. Due to their promisingpharmacological profiles, compounds 9 and 10 were selected for mechanism of action studies (MoA). The MoA studies in L. (L.)infantum revealed that neither of the compounds affected the permeabilization of the plasma membrane. Nevertheless, compound 9induced strong alkalinization of acidocalcisomes, which resulted in a significant and rapid increase in intracellular Ca2+ levels, therebycausing the depolarization of the mitochondrial membrane potential and a reduction in the levels of reactive oxygen species (ROS).In contrast, compound 10 induced a gradual increase in intracellular Ca2+ levels and a similarly gradual reduction in ROS levels, butit caused neither acidocalcisome alkalinization nor mitochondrial membrane potential depolarization. Finally, the MALDI-TOF/MSassessment of protein alterations in L. (L.) infantum treated separately with compounds 9 and 10 revealed changes in mass spectralprofiles from both treatments. These results highlight the anti-L. (L.) infantum potential of these amides especially for compounds9 and 10 and they suggest that these compounds could be promising candidates for future in vivo studies in VL-models.

RESUMEN

Abstract The aim of this clinical, prospective, randomized, and parallel study was to evaluate different in-office treatments for dentin hypersensitivity (DH). One hundred ninety-two teeth with non-cavitated root exposures were treated using different desensitizers: fluoride varnish (Duraphat - FLU); bioactive ceramic solution (Biosilicate - BIOS); universal self-etching adhesive (Single Bond Universal - SBU); bioactive photoactivated varnish (PRG filler - SPRG). The degree of DH was analyzed using a visual analog scale (VAS) and computerized visual scale (CoVAS), before treatments and after 7, 15, and 30 days from the first session. Comparisons among desensitizers were performed using the Kruskal-Wallis and Dunn's tests. Friedman test was used to compare between times (p ≤ 0.05). Comparing desensitizers FLU presented a higher value of DH than BIOS using VAS at 7 days, however, no differences were found using CoVAS analysis. Comparing times, BIOS and SBU showed a reduction in DH after 7 days and SBU showed a reduction at 30 days compared to 7 days using VAS. FLU and SPRG groups reduced DH from 15 days to 30 days using VAS. There was a reduction in DH for FLU, BIOS, and SBU after 7 days and for BIOS this reduction also occurred at 30 days when compared to 15 days using CoVAS. SPRG group showed a reduction from 15 to 30 days. All desensitizers tested were able to reduce the initial sensitivity. The bioactive ceramic solution reduced the DH gradually after 30 days using computerized analysis.

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Resumo O objetivo deste estudo clínico, prospectivo, randomizado e paralelo foi avaliar diferentes tratamentos em consultório para hipersensibilidade dentinária (HD). Cento e noventa e dois dentes com exposições radiculares não cavitadas foram tratados com diferentes dessensibilizantes: verniz fluoretado (Duraphat - FLU); solução cerâmica bioativa (Biosilicato - BIOS); adesivo autocondicionante universal (Single Bond Universal - SBU); verniz fotoativado bioativo (PRG filler - SPRG). O grau de HD foi analisado por meio da escala visual analógica (VAS) e da escala visual computadorizada (CoVAS), antes dos tratamentos e após 7, 15 e 30 dias da primeira sessão. As comparações entre dessensibilizantes foram feitas pelos testes de Kruskal-Wallis e Dunn. O teste de Friedman foi utilizado para comparação entre os tempos (p ≤ 0,05). Comparando os dessensibilizantes, o FLU apresentou um valor de HD maior do que o BIOS usando VAS aos 7 dias, no entanto, nenhuma diferença foi encontrada usando a análise CoVAS. Comparando os tempos, BIOS e SBU apresentaram redução de HD após 7 dias e SBU apresentou redução aos 30 dias em comparação com 7 dias usando VAS. Os grupos FLU e SPRG reduziram a HD em 15 dias a 30 dias usando VAS. Houve redução de HD para FLU, BIOS e SBU após 7 dias e para BIOS essa redução também ocorreu aos 30 dias quando comparada a 15 dias usando CoVAS. O grupo SPRG apresentou redução de 15 para 30 dias. Todos os dessensibilizantes testados foram capazes de reduzir a sensibilidade inicial. A solução de cerâmica bioativa reduziu o HD gradualmente após 30 dias usando análise computadorizada.

RESUMEN

Introdução: na atenção primária à saúde, a atuação de médicos generalistas é importante no rastreamento e no acompanhamento dos estágios iniciais da doença renal crônica devido à alta prevalência, à falta de especialistas, ao impacto na morbimortalidade e qualidade de vida e ao elevado custo aos sistemas de saúde. O uso de aplicativos móveis tornou-se importante na medicina contemporânea por facilitar o acesso à informação, o que otimiza a prestação de cuidados à saúde.Objetivos: desenvolver e validar um aplicativo móvel sobre testes laboratoriais na doença renal crônica para médicos generalistas. Métodos: estudo na modalidade de produção tecnológica, com desenvolvimento de software a partir de revisão da literatura, com validação por especialistas (nefrologistas) e por médicos generalistas. A validação se deu através de respostas a questionários, que avaliaram conteúdo, compreensão, linguagem e apresentação gráfica do aplicativo. Para a análise da consistência interna dos questionários foi utilizado o coeficiente alfa de Cronbach e para avaliar a concordância dos médicos sobre aspectos do aplicativo utilizou-se o Índice de Validade de Conteúdo. O valor adotado para o Índice de Validade de Conteúdo foi maior do que 80%. Resultados: as respostas dos nefrologistas (n=9) e dos médicos generalistas (n=13) foram, respectivamente, medianas de idade de 40 (35-71) anos e 45 (30-50) anos; sexo feminino 5 (55%) e 7 (54%); coeficiente alfa de Cronbach de 0,8025 e 0,9145; Índice de Validade de Conteúdo Global de 95,6% e 92,1%. Conclusão: a confiabilidade das respostas aos questionários mostrou-se boa e houve excelente concordância entre os juízes sobre os aspectos do aplicativo móvel

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Introduction: in primary health care, the performance of general practitioners is important in tracking and monitoring the early stages of chronic kidney disease, due to the high prevalence, lack of specialists, impact on morbidity and quality of life and the high cost to health systems. The use of mobile applications has become important in contemporary medicine because it facilitates access to information, which optimizes the provision of health care. Objectives: to develop and validate a mobile application on laboratory tests in chronic kidney disease, for general practitioners. Methods: study in the technological production modality, with software development based on a literature review, with validation by specialists (nephrologists) and by general practitioners. Validation took place through answers to questionnaires, which evaluated content, understanding, language and graphic presentation of the mobile application. Cronbach's alpha coefficient was used to analyze the internal consistency of the questionnaires, and the Content Validity Index was used to assess physicians' agreement on aspects of the app. The value adopted for the mobile application was greater than 80%. Results: the responses of nephrologists (n=9) and general practitioners (n=13) were, respectively, median age 40 (35-71) years and 45 (30-50) years, 5 (55%) and 7 (54%) were female, Cronbach's alpha coefficient of 0.8025 and 0.9145, Global Content Validity Index of 95.56% and 92.13%. Conclusion: the reliability of the responses to the questionnaires was good and there was excellent agreement between the judges on aspects of the mobile application

RESUMEN

Tegumentary leishmaniasis (TL) is the main clinical manifestation of leishmaniasis, and it can cause the infected hosts to self-healing cutaneous lesions until mutilating scars in mucosal membranes, particularly in the nose and throat. The treatment against disease presents problems, and the diagnosis is hampered by variable sensitivity and/or specificity of the tests. In this context, the development of prophylactic vaccines could be considered as a strategy to control the disease. Previously, we showed that the recombinant LiHyp1 protein plus adjuvant protected mice from infection with Leishmania infantum, which causes visceral leishmaniasis. In the present study, we tested whether rLiHyp1 could induce protection against infection with L. amazonensis, a parasite species able to cause TL. We immunized BALB/c mice with rLiHyp1 plus saponin (rLiHyp1/S) or incorporated in micelles (rLiHyp1/M) as adjuvants and performed parasitological and immunological evaluations before and after infection. Results showed that after in vitro stimulation from spleen cell cultures using rLiHyp1 or a Leishmania antigenic extract (SLA), rLiHyp1/S and rLiHyp1/M groups developed a Th1-type immune response, which was characterized by high levels of IFN-γ, IL-2, TNF-α and IL-12 cytokines, nitrite, and IgG2a isotype antibodies when compared to values found in the control (saline, saponin, micelles alone) groups, which showed higher levels of anti-SLA IL-4, IL-10, and IgG1 antibodies before and after challenge. In addition, mice receiving rLiHyp1/S or rLiHyp1/M presented significant reductions in the lesion average diameter and parasite load in the infected tissue and internal organs. Blood samples were collected from healthy subjects and TL patients to obtain PBMC cultures, which were in vitro stimulated with rLiHyp1 or SLA, and results showed higher lymphoproliferation and IFN-γ production after stimulus using rLiHyp1, as compared to values found using SLA. These results suggest that rLiHyp1 plus adjuvant was protective against experimental TL and could also be considered for future studies as a vaccine candidate against human disease.

Asunto(s)

Leishmania infantum , Leishmaniasis Visceral , Leishmaniasis , Saponinas , Humanos , Animales , Ratones , Micelas , Leucocitos Mononucleares/metabolismo , Proteínas Recombinantes , Leishmaniasis Visceral/parasitología , Adyuvantes Inmunológicos , Citocinas/metabolismo , Vacunación , Ratones Endogámicos BALB C , Antígenos de Protozoos/genética

RESUMEN

The impact of the COVID-19 pandemic caused by the SARS-CoV-2 virus underscored the crucial role of laboratorial tests as a strategy to control the disease, mainly to indicate the presence of specific antibodies in human samples from infected patients. Therefore, suitable recombinant antigens are relevant for the development of reliable tests, and so far, single recombinant proteins have been used. In this context, B-cell epitopes-based chimeric proteins can be an alternative to obtain tests with high accuracy through easier and cheaper production. The present study used bioinformatics tools to select specific B-cell epitopes from the spike (S) and the nucleocapsid (N) proteins from the SARS-CoV-2 virus, aiming to produce a novel recombinant chimeric antigen (N4S11-SC2). Eleven S and four N-derived B-cell epitopes were predicted and used to construct the N4S11-SC2 protein, which was analyzed in a recombinant format against serum and urine samples, by means of an in house-ELISA. Specific antibodies were detected in the serum and urine samples of COVID-19 patients, which were previously confirmed by qRT-PCR. Results showed that N4S11-SC2 presented 83.7% sensitivity and 100% specificity when using sera samples, and 91.1% sensitivity and 100% specificity using urine samples. Comparable findings were achieved with paired urine samples when compared to N and S recombinant proteins expressed in prokaryotic systems. However, better results were reached for N4S11-SC2 in comparison to the S recombinant protein when using paired serum samples. Anti-N4S11-SC2 antibodies were not clearly identified in Janssen Ad26.COV2.S COVID-19-vaccinated subjects, using serum or paired urine samples. In conclusion, this study presents a new chimeric recombinant antigen expressed in a prokaryotic system that could be considered as an alternative diagnostic marker for the SARS-CoV-2 infection, with the potential benefits to be used on serum or urine from infected patients.

Asunto(s)

COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , Epítopos de Linfocito B , Ad26COVS1 , Pandemias , COVID-19/diagnóstico , Proteínas Recombinantes/genética , Proteínas Recombinantes de Fusión/genética

RESUMEN

Treatment of visceral leishmaniasis (VL) is compromised by drug toxicity, high cost and/or the emergence of resistant strains. Though canine vaccines are available, there are no licensed prophylactic human vaccines. One strategy to improve clinical outcome for infected patients is immunotherapy, which associates a chemotherapy that acts directly to reduce parasitism and the administration of an immunogen-adjuvant that activates the host protective Th1-type immune response. In this study, we evaluated an immunotherapy protocol in a murine model by combining recombinant (r)LiHyp1 (a hypothetical amastigote-specific Leishmania protein protective against Leishmania infantum infection), with monophosphoryl-lipid A (MPLA) as adjuvant and amphotericin B (AmpB) as reference antileishmanial drug. We used this protocol to treat L. infantum infected-BALB/c mice, and parasitological, immunological and toxicological evaluations were performed at 1 and 30 days after treatment. Results showed that mice treated with rLiHyp1/MPLA/AmpB presented the lowest parasite burden in all organs evaluated, when both a limiting dilution technique and qPCR were used. In addition, these animals produced higher levels of IFN-γ and IL-12 cytokines and IgG2a isotype antibody, which were associated with lower production of IL-4 and IL-10 and IgG1 isotype. Furthermore, low levels of renal and hepatic damage markers were found in animals treated with rLiHyp1/MPLA/AmpB possibly reflecting the lower parasite load, as compared to the other groups. We conclude that the rLiHyp1/MPLA/AmpB combination could be considered in future studies as an immunotherapy protocol to treat against VL.

Asunto(s)

Adyuvantes Inmunológicos , Amebicidas , Anfotericina B , Leishmaniasis Visceral , Lípido A , Proteínas Protozoarias , Leishmaniasis Visceral/terapia , Animales , Ratones , Anfotericina B/uso terapéutico , Amebicidas/uso terapéutico , Inmunoterapia , Adyuvantes Inmunológicos/uso terapéutico , Ratones Endogámicos BALB C , Modelos Animales de Enfermedad , Proteínas Recombinantes/uso terapéutico , Proteínas Protozoarias/uso terapéutico , Quimioterapia Combinada , Lípido A/uso terapéutico , Protocolos Clínicos , Femenino

RESUMEN

Visceral leishmaniasis (VL) in the Americas is a chronic systemic disease caused by infection with Leishmania infantum parasites. The toxicity of antileishmanial drugs, long treatment course and limited efficacy are significant concerns that hamper adequate treatment against the disease. Studies have shown the promise of an immunotherapeutics approach, combining antileishmanial drugs to reduce the parasitism and vaccine immunogens to activate the host immune system. In the current study, we developed an immunotherapy using a recombinant T cell epitope-based chimeric protein, ChimT, previously shown to be protective against Leishmania infantum, with the adjuvant monophosphoryl lipid A (MPLA) and amphotericin B (AmpB) as the antileishmanial drug. BALB/c mice were infected with L. infantum stationary promastigotes and later they received saline or were treated with AmpB, MPLA, ChimT/Amp, ChimT/MPLA or ChimT/MPLA/AmpB. The combination of ChimT/MPLA/AmpB significantly reduced the parasite load in mouse organs (p < 0.05) and induced a Th1-type immune response, which was characterized by higher ratios of anti-ChimT and anti-parasite IgG2a:IgG1 antibodies, increased IFN-γ mRNA and IFN-γ and IL-12 cytokines and accompanied by lower levels of IL-4 and IL-10 cytokines, when compared to other treatments and controls (all p < 0.05). Organ toxicity was also lower with the ChimT/MPLA/AmpB immunotherapy, suggesting that the inclusion of the vaccine and adjuvant ameliorated the toxicity of AmpB to some degree. In addition, the ChimT vaccine alone stimulated in vitro murine macrophages to significantly kill three different internalized species of Leishmania parasites and to produce Th1-type cytokines into the culture supernatants. To conclude, our data suggest that the combination of ChimT/MPLA/AmpB could be considered for further studies as an immunotherapy for L. infantum infection.

RESUMEN

The treatment against leishmaniasis presents problems, mainly due to their toxicity of the drugs, high cost and/or by the emergence of parasite resistant strains. In this context, new therapeutics should be searched. In this study, two novel synthetic derivatives from vanillin: [4-(2-hydroxy-3-(4-octyl-1H-1,2,3-triazol-1-yl)propoxy)-3-methoxybenzaldehyde] or 3s and [4-(3-(4-decyl-1H-1,2,3-triazol-1-yl)-2-hydroxypropoxy)-3-methoxybenzaldehyde] or 3t, were evaluated regarding their antileishmanial activity against distinct parasite species able to cause cutaneous and visceral leishmaniasis. Results showed that compounds 3s and 3t were effective against Leishmania infantum, L. amazonensis and L. braziliensis promastigote and amastigote-like forms, showing selectivity index (SI) of 25.1, 18.2 and 22.9, respectively, when 3s was used against promastigotes, and of 45.2, 7.5 and 15.0, respectively, against amastigote-like stage. Using the compound 3t, SI values were 45.2, 53.0 and 80.0, respectively, against promastigotes, and of 35.9, 46.0 and 58.4, respectively, against amastigote-like forms. Amphotericin B (AmpB) showed SI values of 5.0, 7.5 and 15.0, respectively, against promastigotes, and of 3.8, 5.0 and 7.5, respectively, against amastigote-like stage. The treatment of infected macrophages and inhibition of the infection upon pre-incubation with the molecules showed that they were effective in reducing the infection degree and inhibiting the infection in pre-incubated parasites, respectively, as compared to data obtained using AmpB. The mechanism of action of 3s and 3t was evaluated in L. infantum, revealing that both 3s and 3t altered the parasite mitochondrial membrane potential leading to reactive oxygen species production, increase in lipid corps and changes in the cell cycle, causing the parasite' death. A preliminary assay using the cell culture supernatant from treated and infected macrophages showed that 3s and 3t induced higher IL-12 and lower IL-10 values; suggesting the development of an in vitro Th1-type response in the treated cells. In this context, data indicated that 3s and 3t could be considered therapeutic agents to be tested in future studies against leishmaniasis.

Asunto(s)

Antiprotozoarios , Leishmania infantum , Leishmaniasis Visceral , Leishmaniasis , Animales , Ratones , Leishmaniasis Visceral/tratamiento farmacológico , Leishmaniasis Visceral/parasitología , Antiprotozoarios/toxicidad , Antiprotozoarios/uso terapéutico , Anfotericina B/toxicidad , Anfotericina B/uso terapéutico , Leishmaniasis/tratamiento farmacológico , Ratones Endogámicos BALB C

RESUMEN

Serological assays have been widely used to detect anti-SARS-CoV-2 antibodies, which are generated from previous exposure to the virus or after vaccination. The presence of anti-SARS-CoV-2 Nucleocapsid antibodies was recently reported in patients´ urine using an in-house urine-based ELISA-platform, allowing a non-invasive way to collect clinical samples and assess immune conversion. In the current study, we evaluated and validated another in-house urine-based ELISA for the detection of anti-SARS-CoV-2 Spike antibodies. Three partial recombinant SARS-CoV-2 Spike proteins comprising the Receptor Binding Domain, expressed in eukaryotic or prokaryotic systems, were tested in an ELISA platform against a panel of over 140 urine and paired serum samples collected from 106 patients confirmed positive for SARS-CoV-2 by qRT-PCR. The key findings from our study were that anti-SARS-CoV-2 Spike antibodies could be detected in urine samples and that the prokaryotic expression of the rSARS-CoV-2 Spike protein was not a barrier to obtain relatively high serology efficiency for the urine-based assay. Thus, use of a urine-based ELISA assay with partial rSARS-CoV-2 Spike proteins, expressed in a prokaryotic system, could be considered as a convenient tool for screening for the presence of anti-SARS-CoV-2 Spike antibodies, and overcome the difficulties arising from sample collection and the need for recombinant proteins produced with eukaryotic expression systems.

Asunto(s)

COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , COVID-19/diagnóstico , Glicoproteína de la Espiga del Coronavirus/genética , Anticuerpos Antivirales , Ensayo de Inmunoadsorción Enzimática , Sensibilidad y Especificidad

RESUMEN

Vaccination against visceral leishmaniasis (VL) should be considered as a safe and effective measure to disease control; however, few vaccines are available against canine VL and there is no an approved human vaccine. In this context, in the present study, we evaluated the endonuclease III (ENDO) protein, which was recently showed to be antigenic for human disease, as a vaccine candidate against Leishmania infantum infection. The recombinant protein (rENDO) was administered in BALB/c mice alone or associated with saponin (rENDO/Sap) or micelles (rENDO/Mic) as adjuvants. Controls received saline, saponin or empty micelles. Results showed that both rENDO/Sap and rENDO/Mic compositions induced higher levels of IFN-γ, IL-12, TNF-α, and GM-CSF cytokines, besides nitrite and IgG2a isotype antibodies, before and after challenge infection, which were related to both CD4+ and CD8+ T cell subtypes. The immunological results contributed to significant reductions in the parasite load found in the spleens, livers, bone marrows and draining lymph nodes of the vaccinated animals. In general, mice immunized with rENDO/Mic presented a slightly higher Th1-type cellular and humoral immune response, as compared to those receiving rENDO/Sap. In addition, saponin caused a slight to moderate inflammatory edema in their vaccinated footpads, which was not observed when micelles were used with rENDO. In addition, a preliminary analysis showed that the recombinant protein was immunogenic to human cells cultures, since PBMCs from treated VL patients and healthy subjects showed higher lymphoproliferation and IFN-γ production in the culture supernatants. In conclusion, data suggest that rENDO could be considered as a candidate to be evaluated in future studies as vaccine to protect against VL.

Asunto(s)

Leishmania infantum , Vacunas contra la Leishmaniasis , Leishmaniasis Visceral , Leishmaniasis , Saponinas , Humanos , Animales , Perros , Ratones , Micelas , Proteínas Recombinantes , Leishmaniasis/prevención & control , Adyuvantes Inmunológicos , Ratones Endogámicos BALB C , Antígenos de Protozoos

RESUMEN

Leishmania amazonensis can cause a wide spectrum of the clinical manifestations of leishmaniasis in humans. The development of new therapeutics is a long and expensive task; in this context, drug repositioning could be considered a strategy to identify new biological actions of known products. In the present study, ivermectin (IVE) was tested against distinct Leishmania species able to cause disease in humans. In vitro experiments showed that IVE was effective to reduce the infection degree and parasite load in Leishmania donovani- and L. amazonensis-infected macrophages that were treated with it. In addition, using the culture supernatant of treated macrophages, higher production of IFN-γ and IL-12 and lower levels of IL-4 and IL-10 were found. Then, IVE was used in a pure form or incorporated into Poloxamer 407-based polymeric micelles (IVE/M) for the treatment of L. amazonensis-infected BALB/c mice. Animals (n = 16 per group) were infected and later received saline, empty micelles, amphotericin B (AmpB), IVE, or IVE/M. They were euthanized at one (n = 8 per group) and 30 (n = 8 per group) days after treatment and, in both endpoints, immunological, parasitological, and biochemical evaluations were performed. Results showed that both IVE and IVE/M induced higher levels of IFN-γ, IL-12, GM-CSF, nitrite, and IgG2a antibodies, as well as higher IFN-γ expression evaluated by RT-qPCR in spleen cell cultures. Such animals showed low organic toxicity, as well as significant reductions in the lesion's average diameter and parasite load in their infected tissue, spleen, liver, and draining lymph node. The efficacy was maintained 30 days post-therapy, while control mice developed a polarized Th2-type response and high parasite load. In this context, IVE could be considered as a new candidate to be applied in future studies for the treatment against distinct Leishmania species.

Asunto(s)

Antiprotozoarios , Leishmania , Leishmaniasis Visceral , Leishmaniasis , Humanos , Ratones , Animales , Micelas , Ivermectina/farmacología , Ivermectina/uso terapéutico , Antiprotozoarios/farmacología , Antiprotozoarios/uso terapéutico , Reposicionamiento de Medicamentos , Leishmaniasis/tratamiento farmacológico , Leishmaniasis/parasitología , Interleucina-12/farmacología , Ratones Endogámicos BALB C , Leishmaniasis Visceral/tratamiento farmacológico

RESUMEN

BACKGROUND: visceral leishmaniasis (VL) is a critical public health problem in over ninety countries. The control measures adopted in Brazil have been insufficient when it comes to preventing the spread of this overlooked disease. In this context, a precise diagnosis of VL in dogs and humans could help to reduce the number of cases of this disease. Distinct studies for the diagnosis of VL have used single recombinant proteins in serological assays; however, the results have been variable, mainly in relation to the sensitivity of the antigens. In this context, the development of multiepitope-based proteins could be relevant to solving such problem. METHODS: a chimeric protein (rMELEISH) was constructed based on amino acid sequences from kinesin 39 (k39), alpha-tubulin, and heat-shock proteins HSP70 and HSP 83.1, and tested in enzyme-linked immunosorbent (ELISA) for the detection of L. infantum infection using canine (n = 140) and human (n = 145) sera samples. RESULTS: in the trials, rMELEISH was able to discriminate between VL cases and cross-reactive diseases and healthy samples, with sensitivity and specificity values of 100%, as compared to the use of a soluble Leishmania antigenic extract (SLA). CONCLUSIONS: the preliminary data suggest that rMELEISH has the potential to be tested in future studies against a larger serological panel and in field conditions for the diagnosis of canine and human VL.