Accuracy improvement enzyme-linked immunosorbent assay using superparamagnetic/polyethylene glycol) nanoparticles for leishmaniasis diagnostic.

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.

Nuxcell Neo® improves vaccine efficacy in antibody response.

Current vaccination protocols raise concerns about the efficacy of immunization. There is evidence that changes in the gut microbiota can impact immune response. The formation of the gut microbiota in newborns plays a crucial role in immunity. Probiotic bacteria and prebiotics present important health-promoting and immunomodulatory properties. Thus, we hypothesize that pro and prebiotic supplementation can improve the efficacy of vaccination in newborns. In this protocol, newborn mice were used and treated with a single-dose rabies vaccine combined with Nuxcell Neo® (2 g/animal/week) for 3 weeks. Samples were collected on days 7, 14, and 21 after vaccination for analysis of cytokines and concentration of circulating antibodies. Our results show an increased concentration of antibodies in animals vaccinated against rabies and simultaneously treated with Nuxcell Neo® on days 14 and 21 when compared to the group receiving only the vaccine. In the cytokine levels analysis, it was possible to observe that there weren't relevant and significant changes between the groups, which demonstrates that the health of the animal remains stable. The results of our study confirm the promising impact of the use of Nuxcell Neo® on the immune response after vaccination.

The combined treatment of gold nanoparticles associated with photobiomodulation accelerate the healing of dermonecrotic lesion.

Introduction: The search for fast and efficient treatment for dermonecrotic lesions caused by the venom of the spider from the Loxosceles simillis, is a demand in health. Prednisolone is one of the most used drugs, however it has side effects. In this context, addictionally gold nanoparticles (GNPs) have anti-inflammatory, antioxidant, and antibacterial properties. The use of photobiomodulation has show to be efficient in the process of tissue repair. Therefore, the purpose of this study was to investigate the anti-inflammatory effect of photobiomodulation and GNPs associated or not with a low concentration of prednisolone in animal models of dermonecrotic lesion.Methodology: For this, rabbits with venon-induced dermonecrotic lesion were subjected to topical treatment with prednisolone + laser or GNPs + laser or Pred-GNPs + laser. The area of edema, necrosis and erythema were measured. On the last day of treatment, the animals were euthanized to remove the organs for histopathological and biochemical analysis.Results: All treatments combinations were effective in promoting the reduction of necrotic tissue and erythema.Conclusion: With this results, we suggest that the use of laser and nanoparticles, associated or not with prednisolone, should be considered for the treatment of dermonecrotic injury.

Immunoproteomics approach for the discovery of antigens applied to the diagnosis of canine visceral leishmaniasis.

In the present study, an immunoproteomic approach using Leishmania infantum parasites isolated from naturally infected dogs from an endemic region of the disease, was carried out to identify new antigens to be used in the diagnosis of canine visceral leishmaniasis (CVL). Protein extracts, obtained from parasites isolated from asymptomatic (CanLA) and symptomatic (CanLS) dogs, were used to perform the two-dimensional gels. Western Blotting assays were carried out by employing a pool of sera from dogs with visceral leishmaniasis (CanLA or CanLS), healthy dogs from an endemic area, or dogs with similar diseases associated with cross-reactions (babesiosis and ehrlichiosis). With these results, it was possible to exclude the spots that showed a cross-reactivity of the sera from groups of healthy dogs, and those with babesiosis or ehrlichiosis. Taken together, 20 proteins were identified, 15 of which have already been described in the literature and 5 of which are hypothetical. An immunogenomic screen strategy was applied to identify conserved linear B-cell epitopes in the identified hypothetical proteins. Two peptides were synthesized and tested in ELISA experiments as a proof of concept for the validation of our immunoproteomics findings. The results demonstrated that the antigens presented sensitivity and specificity values ranging from 81.93% to 97.59% and 78.14 to 85.12%, respectively. As a comparative antigen, a preparation of a Leishmania extract showed sensitivity and specificity values of 75.90% and 74.88%, respectively. The present study was able to identify proteins capable of being used for the serodiagnosis of canine visceral leishmaniasis.

Desenvolvimento e avaliação de uma proteína quimérica recombinante para o diagnóstico e prognóstico das leishmanioses tegumentar e visceral / Development and evaluation of a recombinant chimeric protein for the diagnosis and prognosis of tegumentary and visceral leishmaniasis

As leishmanioses são doenças tropicais negligenciadas com alta endemicidade e que afetam milhares de pessoas no mundo. Sua infecção é causada por parasitos protozoários do gênero Leishmania. A diversidade biológica entre as espécies é quem permite determinar as manifestações clínicas, sendo elas na forma de leishmaniose visceral (LV) ou leishmaniose tegumentar (LT). Dentre estas manifestações, a LV é considerada a mais grave, devido sua alta letalidade e grande emergência em indivíduos com a infecção provocada pelo vírus da imunodeficiência humana (HIV). Atualmente, as medidas de controle e prevenção adotadas pela Organização Mundial da Saúde (OMS), baseiam-se em uma combinação de estratégias de intervenção contra a infecção, uma vez que o diagnóstico eficaz e precoce é indispensável para que se possa intervir com o tratamento adequado, diminuindo índices de mortalidade e a evolução de complicações clínicas. Entretanto, os testes sorológicos utilizados apresentam sensibilidade e especificidade prejudicadas em pacientes com leishmanioses e/ou coinfectados LV/HIV, devido a baixos ní-veis de anticorpos antileishmanial ou pela presença de doenças que causem reação cruzada, levando a resultados falso-positivos. A sensibilidade torna-se também variável em pacientes tratados, uma vez que a sorologia pode manter-se positiva por meses ou anos após o fim do tratamento e cura da doença. Buscando resolver tal problemática, a identificação de novos antígenos, por meio de análises de bioinformática associadas à imunoproteômica, tem permitido a detecção de novas proteínas com potencial aplicação diagnóstica. Em estudos anteriores, as proteínas hipotéticas LiHyT, LiHyD, LiHyV e LiHyP foram encontradas em espécies de Leishmania spp, e avaliadas em suas versões recombinantes por meio de ensaios de ELISA, obtendo resultados satisfatórios para a detecção da LV humana e canina. Com base nessas informações, o presente trabalho teve como objetivo desenvolver uma proteína quimera recombinante base-ada na predição de epítopos lineares específicos de células B das quatro proteínas antigênicas de L. infantum citadas e avaliar o potencial diagnóstico, assim como dos peptídeos individuais que a constituíram, frente à leishmaniose humana, bem como com a coinfecção com HIV, além de testar os antígenos como marcadores prognóstico após o tratamento da LV e LT. As sequências de aminoácidos das proteínas foram avaliadas e oito epítopos de células B foram preditos e utilizados na construção de uma nova proteína quimérica. A proteína foi expressa, purificada e avaliada como antígeno recombinante em ELISA para o diagnóstico de LV, LT, coinfecção LV/HIV e prognóstico em amostras de pacientes tratados de LV e LT. Os epítopos de células B usados na construção da quimera foram sintetizados e também testados em ELISA frente às mesmas amostras, assim como um extrato antigênico solúvel de Leishmania braziliensis (SLA). Os resultados mostraram que a proteína quimera apresentou sensibilidade e especificidade de 100% para diagnosticar a LV, LT e LV/HIV, enquanto os peptídeos sintéticos apresentaram sensibilidade variando entre eles de 9,1% a 90,9% para amostras de LT e 76,8% a 99,2% para amostras de LV e LV/HIV, já os valores de especificidade atingiram 98,3% a 99,1% para LT e 67,1% a 95,7% para LV e LV/HIV. O SLA apresentou sensibilidade e especificidade de 18,2% e 98,3% para LT, e 56,8% a 69,5% para amostras de LV e LV/HIV, respectivamente. Uma avaliação prognóstica preliminar mostrou ainda que os anticorpos anti-quimera diminuíram em níveis significativos, quando comparada a reatividade sorológica antes e seis meses após o tratamento, sugerindo um possível papel prognóstico da quimera para as leishmanioses. O presente estudo, mostrou-se eficaz na construção e avaliação de novos candidatos, que demonstram ter um bom desempenho na detecção diagnóstica e prognóstica para as leishmanioses e dos casos de coinfecção LV/HIV.

Leishmaniasis are neglected tropical diseases with high endemicity that affect thousands of people in the world. Infection is caused by protozoan parasites of the genus Leishmania. The biological diversity between species is what allows determining the clinical manifestations, either in the form of visceral leishmaniasis (VL) or tegumentary leishmaniasis (TL). Among these clinical manifestations, VL is considered the most serious, due to its high lethality and great emergence in individuals with infection caused by the human immunodeficiency virus (HIV). Currently, the control and prevention measures adopted by the World Health Organiza-tion (WHO) are based on a combination of intervention strategies against the infection, since an effective and early diagnosis is essential to intervene with the appropriate treatment, decrea-sing mortality rates and evolution of clinical complications. However, the serological tests used show impaired sensitivity and specificity in patients with leishmaniasis and/or coinfected with VL/HIV, due to low levels of anti-leishmanial antibodies or the presence of diseases that cause cross-reaction, leading to false-positive results. The sensitivity also becomes variable in treated patients, since the serology can remain positive for months or years after the end of the trea-tment and cure of the disease. Seeking to solve this problem, the identification of new antigens, through bioinformatic analysis associated with immunoproteomic, has allowed the detection of new proteins with potential diagnostic application. In previous studies, the hypothetical proteins LiHyT, LiHyD, LiHyV and LiHyP were found in species of Leishmania spp, and evaluated in their recombinant versions through ELISA assays, and satisfactory results were obtained for the detection of human and canine VL. Based on this information, the present work aimed to develop a recombinant chimera protein through on the prediction of specific linear epitopes of B cells derived from these four antigenic proteins of L. infantum and to evaluate its diagnostic potential, as well as the individual peptides that constitute it, against human leishmaniasis, as well as co-infection with HIV, in addition to testing them as possible prognostic markers of patients after VL and TL treatment. The amino acid sequences of the proteins were evaluated and eight B cell epitopes were predicted and used in the construction of a new chimeric protein. The protein was expressed, purified and evaluated as a recombinant antigen in ELISA for the diagnosis of VL, TL, VL/HIV co-infection and prognosis in samples from patients treated for VL and TL. The B cell epitopes used in the construction of the chimera were synthesized and also tested in ELISA against the same samples, as well as a soluble Leishmania braziliensis antigenic extract (SLA). The results showed that the chimera protein apresented sensitivity and specificity of 100% for diagnosing VL, TL and VL/HIV, while the synthetic peptides showed sensitivity ranging from 9.1% to 90.9% for TL samples and 76.8 % to 99.2% for VL and VL/HIV samples, while the specificity values reached from 98.3% to 99.1% for TL and 67.1% to 95.7% for VL and VL/HIV. The SLA showed sensitivity and specificity of 18.2% and 98.3% for TL, and 56.8% to 69.5% for VL and VL/HIV samples, respectively. A preliminary prog-nostic evaluation also showed that anti-chimera antibodies significantly decreased when com-pared to serological reactivity before and six months after treatment, suggesting a possible prognostic role of the antigen for leishmaniasis. The present study proved to be effective in the construction and evaluation of new candidates, who demonstrate good performance in diagnos-tic and prognostic detection for leishmaniasis and VL/HIV co-infection.

Peptide-Integrated Superparamagnetic Nanoparticles for the Identification of Epitopes from SARS-CoV-2 Spike and Nucleocapsid Proteins.

The COVID-19 pandemic, caused by the fast transmission and spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is currently considered a serious health problem, requiring an effective strategy to contain SARS-CoV-2 dissemination. For this purpose, epitopes of the SARS-CoV-2 spike (S) and sucleocapsid (N) proteins were identified by bioinformatics tools, and peptides that mimic these epitopes were chemically synthesized and then conjugated to superparamagnetic nanoparticles (SPMNPs). Three peptides from S protein and three from N protein were used as antigens in a conventional enzyme-linked immunosorbent assay (ELISA) against serum samples from COVID-19-positive patients, or from healthy donors, collected before the pandemic. Three peptides were effective as antigens in conventional peptide-based ELISA, achieving 100% sensitivity and specificity, with high accuracy. The best-performing peptides, p2pS, p1pN, and p3pN, were associated with superparamagnetic nanoparticles (SPMNPs) and were used to perform nanomagnetic peptide-based ELISA. The p2pS-SPMNP conjugate presented 100% sensitivity and specificity and excellent accuracy (area under the curve (AUC) = 1.0). However, p1pN and p3pN peptides, when conjugated to SPMNPs, did not preserve the capacity to differentiate positive sera from negative sera in all tested samples, yet both presented sensitivity and specificity above 80% and high accuracy, AUC > 0.9. We obtained three peptides as advantageous antigens for serodiagnosis. These peptides, especially p2pS, showed promising results in a nanomagnetic peptide-based ELISA and may be suitable as a precoated antigen for commercial purposes, which would accelerate the diagnosis process.

Ecotoxic, genotoxic, and cytotoxic potential of leachate obtained from chromated copper arsenate-treated wood ashes.

Preservative treatments increase the durability of wood, and one of the alternative treatments involves the use of chromated copper arsenate (CCA). Due to the toxicity of CCA, the disposal of CCA-treated wood residues is problematic, and burning is considered to provide a solution. The ecotoxicological potential of ash can be high when these components are toxic and mutagenic. The aim of this study was to evaluate the toxicity and genotoxicity of bottom ash leachates originating from CCA-treated wood burning. Physical-chemical analysis of the leachates revealed that in treated wood ashes leachate (CCA-TWBAL), the contents of arsenic and chromium were 59.45 mg.L-1 and 54.28 mg.L-1, respectively. In untreated wood ashes leachate (UWBAL), these contents were 0.70 mg.L-1 and 0.30 mg.L-1, respectively. CCA-TWBAL caused significant toxicity in Lactuca sativa, Allium cepa, and microcrustacean Artemia spp. (LC50 = 12.12 mg.mL-1). Comet assay analyses using NIH3T3 cells revealed that concentrations ranging from 1.0 and 2.5 mg.mL-1 increase the damage frequency (DF) and damage index (DI). According to MTT assay results, CCA-TWBAL at concentrations as low as 1 mg.mL-1 caused a significant decrease in cellular viability. Hemolysis assay analyses suggest that the arsenic and chromium leachate contents are important for the ecotoxic, cytotoxic, and genotoxic effects of CCA-TWBAL.

Evaluation of the in vivo acute toxicity of poly(thioether-ester) and superparamagnetic poly(thioether-ester) nanoparticles obtained by thiol-ene miniemulsion polymerization.

Poly(thioether-ester) (PTEe) nanoparticles obtained by thiol-ene polymerization have received attention of many researchers due to several advantages, including, biocompatibility and biodegradability. The search for new nanomaterials requires toxicity studies to assess potential toxic effects of their administration. Therefore, the aim of this study was to evaluate the in vivo acute toxicity of PTEe and poly(thioether-ester)-coated magnetic nanoparticles prepared by thiol-ene polymerization in miniemulsion. These nanoparticles presented a mean size of approximately 120 nm, spherical morphology, and negative surface charge. Doses of 40 mg/kg were administered intraperitoneally to Swiss mice and nociceptive, behavioral and biochemical parameters were investigated in five different organs. None of the nanoparticles led to any alterations in the nociceptive and behavioral responses. Biochemical alterations were observed in liver, decreasing the sulfhydryl and glutathione (GSH) levels, suggesting the dependence of the GSH metabolism in the elimination of the nanoparticles. In general, both nanoparticle types did not cause disturbances in biochemical parameters analyzed in others organs. These results suggest that both nanoparticle types did not induce acute toxicity to the different organs evaluated, reinforcing the biocompatibility of PTEe nanoparticles synthetized by thiol-ene polymerization.

Antinociceptive activity of Copaifera officinalis Jacq. L oil and kaurenoic acid in mice.

Copaifera officinalis L. possesses traditional uses as an analgesic, anti-inflammatory, and antiseptic. However, until now the antinociceptive effect and the mechanism of action were not described for Copaifera officinalis L. oil and no compound present in this oil was identified to be responsible for its biological effects. The goal of this study was to identify the presence of kaurenoic acid in Copaifera officinalis oil and investigate its antinociceptive effect, mechanism of action, and possible adverse effects in mice. The quantification of kaurenoic acid in Copaifera officinalis oil was done by HPLC-DAD technique. Male and female albino Swiss mice (25-35 g) were used to test the antinociceptive effect of Copaifera officinalis (10 mg/kg, intragastric) or kaurenoic acid (1 mg/kg) in the tail-flick test, intraplantar injection of capsaicin, allyl isothiocyanate (AITC) or complete Freund's adjuvant (CFA). Copaifera officinalis oil and kaurenoic acid caused the antinociceptive effect in the tail-flick test in a dose-dependent manner, and their effect was reversed by naloxone (an opioid antagonist). Copaifera officinalis oil or kaurenoic acid reduced the nociception caused by capsaicin or AITC and produced an anti-allodynic effect in the CFA model (after acute or repeated administration for 7 days). Possible adverse effects were also observed, and non-detectable adverse effect was observed for the intragastric administration of Copaiba officinalis oil or kaurenoic acid and in the same way, the treatments were neither genotoxic nor mutagenic at the doses tested. Thus, Copaiba officinalis oil, and kaurenoic acid possess antinociceptive action without adverse effects.

Genotoxicity evaluation induced by Tityus serrulatus scorpion venom in mice.

Tityus serrulatus is the scorpion associated with the most severe cases of scorpion envenoming in Brazil. However, there are no studies reporting the genotoxic effects of this venom in natural or experimental envenomations. It is well known that DNA-damage responses are providing opportunities for improving disease detection and management. In this study was evaluating the genotoxicity of the T. serrulatus venom in different organs (hippocampus, cortex, striatum, blood, heart, lung, liver and kidney) and periods in mice experimentally envenomed. ELISA and the Comet assays were used to quantification of venoms antigens and DNA damage, respectively. Forty-eight Swiss mice were divided into five groups and 0.5 DL50 of T. serrulatus venom (0.90 mg/kg) was administered intraperitoneally in each animal. Euthanasia was performed by cervical dislocation in the period of 0h (control group) 1h, 2h, 6h and 12h, where it the tissues were removed. The results showed high DNA damage in all structures analyzed, suggesting that T. serrulatus venom presented genotoxic activity or some secondary effect generated by venom injection. In the ELISA test, toxic circulant antigens were verified in practically all organs at the time intervals analyzed. Therefore, the distribution of the venom changes from organ to organ. We conclude that scorpion envenoming affects DNA in all organs analyzed even when the venom concentration is lower or no detectable, DNA damage persists.