Characterizing the differential susceptibility and resistance to insecticides in populations of Chrysodeixis includens and Rachiplusia nu (Lepidoptera: Noctuidae) in Brazil.

BACKGROUND: Chrysodeixis includens (Walker) and Rachiplusia nu (Guenée) are major Plusiinae pests of soybean in the Southern Cone region of South America. In recent decades, C. includens was the main defoliator of soybean in Brazil, but from 2021 onwards, R. nu emerged as an important soybean pest in various regions of the country. Here, we characterize the differential susceptibility and resistance to insecticides in these Plusiinae pests from two soybean regions of Brazil. RESULTS: Except for spinetoram and chlorfenapyr (comparable lethality against both species) and a Bt-based biopesticide (more lethal for C. includens), the tested insecticides showed higher lethality against R. nu than against C. includens, but populations of the same species, even separated by long distances, presented similar resistance levels. For both species, the 90% lethal concentration (LC90) values of most insecticides were higher than the field-recommended dose. Nevertheless, the field-recommended doses of spinetoram, metaflumizone, emamectin benzoate, cyclaniliprole and chlorfenapyr showed comparable control efficacy against both species, whereas indoxacarb, chlorantraniliprole, flubendiamide, teflubenzuron and chlorfluazuron were more lethal for R. nu, and methoxyfenozide and the Bt-based insecticide were more lethal for C. includens. Thiodicarb, methomyl and lambda-cyhalothrin showed low lethality against both species. CONCLUSIONS: Large interspecific differences in the susceptibility to insecticides was found in major Plusiinae pests of soybean in Brazil. Furthermore, variations in susceptibility to insecticides occurred consistently among species and populations, regardless of the collection site and thus despite unequal temporal and spatial exposure to insecticides. These results demonstrate that accurate species identification is essential for effective control of Plusiinae in soybean. © 2024 Society of Chemical Industry.

Synthetic Peptides Selected by Immunoinformatics as Potential Tools for the Specific Diagnosis of Canine Visceral Leishmaniasis.

Diagnosing canine visceral leishmaniasis (CVL) in Brazil faces challenges due to the limitations regarding the sensitivity and specificity of the current diagnostic protocol. Therefore, it is urgent to map new antigens or enhance the existing ones for future diagnostic techniques. Immunoinformatic tools are promising in the identification of new potential epitopes or antigen candidates. In this study, we evaluated peptides selected by epitope prediction for CVL serodiagnosis in ELISA assays. Ten B-cell epitopes were immunogenic in silico, but two peptides (peptides No. 45 and No. 48) showed the best performance in vitro. The selected peptides, both individually and in combination, were highly diagnostically accurate, with sensitivities ranging from 86.4% to 100% and with a specificity of approximately 90%. We observed that the combination of peptides showed better performance when compared to peptide alone, by detecting all asymptomatic dogs, showing lower cross-reactivity in sera from dogs with other canine infections, and did not detect vaccinated animals. Moreover, our data indicate the potential use of immunoinformatic tools associated with ELISA assays for the selection and evaluation of potential new targets, such as peptides, applied to the diagnosis of CVL.

Ivabradine restores tonic cardiovascular autonomic control and reduces tachycardia, hypertension and left ventricular inflammation in post-weaning protein malnourished rats.

Malnutrition results in autonomic imbalance and heart hypertrophy. Overexpression of hyperpolarization-activated cyclic nucleotide-gated channels (HCN) in the left ventricles (LV) is linked to hypertrophied hearts and abnormal myocardium automaticity. Given that ivabradine (IVA) has emerging pleiotropic effects, in addition to the widely known bradycardic response, this study evaluated if IVA treatment could repair the autonomic control and cardiac damages in malnourished rats. AIM: Assess the impact of IVA on tonic cardiovascular autonomic control and its relationship with hemodynamics regulation, LV inflammation, and HCN gene expression in post-weaning protein malnutrition condition. MAIN METHODS: After weaning, male rats were divided into control (CG; 22 % protein) and malnourished (MG; 6 % protein) groups. At 35 days, groups were subdivided into CG-PBS, CG-IVA, MG-PBS and MG-IVA (PBS 1 ml/kg or IVA 1 mg/kg) received during 8 days. We performed jugular vein cannulation and electrode implant for drug delivery and ECG registration to assess tonic cardiovascular autonomic control; femoral cannulation for blood pressure (BP) and heart rate (HR) assessment; and LV collection to evaluate ventricular remodeling and HCN gene expression investigation. KEY FINDINGS: Malnutrition induced BP and HR increases, sympathetic system dominance, and LV remodeling without affecting HCN gene expression. IVA reversed the cardiovascular autonomic imbalance; prevented hypertension and tachycardia; and inhibited the LV inflammatory process and fiber thickening caused by malnutrition. SIGNIFICANCE: Our findings suggest that ivabradine protects against malnutrition-mediated cardiovascular damage. Moreover, our results propose these effects were not attributed to HCN expression changes, but rather to IVA pleiotropic effects on autonomic control and inflammation.

Diagnosis of Canine Visceral Leishmaniasis by Flow Cytometry Serology using the rMELEISH Multiepitope Antigen Coupled in a Functional Bead.

BACKGROUND: Visceral leishmaniasis (VL) is a zoonotic disease, with dogs being the main reservoir of the Leishmania infantum parasite. OBJECTIVE: To develop a new flow cytometry test to diagnosis canine VL (CVL) diagnosis. METHODS: The current study addresses a new flow cytometry test using beads coupled to the multiepitope antigen rMELEISH. RESULTS: In the study set of samples a sensitivity (87.1%) and specificity (89.9%) was observed. Considering the dogs' clinical status, 20/20 (100.0%) of the symptomatic sera tested positive, while 19/22 (86.4%) of the oligosymptomatic and 16/20 (80.0%) of asymptomatic were positive. In the non-infected control, all samples (0/30) tested as negative. In the cross-reaction control, the test was more efficient in dogs infected with L. braziliensis (2/10) and Trypanosoma cruzi (0/10), than those with Babesia canis (4/10) and Ehrlichia canis (4/10). Dogs immunized with different vaccines (Leishmune, Leish-Tec®, or LBSap) did not present serological reactivity. CONCLUSION: The flow cytometry serology through coupling the antigen rMELEISH in functional beads showed high accuracy in diagnosing CVL.

Comparative Study of a Novel Lateral Flow Rapid Test with Conventional Serological Test Systems for the Diagnosis of Canine Leishmaniosis in Croatia and Brazil.

Control of canine infections with Leishmania infantum (L. infantum), a major zoonotic disease in Brazil and southern Europe, is becoming increasingly important due to its close proximity to humans, the increasing import of dogs from endemic regions and the impact of climate change on vector spreading. Simple, rapid and reliable diagnostic tests are therefore needed to detect infected dogs. Here, we re-evaluated different serological methods for the diagnosis of canine leishmaniosis (CanL) in Croatia and Brazil. The diagnostic performance of the indirect fluorescent antibody test (IFAT) and the VetLine® Leishmania ELISA (GSD Frankfurt, Germany) was compared with three rKLi8.3-based diagnostic test systems, the rKLi8.3 ELISA (GSD Frankfurt, Germany), the INgezim® Leishma CROM (GSD Madrid, Spain) lateral flow test (LFT) and the VetBlot®Leishmania LineBlot (GSD Frankfurt, Germany). CanL symptomatic dogs were efficiently diagnosed by all tests, except the VetLine® Leishmania ELISA, which is based on whole Leishmania antigens. The advantage of rKLi8.3 was also observed in oligo- and asymptomatic dogs from Brazil and Croatia, although with reduced diagnostic efficiency compared to symptomatic dogs. Similar to IFAT and rKLi8.3 ELISA, the LFT did not cross-react with other common canine pathogens; it showed very high specificity for healthy dogs from endemic regions in both countries and did not react with healthy, vaccinated dogs in Brazil. In conclusion, serodiagnostic tests based on the rKLi8.3 antigens are superior to whole parasite antigens, and the LFT has the advantage of providing a laboratory-independent, rapid and specific diagnosis of CanL.

ASP-2/Trans-sialidase chimeric protein induces robust protective immunity in experimental models of Chagas' disease.

Immunization with the Amastigote Surface Protein-2 (ASP-2) and Trans-sialidase (TS) antigens either in the form of recombinant protein, encoded in plasmids or human adenovirus 5 (hAd5) confers robust protection against various lineages of Trypanosoma cruzi. Herein we generated a chimeric protein containing the most immunogenic regions for T and B cells from TS and ASP-2 (TRASP) and evaluated its immunogenicity in comparison with our standard protocol of heterologous prime-boost using plasmids and hAd5. Mice immunized with TRASP protein associated to Poly-ICLC (Hiltonol) were highly resistant to challenge with T. cruzi, showing a large decrease in tissue parasitism, parasitemia and no lethality. This protection lasted for at least 3 months after the last boost of immunization, being equivalent to the protection induced by DNA/hAd5 protocol. TRASP induced high levels of T. cruzi-specific antibodies and IFNγ-producing T cells and protection was primarily mediated by CD8+ T cells and IFN-γ. We also evaluated the toxicity, immunogenicity, and efficacy of TRASP and DNA/hAd5 formulations in dogs. Mild collateral effects were detected at the site of vaccine inoculation. While the chimeric protein associated with Poly-ICLC induced high levels of antibodies and CD4+ T cell responses, the DNA/hAd5 induced no antibodies, but a strong CD8+ T cell response. Immunization with either vaccine protected dogs against challenge with T. cruzi. Despite the similar efficacy, we conclude that moving ahead with TRASP together with Hiltonol is advantageous over the DNA/hAd5 vaccine due to pre-existing immunity to the adenovirus vector, as well as the cost-benefit for development and large-scale production.

Oropouche virus infection induces ROS production and oxidative stress in liver and spleen of mice.

Oropouche virus (OROV) is the aetiological agent of Oropouche fever, the symptoms of which are common to most arboviruses, such as fever, headache, malaise, nausea and vomiting. More than half a million people have been infected with OROV since its isolation in 1955. Although Oropouche fever is classified as a neglected and emerging disease, to date, there are no antiviral drugs or vaccines available against the infection and little is known about its pathogenicity. Therefore, it is essential to elucidate the possible mechanisms involved in its pathogenesis. Since oxidative stress plays a pivotal role in the progression of various viral diseases, in this study, redox homeostasis in the target organs of OROV infection was evaluated using an animal model. Infected BALB/c mice exhibited reduced weight gain, splenomegaly, leukopenia, thrombocytopenia, anaemia, development of anti-OROV neutralizing antibodies, increased liver transaminases, and serum levels of pro-inflammatory cytokines tumour necrosis factor (TNF-α) and interferon-γ (IFN-γ). The OROV genome and infectious particles were detected in the liver and spleen of infected animals, with liver inflammation and an increase in the number and total area of lymphoid nodules in the spleen. In relation to redox homeostasis in the liver and spleen, infection led to an increase in reactive oxygen species (ROS) levels, increased oxidative stress biomarkers malondialdehyde (MDA) and carbonyl protein, and decreased activity of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT). Taken together, these results help elucidate some important aspects of OROV infection that may contribute to the pathogenesis of Oropouche.

The Use of an Adjuvant System Improves Innate and Adaptive Immune Response When Associated with a Leishmania (Viannia) braziliensis Antigen in a Vaccine Candidate against L. (Leishmania) infantum Infection.

BACKGROUND: The adjuvants' optimal dose and the administration route can directly influence the epitope recognition patterns and profiles of innate response. We aimed to establish the effect and the optimal dose of adjuvant systems for proposing a vaccine candidate to be employed with Leishmania (Viannia) braziliensis. METHODS: We evaluated the adjuvants saponin (SAP), monophosphoryl lipid A (MPL) and resiquimod (R-848) isolated and combined as adjuvant systems in a lower dose corresponding to 25%, 33%, and 50% of each adjuvant total dose. Male outbred BALB/c mice were divided into 13 groups, SAP, MPL, and R-848 isolated, and the adjuvant systems SAP plus MPL (SM), SAP plus R-848 (SR), and MPL plus R-848 (MR). RESULTS: SM50 increased levels of all chemokines analyzed and TNF production, while it presented an increased inflammatory cell infiltrate in the skin with macrophage recruitment. Thus, we proposed a vaccine candidate employing L. (V.) braziliensis antigen associated with the SM adjuvant system against experimental L. (Leishmania) infantum challenge. We observed a significant increase in the frequency of cells expressing the central and effector memory CD4+ T cells phenotype in immunized mice with the LBSM50. In the liver, there was a decreased parasite load when mice received LBSM50. CONCLUSIONS: When combined with L. (V.) braziliensis antigen, SM50 increases TNF and IFN-γ, which generates central and effector memory CD4+ T cells. Therefore, using an adjuvant system can promote an effective innate immune response with the potential to compose future vaccines.

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.

Minimum Rim Width and Peripapillary Retinal Nerve Fiber Layer Thickness for Diagnosing Early to Moderate Glaucoma.

PRCIS: In a cross-sectional study from a Brazilian multiracial population, minimum rim width (MRW) and peripapillary retinal nerve fiber layer thickness measurements from OCT showed comparable diagnostic performance in discriminating early to moderate glaucoma from healthy eyes. PURPOSE: The purpose of this study is to compare the ability of MRW and peripapillary retinal nerve fiber layer thickness (RNFLT) measurements in discriminating early to moderate glaucoma from healthy eyes in a Brazilian population. METHODS: A total of 155 healthy controls and 118 patients with mild to moderate glaucoma (mean deviation >-12 dB) underwent MRW and RNFLT measurements with optical coherence tomography. Only 1 eye per patient was included in the analysis. A receiver operating characteristic (ROC) regression model was used to evaluate the diagnostic accuracy of MRW and RNFLT, whereas adjusting for age and Bruch membrane opening area. Sensitivities at fixed specificities of 95% were calculated for each parameter. RESULTS: Global RNFLT and MRW showed comparable area under the ROC curves [0.93 (0.91-0.96) and 0.93 (0.89-0.96), respectively; P =0.973]. Both parameters had similar sensitivities (75% vs. 74%, respectively; P =0.852) at a fixed specificity of 95%. The best sector for diagnosing glaucoma for both parameters was the temporal inferior sector, which showed an area under the ROC curve of 0.93 (0.87-0.96) for RNFLT and 0.91 (0.86-0.95) for MRW ( P =0.320). The temporal inferior sector showed similar sensitivities for RNFLT and MRW measurements (83% vs. 77%, respectively) at a fixed specificity of 95% (P =0.230). CONCLUSIONS: MRW and RNFLT measurements showed comparable diagnostic performance in discriminating early to moderate glaucoma from healthy eyes in a Brazilian multiracial population.

Development and biological evaluation of nanoencapsulated-based pyrethroids with synergists for resistance management of two soybean pests: insights for new insecticide formulations.

BACKGROUND: Chemical control is commonly used against Euschistus heros (F.) and Chrysodeixis includens (Walker) in soybean fields in South America. However, previous studies reported that these pests have reduced susceptibility to pyrethroids in Brazil. On this basis, we developed and evaluated nanoencapsulated-based bifenthrin (BFT) and λ-cyhalothrin (LAM) with the synergists piperonyl butoxide (PBO) and diethyl maleate (DEM) for insect resistance management (IRM). RESULTS: Nanoformulations of BFT and LAM with PBO and DEM presented good physical-chemical characteristics and were stable. The spherical morphology of all systems and the encapsulation efficiency in nanostructured lipid carriers did not change when synergists were added. Nanoencapsulated BFT with DEM applied topically increased the susceptibility of E. heros to BFT by 3.50-fold. Similarly, nanoencapsulated BFT and LAM with PBO in diet-overlay bioassays increased the susceptibility of C. includens to both chemicals by up to 2.16-fold. Nanoencapsulated BFT and LAM with synergists also improve control efficacy of both species, causing higher mortality than commercial products containing these chemistries. CONCLUSIONS: It is possible to develop nanoencapsulated-based formulations of BFT and LAM with PBO or DEM, and these nanoformulations have the potential to improve control of E. heros and C. includens with recognized low susceptibility to pyrethroids. This study provides updates for designing new insecticide formulations for IRM. © 2022 Society of Chemical Industry.

Ivabradine treatment lowers blood pressure and promotes cardiac and renal protection in spontaneously hypertensive rats.

Hypertension is linked to hyperpolarization-activated cyclic nucleotide-gated (HCN) function, expressed in excitable and non-excitable cells. Considering that the reduction in heart rate (HR) improves coronary perfusion and cardiac performance, ivabradine (IVA) emerged as an important drug for the treatment of cardiovascular diseases. AIM: Evaluate if IVA chronic treatment effect can mitigate hypertension and reverse the cardiac and renal damage in SHR. MAIN METHODS: Rats were divided into 4 groups treated for 14 days with PBS (1 ml/kg; i.p) or IVA (1 mg/kg; i.p): 1) WKY PBS; 2) SHR PBS; 3) WKY IVA; and 4) SHR IVA. The systolic blood pressure (SBP) was measured, indirectly, before and during the treatment period with IVA (day 0, 1, 7 and 11). Rats were subjected to artery cannulation for direct blood pressure (BP) measurement. Morphofunctional and gene expression were evaluated in the heart and kidneys. KEY FINDINGS: IVA reduced SBP only in SHR on the 7th day. Direct blood pressure measurement showed that IVA chronic treatment reduced HR in the SHR. Interestingly, mean arterial pressure (MAP) was reduced in SHR IVA when compared to SHR PBS. Serum and urinary biochemical data were not altered by IVA. Moreover, IVA reduced the renal inflammatory infiltrates and increased glomerular density, besides preventing the cardiac inflammatory and hypertrophic responses. SIGNIFICANCE: IVA treatment lowered blood pressure, improved cardiac remodeling and inflammation, as well as decreasing renal damage in SHR. Further, IVA increased renal HCN2 mRNA and reduced cardiac HCN4 mRNA.

A specific Leishmania infantum polyepitope vaccine triggers Th1-type immune response and protects against experimental visceral leishmaniasis.

The development of an immunogenic, effective, and safe vaccine is essential as an alternative for disease control. The present study aimed to evaluate the immunogenicity and efficacy potential of a polyepitope T-cell antigen candidate against visceral leishmaniasis in a murine model. BALB/c mice were immunized with three doses subcutaneously with Poly-T Leish alone or adjuvanted with Saponin plus Monophosphoryl lipid A, with 15-day intervals between doses, and challenged with 107 stationary-phase Leishmania infantum promastigotes via tail vein. Immunogenicity and parasitism in spleen and liver of immunized mice were evaluated 45 days post-challenge. Our results revealed that the immunization with Poly-T Leish and Poly-T Leish/SM increases the percentage of specific T (CD4+ and CD8+) lymphocytes proliferation in vitro after antigen-specific stimulation. Also, Poly-T Leish and Poly-T Leish/SM groups showed a high percentage of IFN-γ and TNF-α-producing T cells, meanwhile, the Poly-T Leish/SM group also showed an increased percentage of multifunctional T cells producing double and triple-positive (IFN-γ+TNF-α+IL-2+) cytokines. The immunization with Poly-T Leish or Poly-T Leish/SM stimulated a decreased IL-4 and IL-10 compared to the Saline and adjuvant group. Poly-T Leish/SM immunized mice exhibit a noteworthy reduction in the parasite burden (spleen and liver) through real-time PCR (96%). Moreover, we observed higher nitrite secretion in 120-hour stimulated-culture supernatant using Griess method. We demonstrated that the Poly-T Leish/SM candidate was potentially immunogenic, providing enhancement of protective immune mechanisms, and conferred protection reducing parasitism. Our candidate was considered potential against visceral leishmaniasis, and eventually, could be tested in phase I and II clinical trials in dogs.

CD4+ T-lymphocytes from asymptomatic dogs infected with Leishmania infantum are able to activate macrophages for higher leishmanicidal ability in an in vitro co-culture experiment.

Dogs are the most common domestic reservoir of Leishmania infantum, making canine visceral leishmaniasis (CVL) a serious public health issue. Identifying new methodologies that can mimic lymphoid and myeloid competence in naturally infected dogs could lower costs and save time in preliminary screenings of potential immunotherapeutic agents and vaccines against CVL. For that, we established a cell-to-cell communication approach between lymphocytes and myeloid cells from healthy, asymptomatic (infected, without apparent clinical signs) and symptomatic (infected with apparent clinical signs) dogs. Peripheral blood mononuclear cells (PBMC) from these dogs were used as source of CD4+, CD8+ T lymphocytes and macrophages, that were posteriorly infected with L. infantum GFP+ promastigotes (green fluorescent protein). Macrophages co-cultured with purified lymphocytes were tested for the ability to control cellular parasitism, and their microbicidal function by producing nitric oxide (NO) and reactive oxygen species (ROS). The kind of T cell response within the co-culture was also evaluated, by assessing their ability to produce interferon-gamma (IFN-γ) and interleukin 4 (IL-4). The data suggests that T lymphocytes from symptomatic dogs are more prone to produce IL-4 than the ones from asymptomatic dogs. Macrophages from asymptomatic dogs also demonstrated a higher microbicidal potential, with increased levels of NO and ROS production, compared to symptomatic dogs, mainly in highly parasitized cells. Together, our results identify the ratio of IL-4/IFN-γ produced by CD4+ and CD8+ T cells, as well as, the ratio between parasite GFP signal/NO and ROS signal in macrophages as potential immunological biomarkers of failure and success of the screened agents. Our findings also propose a reliable methodology that can be used to follow the immune response in trials of potential drugs or vaccines targeting CVL.

LBMPL Vaccine Therapy Induces Progressive Organization of the Spleen Microarchitecture, Improved Th1 Adaptative Immune Response and Control of Parasitism in Leishmania infantum Naturally Infected Dogs.

The spleen plays a central role in human and canine visceral leishmaniasis, where the activation of the immune response occurs in one of the tissues where Leishmania infantum reproduces. Therefore, this organ is both a target to understand the mechanisms involved in the parasite control and a parameter for assessing the therapeutic response. In this sense, this study aimed to evaluate the main histological, immunological and parasitological aspects in the spleen of symptomatic dogs naturally infected by L. infantum treated with the therapeutic vaccine LBMPL. For this, dogs were divided into four groups: dogs uninfected and untreated (NI group); L. infantum-infected dogs that were not treated (INT group); L. infantum-infected dogs that received treatment only with monophosphoryl lipid A adjuvant (MPL group); and L. infantum-infected dogs that received treatment with the vaccine composed by L. braziliensis promastigote proteins associated with MPL adjuvant (LBMPL group). Ninety days after the therapeutics protocol, the dogs were euthanized and the spleen was collected for the proposed evaluations. Our results demonstrated a reduction of hyperplasia of red pulp and follicular area of white pulp, increased mRNA expression of IFN-γ, TNF-α, IL-12 and iNOS, and decreased IL-10 and TGF-ß1, and intense reduction of splenic parasitism in dogs treated with the LBMPL vaccine. These results possibly suggest that the pro-inflammatory environment promoted the progressive organization of the splenic architecture favoring the cellular activation, with consequent parasite control. Along with previously obtained data, our results propose the LBMPL vaccine as a possible treatment strategy for canine visceral leishmaniasis (CVL).

Immunoprophylaxis using polypeptide chimera vaccines plus adjuvant system promote Th1 response controlling the spleen parasitism in hamster model of visceral leishmaniasis.

In recent years, several advances have been observed in vaccinology especially for neglected tropical diseases (NTDs). One of the tools employed is epitope prediction by immunoinformatic approaches that reduce the time and cost to develop a vaccine. In this scenario, immunoinformatics is being more often used to develop vaccines for NTDs, in particular visceral leishmaniasis (VL) which is proven not to have an effective vaccine yet. Based on that, in a previous study, two predicted T-cell multi-epitope chimera vaccines were experimentally validated in BALB/c mice to evaluate the immunogenicity, central and effector memory and protection against VL. Considering the results obtained in the mouse model, we assessed the immune response of these chimeras inMesocricetus auratushamster, which displays, experimentally, similar pathological status to human and dog VL disease. Our findings indicate that both chimeras lead to a dominant Th1 response profile, inducing a strong cellular response by increasing the production of IFN-γ and TNF-α cytokines associated with a decrease in IL-10. Also, the chimeras reduced the spleen parasite load and the weight a correlation between protector immunological mechanisms and consistent reduction of the parasitic load was observed. Our results demonstrate that both chimeras were immunogenic and corroborate with findings in the mouse model. Therefore, we reinforce the use of the hamster as a pre-clinical model in vaccination trials for canine and human VL and the importance of immunoinformatic to identify epitopes to design vaccines for this important neglected disease.

Leishmania amazonensis from distinct clinical forms/hosts has polymorphisms in Lipophosphoglycans, displays variations in immunomodulatory properties and, susceptibility to antileishmanial drugs.

Lipophosphoglycan (LPG), the major Leishmania glycoconjugate, induces pro-inflammatory/immunosuppressive innate immune responses. Here, we evaluated functional/biochemical LPG properties from six Leishmania amazonensis strains from different hosts/clinical forms. LPGs from three strains (GV02, BA276, and LV79) had higher pro-inflammatory profiles for most of the mediators, including tumor necrosis factor alpha and interleukin 6. For this reason, glycoconjugates from all strains were biochemically characterized and had polymorphisms in their repeat units. They consisted of three types: type I, repeat units devoid of side chains; type II, containing galactosylated side chains; and type III, containing glucosylated side chains. No relationship was observed between LPG type and the pro-inflammatory properties. Finally, to evaluate the susceptibility against antileishmanial agents, two strains with high (GV02, BA276) and one with low (BA336) pro-inflammatory activity were selected for chemotherapeutic tests in THP-1 cells. All analyzed strains were susceptible to amphotericin B (AmB) but displayed various responses against miltefosine (MIL) and glucantime (GLU). The GV02 strain (canine visceral leishmaniasis) had the highest IC50 for MIL (3.34 µM), whereas diffuse leishmaniasis strains (BA276 and BA336) had a higher IC50 for GLU (6.87-12.19 mM). The highest IC50 against MIL shown by the GV02 strain has an impact on clinical management. Miltefosine is the only drug approved for dog treatment in Brazil. Further studies into drug susceptibility of L. amazonensis strains are warranted, especially in areas where dog infection by this species overlaps with those caused by Leishmania infantum.

Replacement of Leishmania (Leishmania) infantum Populations in an Endemic Focus of Visceral Leishmaniasis in Brazil.

Visceral leishmaniasis is an important global health problem with an estimated of 50,000 to 90,000 new cases per year. VL is the most serious form of leishmaniasis as it can be fatal in 95% of the cases if it remains untreated. VL is a particularly acute problem in Brazil which contributed with 97% of all cases reported in 2020 in the Americas. In this country, VL affects mainly the poorest people in both urban and rural areas and continues to have a high mortality rate estimated around 8.15%. Here, we performed a temporal parasite population study using whole genome sequence data from a set of 34 canine isolates sampled in 2008, 2012 and 2015 from a re-emergent focus in Southeastern Brazil. Our study found the presence of two distinct sexual subpopulations that corresponded to two isolation periods. These subpopulations diverged hundreds of years ago with no apparent gene flow between them suggesting a process of rapid replacement during a two-year period. Sequence comparisons and analysis of nucleotide diversity also showed evidence of balancing selection acting on transport-related genes and antigenic families. To our knowledge this is the first population genomic study showing a turn-over of parasite populations in an endemic region for leishmaniasis. The complexity and rapid adaptability of these parasites pose new challenges to control activities and demand more integrated approaches to understand this disease in New World foci.

The entrance route: Oral, mucous, cutaneous, or systemic has a marked influence on the outcome of Trypanosoma cruzi experimental infection.

In recent decades, the oral infection of Trypanosoma cruzi has gathered increased attention due to frequent outbreaks that can lead to more severe clinical signs than those usually found in the areas of vector transmission. This study addresses the main routes of infection using metacyclic trypomastigotes (MT) and blood trypomastigotes (BT). Herein, BALB/c mice were infected with the Colombian (TcI) strain via intraperitoneal (IP), oral, intragastric (IG), ocular (OC) and cutaneous (CT) routes with 106 culture-derived MT or BT. Parasitemia was intermittent and low in animals inoculated with MT, in contrast, high parasitemia levels were found in BT-mice. A tropism for the muscles was observed in oral or IG infection with BT. Differently, the parasite was widely distributed in the tissues of mice infected with MT. However, the intensity of the inflammation infiltrating the tissues was higher in oral or IG infection with BT. Animals inoculated with BT via the IG route had similar serum levels of IFN-γ and smaller IL-10 compared to those infected with MT via the IG route. TNF-α levels were higher in the serum from BT-animals, which could explain the higher intensity of heart inflammation in these animals. Our results suggest that the infective form and the route of infection differentially modulated the outcome of Trypanosoma cruzi mice infection.

Quercetin prevents insulin dysfunction in hypertensive animals.

Angiotensin II induced increase in hypertension enhances oxidative stress and compromises insulin action and pancreatic function. Quercetin-rich foods are beneficial for hypertensive and diabetic animals owing to their antioxidant function. The aim of this study was to evaluate the antioxidant effects of quercetin in hypertensive rats on insulin action, signaling, and secretion. Wistar rats were randomly divided into three groups: sham, hypertensive rats (H), and hypertensive rats supplemented with quercetin (HQ). After three months of initial hypertension, quercetin was administered at 50 mg/kg/day for 30 days. Our results indicate that hypertension and serum lipid peroxidation levels were reduced by quercetin supplementation. We observed increased insulin sensitivity in adipose tissue, corroborating the insulin tolerance test, HOMA index, and improvements in lipid profile. Despite normal insulin secretion at 2.8 and 20 mM of glucose, animals treated with quercetin exhibited increased number of islets per section; increased protein expression of muscarinic receptor type 3, VEGF, and catalase in islets; and hepatic mRNA levels of Ide were normalized. In conclusion, supplementation with quercetin improved insulin action and prevented pancreatic and metabolic dysfunction.