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Worldwide, both cultivated and wild plants are pollinated by the honey bee, Apis mellifera. Bee numbers are declining as a result of a variety of factors, including increased pesticide use. Cyflumetofen controls pest mites in some plantations pollinated by bees, which may be contaminated with residual sublethal concentrations of this pesticide, in nectar and pollen. We evaluated the effects of a sublethal concentration of a cyflumetofen formulation on the midgut, hypopharyngeal gland, and fat body of A. mellifera workers orally exposed for 72 h or 10 days. The midgut epithelium of treated bees presented digestive cells with cytoplasm vacuoles and some cell fragmentation, indicating autophagy and cell death. After being exposed to the cyflumetofen formulation for 72 h, the midgut showed a higher injury rate than the control bees, but after 10 days, the organs had recovered. In the hypopharyngeal gland of treated bees, the end apparatus was filled with secretion, suggesting that the acaricide interferes with the secretory regulation of this gland. Histochemical tests revealed differences in the treated bees in both exposure periods in the midgut and hypopharyngeal glands. The acaricide caused cytotoxic effects on the midgut digestive cells, with apical protrusions, plasma membrane rupture, and several vacuoles in the cytoplasm, features of cell degeneration. In the hypopharyngeal glands of the treated bees, the secretory cells presented small electron-dense and large electron-lucent secretory granules. The fat body cells had no changes in comparison with the control bees. In conclusion, the cyflumetofen formulation at sublethal concentrations causes damage to the midgut and the hypopharyngeal glands of honey bee, which may compromise the functions of these organs and colony fitness. Environ Toxicol Chem 2024;43:2455-2465. © 2024 SETAC., (© 2024 SETAC.)

Dogs are important reservoir hosts for Leishmania infantum, the causative agent of visceral leishmaniasis. The complement system, as part of the innate immune defense, is responsible for initiating the fight against pathogens that may invade an organism. A failure of the complement to combat L. infantum may explain, at least in part, why a mammal species is more or less susceptible to visceral leishmaniasis. The objective of this study was to compare the effectiveness of human and dog complement systems against L. infantum parasites. The results showed that dog serum was less effective than human serum at killing promastigote and amastigote-like forms. We also compared the efficiency of human and canine sera in classic and alternative hemolytic assays, as well as the serum efficiency of non-infected and Leishmania-infected dogs. Serum from dogs was less hemolytic than human serum in both pathways tested, but the efficiency of serum from infected dogs was higher than that of non-infected dogs. When testing C3b deposition assays on parasite surfaces, serum from infected dogs was more effective against amastigote-like forms than serum from non-infected dogs. However, both types of serum proved equally effective on promastigotes, while serum from infected dogs was more effective on amastigote-like forms. Considering the efficiency of the complement system, our results indicate that dogs are more susceptible to visceral leishmaniasis than humans are., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Nelder Figueiredo Gontijo reports financial support was provided by Conselho Nacional de Desenvolvimento Científico e Tecnológico - CNPq. Nelder Figueiredo Gontijo reports financial support was provided by Fundação de Amparo à Pesquisa de Minas Gerais - Fapemig. There is no other relationship or activity that may be interpreted as a conflict of interest by the reader. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Objective: To evaluate the correlation between serum anti-Müllerian hormone (AMH) and the number of oocytes retrieved after controlled ovarian stimulation for in vitro fertilization treatments and determine cut-off values predictive of poor and high response to stimulation., Methods: It was performed a retrospective observational study that included 1003 cycles of controlled ovarian stimulation carried between February 2017 and December 2023 at a Medically Assisted Procreation Centre. The exclusion criteria were the following: serum AMH levels obtained more than 6 months prior to the start of the ovarian stimulation, the presence of a single ovary, non-Caucasian ethnicity, a controlled ovarian stimulation cycle performed for the purpose of oocyte donation or fertility preservation, a documented diagnosis of endometriosis, a documented history of ovarian surgery and the absence of essential data for the study in the medical records (absence of the number of oocytes obtained or the AMH value). Poor response to stimulation was defined as ≤ 3 oocytes retrieved, and high response was defined as > 15 oocytes. The correlation between variables was calculated using Spearman's correlation test and cut-off values were determined using ROC (Receiver Operating Characteristic) curves., Results: AMH exhibited a significantly positive correlation with the number of oocytes retrieved (Spearman's correlation coefficient = 0.60, p<0.01). The predictive cut-off for poor ovarian response was 0.72 ng/mL (specificity of 95.13%, sensitivity of 43.23%), and the predictive cut-off for high ovarian response was 4.77 ng/mL (specificity of 89.86%, sensitivity of 38.22%)., Conclusions: Serum AMH proved to be a good predictor of the ovarian response to controlled ovarian stimulation for in vitro fertilization treatments, which makes it useful in supporting clinical decision-making. However, it should not be used as an absolute discriminator of poor or high ovarian response.

The honey bee Apis mellifera plays a significant role as a pollinator of native and cultivated plants, by increasing the productivity of several cultures, preserving the flora, and producing forest seeds. However, bee populations are declining worldwide, including A. mellifera, due to Colony Collapse Disorder, mainly resulting from the constant use of pesticides in the crops. Teflubenzuron is a physiological insecticide that belongs to the benzoylurea group, which inhibits chitin synthesis, the main component of the insect integument classified as safe for non-target insects, including bees. However, its effect on non-target organs of insects remains unknown. The midgut is the main organ of the digestive tract, which works in digestion and absorption and may be exposed to pesticides that contaminate food resources. The present work aimed to verify if the insecticide teflubenzuron is toxic and has histopathological effects on the midgut of A. mellifera adult workers. Workers exposed orally and chronically to the field-realistic concentration of teflubenzuron present 81.54% mortality. The epithelium of the midgut of these bees presents high vacuolization, spherocrystals, cell fragments released to the organ lumen, apocrine secretion, nuclear pyknosis, loss of cell-cell contact, and damage to regenerative cell nests and to the peritrophic matrix. These results indicate that the chitin synthesis-inhibiting insecticide teflubenzuron is toxic to A. mellifera after chronic oral exposure, at realistic field concentration, although it is classified as non-toxic to adult and non-target insects., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Bees are important for maintaining ecosystems, pollinating crops and producing marketable products. In recent years, a decline in bee populations has been reported, with multifactorial causes, including the intensification of pesticide use in agriculture. Among pesticides, cyflumetofen is an insecticide and acaricide used in apple, coffee and citrus crops, whose main pollinator is the honey bee Apis mellifera. Therefore, this bee is a potential target of cyflumetofen during foraging. This study evaluated the histopathological and cytological damage in the midgut, hypopharyngeal glands and fat body of A. mellifera workers exposed to LC 50 of cyflumetofen. The midgut epithelium of exposed bees presented cytoplasmic vacuolization, release of vesicles and cell fragments, which indicate autophagy, increased production of digestive enzymes and cell death, respectively. The cytological analysis of the midgut revealed the dilation of the basal labyrinth and the presence of spherocrystals in the digestive cells. The hypopharyngeal glands produced greater amounts of secretion in treated bees, whereas no changes were observed in the fat body. The results indicate that acute exposure to cyflumetofen negatively affect A. mellifera, causing damage to the midgut and changes in the hypopharyngeal glands, which may compromise the survival and foraging of this pollinator., Competing Interests: Declaration of competing interest The authors confirm that they have no conflicts of interest with respect to the work described in this manuscript., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of competing interest Authors have no conflicts of interest to disclose., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Introduction: Patient and public involvement (PPI) initiatives involving patients with chronic respiratory disease (CRD) are rare. Therefore, this study aimed to explore the perspectives of patients with CRD, carers and interested citizens regarding the relevance and need for a PPI network and suggestions for its implementation., Methods: A qualitative study based on focus groups was conducted. Recruitment occurred through invitations on social media platforms and to patients who have participated in previous asthma studies of the team. Three focus groups were conducted, via video conference, using a semi-structured guide. Thematic analysis was performed by two independent researchers and discussed with the extended team., Results: Fifteen patients with CRD, one carer and one interested citizen (13 females, median 36 (range: 18-72) years) participated. All participants acknowledged the importance of implementing a collaborative network and demonstrated interest in being integrated. Participants acknowledged the importance of their involvement in several phases of the research cycle. The main aim identified for this network was to facilitate communication between patients and researchers. Participants regarded the integration of patients, carers, researchers and healthcare professionals from different scientific areas as relevant. The use of digital platforms to attract members and support the work, together with group dynamics and regular meetings, were some of the most relevant practical considerations for implementing the network. The identified facilitators for their engagement were sharing experiences, researchers' and healthcare professionals' support and feedback and schedule flexibility. The identified barriers included the amount of time dedicated, low health/digital literacy and the potential detachment of nondiagnosed patients or those with low symptom impact in daily life., Conclusion: Patients, carers and citizens acknowledged the relevance of implementing a collaborative network and demonstrated interest in active participation in every stage of the health research cycle. A deeper knowledge of the barriers and facilitators identified in this study could support implementing these initiatives in Portugal., Patient or Public Contribution: This study was designed by a research team that included one patient with asthma and one carer. They were specifically involved in building the study protocol and the interview guide. They also gave feedback regarding the electronic consent form and the short sociodemographic questionnaire created, namely by removing noncontributing words or phrases and rewording expressions. The lay summary was written by another patient with asthma. All participants of this study were invited to implement and integrate the ConectAR network-a collaborative network of research in respiratory health., Public Summary: In Portugal, chronic respiratory patients do not have an active role as 'coinvestigators'. This study aimed to acknowledge if patients and citizens considered a patient and public involvement network useful, whose main purpose would be to facilitate communication between patients and researchers. A study based on online group interviews was carried out with patients with chronic respiratory diseases and interested citizens, both recruited on social media platforms. Participants considered that bringing together patients, carers, researchers and healthcare professionals is valuable because sharing different experiences and perspectives may help patients to improve their daily lives and increase research quality. In conclusion, patients agree that implementing a collaborative network with researchers and healthcare professionals and participating in the health research cycle is quite preponderant. Acknowledging what can help and deter this network may be beneficial to implementing this type of initiative in Portugal., (© 2023 The Authors. Health Expectations published by John Wiley & Sons Ltd.)

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.

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., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

This article aims to introduce the Safe Brain Initiative (SBI) approach, focusing on collecting and leveraging Patient-Reported Outcome Measures (PROMs) to enhance patient-centred precision anaesthesia and prevent postoperative delirium (POD) and neurocognitive disorders (NCD). The SBI was implemented to systematically address the feedback gap in perioperative care by collecting and analysing real-world data. The initiative focuses on monitoring and preventing POD and NCD, providing effective anaesthesia care, assessing patient and team satisfaction, and evaluating environmental sustainability impact. Based on international guidelines, 18 core recommendations were established to address potential complications and challenges associated with anaesthesia. Preliminary results showed a notable reduction in POD and increased awareness among anaesthesia team members regarding PROMs. The SBI approach demonstrated significant benefits during emergency situations, such as the February 2023 earthquake in Turkey, by providing crucial support and comfort to victims requiring multiple surgical interventions. The SBI presents an innovative, cost-effective, and patient-centred approach to perioperative care. By integrating PROMs and systematic feedback mechanisms, the SBI aims to expedite the advancement of efficient, patient-centered precision perioperative care, improve patient outcomes, and elevate the quality of care. The initiative has shown promising results, and its adoption is growing globally. Collaboration among healthcare providers, researchers, and patients is crucial in shaping the future of anaesthesia practice and further improving patient outcomes. Turkish hospitals are encouraged to join the SBI to benefit from international collaborations and contribute to positive change in perioperative care standards. The SBI project significantly advances precision anaesthesia, emphasising personalised care and patient well-being.

Objective: Considering that glucose is an important component of seminal plasma and is a cryoprotectant at high concentrations, the aim of this study was to investigate the possible association of glucose levels in fresh semen with the sperm survival and motility rates following cryopreservation., Methods: This was a prospective study including 149 men undergoing semen analysis due to male and/or female infertility. The seminal samples were analyzed according to the World Health Organization standards and glucose concentrations were measured using a dipstick glucometer. Samples were cryopreserved with Test Yolk Buffer-Gentamicine freezing medium under liquid nitrogen for an average of 120 days. The frozen aliquots were thawed at 37°C for 10 minutes and analyzed using the same methods and protocols used pre-freezing., Results: Glucose levels ranged from 14 to 99 mg/dL and were similar in individuals with normal (n=100) vs. abnormal (n=49) semen analysis. The rates of sperm recovery (total, alive or motile sperm) in the cryopreserved samples did not change among samples with different glucose levels (p>0.05, Kruskal-Wallis ANOVA and Spearman's correlation coefficient)., Conclusions: There appears to be no association between glucose levels in human semen samples and their resistance to cryopreservation.

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., (© 2023. The Author(s).)

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.

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2023. Published by Elsevier B.V.)

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.

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., Competing Interests: Declaration of competing interest I hereby declare that the disclosed information is correct and that no other situation of real, potential or apparent conflict of interest is known to me. I undertake to inform you of any change in these circumstances, including if an issue arises during the course of the publication or work itself., (Copyright © 2022 Elsevier Inc. All rights reserved.)

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 IC 50 for MIL (3.34 μM), whereas diffuse leishmaniasis strains (BA276 and BA336) had a higher IC 50 for GLU (6.87-12.19 mM). The highest IC 50 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., (© 2022 The Authors. Cell Biology International published by John Wiley & Sons Ltd on behalf of International Federation of Cell Biology.)

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., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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 10 6 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., (Copyright © 2022. Published by Elsevier B.V.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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-β1, 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).

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Valdivia, Roatt, Baptista, Ottino, Coqueiro-dos-Santos, Sanders, Reis, Cotton and Bartholomeu.)

The control of human visceral leishmaniasis (VL) is hard since there are no vaccines available as well as the treatment is hampered by toxicity and resistant parasites. Furthermore, as human, and canine VL causes immunosuppression, the combination of drugs with immunostimulatory agents is interesting to upregulate the immunity, reducing side-effects, improving treatment approaches against disease. Herein, we assessed the immunochemotherapy using miltefosine along with a vaccine formulated by Leishmania braziliensis antigens + saponin + monophosphoryl lipid-A (LBSapMPL) in L. infantum-infected hamsters. Two months after infection, the animals received treatments, and after 15 days they were evaluated for the treatment effect. The potential anti-Leishmania effect of miltefosine + LBSapMPL-vaccine was revealed by a specific immune response activation reflecting in control of spleen parasitism using half the miltefosine treatment time. The treated animals also showed an increase of total and T-CD4 splenocytes producing IFN-γ and TNF-α and a decrease of interleukin-10 and anti-Leishmania circulating IgG. In addition, it was demonstrated that the control of spleen parasitism is related to the generation of a protective Th1 immune response. Hence, due to the combinatorial action of miltefosine with LBSapMPL-vaccine in immunostimulating and controlling parasitism, this immunochemotherapy protocol can be an important alternative option against canine and human VL.

One hundred years after the flu pandemic of 1918, the world faces an outbreak of a new severe acute respiratory syndrome, caused by a novel coronavirus. With a high transmissibility, the pandemic has spread worldwide, creating a scenario of devastation in many countries. By the middle of 2021, about 3% of the world population had been infected and more than 4 million people had died. Different from the H1N1 pandemic, which had a deadly wave and ceased, the new disease is maintained by successive waves, mainly produced by new virus variants and the small number of vaccinated people. In the present work, we create a version of the SIR model using the spatial localization of persons, their movements, and considering social isolation probabilities. We discuss the effects of virus variants, and the role of vaccination rate in the pandemic dynamics. We show that, unless a global vaccination is implemented, we will have continuous waves of infections.

This study compared the therapeutic potential of the chemotherapy using meglumine antimoniate encapsulated in a mixture of conventional and PEGylated liposomes (Nano Sb v ) and immunotherapy with anti-canine IL-10 receptor-blocking monoclonal antibody (Anti IL-10R) on canine visceral leishmaniasis (CVL). Twenty mongrel dogs naturally infected by L. infantum, displaying clinical signs of visceral leishmaniasis were randomly divided in two groups. In the first one, nine dogs received six intravenous doses of a mixture of conventional and PEGylated liposomes containing meglumine antimoniate at 6.5 mg Sb/kg/dose. In the second one, eleven dogs received two intramuscular doses of 4 mg of anti-canine IL-10 receptor-blocking monoclonal antibody. The animals were evaluated before (T0) and 30, 90, and 180 days after treatments. Our major results demonstrated that both treatments were able to maintain hematological and biochemical parameters, increase circulating T lymphocytes subpopulations, increase the IFN-γ producing T-CD4 lymphocytes, restore the lymphoproliferative capacity and improve the clinical status. However, although these improvements were observed in the initial post-treatment times, they did not maintain until the end of the experimental follow-up. We believe that the use of booster doses or the association of chemotherapy and immunotherapy (immunochemotherapy) is promising to improve the effectiveness of treating CVL for improving the clinical signs and possibly reducing the parasite burden in dogs infected with Leishmania infantum., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Visceral leishmaniasis (VL) is a serious and neglected disease present worldwide. Chemotherapy using pentavalent antimony (Sb V ) is the most practical and inexpensive strategy available for the VL treatment today, however, it has high toxicity. Alternatively, other drugs are used as viable leishmanicidal therapeutic options. Miltefosine is the only anti-leishmanial agent administered orally, however, it has been reducing its effectiveness. In this sense, there is no ideal therapy for VL since the drugs currently used trigger severe side effects causing discontinuation of treatment, which carries an imminent risk for the emergence of parasite resistance. With that, other therapeutic strategies are gaining prominence. Among them, immunotherapy and/or immunochemotherapy, which the activation/modulation of the immune system can redirect the host's immune response to an effective therapeutic result. Therefore, this work was designed to assess an immunochemotherapy protocol composed of half course of Miltefosine associated with LBSap vaccine (Milt+LBSap) using the hamster Mesocricetus auratus as an experimental model for VL treatment. When evaluating the main hematobiochemical, immunological and therapeutic efficacy parameters, it was demonstrated that the treatment with Milt+LBSap showed restoration of hematobiochemical condition and reduced serum levels of IgG-anti- Leishmania compared to animals infected non treated (INT). Beyond that, an increase in the number of CD4 + lymphocytes producers of IFN-γ in relation to INT or to animals treated with miltefosine during 28 days, and TNF-α increased compared to INT were observed. Also, it was found a reduction of IL-10-production in relation to INT, or animals that received LBSap vaccine only, or miltefosine, following by a reduction in the splenic parasitic burden. These results demonstrate that the immunochemotherapy protocol used can stimulate the immune response, inducing an expressive cellular response sufficient to control spleen parasitism, standing out as a promising proposal for the VL treatment., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 Published by Elsevier B.V.)

An important strategy to reduce the risk of visceral leishmaniasis (VL) in humans is to control the infection and disease progression in dogs, the domestic reservoir of Leishmania infantum parasites. Certain therapeutic strategies that modulate the host immune response show great potential for the treatment of experimental VL, restoring the impaired effector functions or decreasing host excessive responses. It is known that the overproduction of interleukin-10 (IL-10) promotes parasite replication and disease progression in human VL as well as in canine visceral leishmaniasis (CVL). Thus, in the present study we investigated the potential of the anti-canine IL-10 receptor-blocking monoclonal antibody (Bloq IL-10R) to control and reduce in vitro infectivity of L. infantum and improve the ability of PBMC isolated from VL dogs to alter the lymphoproliferative response and intracytoplasmic cytokines. Overall, GFP + Leishmania showed lower capacity of in vitro infectivity in the presence of Bloq IL-10R. Moreover, addition of Bloq IL-10R in cultured PBMC enhanced T-CD4 and CD8 proliferative response and altered the intracytoplasmic cytokine synthesis, reducing CD4 + IL-4 + cells and increasing CD8 + IFN-γ + cells after specific antigen stimulation in PBMC of dogs. Furthermore, we observed an increase of TNF-α levels in supernatant of cultured PBMC under IL-10R neutralizing conditions. Together, our findings are encouraging and reaffirm an important factor that could influence the effectiveness of immune modulation in dogs with VL and suggest that blocking IL-10R activity has the potential to be a useful approach to CVL treatment., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: Data on how SARS-CoV-2 enters and spreads in a population are essential for guiding public policies., Objective: This study seeks to understand the transmission dynamics of SARS-CoV-2 in small Brazilian towns during the early phase of the epidemic and to identify core groups that can serve as the initial source of infection as well as factors associated with a higher risk of COVID-19., Methods: Two population-based seroprevalence studies, one household survey, and a case-control study were conducted in two small towns in southeastern Brazil between May and June 2020. In the population-based studies, 400 people were evaluated in each town; there were 40 homes in the household survey, and 95 cases and 393 controls in the case-control study. SARS-CoV-2 serology testing was performed on participants, and a questionnaire was applied. Prevalence, household secondary infection rate, and factors associated with infection were assessed. Odds ratios (ORs) were calculated by logistic regression. Logistics worker was defined as an individual with an occupation focused on the transportation of people or goods and whose job involves traveling outside the town of residence at least once a week., Results: Higher seroprevalence of SARS-CoV-2 was observed in the town with a greater proportion of logistics workers. The secondary household infection rate was 49.1% (55/112), and it was observed that in most households (28/40, 70%) the index case was a logistics worker. The case-control study revealed that being a logistics worker (OR 18.0, 95% CI 8.4-38.7) or living with one (OR 6.9, 95% CI 3.3-14.5) increases the risk of infection. In addition, having close contact with a confirmed case (OR 13.4, 95% CI 6.6-27.3) and living with more than four people (OR 2.7, 95% CI 1.1-7.1) were also risk factors., Conclusions: Our study shows a strong association between logistics workers and the risk of SARS-CoV-2 infection and highlights the key role of these workers in the viral spread in small towns. These findings indicate the need to focus on this population to determine COVID-19 prevention and control strategies, including vaccination and sentinel genomic surveillance., (©Breno Bernardes-Souza, Saulo Ricardo Costa Júnior, Carolina Ali Santos, Raimundo Marques Do Nascimento Neto, Fernando De Carvalho Bottega, Daiana Carolina Godoy, Bruno Lourençoni Freitas, Daniela Leite Garcia Silva, Titus Josef Brinker, Raiza Aranha Nascimento, Unaí Tupinambás, Alexandre Barbosa Reis, Wendel Coura-Vital. Originally published in JMIR Public Health and Surveillance (https://publichealth.jmir.org), 01.09.2021.)

A second deadlier wave of COVID-19 and the causes of the recent public health collapse of Manaus are compared with the Spanish flu events in that city, and Brazil. Historic sanitarian problems, and its hub position in the Brazilian airway network are combined drivers of deadly events related to COVID-19. These drivers were amplified by misleading governance, highly transmissible variants, and relaxation of social distancing. Several of these same factors may also have contributed to the dramatically severe outbreak of H1N1 in 1918, which caused the death of 10% of the population in seven months. We modelled Manaus parameters for the present pandemic and confirmed that lack of a proper social distancing might select the most transmissible variants. We succeeded to reproduce a first severe wave followed by a second stronger wave. The model also predicted that outbreaks may last for up to five and half years, slowing down gradually before the disease disappear. We validated the model by adjusting it to the Spanish Flu data for the city, and confirmed the pattern experienced by that time, of a first stronger wave in October-November 1918, followed by a second less intense wave in February-March 1919.

The present study was designed to evaluate whether tissue preparation by glutaraldehyde and glycol methacrylate (G/GMA) improves the diagnostic assessment of testicular biopsies from azoospermic men when compared to the standard tissue preparation using Bouin's solution and paraffin. We prospectively included a total of 21 testicular biopsies of sexually mature men aged 29-50 years with infertility and azoospermia. One testicular biopsy fragment from each patient was processed by the G/GMA method, whereas another tissue fragment was contemporarily processed by the conventional Bouin/paraffin (B/P) method. The G/GMA method provided better resolution of cytological details of the seminiferous epithelium, changing the final diagnosis in four cases. The medians of Bergmann's spermatogenesis scores were 0.25 (interquartile range 0.04-0.88) for B/P preparations and 0.79 (interquartile range 0.17-0.96) for G/GMA preparations. Both techniques allowed accurate prediction of sperm recovery from the biopsies (B/P, area under the receiver operating characteristics [ROC] curve 0.88, 95% confidence interval [CI] 0.75-1.00; G/GMA, area under the ROC curve 0.94, 95% CI 0.86-1.00). We conclude that human testicular biopsy preparation with G/GMA improved image resolution under light microscopy and produced more reliable results for the evaluation of spermatogenesis in comparison with B/P, allowing a more precise fertility-oriented diagnosis in azoospermic men. Abbreviations : B/P: Bouin/paraffin; GMA: glycol methacrylate; G/GMA: glutaraldehyde and glycol methacrylate; ICSI: intracytoplasmic sperm injection; OA: obstructive azoospermia; NOA: nonobstructive azoospermia; TESE: testicular sperm extraction.

Standards of care for human visceral leishmaniasis (VL) are based on drugs used parenterally, and oral treatment options are urgently needed. In the present study, a repurposing strategy was used associating tamoxifen (TMX) with polyethylene glycol- block -polylactide nanocapsules (NC) and its anti-leishmanial efficacy was reported in vivo. Stable surface modified-NC (5 mg/mL of TMX) exhibited 200 nm in size, +42 mV of zeta potential, and 98% encapsulation efficiency. Atomic force microscopy evidenced core-shell-NC. Treatment with TMX-NC reduced parasite-DNA quantified in liver and spleen compared to free-TMX; and provided a similar reduction of parasite burden compared with meglumine antimoniate in mice and hamster models. Image-guided biodistribution showed accumulation of NC in liver and spleen after 30 min post-administration. TMX-NC reduced the number of liver granulomas and restored the aspect of capsules and trabeculae in the spleen of infected animals. TMX-NC was tested for the first time against VL models, indicating a promising formulation for oral treatment.

Purpose: Vascular targeted photodynamic therapy (VTP) is a nonsurgical tumor ablation approach used to treat early-stage prostate cancer and may also be effective for upper tract urothelial cancer (UTUC) based on preclinical data. Toward increasing response rates to VTP, we evaluated its efficacy in combination with concurrent PD-1 inhibitor/OX40 agonist immunotherapy in a urothelial tumor-bearing model., Experimental Design: In mice allografted with MB-49 UTUC cells, we compared the effects of combined VTP with PD-1 inhibitor/OX40 agonist with those of the component treatments on tumor growth, survival, lung metastasis, and antitumor immune responses., Results: The combination of VTP with both PD-1 inhibitor and OX40 agonist inhibited tumor growth and prolonged survival to a greater degree than VTP with either immunotherapeutic individually. These effects result from increased tumor infiltration and intratumoral proliferation of cytotoxic and helper T cells, depletion of Treg cells, and suppression of myeloid-derived suppressor cells., Conclusions: Our findings suggest that VTP synergizes with PD-1 blockade and OX40 agonist to promote strong antitumor immune responses, yielding therapeutic efficacy in an animal model of urothelial cancer.

In Brazil, canine visceral leishmaniasis is an important public health problem due to its alarming growth. The high prevalence of infected dogs reinforces the need for a vaccine for use in prophylactic vaccination campaigns. In the present study, we evaluate the immunogenicity and protection of the best dose of Chimera A selected through the screening of cytokines production important in disease. BALB/c mice were vaccinated subcutaneously with three doses and challenged intravenously with 1 × 10 7 L. infantum promastigotes. Spleen samples were collected to assess the intracellular cytokine profile production, T cell proliferation and parasite load. At first, three different doses of Chimera A (5 μg, 10 μg and 20 μg) were evaluated through the production of IFN-γ and IL-10 cytokines. Since the dose of 20 μg showed the best results, it was chosen to continue the study. Secondarily, Chimera A at dose of 20 μg was formulated with Saponin plus Monophosphoryl lipid A. Vaccination with Chimera A alone and formulated with SM adjuvant system was able to increase the percentage of the proliferation of specific T lymphocytes and stimulated a Th1 response with increased levels of IFN-γ, TNF-α and IL-2, and decreased of IL-4 and IL-10. The vaccine efficacy through real-time PCR demonstrated a reduction in the splenic parasite load in animals that received Chimera A formulated with the SM adjuvant system (92%). Additionally, we observed increased levels of nitric oxide in stimulated-culture supernatants. The Chimera A formulated with the SM adjuvant system was potentially immunogenic, being able to induce immunoprotective mechanisms and reduce parasite load. Therefore, the use of T-cell multi-epitope vaccine is promising against visceral leishmaniasis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Purpose: To evaluate the chemical composition and morphological properties of eroded dentin after biomodification with phosphorylated chitosan (P-Chi) and carbodiimide (EDC)., Methods: 42 bovine dentin specimens were used; 21 of these specimens were subjected to erosive challenge with 0.3% citric acid (pH = 3.2) for 2 hours. The specimens were randomly divided into six groups according to dentin substrate (sound or eroded) and biomodification [with 2.5% P-Chi, with 0.5 mol/L EDC, or no biomodification (control)]. The specimens were analyzed by Fourier-transform infrared spectroscopy (FTIR, n= 5, in triplicate) and atomic force microscopy (AFM, n= 2) to verify the phosphate, carbonate, and organic matrix absorption peaks and to investigate surface morphology, respectively. The data were analyzed with Origin 6.0., Results: Dentin erosion reduced the intensity of the phosphate (1,100 cm⁻¹) and carbonate (872 cm⁻¹) related bands, which evidenced demineralization. Eroded dentin consisted of a more irregular surface containing slightly more open tubules. Modification with P-Chi removed intertubular dentin, which was compatible with surface demineralization; however, this modification obliterated dentin tubules. EDC did not promote demineralization. Biomodified dentin had a more irregular surface, irrespective of substrate type., Clinical Significance: Eroded dentin demineralization promoted by biomodification with 2.5% phosphorylated chitosan (P-Chi) is a promising indicator for further studies and highlights the dentin intrinsic characteristics. From the point of view of dentin surface chemical analysis, more studies with P-Chi should be conducted to achieve greater interactions with surfaces and to improve the adhesive interface., Competing Interests: The authors declared no conflict of interest. FAPESP provided the research scholarship (2017/27134-8)., (Copyright©American Journal of Dentistry.)

Syrian hamsters (Mesocricetus auratus) are largely used as a model for infectious diseases because it is very susceptible to several pathogens, including Leishmania spp. parasites. However, the research community faces limitations in its use due to the lack of immunological reagents and tools to study the immune system in this model. In this context, we proposed the validation of some important commercially anti-mouse mAbs (CD4, TNF-α, IFN-γ and IL-10) and how this could be useful to evaluate a specific cellular immune response in Leishmania-infected hamster using flow cytometry experiments. Our data demonstrated a cross-reactivity between these anti-mouse mAbs and hamster molecules that were herein studied. Beyond that, it was able to characterize the development of a specific cellular immune response through cytokine production in L infantum-infected hamsters when compared to uninfected ones. These data not only aid the usage of hamsters as experimental model to investigate various infectious diseases, but they contribute to the design of novel approaches to further investigate the immunological mechanisms associated to pathogen infections., (© 2021 John Wiley & Sons Ltd.)

The sponge implant has been applied as an important in vivo model for the study of inflammatory processes as it induces the migration, proliferation, and accumulation of inflammatory cells, angiogenesis, and extracellular matrix deposition in its trabeculae. The characterization of immune events in sponge implants would be useful in identifying the immunological events that could support the selection of an appropriate experimental model (mouse strain) and time post-implant analysis in optimized protocols for novel applications of this model such as in biomolecules screening. Here, the changes in histological/morphometric, immunophenotypic and functional features of infiltrating leukocytes (LEU) were assessed in sponge implants for Swiss, BALB/c, and C57BL/6 mice. A gradual increase of fibrovascular stroma and a progressive decrease in LEU infiltration, mainly composed of polymorphonuclear cells with progressive shift toward mononuclear cells at late time-points were observed over time. Usually, Swiss mice presented a more prominent immune response with late mixed pattern (pro-inflammatory/anti-inflammatory: IL-2/IFN-γ/IL-4/IL-10/IL-17) of cytokine production. While BALB/c mice showed an early activation of the innate response with a controlled cytokine profile (low inflammatory potential), C57BL/6 mice presented a typical early pro-inflammatory (IL-6/TNF/IFN-γ) response with persistent neutrophilic involvement. A rational selection of the ideal time-point/mouse-lineage would avoid bias or tendentious results. Criteria such as low number of increased biomarkers, no recruitment of cytotoxic response, minor cytokine production, and lower biomarker connectivity (described as biomarker signature analysis and network analysis) guided the choice of the best time-point for each model (Day5/Swiss; Day7/BALB/c; Day6/C57BL/6) with wide application for screening purposes, such as identification of therapeutic biomolecules, selection of antigens/adjuvants, and follow-up of innate and adaptive immune response to vaccines candidates., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Lanna, Resende, Aguiar-Soares, de Miranda, de Mendonça, Melo Júnior, Mariano, Leite, Silveira, Corrêa-Oliveira, Dutra, Reis, Martins-Filho, de Moura, Silveira-Lemos and Giunchetti.)

In this work, a dual detection system based on an impedimetric immunosensor was developed for the first time for the simultaneous detection of anti-Trypanosoma cruzi and anti-Leishmania infantum antibodies in human and dog serum samples. The IBMP 8.1 and rLci1A/rLci2B recombinant antigens were immobilized over the surface of dual screen-printed carbon electrodes (W1 and W2) modified with poly (4-hydroxyphenylacetic acid). Under optimized conditions, the immunosensor recognized specific interactions for anti-T. cruzi antibodies up to a dilution of 1:10,240 and for anti-L. infantum up to 1:5120 in canine serum samples. Relative standard deviation (RSD) values of 2.8% for W1 and 3.6% for W2 were obtained for T. cruzi (W1) and L. infantum antigen (W2) samples in three different electrodes for 3 days (n = 9). The immunosensor was stored at 4 °C for 8 weeks, with activity retention of 70.2% in W1 and 78.2% in W2. The results using the recombinant proteins revealed that all antigens discriminated between negative and positive samples (p < 0.0001) in both dog and human groups, as well as no cross-reactivity could be detected among sera with other infections. With this approach, immunosensor-based diagnostic tests achieved 100% accuracy, suggesting that the antigens are eligible to enter Phase-II studies., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Leishmania spp. parasites have a complex biological cycle presenting basically two different morphological stages, the amastigote and promastigote forms. In vitro cultivation allows a more complete study of the biological aspects of these parasites, indicating better conditions for infection, immunoassay tests, drug evaluations, and vaccines. Thus, we evaluated the three most used culture media for Leishmania spp., Grace's insect cell culture medium (Grace's), liver infusion tryptose (LIT), and Schneider's insect medium (Schneider's), without supplementation or supplemented with fetal calf serum (FCS) and bovine serum albumin (Albumin) to evaluate the growth, viability, and infectivity of the L. infantum promastigotes. It was observed that promastigote forms have a better growth in LIT and Schneider's with or without FCS when compared to that in Grace's. The supplementation with albumin promoted greater viability of the parasites independent of the medium. For in vitro infection of J774.A1 macrophages using light microscopy and flow cytometry analyses, FCS-supplemented LIT and Grace's promoted higher percentage of infected macrophages and parasite load compared with Schneider's media. Taken together, our results demonstrated that the supplementation of LIT culture medium with FCS is the most suitable strategy to cultivate Leishmania infantum parasites enabling the maintenance of growth and infective parasites for research uses.

In this study, we performed a phase I and II clinical trial in dogs to evaluate the toxicity and immunogenicity of LBSap-vaccine prototype, in comparison to Leishmune ® and Leish-Tec ® vaccines. Twenty-eight dogs were classified in four groups: (i) control group received 1 mL of sterile 0.9% saline solution; (ii) LBSap group received 600 μg of Leishmania braziliensis promastigotes protein and 1 mg of saponin adjuvant; (iii) Leishmune ® ; and (iv) Leish-Tec ® . The safety and toxicity of the vaccines were measured before and after three immunizations by clinical, biochemical, and hematological parameters. The clinical examinations revealed that some dogs of LBSap and Leishmune ® groups presented changes at the site of vaccination inoculum, such as nodules, mild edema, and local pain, which were transient and disappeared seventy-two hours after vaccination, but these results indicate that adverse changes caused by the immunizations are tolerable. The immunogenicity results demonstrate an increase of B lymphocytes CD21 + regarding the Leishmune ® group and monocytes CD14 + concerning LBSap and Leishmune ® groups. In the in vitro analyses, an increase in lymphoproliferative activity in LBSap and Leishmune ® groups was observed, with an increase of antigen-specific CD4 + and CD8 + T lymphocytes in the LBSap group. A second approach of in vitro assays aimed at evaluating the percentage of antigen-specific CD4 + and CD8 + T lymphocytes producers of IFN-γ and IL-4, where an increase in both IFN-γ producing subpopulations in the LBSap group was observed, also showed an increase in IFN-γ producers in CD8 + lymphocytes in the Leish-Tec ® group. Our data regarding immunogenicity indicate that the vaccination process, especially with the LBSap vaccine, generated a protective immune response compatible with L. infantum parasite control. Based on the foregoing, the LBSap vaccine would be suitable for further studies of phase III clinical trial in endemic areas with high prevalence and incidence of canine visceral leishmaniasis (VL) cases.

Leishmaniasis is one of the most important tropical neglected diseases according to the World Health Organization. Even after more than a century, we still have few drugs for the disease therapy and their great toxicity and side effects put in check the treatment control program around the world. Moreover, the emergence of strains resistant to conventional drugs, co-infections such as HIV/Leishmania spp., the small therapeutic arsenal (pentavalent antimonials, amphotericin B and formulations, and miltefosine), and the low investment for the discovery/development of new drugs force researchers and world health agencies to seek new strategies to combat and control this important neglected disease. In this context, the aim of this review is to summarize new advances and new strategies used on leishmaniasis therapy addressing alternative and innovative treatment paths such as physical and local/topical therapies, combination or multi-drug uses, immunomodulation, drug repurposing, and the nanotechnology-based drug delivery systems.Key points• The treatment of leishmaniasis is a challenge for global health agencies.• Toxicity, side effects, reduced therapeutic arsenal, and drug resistance are the main problems.• New strategies and recent advances on leishmaniasis treatment are urgent.• Immunomodulators, nanotechnology, and drug repurposing are the future of leishmaniasis treatment.

Leishmaniasis is a set of complex and multifaceted syndromes, with different clinical manifestations, caused by different species of the genus Leishmania spp. that can be characterized by at least four syndromes: visceral leishmaniasis (VL, also known as kala-azar), post-kala-azar dermal leishmaniasis (PKDL), cutaneous leishmaniasis (CL), and mucocutaneous leishmaniasis (MCL). Among the most serious clinical forms, VL stands out, which causes the death of around 59,000 people annually. Fast and accurate diagnosis in VL is essential to reduce the disease's morbidity and mortality. There are a large number of diagnostic tests for leishmaniasis, however they do cross-react with other protozoa and their sensitivity changes according to the clinical form of the disease. Thus, it is essential and necessary to provide a diagnosis that is sufficiently sensitive to detect asymptomatic infected individuals and specific to discriminate individuals with other infectious and parasitic diseases, thus enabling more accurate diagnostic tools than those currently used. In this context, the aim of this review is to summarize the conventional diagnostic tools and point out the new advances and strategies on visceral and cutaneous leishmaniasis diagnosis.

The spread of SARS-CoV-2 and the distribution of cases worldwide followed no clear biogeographic, climatic, or cultural trend. Conversely, the internationally busiest cities in all countries tended to be the hardest hit, suggesting a basic, mathematically neutral pattern of the new coronavirus early dissemination. We tested whether the number of flight passengers per time and the number of international frontiers could explain the number of cases of COVID-19 worldwide by a stepwise regression. Analysis were taken by 22 May 2020, a period when one would claim that early patterns of the pandemic establishment were still detectable, despite of community transmission in various places. The number of passengers arriving in a country and the number of international borders explained significantly 49% of the variance in the distribution of the number of cases of COVID-19, and number of passengers explained significantly 14.2% of data variance for cases per million inhabitants. Ecological neutral theory may explain a considerable part of the early distribution of SARS-CoV-2 and should be taken into consideration to define preventive international actions before a next pandemic.

Purpose: To develop and validate a new test of specific technical skills required for microsurgical varicocelectomy., Materials and Methods: An electronic questionnaire was sent to 558 members of the Brazilian Society of Urology for the validation of the task-specific checklist (TSC) for assessment of microsurgical varicocelectomy. Participants who had experience in this procedure were selected as judges. For construct validation, 12 participants including attending urologists and urological residents in training were recruited for voluntary participation. We formed a group of three experts and a group of nine novices, who had to perform the steps of microsurgical varicocelectomy on a simulation model using human placenta. Each participant was filmed and two blinded raters would then evaluate their performance using the TSC of microsurgical varicocelectomy., Results: 14 judges were recruited. The assessment tool was reformulated, according to the judges suggestions and had the content validity achieved. The final version of the TSC was comprised of the task-specific score, a series of 4 items scored in a binary fashion designed for microscopic sub-inguinal varicocelectomy. The differences between the performance of participants with different levels of experience reflected the construct validity. The reliability between the raters was high. The mean time required to complete the training of microsurgical varicocelectomy in simulation model was significantly shorter for experts compared to novices (201 vs. 496 seconds, p=0.01)., Conclusions: This preliminary study suggests that the task-specific checklist of microsurgical varicocelectomy is reliable and valid in assessing microsurgical skills., Competing Interests: None declared., (Copyright® by the International Brazilian Journal of Urology.)

Visceral leishmaniasis (VL) is a severe disease caused by Leishmania infantum . Dogs are the parasite's main reservoir, favoring its transmission in the urban environment. The analysis of L. infantum from infected dogs contributes to the identification of more virulent parasites, thereby supporting basic and applied studies such as vaccinal and therapeutic strategies. We proposed the in vitro and in vivo characterization of L. infantum strains from naturally infected dogs from a VL endemic area based on an infectivity and pathogenicity analysis. DH82 canine macrophages were infected in vitro with different strains for infectivity analysis, showing distinct infectivity profiles. The strains that showed greater and lesser infectivity using in vitro analyses (616 and 614, respectively) were used to infect hamsters for pathogenicity analysis. The group infected with strain 616 showed 100% survival while the group infected with strain 614 showed 50% after seven months of follow up. Furthermore, the 614 strain induced more noticeable clinicopathological changes and biochemical abnormalities in liver function, along with high inflammation and parasite load in the liver and spleen. We confirmed high variability of infectivity and pathogenicity in L. infantum strains from infected dogs. The results support the belief that screening for L. infantum infectivity using in vitro experiments is inadequate when it comes to selecting the most pathogenic strain., (Copyright © 2020 Resende, Aguiar-Soares, Moreira, Ferreira, Lanna, Cardoso, Mathias, Coura-Vital, Mariano, Leite, Silveira, de Carvalho, Santos, Silveira-Lemos, Martins-Filho, Dutra, Reis and Giunchetti.)

Background: We investigated a likely scenario of COVID-19 spreading in Brazil through the complex airport network of the country, for the 90 days after the first national occurrence of the disease. After the confirmation of the first imported cases, the lack of a proper airport entrance control resulted in the infection spreading in a manner directly proportional to the amount of flights reaching each city, following the first occurrence of the virus coming from abroad., Methodology: We developed a Susceptible-Infected-Recovered model divided in a metapopulation structure, where cities with airports were demes connected by the number of flights. Subsequently, we further explored the role of the Manaus airport for a rapid entrance of the pandemic into indigenous territories situated in remote places of the Amazon region., Results: The expansion of the SARS-CoV-2 virus between cities was fast, directly proportional to the city closeness centrality within the Brazilian air transportation network. There was a clear pattern in the expansion of the pandemic, with a stiff exponential expansion of cases for all the cities. The more a city showed closeness centrality, the greater was its vulnerability to SARS-CoV-2., Conclusions: We discussed the weak pandemic control performance of Brazil in comparison with other tropical, developing countries, namely India and Nigeria. Finally, we proposed measures for containing virus spreading taking into consideration the scenario of high poverty., Competing Interests: Aristóteles Góes-Neto and Vasco Ariston C Azevedo are Academic Editors for PeerJ., (© 2020 Ribeiro et al.)