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Epoxy-α-lapachone (ELAP), an oxirane-functionalized molecule synthesized from naturally occurring lapachol, has shown promising activity against murine infection with Leishmania (Leishmania) amazonensis. Herein, we report the successful development of oil-in-water-type (o/w) microemulsions (ME) loaded with ELAP (ELAP-ME) using Capmul MCM, Labrasol, and PEG 400. Stability studies revealed that ELAP-ME (100 µg/mL of ELAP), which was comprised of globule size smaller than 120.4 ± 7.7 nm, displayed a good stability profile over 73 days. ELAP-ME had an effect in BALB/c mice infected with L. (L.) amazonensis, causing reductions in paw lesions after two weeks of treatment (∼2-fold) when compared to untreated animals. Furthermore, there was also a reduction in the parasite load both in the footpad (60.3%) and in the lymph nodes (31.5%). Based on these findings, ELAP-ME emerges as a promising treatment for tegumentar 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 © 2023 Elsevier B.V. All rights reserved.)

Leishmaniasis represents a complex of diseases with a broad clinical spectrum and epidemiological diversity, considered a major public health problem. Although there is treatment, there are still no vaccines for cutaneous leishmaniasis. Because Leishmania spp. is an intracellular protozoan with several escape mechanisms, a vaccine must provoke cellular and humoral immune responses. Previously, we identified the Leishmania homolog of receptors for activated C kinase (LACK) and phosphoenolpyruvate carboxykinase (PEPCK) proteins as strong immunogens and candidates for the development of a vaccine strategy. The present work focuses on the in silico prediction and characterization of antigenic epitopes that might interact with mice or human major histocompatibility complex class I. After immunogenicity prediction on the Immune Epitope Database (IEDB) and the Database of MHC Ligands and Peptide Motifs (SYFPEITHI), 26 peptides were selected for interaction assays with infected mouse lymphocytes by flow cytometry and ELISpot. This strategy identified nine antigenic peptides (pL1-H2, pPL3-H2, pL10-HLA, pP13-H2, pP14-H2, pP15-H2, pP16-H2, pP17-H2, pP18-H2, pP26-HLA), which are strong candidates for developing a peptide vaccine against leishmaniasis.

Background: Zika virus (ZIKV) is a flavivirus associated with microcephaly and other fetal anormalities. However, evidence of asymptomatic ZIKV infection in pregnant women is still scarce. This study investigated the prevalence of Zika infection in asymptomatic pregnant women attending two public maternities in Maranhão state, Northeast Brazil., Methods: A total of 196 women were recruited at the time of delivery by convenience sampling from two maternity clinics in São Luís, Maranhão, Brazil, between April 2017 and June 2018. Venous blood, umbilical cord blood and placental fragments from maternal and fetal sides were collected from each subject. ZIKV infection was determined by reverse transcription polymerase chain reaction (RT-qPCR) for ZIKV and by serology (IgM and IgG). Nonspecific laboratory profiles (TORCH screen) were obtained from medical records., Results: The participants were mostly from São Luís and were of 19-35 years of age. They had 10-15 years of schooling and they were of mixed race, married, and Catholic. ZIKV was identified in three umbilical cord samples and in nine placental fragments. Mothers with positive ZIKV RT-qPCR were in the age group older than 19 years. Of the 196 women tested by ZIKV rapid test, 6 and 117 women were positive for anti-ZIKV IgM and anti-ZIKV IgG antibodies, respectively. Placental Immunohistochemistry study detected ZIKV in all samples positive by RT-PCR. The newborns did not show any morphological and/or psychomotor abnormalities at birth., Conclusions: Asymptomatic ZIKV infection is frequent, but it was not associated to morphological and/or psychomotor abnormalities in the newborns up to 6 months post-birth. Although pathological abnormalities were not observed at birth, we cannot rule out the long term impact of apparent asymptomatic congenital ZIKV infection., Competing Interests: The authors have declared that no competing interests exist.

Treatment of leishmaniasis is a challenging subject. Although available, chemotherapy is limited, presenting toxicity and adverse effects. New drugs with antileishmanial activity are being investigated, such as antiparasitic compounds derived from plants. In this work, we investigated the antileishmanial activity of the biflavonoid amentoflavone on the protozoan Leishmania amazonensis . Although the antileishmanial activity of amentoflavone has already been reported in vitro , the mechanisms involved in the parasite death, as well as its action in vivo , remain unknown. Amentoflavone demonstrated activity on intracellular amastigotes in macrophages obtained from BALB/c mice (IC 50 2.3 ± 0.93 μM). No cytotoxicity was observed and the selectivity index was estimated as greater than 10. Using BALB/c mice infected with L. amazonensis we verified the effect of an intralesional treatment with amentoflavone (0.05 mg/kg/dose, in a total of 5 doses every 4 days). Parasite quantification demonstrated that amentoflavone reduced the parasite load in treated footpads (46.3% reduction by limiting dilution assay and 56.5% reduction by Real Time Polymerase Chain Reaction). Amentoflavone decreased the nitric oxide production in peritoneal macrophages obtained from treated animals. The treatment also increased the expression of ferritin and decreased iNOS expression at the site of infection. Furthemore, it increased the production of ROS in peritoneal macrophages infected in vitro . The increase of ROS in vitro , associated with the reduction of NO and iNOS expression in vivo , points to the antioxidant/prooxidant potential of amentoflavone, which may play an important role in the balance between inflammatory and anti-inflammatory patterns at the infection site. Taken together these results suggest that amentoflavone has the potential to be used in the treatment of cutaneous leishmaniasis, working as an ally in the control and development of the lesion., 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 © 2021 Rizk, Santos-Pereira, Gervazoni, Hardoim, Cardoso, de Souza, Pelajo-Machado, Carollo, de Arruda, Almeida-Amaral, Zaverucha-do-Valle and Calabrese.)

Background: Considering the complexity of the factors involved in the immunopathology of Chagas disease, which influence the Chagas' disease pathogenesis, anti- T. cruzi immune response, and chemotherapy outcome, further studies are needed to improve our understanding about these relationships. On this way, in this article we analyzed the host genetic influence on hematological, histopathological and immunological aspects after T. cruzi infection., Methods: BALB/c and A mice were intragastrically infected with T. cruzi SC2005 strain, isolated from a patient of an outbreak of Chagas disease. Parameters such as parasite load, survival rates, cytokines production, macrophages, T and B cell frequencies, and histopathology analysis were carried out., Results: BALB/c mice presented higher parasitemia and mortality rates than A mice. Both mouse lineages exhibited hematological alterations suggestive of microcytic hypochromic anemia and histopathological alterations in stomach, heart and liver. The increase of CD8 + T cells, in heart, liver and blood, and the increase of CD19 + B cells, in liver, associated with a high level of proinflammatory cytokines (IL-6, TNF-α, IFN-γ), confer a resistance profile to the host. Although BALB/c animals exhibited the same findings observed in A mice, the response to infection occurred later, after a considerable parasitemia increase. By developing an early response to the infection, A mice were found to be less susceptible to T. cruzi SC2005 infection., Conclusions: Host genetics background shaping the response to infection. The early development of a cytotoxic cellular response profile with the production of proinflammatory cytokines is important to lead a less severe manifestation of Chagas disease., 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 Domingues, Cardoso, Hardoim, Pelajo-Machado, Bertho and Calabrese.)

Leishmaniases are a complex of diseases with a broad spectrum of clinical forms, which depend on the parasite species, immunological status, and genetic background of the host. In the Leishmania major model, susceptibility is associated with the Th2 pattern of cytokines production, while resistance is associated with Th1 response. However, the same dichotomy does not occur in L. amazonensis -infected mice. Cytokines are key players in these diseases progression, while the extracellular matrix (ECM) components participate in the process of parasite invasion as well as lesion healing. In this article, we analyzed the influence of host genetics on the expression of cytokines, inducible nitric oxide synthase (iNOS), and ECM proteins, as well as the parasite load in mice with different genetic backgrounds infected by L. amazonensis . C57BL/10 and C3H/He mice were subcutaneously infected with 10 6 L. amazonensis promastigotes. Lesion kinetics, parasite load, cytokines, iNOS, and ECM proteins expression were measured by quantitative PCR (qPCR) in the footpad, draining lymph nodes, liver, and spleen at early (24 h and 30 days) and late phase (120 and 180 days) of infection. Analysis of lesion kinetics showed that C57BL/10 mice developed ulcerative lesions at the inoculation site after L. amazonensis infection, while C3H/He showed slight swelling in the footpad 180 days after infection. C57BL/10 showed progressive enhancement of parasite load in all analyzed organs, while C3H/He mice showed extremely low parasite loads. Susceptible C57BL/10 mice showed high levels of TGF-β mRNA in the footpad early in infection and high levels of proinflammatory cytokines mRNA (IL-12, TNF-α, and IFN-γ) and iNOS in the late phase of the infection. There is an association between increased expression of fibronectin, laminin, collagen III and IV, and TGF-β. On the other hand, resistant C3H/He mice presented a lower repertory of cytokines mRNA expression when compared with susceptible C57BL/10 mice, basically producing TNF-α, collagen IV, and laminin early in infection. The findings of our study indicate that L. amazonensis infection induces different cytokine expression in resistant and susceptible mice but not like the L. major model. An organ-compartmentalized cytokine response was observed in our model. Host genetics determine this response, which modulates ECM proteins expression., (Copyright © 2020 Cardoso, Zaverucha-do-Valle, Almeida-Souza, Abreu-Silva and Calabrese.)

The aim of this review was to identify anti-inflammatory and antioxidant therapeutic agents and their effects on patients with chagasic myocarditis. A systematic review of the MEDLINE, EMBASE, WEB OF SCIENCE, SCOPUS, LILACS and CENTRAL databases (Cochrane Library) was carried out without language restrictions. The descriptors used were: 'Chagas cardiomyopathy', 'treatment', 'Chagas disease', 'anti-inflammatory agents', 'Trypanosoma cruzi' and 'antioxidants'. A total of 4,138 articles was identified, six of which were selected for data extraction. Of these, four were related to antioxidant therapy with vitamins C and E supplementation, and two using anti-inflammatory therapy. The studies were carried out in Brazil and were published between 2002 and 2017. Antioxidant therapy with vitamin C and E supplementation increases the activity of antioxidant enzymes and reduces the oxidative markers. There is no conclusive data to support the use of vitamin supplementation and anti-inflammatory therapy in the treatment of chagasic cardiomyopathy. However, the studies indicate the possibility of vitamin supplementation as a new approach to the treatment of Chagas disease. Antioxidant therapy was proven to be a viable alternative for attenuating the oxidative damage caused by chronic chagasic cardiopathy, leading to a better prognosis for patients.

Leishmaniasis encompass a group of infectious parasitic diseases occurring in 97 endemic countries where over one billion people live in areas at risk of infection. It is in the World Health Organization list of neglected diseases and it is considered a serious public health problem, with more than 20,000 deaths a year and high morbidity. Infection by protozoa from the genus Leishmania can cause several forms of the disease, which may vary from a self-healing ulcer to fatal visceral infection. Leishmania species, as well as host immune response and genetics can modulate the course of the disease. Leishmania sp are obligatory intracellular parasites that have macrophages as their main host cell. Depending on the activation phenotype, these cells may have distinct roles in disease development, acting in parasite control or proliferation. Therefore, the purpose of this work was to analyze Leishmania amazonensis infection in primary macrophage cells obtained from mice with two distinct genetic backgrounds, ie. different susceptibility to the infection; evaluating the cause for that difference. After infection, peritoneal macrophages from the resistant C3H/He strain presented lower parasite load when compared to susceptible BALB/c macrophages. The same was also true when cells received a Th2 stimulus after infection, but the difference was abrogated under Th1 stimulus. Nitric oxide production and arginase activity was different between the strains under Th1 or Th2 stimulus, respectively, but iNOS inhibition was unable to suppress C3H/He resistance. Hydrogen peroxide production was also higher in C3H/He than BALB/c under Th1 stimulus, but it could not account for differences in susceptibility. These results led us to conclude that, although they have an important role in parasite control, neither NO nor H2O2 production can explain C3H/He resistance to infection. Other studies are needed to uncover different mechanisms of resistance/susceptibility to L. amazonensis., Competing Interests: The authors have declared that no competing interests exist.

Species of the Vernonia genius are widely distributed across the world. In traditional communities, they are commonly used in popular medicine for the treatment of inflammatory diseases. The objective of the present study was to evaluate the anti-inflammatory activity of Vernonia polysphaera Baker hydroalcoholic extract. A λ-carrageenan-induced paw edema and peritonitis model was established in BALB/c mice. The in vitro activity of the extract was measured on LPS-stimulated RAW 264.7 cells. There was no toxic effect on mice or on the cells treated with the extract. Animals treated with V. polysphaera extract demonstrated inhibition of paw edema in comparison with the untreated animals at all the analyzed doses. In peritonitis, treatment with the extract at a dose of 500 mg/kg resulted in a lower total leukocyte count in the peritoneal fluid and blood and lower levels of IL-1β, IL-6, TNF-α and PGE-2 than the control group. Cells treated with 50 and 100 μg/mL of the extract exhibited lower levels of nitrite and pro-inflammatory cytokine production and lower COX-2, NF-κB expression. The V. polysphaera extract demonstrated an anti-inflammatory effect, interfering with cell migration, reducing pro-inflammatory cytokine levels and COX-2 expression and consequent interference with PGE-2, as well as inhibiting NF-κB transcription., Competing Interests: The authors have declared that no competing interests exist.

Leishmania amazonensis is a major etiological agent of human cutaneous leishmaniasis in the Americas; nevertheless there are some reports of this species causing visceral disease in dogs and men. In the present work we have studied a Leishmania strain isolated from a human case of visceral leishmaniasis. We have infected different mouse strains and analyzed the development of the disease, studying the parasite's ability to visceralize and whether this ability is influenced by host genetics. Female BALB/c, C57BL/6, C57BL/10, CBA, DBA/2, and C3H/He mice were subcutaneously infected with 10⁴ L. amazonensis amastigotes. BALB/c, C57BL/6 and C57BL/10 mice were found to be very susceptible to infection, showing lesions that developed to necrosis and ulceration. CBA mice developed a late but severe lesion. DBA/2 mice developed only discrete lesions, while C3H/He mice did not develop any lesions. All mouse strains except C3H/He showed some degree of visceralization, presenting parasites in the spleen, while BALB/c, C57BL/6 and CBA presented parasites also in the liver. Moreover, most of the strains presented high parasite load at the infection site, whereas DBA and C3H/He mice showed low or no parasite load 90 days after infection, respectively. Histopathology corroborates the results, showing that susceptible mice presented an inflammatory reaction with parasites in the skin, lymph nodes and spleen, while strains that are more resistant presented low parasitism and discrete inflammatory reaction. Results indicate that this isolate is extremely virulent, can easily visceralize and that the pathogenesis of leishmaniasis is, at least in part, related to the genetic background of the host.

Chagas disease is a worldwide public health problem. Although the vectorial transmission of Chagas disease has been controlled in Brazil there are other ways of transmission, such as the ingestion of T. cruzi contaminated food, which ensures the continuation of this zoonosis. Here, we demonstrate the influence of the inoculation route on the establishment and development of the SC2005 T. cruzi strain infection in mice. Groups of Swiss mice were infected intragastrically (IG) or intraperitoneally (IP) with the T. cruzi SC2005 strain derived from an outbreak of oral Chagas disease. The results revealed that 100% of IP infected mice showed parasitemia, while just 36% of IG infected showed the presence of the parasite in blood. The parasitemia peaks were later and less intense in the IG infected mice. Mortality of the IP infected animals was more intense and earlier when compared to the IG infected mice. In the IP infected mice leucopenia occurred in the early infection followed by leucocytosis, correlating positively with the increase of the parasites. However, in the IG infected mice only an increase in monocytes was observed, which was positively correlated with the increase of the parasites. Histopathological analyses revealed a myotropic pattern of the SC2005 strain with the presence of inflammatory infiltrates and parasites in different organs of the animals infected by both routes as well as fibrosis foci and collagen redistribution. The flow cytometric analysis demonstrated a fluctuation of the T lymphocyte population in the blood, spleen and mesenteric lymph nodes of the infected animals. T. cruzi DNA associated with the presence of inflammatory infiltrates was detected by PCR in the esophagus, stomach and intestine of all infected mice. These findings are important for the understanding of the pathogenesis of T. cruzi infection by both inoculation routes.

The best-known cervicopharyngeal pain is Eagle syndrome, in which symptomatic elongation of the stylomandibular process occurs and may be accompanied by stylohyoid ligament calcification. Among the causes of elongation of the styloid process, the following may be mentioned: history of trauma, styloid ligament calcification, and formation of bony tissue in the insertion of the styloid ligament. When there is no history of trauma or surgery, it is called the stylohyoid syndrome. In the current study, the clinical case of 34-year-old woman is reported, complaining of pain in the region of the neck, without any history of neck surgery or trauma. A panoramic radiograph and computed tomographic scan showed bilateral elongation of the styloid process. Extraoral surgical intervention was the treatment of choice. It is important to point out that dentists should be aware of this condition to contribute to a better diagnosis and therapeutic procedure.

Here, we describe the situation of canine visceral leishmaniasis in two villages of São José de Ribamar in Maranhão State/Brazil, where human cases have been registered. Blood samples of 36 household crossbred dogs from Sergio Tamer village and 43 dogs from Quinta village were collected and the serum used for serological diagnosis. An Indirect Fluorescent Antibody Test (IFAT) and enzyme-linked immunosorbent assay (ELISA) were used to detect antibodies against Leishmania. The clinical examination showed that 25% of the canine population of Quinta presented a poor body condition and in 39%, ectoparasites (ticks and fleas) were detected. In both tests, serology revealed that 21% (9 out of 43) of the dogs presented antibodies against Leishmania (55% were asymptomatic and 45% were symptomatic). In the Vila Sérgio Tamer, 25% (9 out of 36) of the dogs were seropositive for Leishmania (66.67% were asymptomatic and 33.33% were symptomatic), 33% presented poor body condition, and 22% have ectoparasites. The clinical signs more frequent were skin lesions. The statistical analysis showed that there was no statistical difference (p>0.05) between the seropositivity of the dogs from the two villages. The same was observed when the clinical signs were compared (p>0.05). Both villages have favorable conditions to maintain the cycle of leishmaniasis.

Canine visceral leishmaniosis (CVL) may be an important factor preceding human outbreaks of the disease. We report that the prevalence of canine visceral leishmaniosis infection has been increasing in recent years in Anastácio town, located in the central western region of Brazil. Serological investigations showed that 75.3% of dogs presented antibody titres ranging from 1/40 to 1/160 in the indirect immunofluorescence antibody test (IFAT). Bone marrow and lymph node aspirates provided positive cultures and furnished parasites for enzymological and serological typing in 42.5% and 41.1% of the cases, respectively. All the strains were typed as Leishmania (L.) chagasi. This is primarily a canine disease that spills over into the human population as a zoonosis. The study showed the epidemiological features of the infection in a region in which the problem of visceral leishmaniosis has been underestimated.

BALB/c, C57BL/6, and DBA/2 mice were subcutaneously infected in the left footpad by injecting 10(4) Leishmania (Leishmania) amazonensis amastigotes. Mice were sacrificed 20, 30, 40, 60 and 90 days post-infection. Fragments of liver, kidney, spleen, skin, and draining lymph node were collected for histological examination. Light microscopy showed that at 20 days after infection BALB/c mice presented discrete inflammatory infiltrates in the skin made up of eosinophils, lymphocytes, and rare parasitized macrophages. Ninety days post-infection, the dermis showed necrotic tissue, large numbers of mononuclear cells and vacuolated macrophages filled with amastigotes. Forty days post-infection, the draining lymph nodes showed hyperplastic germinal centers, increase of high endothelial venules and apoptosis in germinal center cells. After the first 3 months post-infection, the involvement of spleen, kidney and liver was discreet, being characterized by multifocal inflammatory infiltrates. Eight months after infection, the animals presented metastatic lesions in the contralateral footpad and nose. In deep dermis, there was remarkable proliferation of fibroblasts associated with collagen fibers. The liver showed multifocal granulomas and mononuclear infiltrate around the blood vessels, but no parasites were observed, except in one animal. In some mice there were immature cells of the hematopoietic lineage. Both BALB/c and C57BL/6 mice presented osteonecrosis, which is characterized by pycnotic osteocytes and empty lacunae at the point of inoculation and subsequently, replacement of this tissue by fibrous connective tissue and colonization of the bone marrow. A diffuse inflammatory process composed of mononuclear cells and rare parasites were seen in the kidneys. In one mouse, bone marrow cells were observed in the renal medulla along with where free amastigotes. DBA/2 mice developed a mild infection and they did not visceralize. In conclusion, our data demonstrates that in susceptible mice L. (L.) amazonensis, a causative agent of tegumentary leishmaniasis, causes pathological changes similar to those produced by Leishmania (L.) infantum in both humans and canids.

Here we describe extracellular matrix alterations in footpad lesions and draining lymph nodes caused by Leishmania (L.) amazonensis in mouse strains with distinct susceptibilities to this parasite: BALB/c (susceptible), C57BL/6 (intermediate), and DBA/2 (resistant). Changes in ECM were observed mainly in BALB/c mice that, in general, presented tissue damage associated with high parasite burden. Under polarized light, Sirius Red revealed type I collagen that was predominant in the primary lesion in all strains studied at the early phase of infection, but gradually decreased and was replaced by abundant type III collagen fibres in chronic phase lesions. The presence of type III collagen seemed to provide support to inflammatory cells, mainly vacuolated and parasitized macrophages. Laminin expression was not altered during infection by L. (L.) amazonensis in any of the mouse strains studied. Furthermore, the decreased fibronectin expression, in all strains, in areas where amastigotes have been found, indicated that this decline was also not related to the genetic background.