Leishmanicidal and immunomodulatory activity of Terminalia catappa in Leishmania amazonensisin vitro infection.

Leishmaniases are infectious-parasitic diseases that impact public health around the world. Antileishmanial drugs presented toxicity and increase in parasitic resistance. Studies with natural products show an alternative to this effect, and several metabolites have demonstrated potential in the treatment of various diseases. Terminalia catappa is a plant species with promising pharmaceutical properties. The objective of this work was to evaluate the therapeutic potential of extracts and fractions of T. catappa on Leishmania amazonensis and investigate the immunomodulatory mechanisms associated with its action. In anti-Leishmania assays, the ethyl acetate fraction exhibited activity against promastigotes (IC50 86.07 ± 1.09 µg/mL) and low cytotoxicity (CC50 517.70 ± 1.68 µg/mL). The ethyl acetate fraction also inhibited the intracellular parasite (IC50 25.74 ± 1.08 µg/mL) with a selectivity index of 20.11. Treatment with T. catappa ethyl acetate fraction did not alter nitrite production by peritoneal macrophages stimulated with L. amazonensis, although there was a decrease in unstimulated macrophages treated at 50 µg/mL (p = 0.0048). The T. catappa ethyl acetate fraction at 100 µg/mL increased TNF-α levels (p = 0.0238) and downregulated HO-1 (p = 0.0030) and ferritin (p = 0.0002) gene expression in L. amazonensis-stimulated macrophages. Additionally, the total flavonoid and ellagic acid content for ethyl acetate fraction was 13.41 ± 1.86 mg QE/g and 79.25 mg/g, respectively. In conclusion, the T. catappa ethyl acetate fraction showed leishmanicidal activity against different forms of L. amazonensis and displayed immunomodulatory mechanisms, including TNF-α production and expression of pro and antioxidant genes.

In Vitro Antioxidant and Antitrypanosomal Activities of Extract and Fractions of Terminalia catappa.

Chagas disease is a severe infectious and parasitic disease caused by the protozoan Trypanosoma cruzi and considered a public health problem. Chemotherapeutics are still the main means of control and treatment of the disease, however with some limitations. As an alternative treatment, plants have been pointed out due to their proven pharmacological properties. Many studies carried out with Terminalia catappa have shown several biological activities, but its effect against T. cruzi is still unknown. The objective of this work is to evaluate the therapeutic potential of extracts and fractions obtained from T. catappa on the parasite T. cruzi, in addition to analyzing its antioxidant activity. T. catappa ethyl acetate fraction were produced and submitted the chemical characterization by Liquid Chromatography Coupled to Mass Spectrometry (LC-MS). From all T. catappa extracts and fractions evaluated, the ethyl acetate and the aqueous fraction displayed the best antioxidant activity by the 2,2-diphenyl-1-picryl-hydrazyl (DPPH) radical scavenging method (IC50 of 7.77 ± 1.61 and 5.26 ± 1.26 µg/mL respectively), and by ferric ion reducing (FRAP) method (687.61 ± 0.26 and 1009.32 ± 0.13 µM of Trolox equivalent/mg extract, respectively). The ethyl acetate fraction showed remarkable T. cruzi inhibitory activity with IC50 of 8.86 ± 1.13, 24.91 ± 1.15 and 85.01 ± 1.21 µg/mL against epimastigotes, trypomastigotes and intracellular amastigotes, respectively, and showed no cytotoxicity for Vero cells (CC50 > 1000 µg/mL). The treatment of epimastigotes with the ethyl acetate fraction led to drastic ultrastructural changes such as the loss of cytoplasm organelles, cell disorganization, nucleus damage and the loss of integrity of the parasite. This effect could be due to secondary compounds present in this extract, such as luteolin, kaempferol, quercetin, ellagic acid and derivatives. The ethyl acetate fraction obtained from T. catappa leaves can be an effective alternative in the treatment and control of Chagas disease, and material for further investigations.

Calotropis procera (Aiton) Dryand (Apocynaceae): State of the art of its uses and Applications.

Calotropis procera (Aiton) Dryand (Apocynaceae), popularly known as milkweed, has been traditionally used to treat diseases particularly associated with gastric disorders, skin disease and inflammatory processes. The present study aimed to review the current scientific evidence regarding the pharmacological effects of C. procera extracted phytochemicals and possible research opportunities as complementary and alternative medicine. Scientific publications were searched in various electronic databases (PubMed, Scopus, Web of Science, Google Scholar, Springer, Wiley, and Mendeley) using the following search terms: Calotropis procera, medicinal plants, toxicity, phytochemical characterization, and biological effects. Collected data showed that cardenolides, steroid glycoside and flavonoids are the main classes of phytochemicals identified in C. procera latex and leaves. In addition, lignans, terpenes, coumarins, and phenolic acids have been reported. These metabolites have been correlated with their biological activities, including mainly antioxidant, anti-inflammatory, antitumoral, hypoglycemic, gastric protective, anti-microbial, insecticide, anti-fungal, anti-parasitic, among others. However, some of the studies were carried out with only a single dose or with a high dose not achievable under physiological conditions. Therefore, the validity of C. procera biological activity may be questionable. Not less important to highlight are the risks associated with its use and the possibility of accumulation of heavy metals that can be toxic. Furthermore, there are no clinical trials with C. procera to date. In conclusion, the need of bioassayguided isolation of bioactive compounds, bioavailability and efficacy, as well as pharmacological and toxicity studies, are needed using in vivo models and clinical trials in order to support the traditionally claimed health benefits.

Antitumor Effect of Açaí (Euterpe oleracea Mart.) Seed Extract in LNCaP Cells and in the Solid Ehrlich Carcinoma Model.

Euterpe oleracea (açaí) fruit has approximately 15% pulp, which is partly edible and commercialized, and 85% seeds. Although açaí seeds are rich in catechins-polyphenolic compounds with antioxidant, anti-inflammatory, and antitumor effects-almost 935,000 tons/year of seeds are discarded as industrial waste. This work evaluated the antitumor properties of E. oleracea in vitro and in vivo in a solid Ehrlich tumor in mice. The seed extract presented 86.26 ± 0.189 mg of catechin/g of extract. The palm and pulp extracts did not exhibit in vitro antitumor activity, while the fruit and seed extracts showed cytotoxic effects on the LNCaP prostate cancer cell line, inducing mitochondrial and nuclear alterations. Oral treatments were performed daily at 100, 200, and 400 mg/kg of E. oleracea seed extract. The tumor development and histology were evaluated, along with immunological and toxicological parameters. Treatment at 400 mg/kg reduced the tumor size, nuclear pleomorphism, and mitosis figures, increasing tumor necrosis. Treated groups showed cellularity of lymphoid organs comparable to the untreated group, suggesting less infiltration in the lymph node and spleen and preservation of the bone marrow. The highest doses reduced IL-6 and induced IFN-γ, suggesting antitumor and immunomodulatory effects. Thus, açaí seeds can be an important source of compounds with antitumor and immunoprotective properties.

Effects of Passovia ovata Mistletoe on Pro-Inflammatory Markers In Vitro and In Vivo.

New agents that can suppress inflammatory responses are being sought, since chronic inflammation is associated with several pathologies. This work aims to elucidate phytochemicals from the hydroethanolic extract of mistletoe Passovia ovata (POH) and its anti-inflammatory potential. POH is submitted to HPLC-UV, qualitative analysis of chemical constituents, and flavonoid quantification. Cytotoxicity is evaluated in RAW 264.7 macrophages by MTT. LPS-stimulated RAW 264.7 cells are treated with POH and, after 48 h, the nitrite and cytokine levels are quantified. BALB/c mice are treated by gavage with POH and stimulated with λ-carrageenan to induce paw oedema or peritonitis. POH yield is 25% with anthraquinones, tannins, anthocyanins, anthocyanidins, flavonols, catechins and flavanones present and flavonoid content of 4.44 ± 0.157 mg QE/g dry weight. POH exhibits low cytotoxicity and significantly reduced (p < 0.01) nitrite, IL-1ß, IL-6, and TNF-α quantification at 500 µg/mL. POH at 500 mg/kg prevents paw edema increase and also reduces inflammatory infiltrate and mast cells in the footpad. In the peritonitis model, POH does not influence cytokines levels or cell counts. Overall, POH demonstrates a high concentration of flavonoids and prominent effects in the reduction in pro-inflammatory markers in vitro and in the inhibition of paw oedema.

Molecular characterization of Brazilian FeLV strains in São Luis, Maranhão Brazil.

The feline leukemia virus (FeLV) belongs to the Retroviridae family and Gammaretrovirus genus, and causes a variety of neoplastic and non-neoplastic diseases in domestic cats (Felis catus), such as thymic and multicentric lymphomas, myelodysplastic syndromes, acute myeloid leukemia, aplastic anemia, and immunodeficiency. The aim of the present study was to carry out the molecular characterization of FeLV-positive samples and determine the circulating viral subtype in the city of São Luís, Maranhão, Brazil, as well as identify its phylogenetic relationship and genetic diversity. The FIV Ac/FeLV Ag Test Kit (Alere™) and the commercial immunoenzymatic assay kit (Alere™) were used to detect the positive samples, which were subsequently confirmed by ELISA (ELISA - SNAP® Combo FeLV/FIV). To confirm the presence of proviral DNA, a polymerase chain reaction (PCR) was performed to amplify the target fragments of 450, 235, and 166 bp of the FeLV gag gene. For the detection of FeLV subtypes, nested PCR was performed for FeLV-A, B, and C, with amplification of 2350-, 1072-, 866-, and 1755-bp fragments for the FeLV env gene. The results obtained by nested PCR showed that the four positive samples amplified the A and B subtypes. The C subtype was not amplified. There was an AB combination but no ABC combination. Phylogenetic analysis revealed similarities (78% bootstrap) between the subtype circulating in Brazil and FeLV-AB and with the subtypes of Eastern Asia (Japan) and Southeast Asia (Malaysia), demonstrating that this subtype possesses high genetic variability and a differentiated genotype.

Visceral leishmaniasis and HIV coinfection in Brazil: epidemiological profile and spatial patterns.

BACKGROUND: Geographical overlap of human visceral leishmaniasis (HVL) and HIV infection favors occurrences of HVL/HIV coinfection. The increasing numbers of cases of HVL/HIV coinfection are a worldwide concern and knowledge of the factors involved can help in developing preventive measures. METHODS: We aimed to identify spatial patterns and describe the epidemiological profile of HVL/HIV coinfection in Brazil from 2007 to 2015. This was an ecological study, in which thematic maps were created through geoprocessing tools, based on secondary data obtained from open-access platforms, to identify priority areas for interventions for controlling HLV/HIV coinfection, using the TerraView 4.2.2 software. RESULTS: We found spatial autocorrelations between the HVL/HIV rates of neighboring municipalities according to the global Moran index (0.10; p<0.01). The highest HVL/HIV rate was in the central-western region. Among the epidemiological characteristics according to the regions, an increasing trend in the annual variation rate was observed, with positive values over the years and statistical significance (p<0.05) in the North with 1.62 (95% CI 0.57 to 2.69; p=0.02) and Northeast with 6.41 (95% CI 2.60 to 10.37; p=0.006). Similarly, increasing trends were observed in the states of Maranhão with 21.34 (95% CI 13.99 to 9.16; p<0.001) and Sergipe with 5.44 (95% CI 0.61 to 10.50; p=0.04). CONCLUSIONS: Our results showed spatial overlap between occurrences of HVL and HIV with spatial patterns of HVL/HIV coinfection, thus revealing that the numbers of cases reported are increasing. Identifying areas with higher coinfection indices contributes to applying interventions and control measures among targeted populations, to prevent new cases.

Hydrodissection to create conjunctival flaps in dogs with corneal ulcers.

Background and Aim: Hydrodissection is a liquid injection technique that is rarely used in animal ophthalmic procedures. The use of this technique in the creation of conjunctival flaps for the treatment of corneal ulcers in dogs can improve the outcome, task, and comfort for patients. This study aimed to evaluate the use of hydrodissection in the creation of conjunctival flaps in dogs with corneal ulcers. Materials and Methods: This study focused on a surgical procedure for creating conjunctival flaps in the eyes of 17 dogs with deep corneal ulcers. We divided the patients into two groups: Hydrodissection was performed in the first group (G1) and conventional divulsion without hydrodissection in the second group (G2). In G1, the conjunctival flap was created by subconjunctival injection of 1 mL of 0.9% sodium chloride, followed by flap construction. The flap was constructed through conventional divulsion using iris scissors in the G2. The operative time, degree of bleeding, and ease of conjunctival divulsion were evaluated during the procedure. Blepharospasm, hyperemia, edema, and scarring of the conjunctiva were evaluated during the post-operative period. Post-operative complications, notably suture dehiscence, were recorded in each group. Results: Hydrodissection is an easy-to-perform maneuver that optimizes the construction of conjunctival flaps. There were no statistical differences in the parameters used to evaluate the trans- and post-operative period between the groups. The volume of sodium chloride administered in the conjunctiva ranged from 0.5 mL to 1 mL in G1. Dehiscence of the flap sutures was observed in four patients (two in G1 and two in G2), with no significant difference between the groups. Conclusion: Hydrodissection facilitates the construction of conjunctival flaps in dogs with corneal ulcers, affording greater comfort to patients and proving to be an excellent option for ophthalmologists.

Biological Graft as an Innovative Biomaterial for Complex Skin Wound Treatment in Dogs: A Preliminary Report.

Complex wounds in dogs are a recurrent problem in veterinary clinical application and can compromise skin healing; in this sense, tissue bioengineering focused on regenerative medicine can be a great ally. Decellularized and recellularized skin scaffolds are produced to be applied in different and complex canine dermal wounds in the present investigation. Dog skin fragments are immersed in a 0.5% sodium dodecyl sulfate (SDS) solution at room temperature and overnight at 4 °C for 12 days. Decellularized samples are evaluated by histological analysis, scanning electron microscopy (SEM) and gDNA quantification. Some fragments are also recellularized using mesenchymal stem cells (MSCs). Eight adult dogs are divided into three groups for the application of the decellularized (Group I, n = 3) and recellularized scaffolds (Group II, n = 3) on injured areas, and a control group (Group III, n = 2). Wounds are evaluated and measured during healing, and comparisons among the three groups are described. In 30- and 60-day post-grafting, the histopathological analysis of patients from Groups I and II shows similar patterns, tissue architecture preservation, epithelial hyperplasia, hyperkeratosis, edema, and mononuclear inflammatory infiltrate. Perfect integration between scaffolds and wounds, without rejection or contamination, are observed in both treated groups. According to these results, decellularized skin grafts may constitute a potential innovative and functional tool to be adopted as a promising dog cutaneous wound treatment. This is the first study that applies decellularized and recellularized biological skin grafts to improve the healing process in several complex wounds in dogs, demonstrating great potential for regenerative veterinary medicine progress.

Ethyl Acetate Fraction of Bixa orellana and Its Component Ellagic Acid Exert Antibacterial and Anti-Inflammatory Properties against Mycobacterium abscessus subsp. massiliense.

Mycobacterium abscessus subsp. massiliense (Mabs) causes chronic infections, which has led to the need for new antimycobacterial agents. In this study, we investigated the antimycobacterial and anti-inflammatory activities of the ethyl acetate fraction of Bixa orellana leaves (BoEA) and ellagic acid (ElAc). In silico analysis predicted that ElAc had low toxicity, was not mutagenic or carcinogenic, and had antimicrobial and anti-inflammatory activities. Apparently, ElAc can interact with COX2 and Dihydrofolate reductase (DHFR) enzymes, which could explain both activities. In vitro analysis showed that BoEA and ElAc exerted antimicrobial activity against Mabs (minimum inhibitory concentration of 1.56, 1.56 mg/mL and bactericidal concentration of 6.25, 3.12 mg/mL, respectively. Clarithromycin showed MIC and MBC of 1 and 6 µg/mL). Treatment with BoEA or ElAc increased survival of Tenebrio molitor larvae after lethal infection with Mabs and reduced carrageenan-induced paw edema in mice, around 40% of edema volume after the fourth hour, similarly to diclofenac. In conclusion, BoEA and ElAc exert antimicrobial effects against Mabs and have anti-inflammatory effects, making them potential sources of antimycobacterial drugs. The biological activities of ElAc may be due to its high binding affinities predicted for COX2 and DHFR enzymes.

Corrigendum: Pteridium spp. and Bovine Papillomavirus: Partners in Cancer.

[This corrects the article DOI: 10.3389/fvets.2021.758720.].

Diagnosis and phylogenetic analysis of bovine leukemia virus in dairy cattle in northeastern Brazil.

Enzootic bovine leukosis (EBL) is a chronic viral disease of wide distribution in cattle herds and may take several years for the first manifestation of clinical signs. Most animals do not present clinical signs. However, the economic losses are underestimated due to this disease. Thus, this work aimed to detect and characterize BLV in dairy cattle in the Maranhão state, northeastern Brazil. Blood samples were collected from 176 animals from 8 municipalities in the southeastern state of Maranhão. Bovine blood samples were subjected to DNA extraction and molecular diagnosis using nested PCR assays for BLV, targeting gp51 gene. Positive samples were then sequenced and then subjected to phylogenetic inferences. BLV DNA was detected in 16 cattle (16/176, 9.09%) in 4 municipalities. Phylogenetic analyzes showed that the sequence obtained clustered in a clade containing BLV sequences classified as genotype 6, with a high degree of support. Our data shows BLV occurrence in the Northeast of Brazil and the identification of genotype 6 in this region. These findings contribute to the molecular epidemiology of this agent in Brazil.

Pteridium spp. and Bovine Papillomavirus: Partners in Cancer.

Bovine papillomavirus (BPV) are a cause for global concern due to their wide distribution and the wide range of benign and malignant diseases they are able to induce. Those lesions include cutaneous and upper digestive papillomas, multiple histological types of urinary bladder cancers-most often associated with BPV1 and BPV2-and squamous cell carcinomas of the upper digestive system, associated with BPV4. Clinical, epidemiological and experimental evidence shows that exposure to bracken fern (Pteridium spp.) and other related ferns plays an important role in allowing viral persistence and promoting the malignant transformation of early viral lesions. This carcinogenic potential has been attributed to bracken illudane glycoside compounds with immune suppressive and mutagenic properties, such as ptaquiloside. This review addresses the role of BPV in tumorigenesis and its interactions with bracken illudane glycosides. Current data indicates that inactivation of cytotoxic T lymphocytes and natural killer cells by bracken fern illudanes plays a significant role in allowing viral persistence and lesion progression, while BPV drives unchecked cell proliferation and allows the accumulation of genetic damage caused by chemical mutagens. Despite limited progress in controlling bracken infestation in pasturelands, bracken toxins remain a threat to animal health. The number of recognized BPV types has steadily increased over the years and now reaches 24 genotypes with different pathogenic properties. It remains essential to widen the available knowledge concerning BPV and its synergistic interactions with bracken chemical carcinogens, in order to achieve satisfactory control of the livestock losses they induce worldwide.

High level of infection by Toxoplasma gondii in pigs slaughtered in the city of São Luís, Maranhão.

A serological, molecular and histopathological study was carried out in order to investigate occurrences of Toxoplasma gondii in pigs slaughtered with and without inspection service. Serum samples were collected from 60 pigs to detect anti-T. gondii antibody by indirect fluorescent antibody (IFAT). Tongue, masseter and diaphragm fragments were also collected for parasite DNA detection by means of the polymerase chain reaction (PCR) and histopathological analysis. The serological results showed that 77% (44/60) of the pigs were positive. Regarding PCR, 66.67% (40/60) were positive for T. gondii. Among the tissues evaluated, the diaphragm was the one with the highest frequency of positivity (40%; 24/60), followed by the masseter (38.33%; 23/60) and tongue (33.3%; 20/60). Histopathological changes were only observed in the diaphragm, which presented inflammatory infiltrates of lymphohistiocytic and neutrophilic types. These results not only show the potential threat of T. gondii to human health, but also demonstrate the dynamic epidemiological situation of toxoplasmosis in pigs in the city of São Luís, providing support for food security regarding pigs and for T. gondii control programs in Brazil.

Effects of acaricidal essential oils from Lippia sidoides and Lippia gracilis and their main components on vitellogenesis in Rhipicephalus microplus (Canestrini, 1888) (Acari: Ixodidae).

Rhipicephalus microplus is an important cattle tick, and resistant strains to synthetic compounds have been widespread. The combined effects of different essential oil compounds enhance biological activity and reduce selection for the development of target organism resistance. Essential oils of two different genotypes of each of Lippia sidoides and Lippia gracilis and their main components, the isomers thymol and carvacrol, have acted as acaricides against R. microplus. Little is known about the effects of the essential oils of L. sidoides and L. gracilis and thymol and carvacrol on the morphophysiology of R. microplus ovaries. This study aimed to identify the morphological changes in the ovaries of R. microplus females treated with essential oils from two different genotypes of each of L. sidoides (102 and 103) and L. gracilis (106 and 201) and the terpenes thymol and carvacrol through histological techniques. The LC50 and LC75 of essential oils and thymol and carvacrol were used for Adult Immersion Test (AIT) with groups of five fully engorged females of R. microplus. A negative control (DMSO 3% solution) was performed. Seven days after the AIT, the ticks were dissected to collect ovaries and their histologic analysis. Only the group treated with the essential oil of L. gracilis genotype 106 at the LC50 had no change compared with the control. The other groups showed the following changes in oocytes I to V: vacuolation, chorion deformation, disorganization of yolk granules, and irregularities at the cell periphery, causing incomplete process of vitellogenesis. Thus, the essential oils tested in this study may be potent products for the control of cattle ticks and thereby preventing further life cycles.

Nitric Oxide Induction in Peritoneal Macrophages by a 1,2,3-Triazole Derivative Improves Its Efficacy upon Leishmania amazonensis In Vitro Infection.

1,2,3-Triazole is one of the most flexible chemical scaffolds broadly used in various fields. Here, we report the antileishmanial activity of 1,2,3-triazole derivatives, the ultrastructural alterations induced by their treatment, and the nitric oxide (NO) modulation effect on their efficacy against Leishmania amazonensis in vitro infection. After the screening of eleven compounds, compound 4 exhibited better results against L. amazonensis promastigotes (IC50 = 15.52 ± 3.782 µM) and intracellular amastigotes (IC50 = 4.10 ± 1.136 µM), 50% cytotoxicity concentration at 84.01 ± 3.064 µM against BALB/c peritoneal macrophages, and 20.49-fold selectivity for the parasite over the cells. Compound 4 induced ultrastructural mitochondrial alterations and lipid inclusions in L. amazonensis promastigotes, upregulated tumor necrosis factor α, interleukin (IL)-1ß, IL-6, IL-12, and IL-10 messenger RNA expressions, and enhanced the NO production, verified by nitrite (p = 0.0095) and inducible nitric oxide synthase expression (p = 0.0049) quantification, which played an important role in its activity against intramacrophagic L. amazonensis. In silico prediction in association with antileishmanial activity results showed compound 4 as a hit compound with promising potential for further studies of new leishmaniasis treatment options.

Inhibitory Effect of Catechin-Rich Açaí Seed Extract on LPS-Stimulated RAW 264.7 Cells and Carrageenan-Induced Paw Edema.

Açaí berry is a fruit from the tree commonly known as açaízeiro (Euterpe oleracea Mart.) originated from the Amazonian region and widely consumed in Brazil. There are several reports of the anti-inflammatory activity of its pulp and few data about the seed's potential in inflammation control. This work aimed to evaluate the effect of catechin-rich açaí extract on lipopolysaccharide (LPS)-stimulated RAW 264.7 cells and carrageenan-induced paw edema. The treatment with E. oleracea ethyl acetate extract (EO-ACET) was used in an in vitro model performed with macrophages stimulated by LPS, in which pro-inflammatory markers were evaluated, and in an in vivo model of acute inflammation, in which edema inhibition was evaluated. EO-ACET showed an absence of endotoxins, and did not display cytotoxic effects in RAW 264.7 cells. LPS-stimulated cells treated with EO-ACET displayed low levels of nitrite and interleukins (IL's), IL-1ß, IL-6 and IL-12, when compared to untreated cells. EO-ACET treatment was able to inhibit carrageenan-induced paw edema at 500 and 1000 mg/kg, in which no acute inflammatory reaction or low mast cell counts were observed by histology at the site of inoculation of λ-carrageenan. These findings provide more evidence to support further studies with E. oleracea seeds for the treatment of inflammation.