Yeast-amoeba interaction influences murine cryptococcosis.

The virulence of Cryptococcus spp. is modulated in the natural environment through interaction with abiotic and biotic factors, and this can occasionally have implications for the progression of cryptococcosis in mammals. Hence, we evaluated whether the prior interaction of highly virulent Cryptococcus gattii strain R265 with Acanthamoeba castellanii influenced the progression of cryptococcosis. The influence of the capsule on endocytosis was evaluated using amoeba and yeast morphometrics. Mice were intratracheally infected with yeast re-isolated from the amoeba (Interaction), yeast without prior contact with the amoeba (Non-Interaction), or sterile phosphate-buffered saline (SHAM). Morbidity signs and symptoms were monitored during the survival curve, while cytokine and fungal burden measurements and histopathological analysis were performed on the 10th day post infection. Morbidity and mortality parameters in experimental cryptococcosis were influenced by the prior interaction of yeast with amoeba, which led to phenotypic changes in the cryptococcal cells, polysaccharide secretion, and their tolerance to oxidative stress. Our results suggest that a prior yeast-amoeba interaction modulates yeast virulence, which is associated with a greater tolerance to oxidative stress related to the exo-polysaccharide content and influences the progression of cryptococcal infection.

Immunomodulatory and anti-infective effects of Cratylia mollis lectin (Cramoll) in a model of wound infection induced by Staphylococcus aureus.

This work evaluated the immunomodulatory and anti-infective effects of Cratylia mollis lectin (Cramoll) in a model of wound infection induced by S. aureus. Swiss mice were divided into 3 groups (n = 12/group): non-inoculated (Control group); inoculated with S. aureus (Sa group); inoculated with S. aureus and treated with Cramoll (Sa + Cramoll group). In each animal, one lesion (64 mm2) was induced on the back and contaminated with S. aureus (~4.0 × 106 CFU/wound). The treatment with Cramoll (5 µg/animal/day) started 1-day post-infection (dpi) and extended for 10 days. Clinical parameters (wound size, inflammatory aspects, etc.) were daily recorded; while cytokines levels, bacterial load and histological aspects were determined in the cutaneous tissue at 4th dpi or 11th dpi. The mice infected with S. aureus exhibited a delay in wound contraction and the highest inflammatory scores. These effects were impaired by the treatment with Cramoll which reduced the release of key inflammatory mediators (TNF-α, NO, VEGF) and the bacterial load at wound tissue. Histological evaluations showed a restauration of skin structures in the animals treated with Cramoll. Taken together, these results provide more insights about the healing and immunomodulatory properties of Cramoll and suggest this lectin as a lead compound for treatment of wound infection.

Corrigendum: Galloyl-Hexahydroxydiphenoyl (HHDP)-Glucose Isolated From Punica granatum L. Leaves Protects Against Lipopolysaccharide (LPS)-Induced Acute Lung Injury in BALB/c Mice.

[This corrects the article DOI: 10.3389/fimmu.2019.01978.].

Galloyl-Hexahydroxydiphenoyl (HHDP)-Glucose Isolated From Punica granatum L. Leaves Protects Against Lipopolysaccharide (LPS)-Induced Acute Lung Injury in BALB/c Mice.

The hydroalcoholic extract and ethyl acetate fraction of Punica granatum leaves have been known to exhibit anti-inflammatory activities. In this study, we investigated the therapeutic effects of galloyl-hexahydroxydiphenoyl (HHDP)-glucose isolated from pomegranate leaves on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice. Male BALB/c mice were treated with different doses of galloyl-HHDP-glucose (5, 50, and 100 mg/Kg) or dexamethasone at 5 mg/Kg (per os) 6 h after intra-tracheal instillation of LPS. Vehicle-treated mice were used as controls. Twenty-four hours after LPS challenge, bronchoalveolar lavage fluid (BALF), and lung samples were collected for analyses. They were evaluated by monitoring the expression of NF-κB, JNK, and cytokine genes and proteins, as well as cell migration and lung function. All doses of galloyl-HHDP-glucose inhibited LPS-induced JNK and NF-κB activation. Likewise, the galloyl-HHDP-glucose-treated animals presented reduced expression of the TNF-α, IL-6, and IL-1ß genes in the lungs and reduced TNF-α, IL-6, IL-1ß, and IL-8 protein levels when compared with the vehicle-treated LPS-challenged mice. In addition, the ALI mice treated with galloyl-HHDP-glucose also presented reduced lung inflammatory cell accumulation, especially that of neutrophils, in their BALF and lungs. In addition, galloyl-HHDP-glucose treatment markedly ameliorated the LPS-induced pulmonary mechanism complications and attenuated weight loss. Overall, we showed for the first time that galloyl-HHDP-glucose protects against ALI, and may be useful for treating ALI and other inflammatory disorders.

Brucella-associated cervical bursitis in cattle.

Bovine brucellosis poses a risk to human health and causes serious economic losses for the animal industry. This report describes the use of different diagnostic methods for the diagnosis of brucellosis in cattle affected by cervical bursitis from a slaughterhouse located in São Luís, Maranhão, Brazil. Serum samples from a total of 47 cattle with bursitis were collected and submitted to the Rose Bengal Test (RBT), and RBT-positive samples were further confirmed by the 2-mercaptoethanol (2-ME) assay. RBT indicated 85.1% (40/47) of positive samples, from which 78.7% (37/47) were confirmed by 2-ME. Immunohistochemistry detected Brucella spp. in 34.0% (16/47) of tissues with bursitis. PCR and/or bacterial isolation demonstrated that 63.8% (30/47) of samples were positive and morphologically compatible with Brucella sp. All colonies suggestive of Brucella sp. were confirmed by PCR. Isolates were further characterized by PCR Multiplex AMOS-ENHANCED, which indicated that the isolates corresponded to biovar 1, 2, 4 (43.33%). This study evidences an association between cervical bursitis and Brucella spp. infection in cattle, and that different biovars of Brucella circulate in bovine herds in Maranhão.

Hydroalcoholic Extract and Ethyl Acetate Fraction of Bixa orellana Leaves Decrease the Inflammatory Response to Mycobacterium abscessus Subsp. massiliense.

The incidence of infections caused by rapidly growing mycobacteria (RGM), especially Mycobacterium abscessus subsp. massiliense (Mabs), is increasing worldwide. Severe infections are associated with abscess formation and strong inflammatory response. This study evaluated the antimicrobial and anti-inflammatory activities of a hydroalcoholic extract (BoHE) and ethyl acetate fraction (BoEA) of Bixa orellana leaves. Antimicrobial activity was evaluated by broth microdilution to determine the minimum inhibitory (MIC) and the minimum bactericidal (MBC) concentrations. Cytotoxicity was evaluated using erythrocytes and RAW 264.7 cells. Nitric oxide (NO) was assayed in stimulated RAW 264.7 cells, and inflammatory cell migration and acute toxicity were evaluated in a Mabs-induced peritonitis mouse model. The compounds present in BoEA were identified by high performance liquid chromatography and mass spectrometry (HPLC-MS). The MIC and MBC values were 2.34 mg/mL and 37.5 mg/mL for BoHE and 0.39 mg/mL and 6.25 mg/mL for BoEA. The extracts did not induce significant toxicity in erythrocytes and RAW 264.7 cells. High levels of NO induced by Mabs were decreased by treatment with both extracts. The anti-inflammatory activity was confirmed in vivo by significant reduction of the cell migration to the peritoneum following BoHE and BoEA pretreatment. Animals treated with BoHE or BoEA did not show signs of acute toxicity in stomach, liver, and kidney. The chemical characterization of BoEA (the most active extract) revealed that kaempferol-3-O-coumaroyl glucose is its major component. The extract of B. orellana may be effective for treating infections caused by Mabs.

Bone substitute made from a Brazilian oyster shell functions as a fast stimulator for bone-forming cells in an animal model.

Despite their demonstrated biocompatibility and osteogenic properties, oyster shells have been reported as a potential alternative to other commonly used materials for bone substitution. This study evaluated whether an experimental bone substitute (EBS) made from a typical oyster shell of Northeastern Brazil (Crassostrea rhizophora) has effects on bone development using an animal model. Oysters were collected from a biologically assisted vivarium, and their inner layer was used for preparing an EBS. Chemical and surface characterization of EBS was performed using Individually Coupled Plasma Optical Emission Spectrometry (ICP-OES) and Scanning Electron Microscope (SEM), respectively. Seventy-two rats were randomly assigned to groups according to the treatment of bone defects created in the submandibular area: Negative Control (-C), Positive Control (+C; Bio-Oss®) and EBS. Euthanasia occurred at 7, 21, 42 and 56 days postoperatively. The bone pieces were stained with hematoxylin and eosin (H&E). The formation of bone tissue was evaluated histologically and histomorphometrically. Data were analyzed through the Kruskal-Wallis test and ANOVA considering a significant level of 5%. The main element found in EBS was calcium (71.68%), and it presented heterogeneity in the particle size and a porosity aspect at SEM analysis. Histological results revealed the absence of inflammatory cells in all groups, being that EBS presented the most accelerated process of bone formation with a statistically significant difference between this group and the +C and -C groups in the 21-day time-point (p < 0.05). After 21 days, the bone formation process was similar between all groups (p > 0.05), showing an immature lamellar bone pattern after 56 days of experimentation (p > 0.05). Within the limitations of this study, it was possible to conclude that EBS presented good biocompatibility and promoted fast stimulation for bone-forming cells in an animal model.

Punica granatum L. Leaf Extract Attenuates Lung Inflammation in Mice with Acute Lung Injury.

The hydroalcoholic extract of Punica granatum (pomegranate) leaves was previously demonstrated to be anti-inflammatory in a rat model of lipopolysaccharide- (LPS-) induced acute peritonitis. Here, we investigated the anti-inflammatory effects of the ethyl acetate fraction obtained from the pomegranate leaf hydroalcoholic extract (EAFPg) on the LPS-induced acute lung injury (ALI) mouse model. Male Swiss mice received either EAFPg at different doses or dexamethasone (per os) prior to LPS intranasal instillation. Vehicle-treated mice were used as controls. Animals were culled at 4 h after LPS challenge, and the bronchoalveolar lavage fluid (BALF) and lung samples were collected for analysis. EAFPg and kaempferol effects on NO and cytokine production by LPS-stimulated RAW 264.7 macrophages were also investigated. Pretreatment with EAFPg (100-300 mg/kg) markedly reduced cell accumulation (specially neutrophils) and collagen deposition in the lungs of ALI mice. The same animals presented with reduced lung and BALF TNF-α and IL-1ß expression in comparison with vehicle controls (p < 0.05). Additionally, incubation with either EAFPg or kaempferol (100 µg/ml) reduced NO production and cytokine gene expression in cultured LPS-treated RAW 264.7 macrophages. Overall, these results demonstrate that the prophylactic treatment with EAFPg attenuates acute lung inflammation. We suggest this fraction may be useful in treating ALI.

Sulforaphane Modulates Joint Inflammation in a Murine Model of Complete Freund's Adjuvant-Induced Mono-Arthritis.

Rheumatoid arthritis (RA) is characterized by inflammation of one or more joints, and affects ~1% of the adult population worldwide. Sulforaphane (SFN) is a natural compound that has been suggested as an antioxidant. Here, SFN's effects were evaluated in a murine mono-arthritis model. Mono-arthritis was induced in mice by a single intra-articular injection of Complete Freund's Adjuvant (CFA-10 µg/joint, in 10 µL) into the ipsilateral joint. The contralateral joint received an equal volume of PBS. On the 4th day post-joint inflammation induction, animals received either SFN (10 mg/kg) or vehicle (3% DMSO in saline), intraperitoneally (i.p.), twice a day for 3 days. Joint swelling and secondary mechanical allodynia and hyperalgesia were evaluated over 7 days post-CFA. After this period, animals were culled and their blood and synovial fluid samples were collected for analysis of cell populations, cytokine release and thioredoxin reductase (TrxR) activity. Knee joint samples were also collected for histology. SFN reduced joint swelling and damage whilst increasing the recruitment of Ly6C⁺ and Ly6G⁺ cells to CFA-injected joints. SFN-treated animals presented down-regulation of CD11b and CD62L on synovial fluid Ly6G⁺ cells. Synovial fluid samples obtained from CFA-injected joints and plasma samples of SFN-treated mice presented higher levels of IL-6 and increased activity of TrxR, in comparison with controls. These results indicate that SFN reduces knee joint damage by modulating cell activation/migration to the joints, cytokine production and increasing the activity of TrxR, and therefore, may represent an alternative treatment to joint inflammation.

Molecular and serological detection of Theileria equi, Babesia caballi and Anaplasma phagocytophilum in horses and ticks in Maranhão, Brazil / Detecção molecular e serológica de Theileria equi, Babesia caballi e Anaplasma phagocytophilum em equinos e carrapatos no Maranhão

Equine piroplasmosis is a tick-borne disease caused by the intraeytrhocytic protozoans Babesia caballi and Theileria equi. It has been reported as a main equine parasitic disease. In addition, Anaplasma phagocytophilum, the causative agent of granulocytic ehrlichiosis, causes a seasonal disease in horses. Both diseases, can be detrimental to animal health. In this sense, blood samples and ticks were collected from 97 horses raised in the microregion of Baixada Maranhense, Maranhão State, Brazil. Serum samples were subjected to Indirect Fluorescence Antibody Test (IFAT) and blood samples and ticks to Polymerase Chain Reaction (PCR) to evaluate the infection by Theileria equi, Babesia caballi and Anaplasma phagocytophilum. The overall seroprevalence was 38.14%, 18.55% and 11.34% for T. equi, B. caballi and A. phagocytophilum, respectively. The results of PCR from blood samples showed 13.40% and 3.09% positive samples to T. equi and B. caballi, respectively. A total of 170 tick specimens were collected and identified as Dermacentor nitens, Amblyomma cajennense sensu lato and Rhipicephalus (Boophilus) microplus. It was detected 2.35% (4/170) and 0.59% (1/170) positive tick samples by PCR for T. equi and B. caballi, respectively. All samples were negative to A. phagocytophilum. No statically difference (p>0.05) was observed when gender, age, use of ectoparasiticide and tick presence were analyzed. A BLASTn analysis of the sequenced samples indicated 97 to 100% similarity with T. equi 18S rRNA gene sequences in GenBank and 98 to 100% with B. caballi. Genetic analysis classified the obtained sequences as T. equi and B. caballi cluster, respectively. It can be concluded that these pathogens occur and are circulating in the studied area.(AU)

A piroplasmose equina é uma doença transmitida por carrapatos causada pelos protozoários intraeritrocitários Babesia caballi e Theileria equi. É relatada como uma doença parasitária comum em equinos. Além disso, Anaplasma phagocytophilum, o agente causal da ehrlichiose granulocítica, causa uma doença sazonal em equinos. Ambas as doenças, podem ser prejudiciais para a saúde animal. Nesse sentido, amostras de sangue e carrapatos foram coletadas de 97 cavalos criados na microrregião da Baixada Maranhense, estado do Maranhão, Brasil. As amostras de soro foram submetidas ao Teste de Imunofluorescência Indireta (RIFI) e amostras de sangue e os carrapatos a Reação da Polimerase em Cadeia (PCR) para avaliar a infecção por Theileria equi, Babesia caballi e Anaplasma phagocytophilum. A prevalência foi de 38,14%, 18,55% e 11,34% para T. equi, B. caballi e A. phagocytophilum, respectivamente. Os resultados da PCR para as amostras de sangue demonstraram 13,40% e 3,09% de positividade para T. equi e B. caballi, respectivamente. Um total de 170 specimens de carrapatos foi coletado e foram identificados Dermacentor nitens, Amblyomma cajennense sensu lato and Rhipicephalus (Boophilus) microplus. Obteve-se 2,35% (4/170) e 0,59% (1/170) positivos por PCR para T. equi e B. caballi, respectivamente. Todas as amostras foram negativas para A. phagocytophilum. Não houve diferença estatística significativa (p>0.05) em relação ao sexo, idade, uso de ectoparasiticida e presença de carrapatos. A análise BLASTn das amostras sequenciadas para gene 18S rRNA indicaram 97 a 100% de similaridade com T. equi e 98-100% com B. caballi no GenBank. Análises genéticas classificaram as sequencias obtidas no mesmo clado que T. equi e B. caballi, respectivamente. Podemos concluir que estes patógenos estão circulando na área de estudo.(AU)

One Plant, Many Uses: A Review of the Pharmacological Applications of Morinda citrifolia.

Morinda citrifolia, also known as noni, is commonly used in popular medicine in Brazil. Many parts of the noni tree are utilized in such practices, including the roots, leaves and seeds. Through a search of online databases, the present article reviews 92 research studies on the biological actions of M. citrifolia. The paper will discuss the therapeutic effects of noni and its compounds in a variety of forms of presentation, focusing on studies that support its traditional use. A large and diverse number of properties were identified, which were divided into immunostimulatory, antitumor, antidiabetic, anti-obesity, antibacterial and anti-septic, antifungal, antiviral, leishmanicidal, antiinflammatory, antinociceptive and analgesic, antioxidant, neuroprotective, wound healing, antiallergic, antiangiogenic, antiemetic and anti-nausea, anti-gastric ulcer and oesophagitis, anthelmintic, antimutagenic, antipsychotic, anxiolytic, photoprotective, anti-wrinkle and periodontal tissue regeneration activities. While it was concluded that although M. citrifolia is widely and successfully used for the treatment or prevention of various diseases, it should be consumed carefully, and only after exhaustive studies into its chemical constituents and mechanisms of action, both in in vitro and in vivo models, as well as clinical trials. Copyright © 2017 John Wiley & Sons, Ltd.

Detection of Brucella sp. infection through serological, microbiological, and molecular methods applied to buffaloes in Maranhão State, Brazil.

The aim of the current study is to diagnose Brucella spp. infection using methods such as serology, bacterial isolation, and molecular analysis in buffaloes bred in Maranhão State. In order to do so, 390 samples of buffalo serum were subjected to serological tests, to Rose Bengal Plate Test (RBPT) and to 2-mercaptoethanol (2-ME) combined with slow agglutination test (SAT). Vaginal swabs were collected from seropositive animals and subjected to bacterial isolation and to generic PCR. According to the serological test, 16 animals had a positive reaction to the confirmatory test (2-ME/SAT). As for bacterial isolation, three samples resulted in the isolation of Brucella spp.-characteristic colonies, which were confirmed through PCR. These results confirmed Brucella spp. infection in the buffalo herd from Maranhão State.

Morinda citrifolia Linn. Reduces Parasite Load and Modulates Cytokines and Extracellular Matrix Proteins in C57BL/6 Mice Infected with Leishmania (Leishmania) amazonensis.

The absence of an effective vaccine and the debilitating chemotherapy for Leishmaniasis demonstrate the need for developing alternative treatments. Several studies conducted with Morinda citrifolia have shown various biological activities, including antileishmanial activity, however its mechanisms of action are unknown. This study aimed to analyze the in vivo activity of M. citrifolia fruit juice (Noni) against Leishmania (Leishmania) amazonensis in C57BL/6 mice. M. citrifolia fruit juice from the Brazilian Amazon has shown the same constitution of other juices produced around the world and liquid chromatography-mass spectrometry analysis identified five compounds: deacetylasperulosidic acid, asperulosidic acid, rutin, nonioside B and nonioside C. Daily intragastric treatment with Noni was carried out after 55 days of L. (L.) amazonensis infection in C57BL/6 mice. Parasitic loads, cytokine and extracellular protein matrix expressions of the lesion site were analyzed by qPCR. Histopathology of the lesion site, lymph nodes and liver were performed to evaluate the inflammatory processes. Cytokines and biochemical parameters of toxicity from sera were also evaluated. The Noni treatment at 500 mg.kg-1.day-1 for 60 days decreased the lesion size and parasitic load in the footpad infected with L. (L.) amazonensis. The site of infection also showed decreased inflammatory infiltrates and decreased cytokine expressions for IL-12, TNF-α, TGF-ß and IL-10. On the other hand, Noni treatment enhanced the extracellular matrix protein expressions of collagen IV, fibronectin and laminin in the infected footpad as well collagen I and II, fibronectin and laminin in the mock-infected footpads. No toxicity was observed at the end of treatment. These data show the efficacy of Noni treatment.

Putative ATP-binding cassette transporter is essential for Brucella ovis pathogenesis in mice.

Brucella ovis is a major cause of reproductive failure in sheep, which is associated with epididymitis and infertility in rams. Importantly, B. ovis is one of the few Brucella species that is not zoonotic. Due to the scarcity of studies on B. ovis infection, a murine model of infection was developed. The roles of B. ovis genes encoding a putative hemagglutinin and an ABC transporter were investigated in the mouse model. The kinetics of B. ovis infection were similar in BALB/c and C57BL/6 mice, and both strains of mice developed multifocal microgranulomas in the liver and spleen, but only minimal colonization and histopathological changes were observed in the genital tract. Therefore, the mouse was considered a suitable infection model for B. ovis but not for B. ovis-induced genital disease. Two mutant strains were generated in this study (the ΔabcAB and Δhmg strains). The B. ovis ΔabcAB strain was attenuated in the spleens and livers of BALB/c mice compared to the wild-type (WT) strain (P < 0.001). Conversely, the Δhmg strain infected mice at the same level as WT B. ovis, suggesting that a putative hemagglutinin is not required for B. ovis pathogenesis. Additionally, the ΔabcAB strain did not survive in peritoneal macrophages, extracellularly in the peritoneal cavity, or in RAW 264.7 macrophages. Moreover, infection with the ΔabcAB strain was not lethal for male regulatory factor 1-knockout mice, whereas infection with the B. ovis WT strain was 100% lethal within 14 days postinfection. These results confirm that the predicted ABC transporter is required for the full virulence and survival of B. ovis in vivo.