Inhibition of murine colorectal cancer metastasis by targeting M2-TAM through STAT3/NF-kB/AKT signaling using macrophage 1-derived extracellular vesicles loaded with oxaliplatin, retinoic acid, and Libidibia ferrea.

Colorectal cancer is still unmanageable despite advances in target therapy. However, extracellular vesicles (EVs) have shown potential in nanomedicine as drug delivery systems, especially for modulating the immune cells in the tumor microenvironment (TME). In this study, M1 Macrophage EVs (M1EVs) were used as nanocarriers of oxaliplatin (M1EV1) associated with retinoic acid (M1EV2) and Libidibia ferrea (M1EV3), alone or in combination (M1EV4) to evaluate their antiproliferative and immunomodulatory potential on CT-26 and MC-38 colorectal cancer cell lines and prevent metastasis in mice of allograft and peritoneal colorectal cancer models. Tumors were evaluated by qRT-PCR and immunohistochemistry. The cell death profile and epithelial-mesenchymal transition process (EMT) were analyzed in vitro in colorectal cancer cell lines. Polarization of murine macrophages (RAW264.7 cells) was also carried out. M1EV2 and M1EV3 used alone or particularly M1EV4 downregulated the tumor progression by TME immunomodulation, leading to a decrease in primary tumor size and metastasis in the peritoneum, liver, and lungs. STAT3, NF-kB, and AKT were the major genes downregulated by of M1EV systems. Tumor-associated macrophages (TAMs) shifted from an M2 phenotype (CD163) to an M1 phenotype (CD68) reducing levels of IL-10, TGF-ß and CCL22. Furthermore, malignant cells showed overexpression of FADD, APAF-1, caspase-3, and E-cadherin, and decreased expression of MDR1, survivin, vimentin, and PD-L1 after treatment with systems of M1EVs. The study shows that EVs from M1 antitumor macrophages can transport drugs and enhance their immunomodulatory and antitumor activity by modulating pathways associated with cell proliferation, migration, survival, and drug resistance.

Molecular identification of Trypanosoma cruzi in domestic animals in municipalities of the State of Rio Grande do Norte, Brazil.

Trypanosoma cruzi, the etiologic agent of American trypanosomiasis, is a vector-borne zoonotic parasite which has been little studied regarding its infection in domestic animals. In this study, we evaluated the occurrence of natural infection by T. cruzi in farm animals using molecular markers and phylogenetic analysis in blood clot samples of 60 sheep (Ovis aires), 22 goats (Capra hircus), and 14 horses (Equus caballus) in eight municipalities located in an infection risk area in the state of Rio Grande do Norte (RN), Northeast Region of Brazil. Trypanosoma spp. infection was identified by amplifying the rRNA 18S SSU gene in 48.9% of the samples. The SH022 sample showed 99.8% similarity with the Y strain of T. cruzi in phylogeny, grouped in the DTU II clade. Blood clots of sheep, goats, and horses detected T. cruzi kDNA in 28.3% (17/60), 22.7% (5/22), and 15.4% (2/14) of the samples, respectively. These animals were distributed in the three studied mesoregions throughout the state of RN. The identification of natural infection in domestic animals contributes to expand the epidemiological transmission scenario in an area where T. brasiliensis is the main vector.

Trypanosoma cruzi circulating among dogs and triatomines in the endemic countryside of the State of Rio Grande do Norte, Brazil.

Vector transmission of Trypanosoma cruzi occurs in several areas of Brazil, including the northeastern region, and domestic animals can serve as reservoirs of the parasite. The aim of this study was to monitor dogs as domestic reservoirs for infection by T. cruzi, and the main triatomine species involved in parasite transmission in rural areas of municipalities in the State of Rio Grande do Norte, in northeastern, Brazil. Blood samples from dogs (n = 40) and manual triatomine capture were performed in domiciliary and peridomiciliary environments in rural areas of the towns of Acari, Caraúbas and Marcelino Vieira, between 2013 and 2016. Subsequently, infection of dogs was determined by Polymerase Chain Reaction (PCR), Enzyme-Linked Immunosorbent Assay (ELISA) for the detection of IgM and IgG isotypes and Indirect Immunofluorescence (IIF) reactions for detection of IgG. Triatomine infection was determined by PCR. Forty (16/40) percent of the dogs were seropositive for T. cruzi; 20.0% (8/40) of such reactivity indicated the acute phase, and 20.0% (8/40), the chronic phase. PCR was positive in 42.5% (17/40) of the dogs' blood samples. Specimens of Triatoma brasiliensis, Triatoma pseudomaculata, Rhodnius nasutus and Panstrongylus lutzi were found to be infected; however only T. brasiliensis nymphs and adults were infected in both environments. Triatomines evaluation showed 82.5% (94/114) of PCR positivity. Taken together, our results confirm that dogs are domestic reservoirs of T. cruzi in northeastern Brazil and T. brasiliensis is the main triatomine species correlated with parasite transmission in domiciliary environments. There is a continuing need to control peridomiciliary populations of triatomines and to implement continuous surveillance strategies for reservoirs with the help from the community.

Myocardial scars correlate with eletrocardiographic changes in chronic Trypanosoma cruzi infection for dogs treated with Benznidazole.

OBJECTIVES: The cardiac form of Chagas disease is evidenced by a progressive cardiac inflammation that leads to myocarditis, fibrosis and electrocardiographic (ECG) conduction abnormalities. Considering these characteristics, the aim of this study was to prospectively evaluate the early ECG changes in dogs that were experimentally inoculated with Benznidazole (Bz)-susceptibly (Berenice-78) and Bz-resistant (VL-10, and AAS) Trypanosoma cruzi strains and, later, evaluate the efficacy of Bz treatment for preventing these ECG alterations. METHODS: Electrocardiographic changes of treated and untreated animals were prospectively evaluated for up to 270 days after infection, at which point collagen (right atrium) quantification was performed. RESULTS: All infected dogs had a high intensity of heart fibrosis (4616.00 ± 1715.82 collagen/74931 µm(2) in dogs infected with Berenice-78 strain, 5839.2 ± 1423.49 collagen/74931 µm(2) in infected by AAS and 6294.40 ± 896.04 collagen/74931 µm(2) in animals infected with VL-10 strain), while 78.57% of all infected dogs showed ECG alterations. Bz Therapy reduced or prevented fibrosis in Bz-susceptible Berenice-78 (2813.00 ± 607.13 collagen/74931 µm(2) ) and Bz-resistant AAS strains (4024 ± 1272.44 collagen/74931 µm(2) ), coincident with only 10% de ECG alterations at 270 days. However, in those animals infected with a Bz-resistant VL-10 strain, specific treatment did not alter collagen deposition (6749.5 ± 1596.35 collagen/74931 µm(2) ) and there was first atrioventricular block and chamber overload at 120 and 270 days after infection, with 75% abnormal ECG exams. CONCLUSIONS: These findings indicate that an effective antiparasitic treatment in the early stage of Chagas disease can lead to a significant reduction in the frequency and severity of the parasite-induced cardiac disease, even if parasites are not completely eliminated.

Immunocompetent mice model for dengue virus infection [corrected].

Dengue fever is a noncontagious infectious disease caused by dengue virus (DENV). DENV belongs to the family Flaviviridae, genus Flavivirus, and is classified into four antigenically distinct serotypes: DENV-1, DENV-2, DENV-3, and DENV-4. The number of nations and people affected has increased steadily and today is considered the most widely spread arbovirus (arthropod-borne viral disease) in the world. The absence of an appropriate animal model for studying the disease has hindered the understanding of dengue pathogenesis. In our study, we have found that immunocompetent C57BL/6 mice infected intraperitoneally with DENV-1 presented some signs of dengue disease such as thrombocytopenia, spleen hemorrhage, liver damage, and increase in production of IFNγ and TNFα cytokines. Moreover, the animals became viremic and the virus was detected in several organs by real-time RT-PCR. Thus, this animal model could be used to study mechanism of dengue virus infection, to test antiviral drugs, as well as to evaluate candidate vaccines.

IL-17 produced during Trypanosoma cruzi infection plays a central role in regulating parasite-induced myocarditis.

BACKGROUND: Chagas disease is a neglected disease caused by the intracellular parasite Trypanosoma cruzi. Around 30% of the infected patients develop chronic cardiomyopathy or megasyndromes, which are high-cost morbid conditions. Immune response against myocardial self-antigens and exacerbated Th1 cytokine production has been associated with the pathogenesis of the disease. As IL-17 is involved in the pathogenesis of several autoimmune, inflammatory and infectious diseases, we investigated its role during the infection with T. cruzi. METHODOLOGY/PRINCIPAL FINDINGS: First, we detected significant amounts of CD4, CD8 and NK cells producing IL-17 after incubating live parasites with spleen cells from normal BALB/c mice. IL-17 is also produced in vivo by CD4(+), CD8(+) and NK cells from BALB/c mice on the early acute phase of infection. Treatment of infected mice with anti-mouse IL-17 mAb resulted in increased myocarditis, premature mortality, and decreased parasite load in the heart. IL-17 neutralization resulted in increased production of IL-12, IFN-gamma and TNF-alpha and enhanced specific type 1 chemokine and chemokine receptors expression. Moreover, the results showed that IL-17 regulates T-bet, RORgammat and STAT-3 expression in the heart, showing that IL-17 controls the differentiation of Th1 cells in infected mice. CONCLUSION/SIGNIFICANCE: These results show that IL-17 controls the resistance to T. cruzi infection in mice regulating the Th1 cells differentiation, cytokine and chemokine production and control parasite-induced myocarditis, regulating the influx of inflammatory cells to the heart tissue. Correlations between the levels of IL-17, the extent of myocardial destruction, and the evolution of cardiac disease could identify a clinical marker of disease progression and may help in the design of alternative therapies for the control of chronic morbidity of chagasic patients.

Benznidazole therapy during acute phase of Chagas disease reduces parasite load but does not prevent chronic cardiac lesions.

The goals of this study were to evaluate the efficacy of benznidazole (Bz) treatment in decreasing of the parasitic load during the acute phase of experimental Chagas disease and to analyze its influence in the development of cardiac chronic alterations in mice inoculated with drug-resistant Trypanosoma cruzi strains. Our results showed that the early Bz treatment (started at 4th day of infection) was efficient in reducing the parasite load in animals from both acute and chronic phase of the infection. Moreover, this reduction in the parasite load could not be associated with the intensity of the cardiac chronic lesions. The histopathological evaluation of cardiac tissue of Bz-treated mice showed three different patterns of response: (1) presence of a small number of inflammatory cells and fibrotic area similar to noninfected mice; (2) similar intensity of inflammatory infiltrate and smaller fibrotic area in relation to nontreated animals; (3) similar intensity of inflammatory infiltrated and fibrosis area among the Bz-treated and nontreated animals. Each specific pattern was obtained with different T. cruzi strain, suggesting that the pattern of the heart lesions in chronic phase of Bz-treated animals was T. cruzi strain dependent but not related with drug resistance levels.