miR-148a regulation interferes in inflammatory cytokine and parasitic load in canine leishmaniasis.

Canine leishmaniasis (CanL) is a severe public health threat. Infected animals mediate transmission of the Leishmania protozoan to humans via the sandfly's bite during a blood meal. CanL progression depends on the degree of suppression of the immune response, possibly associated with microRNAs (miR), which can modulate mRNA translation into proteins and (consequently) regulate cell function. Increased miR-148a in splenic leukocytes (SL) of dogs with CanL was observed in previous studies, and in silico analysis, identified possible pathways involved in immune response regulation that are affected by this miR. Therefore, we evaluated the involvement of miR-148a in the regulation of TNF-α, IL-6, IL-12, IL-1ß, iNOS, MHCII, CD80, CD3, T-bet, and GATA-3 transcription factors and their relationship with parasite load in SL of dogs with CanL. Splenic leukocytes obtained from healthy and diseased dogs were transfected with miR-148a mimic and inhibitor oligonucleotides. After 48 hours, expression levels of MHCII, CD80, iNOS, CD3, T-bet, and GATA-3 were evaluated by flow cytometry, and concentrations of TNF-α, IL-12, IL-6, and IL-1ß were measured in culture supernatants by capture enzyme-linked immunosorbent assays. Transfection of SL with miR-148a mimics decreased iNOS levels in cells and TNF-α, IL-6, and IL-12 in the supernatants of cultured SL from CanL dogs. Interestingly, transfection with miR-148a inhibitor decreased parasite load in SL cells. These results suggest a direct or not regulatory role of this miR in the immune response to Leishmania infantum infection. We conclude that miR-148a can modulate immune responses by regulating inflammatory cytokines during CanL. Our results contribute to understanding the complex host/parasite interaction in CanL and could assist the development of treatments.

Regulatory effect of PGE2 on microbicidal activity and inflammatory cytokines in canine leishmaniasis.

Canine leishmaniasis (CanL) is caused by the intracellular parasite Leishmania infantum. Prostaglandin E2 (PGE2 ) exerts potent regulatory effects on the immune system in experimental model Leishmania infection, but this influence has not yet been studied in CanL. In this study, PGE2 and PGE2 receptor levels and the regulatory effect of PGE2 on arginase activity, NO2 , IL-10, IL-17, IFN-γ, TNF-α and parasite load were evaluated in cultures of splenic leucocytes obtained from dogs with CanL in the presence of agonists and inhibitors. Our results showed that splenic leucocytes from dogs with CanL had lower EP2 receptor levels than those of splenic leucocytes from healthy animals. We observed that NO2 levels decreased when the cells were treated with a PGE2 receptor agonist (EP1/EP2/EP3) or COX-2 inhibitor (NS-398) and that TNF-α, IL-17 and IFN-γ cytokine levels decreased when the cells were treated with a PGE2 receptor agonist (EP2) or PGE2 itself. The parasite load in splenic leucocyte cell cultures from dogs with CanL decreased after stimulation of the cells with PGE2 . We conclude that Leishmania infection of dogs modulates PGE2 receptors and speculate that the binding of PGE2 to its receptors may activate the microbicidal capacity of cells.

The effects of increased heme oxygenase-1 on the lymphoproliferative response in dogs with visceral leishmaniasis.

Canine visceral leishmaniasis (CVL) is known to affect the cellular immunity of infected dogs, through impairing lymphoproliferation and microbicidal mechanisms. This study examined heme oxygenase-1 (HO-1) and its metabolites, oxidative stress and IL-10 levels in CVL and investigated correlations between these parameters. Additionally, the effects of HO-1 inhibition on the lymphoproliferative response and cytokine production in lymph node cells (LNCs) from infected dogs were evaluated. Forty-four dogs, 24 controls and 20 dogs with CVL were selected. Plasma and splenic levels of HO-1, haptoglobin, soluble CD163 receptor, ferritin and IL-10 were determined using capture ELISA. The HO-1 levels and relative gene expression in peripheral blood and bone marrow mononuclear cells were also determined. LNCs proliferation was evaluated with an HO-1 activator and with an HO-1 inhibitor, in the presence of the Leishmania infantum soluble antigen (SAgL), using flow cytometry. HO-1, IL-2, IFN-gamma and IL-10 were also determined in these cultures using capture ELISA. Infected dogs presented oxidative stress and increased HO-1 levels and relative gene expression, with correlation between oxidative stress and HO-1. The substances from heme metabolism and IL-10 were also elevated in the plasma and spleens of infected dogs. IL-10 and HO-1 levels were positively correlated with one another. Inhibition of HO-1 increased LNCs proliferation and decreased IL-10 and IL-2 production in the presence of SAgL. The increased HO-1 metabolism observed in CVL is probably associated with oxidative stress and increased IL-10, which could be one of the mechanisms responsible for inhibition of the lymphoproliferative response in sick dogs.

Alteration of sFAS and sFAS ligand expression during canine visceral leishmaniosis.

Visceral leishmaniosis (VL) is caused by intracellular parasites of the genus Leishmania that affect humans and several animal species. Dogs are one of the main urban reservoirs of Leishmania infantum and play a central role in the transmission cycle to humans via sandflies. CD3+ cells apoptosis is involved in the immune response in VL. Dysregulation of apoptosis has been implicated in various disease states. An important regulator of apoptosis is the FAS-FAS-associated death domain protein (cluster of differentiation 95 - CD95) and FASL-FAS ligand protein (cluster of differentiation 178 - CD178) system involved in the down-regulation of immune reactions and in T cell-mediated cytotoxicity. FAS is a member of the tumor necrosis factor (TNF) receptor super family, which can be expressed in transmembrane or soluble forms. The soluble levels of FAS (sFAS), FASL (sFASL) and active Caspase-3, this last related to apoptotic cascade, were investigated in the spleen of 19 symptomatic dogs presenting moderate VL and 6 healthy dogs, determined by ELISA assay. The splenic parasite load was determined by real-time PCR monitoring of amplification of the intergenic internal transcribed spacer (ITS1) gene of parasite rRNA. sFAS levels were lower (p<0.05). sFASL and active Caspase-3 levels were higher (p<0.05) in dogs with VL compared with controls. Negative correlation was observed between parasite burden and sFASL levels. The increase in sFASL could be related to the mechanism involved in the elimination of the parasite.

CD95 (FAS) and CD178 (FASL) induce the apoptosis of CD4+ and CD8+ cells isolated from the peripheral blood and spleen of dogs naturally infected with Leishmania spp.

Infected dogs are urban reservoirs of Leishmania chagasi, which is a causative agent of visceral leishmaniasis (VL). Dogs exhibit immune suppression during the course of this disease, and lymphocyte apoptosis is involved in this process. To investigate apoptosis and the expression levels of FAS-FAS-associated death domain protein (CD95 or APO-1), FASL-FAS ligand protein (CD178), and TRAIL-TNF-related apoptosis-inducing ligand (CD253) receptors in peripheral blood mononuclear cells and spleen leukocytes from 38 symptomatic dogs with moderate VL and 25 healthy dogs were evaluated by flow cytometry. The apoptosis rate of blood and splenic CD4+ and CD8+ cells was higher in infected dogs than in healthy dogs. The expression levels of FAS and FASL in blood and splenic CD4+ cells were lower in infected dogs than in healthy dogs. FAS expression in CD8+ cells was higher in infected dogs than in healthy dogs; in contrast, FASL expression was lower in infected dogs. The expression of the TRAIL receptor increased only in splenic CD8+ cells from infected dogs. The FAS and FAS-L blocking antibodies confirmed the importance of these receptors in apoptosis. Our results enhance the current understanding of the immune response in dogs infected with L. chagasi, facilitating the future development of therapeutic interventions to reduce lymphocyte depletion.

Repair of lamellar scleral lesions in dogs with preserved equine renal capsule: short report

PURPOSE: To evaluate the use of equine renal capsule preserved in glycerin to repair lamellar scleral lesions in dogs. METHODS: Twelve healthy mongrel dogs, male and female, weighing 12 kg were used. The study was both clinical and morphological, and performed on the first, third, seventh, 15th 30th and 60th day after surgery. Temporal canthotomy was performed after standard preoperative and general anesthesia. Conjunctival and sclera square incisions of 0.5x0.5 cm were carried out in a one o' clock position, near the limbus. A fragment of hydrated biological prosthesis, of the saro shape, was sutured with 7-0 Vicryl® in an interrupted suture. RESULTS: The clinical evaluation showed blepharospasm/photophobia until the 7th day after surgery. Conjunctival edema appeared up to the 5th day after surgery. Mucoid ocular discharge was sustained until the 10th day after surgery. Hyperemia was observed until the end of the evaluation period. There were no signs of graft extrusion in all animals. The anterior and posterior segmente did not show clinical signs of inflammation. The optical microscopy mor phological evaluation showed an inflammatory exudation with acute aspects in the early and intermediate periods, and inflammatory exudation with chronic aspects in the late periods. There was incorporation of the implant by the recipient's sclera. CONCLUSION: These results suggest that the equine renal preserved capsule could be a useful alternative tissue to repair lamellar corneal lesions in dogs and humans.