Graça Alexandre-Pires

Canine leishmaniosis. Immunophenotypic profile of leukocytes in different compartments of symptomatic, asymptomatic and treated dogs

Canine visceral leishmaniasis (CanL) is an emerging disease, expanding in various parts of the world. The infection caused by Leishmania, an intracellular protozoan parasite, can show different clinical manifestations, from asymptomatic or subclinical to symptomatic dogs, in which a wide spectrum of clinical signs is evident. The fact that the parasite replicates in different organs raises the hypothesis that each organ may have a specific immune response. The local immune responses should be evaluated and taken into consideration when developing prophylactic tools. Therefore, phenotypic characterization of peripheral blood, lymph node and bone marrow lymphocyte populations and the expression of class II molecules of major histocompatibility complex (MHCII) were performed in asymptomatic and symptomatic dogs and in dogs that had been diagnosed and treated for leishmaniasis. Our findings showed that blood and bone marrow lymphocytes from symptomatic dogs were highly activated. In bone marrow of asymptomatic and treated dogs, a high frequency of MHCII(+) lymphocytes was observed, as well as MHCII(+) monocytes in the treated group. These results show increased expression of MHCII molecules giving evidence for antigenic presentation mainly by lymphocytes. The symptomatic and treated dogs showed an expansion of CD4(+) T cells subpopulations in lymph nodes, revealing an important contribution of these cells in controlling local parasite replication. This study also underlines the eventual importance of CD3(+)CD4(-)CD8(-) (double negative) and CD3(+)CD4(+)CD8(+) (double positive) T cell subsets in sensing and controlling latent infections and their possible function in the immune dynamics during CanL. The specific cellular immune responses raised in different compartments where the parasite replicates seem to have variable effects on local parasite control, highlighting the complexity of the cellular immune response developed by the dog infected by Leishmania infantum.

Scavenger function of resident autofluorescent perivascular macrophages and their contribution to the maintenance of the blood-retinal barrier.

PURPOSE The retina contains two distinct populations of monocyte-derived cells: perivascular macrophages, and microglia. The present study was undertaken to evaluate the presence and function in mouse and human retinas of a subtype of resident perivascular macrophages with scavenger function, different from microglia, in physiological conditions and during retinopathy. METHODS Perivascular macrophages were characterized by means of confocal microscopy, electron microscopy, and flow cytometry analyses. Two murine models of blood-retinal barrier breakdown and photoreceptor degeneration were used to analyze the role of these macrophages during retinopathy. RESULTS The macrophages analyzed constituted a small population of resident perivascular cells different from microglia, since they were Iba-1 negative. Although these cells expressed F4/80 and CD11b antigens in common with microglia, they also expressed BM8 and MOMA-2 epitopes, which are macrophagic markers not expressed by microglia. Perivascular macrophages emitted autofluorescence due to cytoplasmic inclusions containing protein-bound oxidized lipids. They constitutively expressed the scavenger receptor class A and moved along blood vessels, providing an additional coating to thinner areas of the basement membrane. Moreover, they accumulated blood-borne horseradish peroxidase and acetylated low-density lipoprotein in healthy retinas. In addition, during blood-retinal barrier breakdown and photoreceptor degeneration, these cells migrated to the lesion site. CONCLUSIONS All these morphologic and functional features are consistent with those described for brain Mato cells. Thus, this study showed the presence of autofluorescent perivascular macrophages, different from microglia, with a scavenger function that may contribute to the maintenance of the blood-retinal barrier in healthy conditions and that are also involved in retinopathy.

Cytokine gene expression in the tissues of dogs infected by Leishmania infantum.

Canine leishmaniosis (CanL) caused by the protozoan parasite Leishmania infantum is a chronic systemic disease that is endemic in certain parts of the world. The domestic dog is the most important reservoir of L. infantum and is the main source of infection for other animals and for the human population. The aim of this study was to evaluate and compare the level of expression of genes encoding particular cytokines (interleukin [IL]-12, interferon [IFN]-γ, IL-2 and IL-4) in different tissues and organs of 53 adult dogs with or without clinical signs of leishmaniosis and after treatment for the disease. Asymptomatic dogs showed high expression of genes encoding IL-4 in blood leucocytes and of genes encoding IL-12 and IL-2 in lymph nodes. Blood leucocytes from symptomatic dogs had a mixed Th1 and Th2 cytokine gene expression profile, but lymph nodes from these animals had dominant IL-2 and IFN-γ gene expression, while bone marrow appeared to be unresponsive. The predominance of IL-4 gene expression in the blood of asymptomatic dogs may favour parasite replication, while the balance between Th1 and Th2 cytokine gene expression in the blood of symptomatic dogs may be important in reducing parasite replication and delaying the dissemination of Leishmania to other organs. The drugs used to treat CanL do not completely eliminate the parasite, so the high expression of the gene encoding IL-4 in blood leucocytes and the high expression of IL-12 and IL-4 mRNA in lymph nodes may reflect the persistence of residual Leishmania amastigotes. L. infantum appears able to regulate the host immune response in order to ensure its survival, but also to prevent the host from succumbing to infection. This guarantees its transmission and the completion of its life cycle.

Immunization with the Leishmania infantum recombinant cyclophilin protein 1 confers partial protection to subsequent parasite infection and generates specific memory T cells.

Control of zoonotic visceral leishmaniosis can be achieved using several available drugs. These drugs present high toxicity and require longer treatment regimens which complicate compliance to the treatment. Other control measures directed to the vector or the reservoirs are useful tools to restrain the spreading of this disease but the effects are transitory. A safe, affordable and efficient vaccine conferring long lasting immunity should be the most cost effective way of controlling zoonotic visceral leishmaniosis. The present study aims at characterizing a cyclophilin protein 1 of Leishmania infantum (LiCyP1) and investigating whether recombinant LiCyP1 (LirCyP1) is able to confer protection against infection by evaluating viable parasite load and the generation of specific CD4(+) and CD8(+) effector and central memory T cells in rodent model. LiCyP1 is present in the cytoplasm of L. infantum amastigotes and promastigotes. Immunization of BALB/c mice with LirCyP1 confers high protection to L. infantum infection, causing a marked reduction in parasite replication in the liver and spleen. Furthermore, helper and cytotoxic memory T cell subsets able to specifically recognize parasite antigens expanded in immunized and in challenged mice. CD4(+) T cell subpopulation of intermediate phenotype (CD62L(high)CD127(low)) of challenging mice also presented an accentuated expansion after the recall. This study demonstrated that LirCyP1 confers partial protection to L. infantum infection, promoting the generation of a desired long lasting immunity. LirCyP1 can be considered a potential candidate for the design of a vaccine against zoonotic visceral leishmaniosis.

Canine leishmaniosis. Modulation of macrophage/lymphocyte interactions by L. infantum

Canine leishmaniosis, caused by Leishmania infantum, is a systemic disease with variable clinical signs and a progressive evolution. This disease is characterized by impaired T cell-mediated immune response, which has been associated with disease chronicity and high mortality. Protective immunity against leishmaniosis is thought to be mediated by T cell and cytokine production. The T cell activation requires a primary signal delivered by the major histocompatibility complex (MHC) molecules present on the surface of antigen presenting cells, and a non-specific signal generated by co-stimulatory molecules. To characterize canine immune responses in the presence of L. infantum parasites or their antigens, in vitro cell cultures of canine macrophages and lymphocytes were established, and the macrophages presenting MHC class II molecules were evaluated as well as the expression of IL-12 and CD80-86 co-stimulatory molecules and nitric oxide production. The results showed for the first time the up-regulation of MHC class II molecules on the surface in canine peripheral blood monocyte-derived macrophages during L. infantum infection in the presence of lymphocytes. In addition, a lack of co-stimulatory expression and a reduced release of nitric oxide were observed, suggesting a loss of T cell function and consequently an inactivation of the macrophage oxidative burst which, in turn, favors the survival of Leishmania. These results constitute a new contribution for the understanding of the interactions between L. infantum and the canine immune system.

New insights into neutrophil and Leishmania infantum in vitro immune interactions

The interaction between polymorphonuclear leukocytes (PMN) or neutrophils and Leishmania became an interesting focus of research, since PMN turn out to be essential cells in transiently hosting the parasites. This study aims to evaluate whether L. infantum, the etiological agent of zoonotic visceral leishmaniasis, influences the in vitro functional activity of murine neutrophils. Phagocytosis, chemotaxis, oxidative burst, degranulation and apoptosis assays were performed. Cytokines, chemokines and toll-like receptors gene expression were evaluated by Real-time PCR. Results indicate that some of the innate features of PMN immunity were activated when in contact with L. infantum. However, parasites might negatively interfere with PMN defense mechanisms compromising the link between innate and acquired immunity. This work provides additional insights on the inflammatory immune interactions between neutrophils and L. infantum highlighting the role of PMN in Leishmania infection.

Seasonal Changes in Testes Vascularisation in the Domestic Cat (Felis domesticus): Evaluation of Microvasculature, Angiogenic Activity, and Endothelial Cell Expression

Some male seasonal breeders undergo testicular growth and regression throughout the year. The objective of this study was to understand the effect of seasonality on: (i) microvasculature of cat testes; (ii) angiogenic activity in testicular tissue in vitro; and (iii) testicular endothelial cells expression throughout the year. Testicular vascular areas increased in March and April, June and July, being the highest in November and December. Testes tissue differently stimulated in vitro angiogenic activity, according to seasonality, being more evident in February, and November and December. Even though CD143 expression was higher in December, smaller peaks were present in April and July. As changes in angiogenesis may play a role on testes vascular growth and regression during the breeding and non-breeding seasons, data suggest that testicular vascularisation in cats is increased in three photoperiod windows of time, November/December, March/April and June/July. This increase in testicular vascularisation might be related to higher seasonal sexual activity in cats, which is in agreement with the fact that most queens give birth at the beginning of the year, between May and July, and in September.

Leishmania infantum antigens modulate memory cell subsets of liver resident T lymphocyte

In the recent years, the liver has been recognized as an important immune organ with major regulatory functions and immune memory, adding to the well-described vital metabolic functions. There are evidences from experimental infections performed with visceral Leishmania species that immune responses to parasite infection can be organ-specific. The liver is the compartment of acute resolving infection, with minimal tissue damage and resistance to reinfection, whereas the spleen is the compartment of parasite persistence. Control of hepatic infection in mice requires a coordinated immune response that involves the development of inflammatory granulomas. It is also described that the liver harbors populations of resident lymphocytes, which may exhibit memory characteristics. Therefore, the present study aims to address the role of the liver as an immune memory organ in the context of Leishmania infantum infection, by characterizing phenotypically resident liver T lymphocytes. The dynamics of memory T cells in L. infantum infected BALB/c mice and the effect of anti-leishmanial treatment in the differentiation of memory cell subsets were analyzed. The potential of recognition, differentiation and selection of memory lymphocytes by three L. infantum recombinant proteins were also explored. L. infantum infection generates effector and central memory T cells, but the cells did not expand when recalled, demonstrating a possible parasite silencing effect. The treatment with a leishmanicidal drug (antimoniate meglumine) increases the levels of memory and effector T cells, eliciting a more robust hepatic immune response. L. infantum parasites with a decreased sensitivity to the leishmanicidal drug favor the expansion of memory CD8+ T cell subset, but inhibit the proliferation of CD8+ T effector cells, possibly assuring their own survival. The recombinant proteins LirCyp1 and LirSOD are strongly recognized by memory cells of treated mice, indicating that these proteins might be used in a prophylactic or therapeutic vaccine formulation. Thus, L. infantum released antigens induce the development of immune memory subsets in the liver resident T cell population that specifically recognized parasite antigens, including recombinant proteins.

Canine neutrophils activate effector mechanisms in response to Leishmania infantum

Canine leishmaniosis caused by L. infantum is a severe zoonotic disease. Although macrophages are the definitive host cells, neutrophils are the first cells to encounter the parasite soon after its inoculation in the dermis by the phlebotomine vector. To study the interaction of dog neutrophils and L. infantum promastigotes, blood neutrophils were isolated from healthy donors and the infection was established in vitro. In the majority of the dogs, L. infantum was efficiently phagocytized by neutrophils, and oxidative (superoxide production) and non-oxidative (neutrophil elastase exocytosis) intracellular effector mechanisms were activated, but the release of neutrophil extracellular traps was minimized. Furthermore, promastigotes and culture supernatants induced neutrophil migration, but the prior contact with Leishmania inhibits chemotaxis, which might contribute to neutrophil retention at the inoculation site. Neutrophil-parasite interaction resulted in a decrease in parasite viability, although some intracellular promastigotes survive and maintain their proliferative capacity. These findings indicate that dog neutrophils are competent effector cells able to control the initial L. infantum infection. However, some parasites evade intracellular effector mechanisms and can be transferred to the definitive host cell, the macrophage, contributing to the development of canine leishmaniosis.

Understanding the inguinal sinus in sheep (Ovis aries)—Morphology, secretion, and expression of progesterone, estrogens, and prolactin receptors

Post-parturient behavior of mammalian females is essential for early parent–offspring contact. After delivery, lambs need to ingest colostrum for obtaining the related immunological protection, and early interactions between the mother and the lamb are crucial. Despite visual and auditory cues, olfactory cues are decisive in lamb orientation to the mammary gland. In sheep, the inguinal sinus is located bilaterally near the mammary gland as a skin pouch (IGS) that presents a gland that secretes a strong-smelling wax. Sheep IGS gland functions have many aspects under evaluation. The objective of the present study was to evaluate sheep IGS gland functional aspects and mRNA transcription and the protein expression of several hormone receptors, such as progesterone receptor (PGR), estrogen receptor 1 (ESR1), and 2 (ESR2) and prolactin receptor (PRLR) present. In addition, another aim was to achieve information about IGS ultrastructure and chemical compounds produced in this gland. All hormone receptors evaluated show expression in IGS during the estrous cycle (follicular/luteal phases), pregnancy, and the post-partum period. IGS secretion is rich in triterpenoids that totally differ from the surrounding skin. They might be essential substances for the development of an olfactory preference of newborns to their mothers.