Kátia da Silva Calabrese

A New Mouse Model Reveals a Critical Role for Host Innate Immunity in Resistance to Rift Valley Fever

Rift Valley fever (RVF) is an arthropod-borne viral disease repeatedly reported in many African countries and, more recently, in Saudi Arabia and Yemen. RVF virus (RVFV) primarily infects domesticated ruminants, resulting in miscarriage in pregnant females and death for newborns and young animals. It also has the ability to infect humans, causing a feverish syndrome, meningoencephalitis, or hemorrhagic fever. The various outcomes of RVFV infection in animals and humans argue for the existence of host genetic determinants controlling the disease. We investigated the susceptibility of inbred mouse strains to infection with the virulent RVFV ZH548 strain. Compared with classical BALB/cByJ mice, wild-derived Mus m. musculus MBT/Pas mice exhibited earlier and greater viremia and died sooner, a result in sharp contrast with their resistance to infection with West Nile virus and influenza A. Infection of mouse embryonic fibroblasts (MEFs) from MBT/Pas mice with RVFV also resulted in higher viral production. Microarray and quantitative RT-PCR experiments showed that BALB/cByJ MEFs displayed a significant activation of the type I IFN pathway. In contrast, MBT/Pas MEFs elicited a delayed and partial type I IFN response to RVFV infection. RNA interference-mediated inhibition of genes that were not induced by RVFV in MBT/Pas MEFs increased viral production in BALB/cByJ MEFs, thus demonstrating their functional importance in limiting viral replication. We conclude that the failure of MBT/Pas murine strain to induce, in due course, a complete innate immune response is instrumental in the selective susceptibility to RVF.

Canine Visceral Leishmaniosis in Anastácio, Mato Grosso do Sul State, Brazil

Canine visceral leishmaniosis (CVL) may be an important factor preceding human outbreaks of the disease. We report that the prevalence of canine visceral leishmaniosis infection has been increasing in recent years in Anastácio town, located in the central western region of Brazil. Serological investigations showed that 75.3% of dogs presented antibody titres ranging from 1/40 to 1/160 in the indirect immunofluorescence antibody test (IFAT). Bone marrow and lymph node aspirates provided positive cultures and furnished parasites for enzymological and serological typing in 42.5% and 41.1% of the cases, respectively. All the strains were typed asLeishmania (L.) chagasi. This is primarily a canine disease that spills over into the human population as a zoonosis. The study showed the epidemiological features of the infection in a region in which the problem of visceral leishmaniosis has been underestimated.

Immunopathological Studies of Leishmania amazonensis Infection in Resistant and in Susceptible Mice

Leishmania amazonensis infection was studied in mice to evaluate the evolution of leishmaniasis. The association of different methods, such as lesion kinetics, limiting dilution analysis, and immunohistochemistry, established different levels of susceptibility and resistance. Mice were arranged in 3 groups: susceptible (C57BL/10 and CBA), relatively resistant (DBA/2), and resistant (C3H.He). The histopathological analysis of primary lesions and draining lymph nodes showed a predominance of eosinophils and mast cells in the initial phase of infection in all mice. However, the most susceptible mice presented a greater number of amastigotes and higher tissue damage. The immunoglobulin analysis showed that susceptible mice produced high levels of antibodies, whereas resistant and relatively resistant mice exhibited low production of antibodies. Resistant mice showed parasite persistency in the skin and lymph nodes, suggesting that the infection in these mice can be sustained through the infection of cells such as dendritic cells, fibroblasts, and other cells present in these organs.

Histopathological analysis of initial cellular response in TLR-2 deficient mice experimentally infected by Leishmania (L.) amazonensis.

Tegumentary leishmaniasis is an important public health problem in several countries. The capacity of the Leishmania species, at the initial moments of the infection, to invade and survive inside the host cells involves the interaction of surface molecules that are crucial in determining the evolution of the disease. Using C57BL/6 wild‐type and TLR‐2−/− mice infected with L. (L.) amazonensis, we demonstrated that TLR‐2−/− mice presented eosinophilic granuloma in the ear dermis, different from C57BL/6 wild‐type mice that presented a cellular profile characterized mainly by mononuclear cell infiltrates, besides neutrophils and eosinophils, during the two first week of infection. When the parasite load was evaluated, we found that the absence of TLR‐2 lead to a significant reduction of the infection in deficient mice, when compared with C57BL/6 mice which were more susceptible to the infection. Using TLR‐2 deficient mice, it was possible to show that the absence of this receptor determined the reduction of the parasite load and the recruitment of inflammatory cells during the two first weeks after L. (L.) amazonensis infection.

Ocular toxoplasmosis: the role of retinal pigment epithelium migration in infection

AbstractOur aim was to study the migration of retinal pigmented epithelium (RPE) into the retinal layer during infection of C57BL/6 mice with Toxoplasma gondii. Eyes from infected and non-infected animals were analyzed on the 60th day of infection by light and transmission electron microscopy. Non-infected eyes showed a normal morphology. In contrast, we observed free parasites in the retinal vasculature, the presence of mononuclear inflammatory infiltrate (MNII) and parasites in the vasculature of choroids in infected eyes. No inflammatory infiltrate was observed; RPE cells were identified near the MNII in nuclear and plexiforme layers. RPE cells were also found on the ganglion cell layer and in the outer segments of the photoreceptor. The morphology showed that RPE cells caused a discontinuity in the nuclear and plexiforme layers. Clusters of parasites were found surrounded by RPE cells and MNII in the inner plexiforme layers. Ultrastructural analysis showed that RPE cells migrated through the epithelium into the inner retinal layers. We did not observe Toxoplasma cysts in many eyes in which pathological changes were detected. Only 8.3% of the animals had Toxoplasma cysts in the inner nuclear layer in the absence of inflammatory cells. The migration of RPE cells can be triggered by a disruption of the RPE monolayer or injury to the neural retina, as in the case of toxoplasmosis.

Hepatic extracellular matrix alterations in dogs naturally infected with Leishmania (Leishmania) chagasi.

The aim of this work was to study alterations in the extracellular matrix of liver in dogs naturally infected with Leishmania (Leishmania) chagasi that are correlated with clinical aspects and with histological, parasitological and immunological findings. The study was carried out on 30 dogs, 10 uninfected (control group) and 20 infected. The infected animals were further divided into two groups: an asymptomatic group of 10 dogs without clinical signs of the disease; and a symptomatic group of 10 dogs with classical clinical signs. All thirty animals were mongrel dogs of undefined age, obtained from the municipality of Belo Horizonte, MG, metropolitan area. During necropsy, liver fragments were collected and fixed in 10% buffered formaldehyde for histological examination. Paraffined sections of the tissues were stained with haematoxylin–eosin, Gomori’s ammoniacal silver stain for reticular fibres and strepto‐avidin peroxidase for immunohistochemical detection of Leishmania amastigotes. Frozen tissue sections were stained by immunofluorescence for fibronectin (FN) and laminin (LN). Liver collagen deposition was significantly greater in the infected than the control animals and differed significantly between the symptomatic and asymptomatic dogs. There was a positive correlation between the parasite load and liver collagen deposition. The increased collagen deposition in infected animal livers may be associated with the parasite burden. Adhesive FN and LN fibres were significantly more highly expressed in the livers of symptomatic than of asymptomatic dogs. Our results demonstrate that canine visceral leishmaniasis causes fibrogenesis in liver, associated with the parasite load and degenerative processes.

Enhancement of Leishmania amazonensis infection in BCG non-responder mice by BCG-antigen specific vaccine

Different patterns of cutaneous leishmaniasis can be induced when a challenge of alike dose of Leishmania amazonensis amastigotes in various inbred strains was applied. Two strains of mice, the Balb/c and C57BL/10J, showed exceptional susceptibility, and 10(6) amastigotes infective dose lead, to ulcerative progressive lesions with cutaneous metastasis and loss by necrosis of leg on which the footpad primary lesion occurred. Lesions were also progressive but in a lower degree when C3H/HeN and C57BL/6 were infected. Lesions progress slowly in DBA/2 mice presenting lesions which reach a discreet peak after 12 weeks, do not heal but do not ulcerate. DBA/2 mice is, therefore, a good model for immunomodulation. In attempt to determine the influence of BCG in vaccination schedule using microsomal fraction, DBA/2 became an excellent model, since it is also a non-responder to BCG. Vaccination of DBA/2 mice, receiving the same 10(6) BCG viable dose and 10 micrograms or 50 micrograms of protein content of microsomal fraction, lead to a progressive disease with time course similar to those observed in susceptible non-vaccinated C57BL/10J mice after 6 months of observation. An enhancement of infection in BCG non-responder mice suggests that use of BCG as immunostimulant in humans could be critical for both vaccination and immunoprophylactic strategies.

Serum and aqueous humour cytokine response and histopathological alterations during ocular Toxoplasma gondii infection in C57BL/6 mice.

Toxoplasma gondii, an obligate intracellular protozoan parasite, infects most species of warm-blooded animals, and in humans it causes toxoplasmosis. Healthy people that become infected rarely present clinical symptoms because the immune system prevents the parasite from causing illness. Congenital toxoplasmosis may result in abortion, hydrocephalus, as well as neurological and ocular disease (most frequently retinochoroiditis) of the newborn. In immunocompromised patients, reactivation of latent disease can cause encephalitis. Cell-mediated immunity to T. gondii antigens involves innate acute inflammatory responses and antigen-specific adaptive immunity. Considering the complexity of the immunological events triggered during toxoplasmosis, systemic and local responses were evaluated by cytokine measurements. Aqueous humour and serum were obtained from non-infected and T. gondii Me-49 strain infected C57BL/6 mice for cytokine quantification. Histopathological analyses were made with eyes enucleated from mice after 30 days of infection. ELISA assays showed an increase of IFN-gamma levels both in serum and aqueous humour of infected mice in opposition to a decrease in IL-10 levels. On the other hand, TGF-beta was high, whereas IL-12 and TNF-alpha were present in small levels in both groups. We also detected higher levels of IL-4 and IL-6 in aqueous humour than in serum of infected mice when compared to the control group. MIP-2 presented no significant differences between the two groups. Fas and Fas-L were also present in similar levels in serum of non-infected and infected mice, but both chemokines were increased in the aqueous humour of infected mice. Histopathological analysis of infected mice showed inflammatory infiltrates around blood vessels and alteration of the outer photoreceptor segments, on the external and inner nuclear layer. Parasites were observed in 82% of eyes, inside the blood vessels associated with inflammatory infiltrate. Edema, characterized by the increase of interstitial spaces between the FTR, forming lacunae was also noted. These alterations take the form of projections (retino-vitreal), characteristic of retinochoroiditis. In conclusion, T. gondii infection of C57BL/6 mice revealed that cytokine patterns alone do not assure susceptibility or resistance against infection, thus reinforcing the notion that it is necessary more than cytokine dosage to determine Th1 or Th2 profile in this model.

Ocular toxoplasmosis in mice: comparison of two routes of infection

This paper aims to test the influence of route of infection (intravitreal and instillation) on the course of ocular toxoplasmosis in mice, using the Toxoplasma gondii Me-49 strain. All mice inoculated intravitreally or by instillation presented the same pattern of infection. Using either route, parasites were observed in the retinal vessel with the formation of a glial reaction in the inner plexiforme layer and discontinuity of the pigmented epithelium of the retina 7 days after infection. However, when the intravitreal route was used a more intense inflammatory infiltrate was observed in the retina. The results suggest that inoculation route remarkably influences the inflammatory pattern in ocular toxoplasmosis and that the instillation route should be preferentially used in experimental infections in the murine ocular model of infection by T. gondii, specially with small animals where there is extensive needle damage, which is not observed in the instillation route.

In Vitro Antileishmanial Activity of Essential Oil of Vanillosmopsis arborea (Asteraceae) Baker

The search for new immunopharmacological chemical agents to treat various diseases caused by bacteria, fungi, and protozoa, such as leishmaniasis, for example, has led to the exploration of potential products from plant species and their main active ingredients. Antimonial drugs are the current treatment for leishmaniasis. These drugs cause major side effects and frequent discontinuation of treatment. In this study, we evaluated the in vitro leishmanicidal activity of essential oil of Vanillosmopsis arborea (VAEO) and its major compound α-bisabolol against Leishmania amazonensis. The essential oil and α-bisabolol showed activity against promastigotes (IC50 7.35 and 4.95 μg/mL resp.) and intracellular amastigotes (IC50 12.58 and 10.70 μg/mL, resp.). Neither product showed any cytotoxicity on treated macrophages. The ultrastructural analysis of promastigotes incubated with VAEO or α-bisabolol at 30 μg/mL, showed morphological changes with the accumulation of vesicles electrodense lipid inclusions. The results give evidence that both VAEO and α-bisabolol have potential as new therapeutic agents against leishmaniasis.