Lucio Ayres Caldas

Phosphatidylserine Exposure by Toxoplasma gondii Is Fundamental to Balance the Immune Response Granting Survival of the Parasite and of the Host

Phosphatidylserine (PS) exposure on the cell surface indicates apoptosis, but has also been related to evasion mechanisms of parasites, a concept known as apoptotic mimicry. Toxoplasma gondii mimics apoptotic cells by exposing PS, inducing secretion of TGF-beta1 by infected activated macrophages leading to degradation of inducible nitric oxide (NO) synthase, NO production inhibition and consequently persisting in these cells. Here PS+ and PS− subpopulation of tachyzoites were separated and the entrance mechanism, growth and NO inhibition in murine macrophages, and mice survival and pathology were analyzed. Infection index in resident macrophages was similar for both PS subpopulations but lower when compared to the total T. gondii population. Growth in resident macrophages was higher for the total T. gondii population, intermediate for the PS+ and lower for the PS− subpopulation. Production of NO by activated macrophages was inhibited after infection with the PS+ subpopulation and the total populations of tachyzoites. However, the PS− subpopulation was not able to inhibit NO production. PS+ subpopulation invaded macrophages by active penetration as indicated by tight-fitting vacuoles, but the PS− subpopulation entered macrophages by phagocytosis as suggested by loose-fitting vacuoles containing these tachyzoites. The entrance mechanism of both subpopulations was confirmed in a non-professional phagocytic cell line where only the PS+ tachyzoites were found inside these cells in tight-fitting vacuoles. Both subpopulations of T. gondii killed mice faster than the total population. Clear signs of inflammation and no tachyzoites were seen in the peritoneal cavity of mice infected with the PS− subpopulation. Moreover, mice infected with the PS+ subpopulation had no sign of inflammation and the parasite burden was intense. These results show that PS+ and PS− subpopulations of T. gondii are necessary for a successful toxoplasma infection indicating that both subpopulations are required to maintain the balance between inflammation and parasite growth.

Microscopic analysis of calcium ionophore activated egress of Toxoplasma gondii from the host cell.

Toxoplasma gondii invades and destroys nucleated cells of warm blooded hosts in a process which involves several steps: recognition, adhesion, penetration, multiplication inside a parasitophorous vacuole (PV) and egress. The last one is the least understood. Parasite egress from LLC-MK2 cells infected with the RH strain of T. gondii was artificially triggered with 4BrA23187 calcium ionophore. The combination of videomicroscopy, field emission scanning electron microscopy (FESEM), and transmission electron microscopy (TEM) showed that egress does not result from host cell rupture due to overloading with tachyzoites. Videomicroscopy showed that upon calcium ionophore administration parasite rosettes disassemble, the contour of the parasitophorous vacuole disappears and each tachyzoite takes a separate route to the extracellular medium. FESEM and TEM showed the fragmentation of the intravacuolar network, the fragmentation of parasitophorous vacuole membrane and individual tachyzoites with extruded conoids migrating through the cytosol, tightly surrounded by remnants of parasitophorous vacuole membrane or free in the cytosol. Both videomicroscopy and FESEM showed that a single parasite can cross the host cell membrane without disrupting it, while a large number of parasites, egressing simultaneously, rupture the membrane and the cell as a whole. These data suggest that invasion and egress share less similarities than previously believed.

Inactivation of classical swine fever virus: association of hydrostatic pressure and ultraviolet irradiation.

Reversible pressure-induced disassembly of several viruses has suggested the idea of using hydrostatic pressure to suppress virus infectivity. In this study, the effects of high hydrostatic pressure and ultraviolet (UV) irradiation were investigated on classical swine fever virus (CSFV) in an attempt to eliminate residual infectivity. The structural modifications were followed by intrinsic fluorescence and biological activity assays. The kinetics of CSFV inactivation showed that pressure-induced inactivation was not enough to eliminate viral infectivity. However, when pressure was applied in association with UV irradiation no infectious focus was observed. The application of these two methods against CSFV can be an attractive inactivation strategy for the development of a vaccine.

Dynamin inhibitor impairs Toxoplasma gondii invasion

The protozoan parasite Toxoplasma gondii infects its host cells through an active mechanism. In this work, we obtained evidence that host cells also play a fundamental role during the infection process. We found that previous incubation of the host cells, but not the parasites, with Dynasore, a small molecule that inhibits dynamin GTPase activity, markedly reduced the penetration of T. gondii tachyzoites into LLC-MK2 cells. In contrast, parasite adhesion to the host cell surface increased, as observed both by light and electron microscopy. Intriguingly, the few parasites internalized by Dynasore-treated cells remained in vacuoles located at the periphery of the cell, in contrast to the perinuclear localization seen in the control.

The effect of kinase, actin, myosin and dynamin inhibitors on host cell egress by Toxoplasma gondii.

Toxoplasma gondii is a protozoan parasite that can infect the nucleated cells of all warm-blooded animals. Despite its medical and veterinary importance, the egress of T. gondii from host cells has not been fully elucidated. This process is usually studied with calcium ionophores, which artificially trigger T. gondii egress. Among the diverse signaling events that take place during egress, kinases appear to play a crucial role. In this work we employed several kinase inhibitors to examine their role in egress: although parasite egress was only slightly impaired by treatment with the PI3K and PKC inhibitors wortmannin and staurosporine, the addition of the tyrosine kinase-specific inhibitor genistein efficiently blocked the exit of parasites by more than 50%. IPA-3, a non-ATP-competitive inhibitor of p21-activated kinases, which play a role in actin cytoskeleton remodeling inhibited egress of T. gondii by only 15%. The myosin motor inhibitor blebbistatin and the actin polymerization inhibitor cytochalasin D also blocked the egress of T. gondii. Nevertheless, dynasore, which is known to block the GTPase activity of dynamin, had little or no effect on T. gondii egress.

Classical Swine Fever in Brazil: study for the survey of classical swine fever outbreaks in Brazil from 1978 to 2004

Os programas oficiais para o controle e erradicacao de pestes suinas forneceram uma oportunidade de levantar o perfil de ocorrencia da Peste Suina Classica (PSC). Independente das estrategias aplicadas durante 26 anos foi demonstrado que o numero de surtos de PSC de 1978 ate 2004 caiu drasticamente em todo pais, especialmente nos quatorze Estados inclusos na “Zona Livre de PSC”. O estudo comparou o numero de surtos de PSC durante a vigencia do Programa de Combate as Pestes Suinas (PCPS) de 1984 a 1991 e o Programa de Controle e Erradicacao da PSC (PCEPSC) de 1992 a 2004. Considerando a evolucao tecnologica nos sistemas de producao de suinos, a diferenca nos resultados obtidos apos a implementacao de cada programa foi avaliada pelo teste estatistico Mann Whitney por meio da ordenacao do numero de surtos ocorridos. Essa analise demonstrou uma diferenca significativa (p

Prostaglandin A1 Inhibits Replication of Classical Swine Fever Virus

Prostaglandins (Pgs) have been shown to inhibit the replication of several DNA and RNA viruses. Here we report the effect of prostaglandin (PgA1) on the multiplication of a positive strand RNA virus, Classical Swine Fever Virus (CSFV) in PK15 cells. PgA1 was found to inhibit the multiplication of CSFV. At a concentration of 5 micrograms/ml, which was nontoxic to the cells, PgA1 inhibitis virus production in 99%. In PgA1 treated cells the size and number of characteristic Classical Swine Fever focus decreased in amount.

Thieno[2,3-b]pyridine derivatives: a new class of antiviral drugs against Mayaro virus

Mayaro virus (MAYV) is an arthropod-borne virus and a member of the family Togaviridae, genus Alphavirus. Its infection leads to an acute illness accompanied by long-lasting arthralgia. To date, there are no antiviral drugs or vaccines against infection with MAYV and resources for the prevention or treatment of other alphaviruses are very limited. MAYV has served as a model to study the antiviral potential of several substances on alphavirus replication. In this work we evaluated the antiviral effect of seven new derivatives of thieno[2,3-b]pyridine against MAYV replication in a mammalian cell line. All derivatives were able to reduce viral production effectively at concentrations that were non-toxic for Vero cells. Molecular modeling assays predicted low toxicity risk and good oral bioavailability of the substances in humans. One of the molecules, selected for further study, demonstrated a strong anti-MAYV effect at early stages of replication, as it protected pre-treated cells and also during the late stages, affecting virus morphogenesis. This study is the first to demonstrate the antiviral effect of thienopyridine derivatives on MAYV replication in vitro, suggesting the potential application of these substances as antiviral molecules against alphaviruses. Additional in vivo research will be needed to expand the putative therapeutic applications.

Prostaglandin A1 triggers Mayaro virus inhibition and heat shock protein 70 expression in an epithelial cell model

INTRODUCTION The Mayaro virus (MAYV), which is an arbovirus closely related to the Chikungunya virus, causes a dengue-like acute illness that is endemic to Central and South America. We investigated the anti-MAYV activity of prostaglandin A1 (PGA1), a hormone which exhibits antiviral activity against both ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) viruses. Further, we examined the effects of inducting the stress protein HSP70 following PGA1 treatment. METHODS Hep-2 cells infected with MAYV were treated with PGA1 (0.1-6μg/ml) 12h before infection and for different periods post-infection. Inhibition of viral replication inhibition was analyzed via viral titer determination, whereas the effect of PGA1 on viral morphogenesis was examined via transmission electron microscopy (TEM). Autoradiography (with 35S methionine labeling) and western blotting were used to assess the effect of PGA1 treatment on viral and cellular protein synthesis, and on HSP70 induction, respectively. RESULTS PGA1 strongly reduced viral replication in Hep-2 cells, particularly when added during the early stages of viral replication. Although PGA1 treatment inhibited viral replication by 95% at 24 hours post-infection (hpi), viral structural protein synthesis was inhibited only by 15%. TEM analysis suggested that PGA1 inhibited replication before viral morphogenesis. Western blot and densitometry analyses showed that PGA1 treatment increased HSP70 protein levels, although this was not detectable via autoradiography. CONCLUSIONS PGA1 inhibits MAYV replication in Hep-2 cells at early stages of viral replication, prior to production of viral structural proteins, possibly via HSP70 induction.

A Window to Toxoplasma gondii Egress

The Toxoplasma gondii cellular cycle has been widely studied in many lifecycle stages; however, the egress event still is poorly understood even though different types of molecules were shown to be involved. Assuming that there is no purpose or intentionality in biological phenomena, there is no such question as “Why does the parasite leaves the host cell”, but “Under what conditions and how?”. In this review we aimed to summarize current knowledge concerning T. gondii egress physiology (signalling pathways), structures, and route.