Claudio Bruno Silva de Oliveira

Comparative Study on the Antioxidant and Anti-Toxoplasma Activities of Vanillin and Its Resorcinarene Derivative

A resorcinarene derivative of vanillin, resvan, was synthesized and characterized by spectroscopic techniques. We measured the cytotoxicity (in vivo and in vitro), antioxidant and anti-Toxoplasma activities of vanillin and the resorcinarene compound. Here we show that vanillin has a dose-dependent behavior with IC50 of 645 µg/mL through an in vitro cytotoxicity assay. However, we could not observe any cytotoxic response at higher concentrations of resvan (IC50 > 2,000 µg/mL). The in vivo acute toxicity assays of vanillin and resvan exhibited a significant safety margin indicated by a lack of systemic and behavioral toxicity up to 300 mg/kg during the first 30 min, 24 h or 14 days after administration. The obtained derivative showed greater antioxidative activity (84.9%) when comparing to vanillin (19.4%) at 1,000 μg/mL. In addition, vanillin presents anti-Toxoplasma activity, while resvan does not show that feature. Our findings suggest that this particular derivative has an efficient antioxidant activity and a negligible cytotoxic effect, making it a potential target for further biological investigations.

Epidemiological and serological profiles of ocular toxoplasmosis in the municipality of Natal, northeastern Brazil

BACKGROUND Toxoplasma gondii is the main culprit in most cases of infectious uveitis, in both acute and recurrent cases of congenital toxoplasmosis and acquired infections. METHODS The ocular toxoplasmosis was evaluated in patients at the the reference unit in ophthalmology, in Rio Grande do Norte State, determining the risk factors, and the epidemic, serological and clinical profiles. The production of IgM and IgG antibodies to T. gondii was evaluated by microparticle enzyme immunoassay (MEIA). The same patients diagnosed with fundoscopic alterations have been subjected to the fundus photography procedure. RESULTS Of the 116 patients with positive serology, 66 patients had bilateral ocular damage and 38 patients showed a higher frequency of lesions of type I. The epidemiological investigation showed that direct contact with cats, the consumption of raw or undercooked meat and direct contact with soil are factors not related to ocular toxoplasmosis development. The characterization of the sample was significant for patients aged 31-40 years. CONCLUSIONS Ocular toxoplasmosis is widely distributed in Natal and other cities in Rio Grande do Norte state, with special relevance for bilateral lesions in 56.9% of the patients assessed, the most frequent being type I with intraocular disposition in the macula.

Pathogenicity and phenotypic sulfadiazine resistance of Toxoplasma gondii isolates obtained from livestock in northeastern Brazil

Toxoplasma gondii is the causative protozoan agent of toxoplasmosis, which is a common infection that is widely distributed worldwide. Studies revealed stronger clonal strains in North America and Europe and genetic diversity in South American strains. Our study aimed to differentiate the pathogenicity and sulfadiazine resistance of three T. gondiiisolates obtained from livestock intended for human consumption. The cytopathic effects of the T. gondii isolates were evaluated. The pathogenicity was determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) using a CS3 marker and in a rodent model in vivo. Phenotypic sulfadiazine resistance was measured using a kinetic curve of drug activity in Swiss mice. IgM and IgG were measured by ELISA, and the dihydropteroate synthase (DHPS) gene sequence was analysed. The cytopathic effects and the PCR-RFLP profiles from chickens indicated a different infection source. The Ck3 isolate displayed more cytopathic effects in vitro than the Ck2 and ME49 strains. Additionally, the Ck2 isolate induced a differential humoral immune response compared to ME49. The Ck3 and Pg1 isolates, but not the Ck2 isolate, showed sulfadiazine resistance in the sensitivity assay. We did not find any DHPS gene polymorphisms in the mouse samples. These atypical pathogenicity and sulfadiazine resistance profiles were not previously reported and served as a warning to local health authorities.

An alternative nested-PCR assay for the detection of Toxoplasma gondii strains based on GRA7 gene sequences.

Toxoplasma gondii is a widespread parasite able to infect virtually any nucleated cells of warm-blooded hosts. In some cases, T. gondii detection using already developed PCR primers can be inefficient in routine laboratory tests, especially to detect atypical strains. Here we report a new nested-PCR protocol able to detect virtually all T. gondii isolates. Analyzing 685 sequences available in GenBank, we determine that GRA7 is one of the most conserved genes of T. gondii genome. Based on an alignment of 85 GRA7 sequences new primer sets that anneal in the highly conserved regions of this gene were designed. The new GRA7 nested-PCR assay providing sensitivity and specificity equal to or greater than the gold standard PCR assays for T. gondii detection, that amplify the B1 sequence or the repetitive 529bp element.

THE INFLUENCE OF SALT INTAKE ON THE COURSE OF EXPERIMENTAL TOXOPLASMOSIS IN OUTBRED OR INBRED MOUSE STRAINS

Many environmental factors contribute to an effective immune response against Toxoplasma gondii (Tg) infection, among which diet is important in triggering the immune response of the host to infection. Emerging reports suggest that salt intake undermines the regulatory mechanisms mediated by innate and adaptive immune cells. Unfortunately, the impact of an Intermediate Salt Diet (ISD) on the pathogenesis and immune response to toxoplasmosis remains unclear. The purpose of this study was to evaluate the susceptibility profile to an ISD (NaCl 2%) of two mouse strains (outbred Swiss and inbred C57BL6) infected by the ME49 strain of Tg. Our data confirm an antagonistic susceptibility to oral Tg infection among the two mouse strains. Sodium intake induced the highest mortality in C57BL6 compared to Swiss mice in the infected groups. A simultaneous ISD with the infection did not induce significant differences in body weight in either mouse strains. Both mouse strains showed an antagonistic response to a sodium intake diet on the number of parasite brain cysts. An increased number of brain cysts in C57BL6 ISD-Tg animals were noted while Swiss ISD-Tg animals presented a decrease in the number of brain cysts compared to NSD-Tg (Normal Salt Diet) for both mouse strains. Furthermore, sodium intake caused a significant reduction in the specific humoral immune response against Tg in inbred C57BL6 mice. Thus, our data reveal that an ISD affects the humoral immune response in the murine model and influences the course of Tg infection.

Polymorphisms in Toxoplasma gondii: role of atypical strains in unusual clinical manifestations of toxoplasmosis

Dear Editor, I recently had the opportunity to read the article entitled “An atypical Toxoplasma gondii genotype in a rural Brazilian dog co-infected with Leishmania (Viannia) braziliensis,” an article published in the Journal of the Brazilian Society of Tropical Medicine by Silva et al.1. I think that it is of great importance that the authors researched and sought possible polymorphisms in parasites isolated from animals. In such cases, it is common to note that these parasites are often also found in humans2. I consider it great because for a long time it was thought that Toxoplasma gondii typically had a clonal lineage, with four main groups scattered mainly in Europe and North America3,4. Recently, several studies have shown that in South America, especially Brazil, there are a considerable number of parasite isolates with atypical features compared to those observed in Europe and North America2,5. Much of this research has been based on the isolation of the parasite from livestock or even pets. This fact, apparently, is not surprising, since these animals live in close proximity to humans, and these farm animals are often consumed without taking the necessary precautions to ensure the destruction of parasite cysts in the meat of infected animals. In the State of Rio Grande do Norte, Brazil, I have observed with great concern the emergence of unusual toxoplasmosis profiles. In a study published in 20146, it was observed that patients from an eye clinic, who were diagnosed with ocular toxoplasmosis had a higher incidence of bilateral ocular lesions, which is a deviation from its classic unilateral ocular presentation. It is also of great concern to note that many of these patients are asymptomatic and associated with congenital infection, which increases the concern about other forms of damage that may arise in patients if diagnosed late. These different clinical patterns generate many questions. For instance, does this happen because of the different populations of the parasites, or are they due to characteristics of the hosts? Recently, in order to seek clarification to some of these questions in this state, some studies have been developed in order to characterize genetically distinct parasite populations circulating in livestock5,7. In these studies, we observed that there is a great genetic variability within protozoan populations in this region. From evaluations performed in a murine model, it was verified that these parasites have different levels of pathogenicity and resistance to standard treatment. Parasites with genetic profiles similar to those observed here were also found in Fernando de Noronha Island and the State of Rondônia, which is almost 5,000 kilometers away, thus demonstrating the great capacity of the parasite to spread to distant regions5. Therefore, there is the need to urgently establish some clinical correlation in this regard, including the locally prevalent strains of the parasite, as well as those in different parts of the country. This information will be critical in high-risk groups, including pregnant women and immunosuppressed patients, who typically experience more severe clinical manifestations of toxoplasmosis. Thus, I reiterate that studies of this kind are essential for broadening our knowledge of this disease in different regions. These studies provide information on parasite biology, which together with the clinical manifestations and characteristics within each region, give clarity about this disease, especially during atypical presentations in high-risk populations. Moreover, I also propose that health authorities prioritize the early diagnosis of the disease in order to reduce the problems associated with congenital toxoplasmosis in asymptomatic cases in higher risk areas.

Anti-Toxoplasma Activity of Estragole and Thymol in Murine Models of Congenital and Noncongenital Toxoplasmosis

Abstract Toxoplasmosis is caused by Toxoplasma gondii, an obligatory intracellular protozoan. Normally benign, T. gondii infections can cause devastating disease in immunosuppressed patients and through congenital infection of newborn babies. Few prophylactic and therapeutic drugs are available to treat these infections. The goal of the present study was to assess the anti-Toxoplasma effects in a congenital and noncongenital model of toxoplasmosis (using ME49 strain), besides assessing immunological changes, in vitro cytotoxicity, and in vivo acute toxicity of commercial estragole and thymol. The congenital experimental model was used with intermediate stages of maternal infection. The serum levels of immunoglobulin (Ig)M, IgG, interleukin (IL)-10, IL-12, and interferon-gamma (IFN-γ) were quantified from infected and treated C57Bl/6 mice. Estragole and thymol respectively exhibited low to moderate in vivo toxicity and cytotoxicity. Animals treated with estragole showed high IFN-γ and strong type 1 helper T cell response. Both compounds were active against T. gondii ME49 strain. Furthermore, orally administered estragole in infected pregnant mice improved the weight of offspring compared with untreated controls. Subcutaneous administration of both compounds also increased the weight of mouse offspring born to infected mothers, compared with untreated controls. Estragole and thymol display important anti-Toxoplasma activity. Further studies are needed to elucidate the mechanism of action of these compounds.